Biologically Active Peptides: From Basic Science to Applications for Human Health 0128213892, 9780128213896

Table of contents :
Front Cover
Biologically Active Peptides
Copyright Page
Contents
List of contributors
Preface
1 Bioactive peptides in health and disease: an overview
1.1 Introduction
1.2 Preparation of bioactive peptides
1.3 Absorption of peptides in the small intestine
1.3.1 Paracellular transport
1.3.2 Transcellular transport
1.3.3 Absorption of peptides in the large intestine (colon)
1.3.4 Approaches for enhancing the absorption of peptides
1.3.5 Structure-activity relationship of bioactive peptides
1.4 Bioactivities of food-derived bioactive peptides focusing on inhibiting chronic diseases
1.4.1 Anticancer activity
1.4.2 Anti-inflammatory effect
1.4.3 Antimicrobial activity
1.4.4 Antihypertensive effect
1.4.5 Immunomodulatory peptides
1.4.6 Antidiabetic effect
1.5 Conclusion
References
2 Enzymatic mechanisms for the generation of bioactive peptides
2.1 Introduction
2.1.1 Enzymatic mechanisms in the hydrolysis of food proteins
2.1.2 Bioactive peptides generated during food processing
2.1.3 Bioactive peptides generated through the hydrolysis of proteins with commercial peptidases
2.2 Degree of hydrolysis
2.2.1 Definition
2.2.2 Precursor techniques and alternative methods/procedures
2.3 Assay of endopeptidase activity
2.3.1 Definition
2.3.1.1 Materials, equipment, and reagents
2.3.1.2 Protocol
2.3.1.3 Analysis
2.3.1.4 Alternative methods/procedures
2.4 Assay of exopeptidase activity
2.4.1 Definition
2.4.2 Materials, equipment, and reagents
2.4.3 Protocol
2.4.3.1 Analysis
2.4.3.2 Alternative methods/procedures
2.4.4 Pros and cons
2.4.5 Summary
References
3 Novel technologies in bioactive peptides production and stability
3.1 Introduction
3.2 Expression of recombinant peptides
3.2.1 Escherichia coli expression vectors and strains for recombinant protein production
3.3 Stability of proteins and peptides
3.4 Definition: production of recombinant bioactive peptides in Escherichia coli
3.4.1 Antihypertensive peptides
3.4.2 Antiangiogenic peptides
3.5 Protocol
3.5.1 Antihypertensive cassette design
3.5.2 Amplification of the encrypted vasoinhibin peptide
3.5.3 DNA cloning into a suitable vector
3.5.3.1 Fragment amplification by PCR and purification of PCR product
3.5.3.2 Ligation of amplified fragments by PCR into transient vectors
3.5.4 Transformation of the host cells
3.5.4.1 Competent cells preparation
3.5.4.2 Transformation
3.5.4.3 Preparation of plasmid DNA
3.5.4.4 Fragment restriction and ligation into expression vector
3.5.5 Induction of the expression of the desired protein under controlled conditions
3.5.6 Recovery and purification of the recombinant product
3.5.7 Preparation and encapsulation of recombinant peptides
3.6 Summary
References
4 Methodologies for extraction and separation of short-chain bioactive peptides
4.1 Introduction
4.2 Definition: Short-chain peptide enrichment
4.3 Materials, equipment and reagents
4.4 Protocols
4.5 Pros and cons
4.6 Alternative methods/procedures
4.7 Troubleshooting & Optimization
4.8 Materials, equipment and reagents
4.9 Protocols
4.10 Pros and cons
4.11 Alternative methods/procedures
4.12 Troubleshooting & Optimization
4.13 Summary
References
5 Methodologies for peptidomics: Identification and quantification
5.1 Introduction
5.2 Identification of naturally generated peptides
5.3 Materials, equipment, and reagents
5.3.1 Protocol
5.3.2 Analysis and statistics
5.3.3 Pros and cons
5.3.4 Alternative methods/procedures
5.3.5 Troubleshooting and optimization
5.4 Label-free relative quantitation of naturally generated peptides
5.4.1 Materials, equipment, and reagents
5.4.2 Protocols
5.4.3 Analysis and statistics
5.4.4 Pros and cons
5.4.5 Alternative methods/procedures
5.4.6 Troubleshooting and optimization
5.5 Absolute quantitation of naturally generated peptides
5.5.1 Materials, equipment, and reagents
5.5.2 Protocols
5.5.3 Analysis and statistics
5.5.4 Pros and cons
5.5.5 Alternative methods/procedures
5.5.6 Troubleshooting and optimization
5.6 Summary
References
6 Methodologies for bioactivity assay: biochemical study
6.1 Introduction
6.2 Antioxidant activity assays
6.2.1 Ferric-reducing antioxidant power assay
6.2.1.1 Definition
6.2.1.2 Materials, equipment, and reagents
6.2.1.3 Protocols
6.2.1.4 Analysis and statistics
6.2.1.5 Safety considerations and standards
6.2.1.6 Pros and cons
6.2.1.7 Precursor techniques and related techniques
6.2.2 Oxygen radical absorbance capacity (ORAC) assay
6.2.2.1 Definition
6.2.2.2 Materials, equipment, and reagents
6.2.2.3 Protocols
6.2.2.4 Analysis and statistics
6.2.2.5 Safety considerations and standards
6.2.2.6 Pros and cons
6.2.2.7 Precursor techniques and related techniques
6.2.3 Trolox-equivalent antioxidant capacity assay
6.2.3.1 Definition
6.2.3.2 Materials, equipment, and reagents
6.2.3.3 Protocol
6.2.3.4 Analysis and statistics
6.2.3.5 Safety considerations and standards
6.2.3.6 Pros and cons
6.2.3.7 Precursor and related techniques
6.2.4 Other antioxidant activity assays
6.3 Enzyme inhibitory assays
6.3.1 Assay of angiotensin-I-converting enzyme inhibition
6.3.1.1 Definition
6.3.1.2 Materials, equipment, and reagents
6.3.1.3 Protocol
6.3.1.4 Analysis and statistics
6.3.1.5 Alternative methods/procedures
6.3.2 Assay of renin inhibition
6.3.2.1 Definition
6.3.2.2 Materials, equipment, and reagents
6.3.2.3 Protocols
6.3.2.4 Analysis and statistics
6.3.2.5 Alternative methods/procedures
6.3.3 Assay of dipeptidyl peptidase IV inhibitory activity
6.3.3.1 Definition
6.3.3.2 Materials, equipment, and reagents
6.3.3.3 Protocols
6.3.3.4 Analysis and statistics
6.3.3.5 Precursor and related techniques
6.3.3.6 Alternative methods/procedures
6.3.4 Assay of α-amylase inhibitory activity
6.3.4.1 Definition
6.3.4.2 Materials, equipment, and reagents
6.3.4.3 Protocols
6.3.4.4 Analysis and statistics
6.3.4.5 Precursor and related techniques
6.3.4.6 Alternative methods/procedures
6.3.5 Assay of α-glucosidase inhibitory activity
6.3.5.1 Definition
6.3.5.2 Materials, equipment, and reagents
6.3.5.3 Protocols
6.3.5.4 Analysis and statistics
6.3.5.5 Precursor and related techniques
6.3.5.6 Alternative methods/procedures
6.3.6 Assay of lipase inhibitory activity
6.3.6.1 Definition
6.3.6.2 Assay A
6.3.6.2.1 Materials, equipment, and reagents
6.3.6.2.2 Protocols
6.3.6.2.3 Analysis and statistics
6.3.6.3 Assay B
6.3.6.3.1 Materials, equipment, and reagents
6.3.6.3.2 Protocols
6.3.6.3.3 Analysis and statistics
6.3.7 Assay of tyrosinase inhibitory activity
6.3.7.1 Definition
6.3.7.2 Materials, equipment, and reagents
6.3.7.3 Protocols
6.3.7.4 Analysis and statistics
6.3.7.5 Precursor and related techniques
6.3.7.6 Alternative methods/procedures
6.3.8 Assay of trypsin inhibitory activity
6.3.8.1 Definition
6.3.8.2 Materials, equipment, and reagents
6.3.8.3 Protocols
6.3.8.4 Analysis and statistics
6.3.8.5 Precursor and related techniques
6.3.8.6 Alternative methods/procedures
6.3.9 Assay of chymotrypsin inhibitory activity
6.3.9.1 Definition
6.3.9.2 Materials, equipment, and reagents
6.3.9.3 Protocols
6.3.9.4 Analysis and statistics
6.3.9.5 Precursor and related techniques
6.3.9.6 Alternative methods/procedures
6.3.10 Assay of acetylcholinesterase inhibitory activity
6.3.10.1 Definition
6.3.10.2 Materials, equipment, and reagents
6.3.10.3 Protocols
6.3.10.4 Analysis and statistics
6.3.10.5 Precursor and related techniques
6.3.10.6 Alternative methods/procedures
6.3.11 Pros and cons
6.3.12 Troubleshooting and optimization
6.4 Summary
Acknowledgments
References
7 Methodologies for bioactivity assay: cell study
7.1 Introduction
7.2 Cell culture basics
7.2.1 Basic equipment for cell culture
7.2.2 Safety aspects of cell culture
7.2.2.1 Risk assessment
7.2.2.2 Biohazards
7.2.2.3 Disinfection
7.2.2.4 Waste disposal
7.2.3 Aseptic technique and contamination control
7.2.3.1 Personal hygiene
7.2.3.2 Sterile work area—biosafety cabinet
7.2.3.3 Sterile reagent and media
7.2.4 Cell types and sourcing of cell lines
7.2.4.1 Primary cultures
7.2.4.2 Continuous cultures
7.2.4.3 Selecting the appropriate cell line
7.2.4.4 Sourcing cell lines
7.2.5 Cell culture conditions
7.2.5.1 Culture media
7.2.5.2 Temperature, pH, CO2, and O2 levels
7.2.5.3 Subculturing
7.3 Basic cell culture protocols
7.3.1 Protocol 1. Subculturing adherent cultures
7.3.2 Protocol 2. Subculturing suspension cultures
7.3.3 Protocol 3. Quantification of total cell number and cell viability
7.3.4 Protocol 4. Freezing cells
7.3.5 Protocol 5. Thawing cryopreserved cells
7.4 Study bone health-promoting peptide
7.4.1 Bone formation cells
7.4.1.1 Protocol 6. In vitro osteoblasts culturing
MC3T3-E1 cell line (ATCC CRL-2593)
Materials, equipment, and reagents
Method
7.4.1.2 Protocol 7. Mineralization assay—Alizarin Red S staining assay
Materials, equipment, and reagents
Method
7.4.2 Bone resorption cells
7.4.2.1 Protocol 8. In vitro macrophage RAW 264.7 cell culture
RAW 264.7 cell line (ATCC TIB-71)
Materials, equipment, and reagents
Method
7.4.2.2 Protocol 9. The generation of osteoclast from macrophage RAW 264.7
Materials, equipment, and reagents
Method
7.4.2.3 Protocol 10. Tartrate resistant acid phosphatase staining
Materials, equipment, and reagents
Method
7.4.2.4 Protocol 11. Osteoclastic resorption assay
Materials, equipment, and reagents
Method
7.5 Biochemical and molecular analysis of cell study
7.5.1 Protocol 12. Western blotting
7.5.1.1 Materials, equipment, and reagents
7.5.1.2 Method
7.5.1.3 Preparation of cell lysate
7.5.1.4 Preparation of SDS polyacrylamide gel [Note 10]
7.5.1.5 Electrophoresis
7.5.1.6 Electrophoretic transfer from gel to membrane
7.5.1.7 Protein detection
7.5.2 Protocol 13. Quantitative reverse transcription polymerase chain reaction
7.5.2.1 Materials, equipment, and reagents
7.5.2.2 Method
7.5.2.3 RNA extraction by TRIzol reagent [Note 2]
7.5.2.4 Reverse transcription
7.5.2.5 Design primers for SYBR Green qPCR assay
7.5.2.6 Perform quantitative reverse transcription polymerase chain reaction using SYBR Green assay
7.5.2.7 Analysis of quantitative reverse transcription polymerase chain reaction data: comparative CT methods [Note 7]
7.6 Summary
References
8 Methodologies for bioactivity assay: animal study
Abbreviations
8.1 Introduction
8.2 Administration of food peptides and animal safety
8.2.1 Safety and toxicological evaluation of peptides
8.2.2 Meal feeding information
8.2.3 Distribution of gender and age
8.2.4 Development of oral and injectable peptides derived from food
8.3 Animal models to evaluate hypertension
8.3.1 Classical animal models to evaluate hypertension
8.3.2 Newfangled animal models to evaluate hypertension and cardiovascular disease
8.4 Animal models to evaluate metabolic dysfunction
8.4.1 Animal models to evaluate metabolic dysfunction
8.4.2 Knockout mice models to evaluate metabolic dysfunction
8.5 Analysis and statistics
8.5.1 Sample size: power analysis
8.5.2 Handling of normal and nonnormal distributed data
8.5.3 Multivariate analysis of animal studies
8.6 Safety considerations and standards during the development of animal models
8.6.1 Bioethics considerations
8.6.2 Clinical evaluation of sick animals
8.7 Summary
References
9 Methodologies for bioavailability assessment of food-derived peptide
9.1 Introduction
9.2 Structure of peptides in foods
9.3 Presence of food-derived peptides with modified amino acid residues in blood
9.4 Direct identification of food-derived peptides in the body
9.5 Detection of exopeptidase-resistant peptides in blood
9.6 Peptides pass through Caco-2 monolayer
9.7 Biological activity of food-derived peptides in body
9.8 Conclusion and future prospects
References
10 Methodologies for studying the structure–function relationship of food-derived peptides with biological activities
10.1 Introduction
10.2 Bioactivity prediction of peptides
10.3 Mapping methods to predict structure–function of bioactive peptides
10.4 In silico methods predicting bioactivity in food-derived peptides
10.5 Methods to analyze the physicochemical feature of bioactive peptide
10.6 Quantitative structure–activity relationship methods to assess food-derived peptide functions
10.7 Artificial neural networking and quantitative structure–activity relationship integrative approach to assess bioactive...
10.8 Limitations of classical bioinformatics and computational biology approach for peptide analysis
10.9 Conclusion and future directions
References
11 Methodologies for investigating the vasorelaxation action of peptides
11.1 Introduction
11.2 Principles
11.2.1 Measurement of vascular tension
11.2.2 Measurement of [Ca2+]i
11.2.3 Assay for Ca2+–CaM complex formation
11.3 Materials, equipments, and reagents
11.3.1 Measurement of vascular tension
11.3.1.1 Materials
11.3.1.2 Equipment
11.3.1.3 Reagents
11.3.2 Measurement of intracellular Ca2+ concentration [Ca2+]i
11.3.2.1 Materials
11.3.2.2 Equipments
11.3.2.3 Reagents
11.3.3 Assay for Ca2+–CaM complex formation
11.3.3.1 Materials
11.3.3.2 Equipments
11.3.3.3 Reagents
11.4 Protocols
11.4.1 Measurement of vascular tension
11.4.1.1 Preparation of aortic rings from rats
11.4.1.2 Measurement of vasorelaxation tension in contracted rat aortic rings
11.4.2 Measurement of [Ca2+]i
11.4.2.1 Cell culture
11.4.2.2 Measurement of [Ca2+]i in vascular smooth muscle cells
11.4.3 Assay for Ca2+–CaM complex formation
11.5 Analysis and statistics
11.5.1 Measurement of vascular tension
11.5.2 Measurement of [Ca2+]i
11.5.3 Percentage of Ca2+–CaM complex formation
11.5.4 The Hill-plot analysis
11.6 Safety considerations and standards
11.6.1 Animal ethics
11.6.1.1 Ethical statement
11.6.1.2 Protocol for euthanasia
11.7 Pros and cons
11.7.1 Measurement of vascular tension
11.7.2 Measurement of [Ca2+]i
11.7.3 Assay for Ca2+–CaM complex formation
11.8 Alternative methods/procedures
11.8.1 Measurement of vascular tension using rat mesenteric arteries
11.8.2 The patch clamp test
11.9 Troubleshooting and optimization
11.9.1 Measurement of vascular tension
11.9.2 Measurement of [Ca2+]i
11.10 Summary
References
12 Methodologies for studying mechanisms of action of bioactive peptides: a multiomic approach
12.1 Introduction
12.2 Investigation of the regulatory properties of dietary peptides in cellular signaling events
12.2.1 In silico approach for characterizing bioactive peptides
12.2.2 In silico approach for investigation of the interaction between bioactive peptides and molecular target
12.2.3 Exploration of the molecular basis of the dietary peptide modulating cellular signaling transduction via an integrat...
12.3 Conclusion
References
13 CRISPR–Cas systems in bioactive peptide research
13.1 Introduction
13.2 Timeline and development of CRISPR–Cas system
13.3 Beyond Cas9
13.4 Advancing biological research
13.5 Bioactive peptides and CRISPR–Cas9
13.5.1 Generating CRISPR-guided targets for peptide-based studies in mammalian cells
13.6 Materials, equipment, and reagents
13.7 Protocols
13.8 Analysis and quality control
13.9 Ethical reflections
13.10 Future directions
13.11 Conclusions
References
14 Databases of bioactive peptides
14.1 Introduction
14.2 General overview of databases and their classification
14.3 Biological and chemical information on peptides in brief
14.4 Some databases of bioactive peptide sequences
14.5 Using bioinformatic databases for the analysis of food proteins and peptides
14.6 Conclusion
Acknowledgments
References
15 Encapsulation technology for protection and delivery of bioactive peptides
15.1 Introduction
15.2 Microparticulate delivery systems
15.2.1 Food-grade microparticulate carrier materials
15.2.1.1 Polysaccharide-based carriers
15.2.1.2 Protein-based carriers
15.2.1.3 Lipid-based carriers
15.2.2 Techniques for fabricating microparticles
15.2.2.1 Spray drying
15.2.2.2 Coacervation
15.2.3 Bitter taste and hygroscopicity of microencapsulated peptides
15.2.3.1 Bitter taste
15.2.3.2 Hygroscopicity
15.2.4 Release characteristics, gastric stability, and bioavailability of microencapsulated peptides
15.3 Hydrogel delivery systems
15.3.1 Fabrication of bioactive peptide-loaded microgels
15.3.1.1 Injection–gelation method
15.3.1.2 Emulsion templating
15.3.2 Encapsulation efficiency of bioactive peptides in microgels
15.3.3 Release behavior and bioactive properties of encapsulated peptides in microgels
15.4 Nanoparticulate delivery systems for bioactive peptides
15.4.1 Liposome-based nanoencapsulation system for bioactive peptides
15.4.2 Polyelectrolyte-based nanoencapsulation system for bioactive peptide delivery
15.4.3 Nanoemulsion-based delivery system for bioactive peptides delivery
15.4.4 Solid lipid nanoparticles for bioactive peptide delivery
15.5 Conclusion and future perspectives
References
16 Plant sources of bioactive peptides
16.1 Introduction
16.2 Plant proteins classification and isolation and extraction methods
16.3 Sources and production of bioactive plant peptides
16.3.1 Naturally occurring bioactive peptides in plants
16.3.2 Plant-derived bioactive peptides through enzymatic hydrolysis
16.3.3 Plant-derived bioactive peptides through fermentation
16.3.4 Unique aspects of plant proteins and preparing bioactive peptides from plant sources
16.4 Mechanistic insights on the biological activities of bioactive peptides from plants
16.4.1 The role of plant-derived peptides in inflammation and immunomodulation
16.4.2 The anticancer effect of plant-derived peptides: prevention, initiation, and progression
16.4.3 The role of plant-derived peptides in metabolic syndrome
16.5 Challenges and opportunities in studying the health benefits of plant-derived peptides
16.6 Conclusion
Acknowledgements
References
17 Generation of bioactivities from proteins of animal sources by enzymatic hydrolysis and the Maillard reaction
17.1 Introduction
17.2 Bioactive peptides from milk
17.2.1 Generation of peptides from milk
17.2.2 Utilization of cheese whey for producing peptides
17.2.3 Evaluation of milk proteins for bioactive peptides
17.3 Bioactive peptides from meat
17.3.1 Generation of peptides by gastrointestinal digestion
17.3.2 Generation of peptides during aging
17.3.3 Generation of peptides during fermentation
17.3.4 Generation of peptides by protease treatments
17.4 Bioactive peptides from animal by-products
17.4.1 Generation of peptides from blood
17.4.2 Generation of peptides from collagen
17.5 Bioactive peptides from marine sources
17.5.1 Generation of peptides from seafood and its by-products
17.5.2 Commercial development of marine-derived peptides
17.6 Bioactive peptides and the Maillard reaction
17.6.1 The Maillard reaction
17.6.2 The Maillard reaction and meat
17.6.3 Bioactivities of Maillard reaction products from peptides
17.6.4 Bioactivities of volatile Maillard reaction products from peptides
17.7 Conclusion
References
18 Sustainable, alternative sources of bioactive peptides
18.1 Introduction
18.2 Fungi
18.2.1 Major fungi protein and mechanisms of extraction
18.2.2 Bioactive properties of peptides derived from fungi
18.3 Edible insects
18.3.1 Extraction of bioactive peptides from insects
18.3.2 Bioactivity of peptides derived from insects
18.4 Marine macroalgae
18.4.1 Mechanisms of extraction of bioactive peptides from marine macroalgae
18.4.2 Bioactive properties of peptides from macroalgae proteins
18.5 Underutilized agricultural by-products
18.5.1 Mechanisms for extraction of bioactive peptides from underutilized agricultural by-products
18.5.2 Bioactivity of peptides derived from underutilized agricultural by-products
18.6 Conclusion
References
19 Application in nutrition: mineral binding
19.1 Introduction
19.2 Importance of minerals for nutrition
19.2.1 Main mineral involved in nutrition and their needs in human
19.2.2 Safety considerations and standards/regulation
19.2.3 Bioavailability and metabolism of minerals
19.3 Evidence of health effects of mineral-binding peptide
19.4 Mineral-binding peptides: potential applications, sources, production, and commercialization
19.4.1 Application of mineral-binding peptides in nutrition
19.4.1.1 In case of mineral deficiency
19.4.1.2 In case of oxidation phenomena
19.4.2 Sources of mineral-binding peptides
19.4.2.1 Mineral-binding peptide in natural resources
19.4.2.2 Production of mineral-binding peptide
19.4.2.2.1 Proteolysis
19.4.2.2.2 Chemical peptide synthesis
19.5 Selective extraction of mineral-binding peptides from complex hydrolyzates
19.5.1 Peptides–metal ion interactions
19.5.2 Mineral-binding peptide screening techniques
19.5.2.1 Spectroscopic techniques
19.5.2.1.1 Principle of spectroscopic techniques
19.5.2.1.2 Use of spectroscopic techniques to understand metal–peptide interactions
19.5.2.2 Isothermal titration calorimetry
19.5.2.2.1 Principle of isothermal titration calorimetry
19.5.2.2.2 Use of ITC for MBP screening
19.5.2.3 Surface plasmon resonance
19.5.2.3.1 Principle of surface plasmon resonance
19.5.2.3.2 Use of SPR for MBP screening
19.5.2.4 Electrically switchable nanolever technology
19.5.2.4.1 Principle of the switchSENSE technology
19.5.2.4.2 Application of switchSENSE for mineral-binding peptide screening
19.5.2.5 Electrospray ionization-mass spectrometry
19.5.2.5.1 Principle of electrospray ionization-mass spectrometry
19.5.2.5.2 Use of ESI-MS for MBP screening
19.5.3 Immobilized metal-ion affinity chromatography separation
19.5.3.1 Principle of immobilized metal-ion affinity chromatography
19.5.3.2 Use of IMAC for MBP screening
19.6 Summary
Acknowledgment
References
20 Applications in nutrition: clinical nutrition
20.1 Introduction
20.1.1 Overview of clinical nutritional support and clinical nutrition therapy
20.1.2 Application of biologically active peptides in clinical nutritional support and therapy
20.2 Application of biologically active peptides in disease treatment
20.2.1 Application of biologically active peptides in the clinical treatment of cardiovascular diseases
20.2.2 Application of biologically active peptides in the clinical treatment of cancer
20.2.3 Application of biologically active peptides in the clinical treatment of liver injury
20.2.4 Application of biologically active peptides in the clinical treatment of diabetes mellitus
20.2.5 Application of biologically active peptides in the clinical treatment of other diseases
20.3 Application of biologically active peptides in clinical nutritional foods
20.3.1 Determination of proportions of biologically active peptides in products with specific nutritional requirements
20.3.1.1 Characteristics of clinical nitrogen supplementation products
20.3.1.2 Nitrogen intake requirements for different patients
20.3.1.3 Design requirements for clinical biologically active peptide products
20.3.2 Source selection of biologically active peptides in products for patients with specific health needs
20.3.3 Product forms
20.4 Summary and prospects
References
21 Applications in nutrition: sport nutrition
21.1 Introduction
21.2 Rationale
21.3 Application in sports nutrition
21.3.1 Bioactive peptides, body composition, and muscular performance
21.3.2 Bioactive peptides and muscle damage
21.3.2.1 Mechanisms
21.3.2.1.1 Effects on protein synthesis
21.3.2.1.2 Antiinflammatory effect
21.3.2.1.3 Antioxidant activity
21.3.2.2 Interim conclusion
21.3.3 Bioactive peptides and connective tissue
21.3.3.1 Tendon
21.3.3.2 Cartilage and functional joint pain
21.3.3.3 Interim conclusion
21.4 Limitations
21.5 Practical applications
21.6 Summary
References
22 Application in nutrition: cholesterol-lowering activity
22.1 Introduction
22.2 Rationale: peptides activity and characterization
22.3 Peptides from plant proteins
22.3.1 Soybean peptides
22.3.2 Lupin peptides
22.3.3 Hempseed peptides
22.4 Hypocholesterolemic peptide from other seeds: amaranth, cowpea, and rice
22.5 Peptides from animal sources
22.5.1 Milk peptides
22.5.2 Meat peptides
22.5.3 Fish peptides
22.5.4 Egg peptides
22.5.5 Royal jelly peptides
22.6 Structure–activity relationship of hypocholesterolemic peptides
22.7 Summary
References
23 Applications in nutrition: Peptides as taste enhancers
23.1 Introduction
23.2 Umami and umami-enhancing peptides
23.2.1 Umami taste
23.2.2 Umami taste receptors
23.2.3 Structural characteristics of umami and umami-enhancing peptides
23.3 Bitter and bitter inhibitory peptides
23.3.1 Bitter taste
23.3.2 Bitter taste receptor
23.3.3 Bitter taste inhibitory peptides
23.4 Salt taste-enhancing peptides
23.4.1 Salt taste
23.4.2 Salty taste receptors
23.4.3 Structural characteristics of salty taste-enhancing peptides
23.5 Kokumi peptides
23.5.1 Kokumi taste
23.5.2 Kokumi taste receptors
23.5.3 The characteristics of kokumi peptides
23.6 Summary
Acknowledgments
References
24 Cardiovascular benefits of food protein-derived bioactive peptides
24.1 Introduction
24.2 Inhibition of the renin–angiotensin–aldosterone system: antihypertensive peptides
24.2.1 ACE- and renin-inhibitory peptides
24.2.1.1 Animal protein-derived hydrolysates and peptides
24.2.1.2 Plant protein-derived hydrolysates and peptides
24.2.2 Foods formulated with antihypertensive protein hydrolysates and peptides
24.3 Conclusions
24.4 Future trends
References
25 Applications in medicine: hypoglycemic peptides
25.1 Introduction
25.2 Carbohydrate digestion and glucose homeostasis
25.3 Pathophysiology of type 2 diabetes
25.4 Clinical diagnosis of diabetes
25.5 Diverse physiological properties of protein hydrolysates and bioactive peptides
25.6 Antidiabetic properties of protein hydrolysates/peptides (in vivo studies)
25.7 Antidiabetic properties of protein hydrolysates/peptides (clinical studies)
25.8 Conclusions
References
26 Application in medicine: obesity and satiety control
Abbreviations
26.1 Introduction
26.2 Synthetic peptides
26.2.1 Synthetic peptides: glucagon-like peptide-1 mimetics
26.2.2 Synthetic peptides: multiple actions mimetics
26.2.3 Safety considerations and limitations for synthetic peptides
26.2.4 Other synthetic peptides in preclinical trials and in vitro development
26.3 Food-derived peptides
26.3.1 Food-derived peptides targeting CCK and GI enzymes with proven in vivo efficacy
26.3.2 Food-derived peptides targeting ghrelin, opioid receptor, and GI transit with proven in vivo efficacy
26.3.3 Food-derived peptides targeting lipid metabolism with proven in vivo efficacy
26.3.4 Food-derived peptides inhibiting protease dipeptidyl peptidase-4
26.3.5 In vitro evidence of food-derived peptides
26.3.6 Limitations: survival of food-derived peptides during gut transit
26.4 Commercial dietary protein hydrolyzates with antiobesity potential
26.5 Summary
Acknowledgments
References
27 Food-derived osteogenic peptides towards osteoporosis
27.1 Introduction
27.2 Evaluation and diagnosis of osteoporosis
27.2.1 Bone formation and resorption biomarkers
27.2.2 Computed tomography diagnosis
27.3 Osteogenic agents
27.3.1 Drugs for osteoporosis
27.3.2 Osteogenic peptides
27.4 Characterization of osteogenic peptides
27.4.1 Preparation of osteogenic peptides
27.4.2 Identification of osteogenic peptides
27.5 Bioavailability of osteogenic peptides
27.5.1 Absorption analysis
27.5.2 Pharmacokinetic analysis
27.6 Conclusions
Acknowledgments
Reference
28 Applications in medicine: mental health
28.1 Introduction
28.1.1 Peptide transport across the blood–brain barrier and use as shuttles
28.2 Peptides as diagnostic tools in brain tumors and CNS disorders
28.2.1 Peptide-based imaging tracers
28.2.2 Peptides as biomarkers
28.3 Therapeutic applications of peptides for mental health
28.3.1 Neurodevelopmental disorders
28.3.2 Psychotic disorders
28.3.3 Depressive, bipolar, and anxiety disorders
28.3.4 Neurocognitive and neurodegenerative disorders
28.3.5 Others
28.4 Conclusion
References
29 Applications in medicine: joint health
29.1 Introduction
29.2 Overview of joint diseases
29.2.1 Osteoarthritis
29.2.2 Rheumatoid arthritis
29.3 Peptides activity and characterization
29.3.1 Natural bioactive peptide sources
29.3.2 Peptidome analysis
29.4 Mechanisms of action
29.4.1 Cartilage proliferation
29.4.2 Antioxidant, antimicrobial, and antiinflammatory activities
29.4.3 Neuroactivity
29.5 Evidence in joint health benefits
29.6 Potential applications, production, and commercialization
29.6.1 Diagnostic
29.6.2 Prophylaxis/therapeutic
29.6.3 Production and commercialization
29.7 Summary
Acknowledgments
References
30 Applications in food technology: antimicrobial peptides
30.1 Introduction
30.2 Classification
30.3 Current and potential food applications
30.3.1 Commercial application of nisin
30.3.2 Commercial application of pediocin
30.3.3 Commercial application of MicroGARD
30.3.4 Commercial application of ε-polylysine
30.3.5 Other antimicrobial peptide preparations received regulatory approval
30.4 Hurdle approach
30.5 Application of antimicrobial peptides for improving human health
30.5.1 Antimicrobial peptides production by probiotic strains
30.5.2 Antiinfective activity of antimicrobial peptides
30.5.3 Antiviral effect of antimicrobial peptides
30.5.4 Bioavailability and metabolism
30.6 Mechanisms of action
30.6.1 Mechanisms of action against bacteria and fungi
30.6.2 Mechanisms of action against viruses
30.7 Safety considerations and regulations
30.7.1 Safety of antimicrobial peptides
30.7.2 Regulatory aspects of using AMPs or AMP producers in food
30.8 Limitations
30.9 Summary
References
Index
Back Cover

Citation preview

Biologically Active Peptides

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Biologically Active Peptides From Basic Science to Applications for Human Health Edited by Fidel Toldra´ Department of Food Science, Institute of Agrochemistry and Food Technology (CSIC), Valencia, Spain

Jianping Wu Faculty of Agricultural Life and Environmental Sciences, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-821389-6 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisitions Editor: Peter B. Linsley Editorial Project Manager: Sam W. Young Production Project Manager: Maria Bernard Cover Designer: Victoria Pearson Typeset by MPS Limited, Chennai, India

Contents List of contributors .............................................................................................xxiii Preface............................................................................................................... xxix Chapter 1: Bioactive peptides in health and disease: an overview .............................. 1 JuDong Yeo and Fereidoon Shahidi 1.1 Introduction ........................................................................................................... 1 1.2 Preparation of bioactive peptides ........................................................................... 2 1.3 Absorption of peptides in the small intestine ......................................................... 3 1.3.1 Paracellular transport ..................................................................................3 1.3.2 Transcellular transport ................................................................................4 1.3.3 Absorption of peptides in the large intestine (colon) ...................................5 1.3.4 Approaches for enhancing the absorption of peptides .................................6 1.3.5 Structure-activity relationship of bioactive peptides ....................................8 1.4 Bioactivities of food-derived bioactive peptides focusing on inhibiting chronic diseases ..................................................................................... 9 1.4.1 Anticancer activity ......................................................................................9 1.4.2 Anti-inflammatory effect...........................................................................11 1.4.3 Antimicrobial activity ...............................................................................12 1.4.4 Antihypertensive effect .............................................................................13 1.4.5 Immunomodulatory peptides .....................................................................15 1.4.6 Antidiabetic effect.....................................................................................16 1.5 Conclusion ........................................................................................................... 18 References ................................................................................................................... 18

Chapter 2: Enzymatic mechanisms for the generation of bioactive peptides .............. 27 Fidel Toldra´ and Leticia Mora 2.1 Introduction ......................................................................................................... 27 2.1.1 Enzymatic mechanisms in the hydrolysis of food proteins ........................27 v

vi Contents 2.1.2 Bioactive peptides generated during food processing ................................30 2.1.3 Bioactive peptides generated through the hydrolysis of proteins with commercial peptidases .........................................................30 2.2 Degree of hydrolysis ............................................................................................ 37 2.2.1 Definition ..................................................................................................37 2.2.2 Precursor techniques and alternative methods/procedures .........................38 2.3 Assay of endopeptidase activity ........................................................................... 38 2.3.1 Definition ..................................................................................................38 2.4 Assay of exopeptidase activity ............................................................................. 40 2.4.1 Definition ..................................................................................................40 2.4.2 Materials, equipment, and reagents ...........................................................40 2.4.3 Protocol ....................................................................................................41 2.4.4 Pros and cons ............................................................................................41 2.4.5 Summary...................................................................................................42 References ................................................................................................................... 42

Chapter 3: Novel technologies in bioactive peptides production and stability ............ 47 Aı´da Jimena Velarde-Salcedo, Gabriela Va´zquez-Rodrı´guez, Antonio De Leo´n-Rodrı´guez and Ana Paulina Barba de la Rosa 3.1 Introduction ......................................................................................................... 47 3.2 Expression of recombinant peptides ..................................................................... 48 3.2.1 Escherichia coli expression vectors and strains for recombinant protein production .....................................................................................50 3.3 Stability of proteins and peptides ......................................................................... 52 3.4 Definition: production of recombinant bioactive peptides in Escherichia coli ................................................................................................... 57 3.4.1 Antihypertensive peptides .........................................................................57 3.4.2 Antiangiogenic peptides ............................................................................58 3.5 Protocol ............................................................................................................... 59 3.5.1 Antihypertensive cassette design ...............................................................59 3.5.2 Amplification of the encrypted vasoinhibin peptide ..................................60 3.5.3 DNA cloning into a suitable vector ...........................................................61 3.5.4 Transformation of the host cells ................................................................63 3.5.5 Induction of the expression of the desired protein under controlled conditions .................................................................................66 3.5.6 Recovery and purification of the recombinant product ..............................68 3.5.7 Preparation and encapsulation of recombinant peptides ............................69 3.6 Summary ............................................................................................................. 70 References ................................................................................................................... 71

Contents vii

Chapter 4: Methodologies for extraction and separation of short-chain bioactive peptides................................................................................ 75 Andrea Cerrato, Sara Elsa Aita, Carmela Maria Montone, Anna Laura Capriotti, Susy Piovesana and Aldo Lagana` 4.1 Introduction ....................................................................................................... 75 4.2 Definition: Short-chain peptide enrichment ........................................................ 77 4.3 Materials, equipment and reagents ..................................................................... 77 4.4 Protocols ............................................................................................................ 78 4.5 Pros and cons ..................................................................................................... 78 4.6 Alternative methods/procedures ......................................................................... 79 4.7 Troubleshooting & Optimization........................................................................ 79 4.8 Materials, equipment and reagents ..................................................................... 80 4.9 Protocols ............................................................................................................ 81 4.10 Pros and cons ..................................................................................................... 82 4.11 Alternative methods/procedures ......................................................................... 82 4.12 Troubleshooting & Optimization........................................................................ 83 4.13 Summary ........................................................................................................... 83 References ................................................................................................................... 84

Chapter 5: Methodologies for peptidomics: Identification and quantification ............ 87 Leticia Mora and Fidel Toldra´ 5.1 Introduction ......................................................................................................... 87 5.2 Identification of naturally generated peptides ...................................................... 88 5.3 Materials, equipment, and reagents ...................................................................... 89 5.3.1 Protocol ....................................................................................................89 5.3.2 Analysis and statistics ...............................................................................90 5.3.3 Pros and cons ............................................................................................90 5.3.4 Alternative methods/procedures ................................................................90 5.3.5 Troubleshooting and optimization .............................................................91 5.4 Label-free relative quantitation of naturally generated peptides ........................... 91 5.4.1 Materials, equipment, and reagents ...........................................................92 5.4.2 Protocols ...................................................................................................92 5.4.3 Analysis and statistics ...............................................................................93 5.4.4 Pros and cons ............................................................................................95 5.4.5 Alternative methods/procedures ................................................................96 5.4.6 Troubleshooting and optimization .............................................................96 5.5 Absolute quantitation of naturally generated peptides .......................................... 96 5.5.1 Materials, equipment, and reagents ...........................................................97 5.5.2 Protocols ...................................................................................................97

viii

Contents 5.5.3 Analysis and statistics ............................................................................... 97 5.5.4 Pros and cons ............................................................................................ 99 5.5.5 Alternative methods/procedures ................................................................ 99 5.5.6 Troubleshooting and optimization ............................................................. 99 5.6 Summary ........................................................................................................... 100 References ................................................................................................................. 101

Chapter 6: Methodologies for bioactivity assay: biochemical study .........................103 Miryam Amigo-Benavent, Mohammadreza Khalesi, Ganesh Thapa and Richard J. FitzGerald 6.1 Introduction ....................................................................................................... 103 6.2 Antioxidant activity assays ................................................................................ 104 6.2.1 Ferric-reducing antioxidant power assay ................................................. 105 6.2.2 Oxygen radical absorbance capacity (ORAC) assay ................................ 108 6.2.3 Trolox-equivalent antioxidant capacity assay .......................................... 110 6.2.4 Other antioxidant activity assays ............................................................. 113 6.3 Enzyme inhibitory assays................................................................................... 114 6.3.1 Assay of angiotensin-I-converting enzyme inhibition ............................ 114 6.3.2 Assay of renin inhibition ....................................................................... 117 6.3.3 Assay of dipeptidyl peptidase IV inhibitory activity ............................. 119 6.3.4 Assay of α-amylase inhibitory activity ................................................. 121 6.3.5 Assay of α-glucosidase inhibitory activity ............................................ 124 6.3.6 Assay of lipase inhibitory activity ......................................................... 126 6.3.7 Assay of tyrosinase inhibitory activity .................................................. 129 6.3.8 Assay of trypsin inhibitory activity ....................................................... 131 6.3.9 Assay of chymotrypsin inhibitory activity ............................................. 133 6.3.10 Assay of acetylcholinesterase inhibitory activity ................................... 135 6.3.11 Pros and cons ........................................................................................ 137 6.3.12 Troubleshooting and optimization ......................................................... 138 6.4 Summary ........................................................................................................... 139 Acknowledgments ...................................................................................................... 140 References ................................................................................................................. 141 Chapter 7: Methodologies for bioactivity assay: cell study .....................................155 Nan Shang, Khushwant S. Bhullar and Jianping Wu 7.1 Introduction ....................................................................................................... 155 7.2 Cell culture basics.............................................................................................. 156 7.2.1 Basic equipment for cell culture ............................................................. 156 7.2.2 Safety aspects of cell culture................................................................... 156

Contents ix 7.2.3 Aseptic technique and contamination control ..........................................159 7.2.4 Cell types and sourcing of cell lines .......................................................160 7.2.5 Cell culture conditions ............................................................................163 7.3 Basic cell culture protocols ................................................................................ 165 7.3.1 Protocol 1. Subculturing adherent cultures ..............................................165 7.3.2 Protocol 2. Subculturing suspension cultures ..........................................167 7.3.3 Protocol 3. Quantification of total cell number and cell viability ............167 7.3.4 Protocol 4. Freezing cells ........................................................................167 7.3.5 Protocol 5. Thawing cryopreserved cells .................................................168 7.4 Study bone health-promoting peptide................................................................. 169 7.4.1 Bone formation cells ...............................................................................169 7.4.2 Bone resorption cells ..............................................................................172 7.5 Biochemical and molecular analysis of cell study .............................................. 177 7.5.1 Protocol 12. Western blotting..................................................................177 7.5.2 Protocol 13. Quantitative reverse transcription polymerase chain reaction..........................................................................................182 7.6 Summary ........................................................................................................... 186 References ................................................................................................................. 186

Chapter 8: Methodologies for bioactivity assay: animal study ................................191 Feiran Xu and Elvira Gonzalez de Mejia Abbreviations ............................................................................................................. 191 8.1 Introduction ....................................................................................................... 191 8.2 Administration of food peptides and animal safety ............................................ 193 8.2.1 Safety and toxicological evaluation of peptides ......................................193 8.2.2 Meal feeding information ........................................................................194 8.2.3 Distribution of gender and age ................................................................195 8.2.4 Development of oral and injectable peptides derived from food .............199 8.3 Animal models to evaluate hypertension............................................................ 199 8.3.1 Classical animal models to evaluate hypertension ...................................200 8.3.2 Newfangled animal models to evaluate hypertension and cardiovascular disease .............................................................................203 8.4 Animal models to evaluate metabolic dysfunction ............................................. 204 8.4.1 Animal models to evaluate metabolic dysfunction ..................................206 8.4.2 Knockout mice models to evaluate metabolic dysfunction ......................208 8.5 Analysis and statistics ........................................................................................ 209 8.5.1 Sample size: power analysis....................................................................209 8.5.2 Handling of normal and nonnormal distributed data ...............................209 8.5.3 Multivariate analysis of animal studies ...................................................209

x

Contents 8.6 Safety considerations and standards during the development of animal models ............................................................................................... 210 8.6.1 Bioethics considerations ..........................................................................210 8.6.2 Clinical evaluation of sick animals .........................................................211 8.7 Summary ........................................................................................................... 211 References ................................................................................................................. 212

Chapter 9: Methodologies for bioavailability assessment of food-derived peptide ...........................................................................221 Kenji Sato 9.1 Introduction ....................................................................................................... 221 9.2 Structure of peptides in foods ............................................................................ 225 9.3 Presence of food-derived peptides with modified amino acid residues in blood ................................................................................................ 226 9.4 Direct identification of food-derived peptides in the body ................................. 227 9.5 Detection of exopeptidase-resistant peptides in blood ........................................ 229 9.6 Peptides pass through Caco-2 monolayer ........................................................... 231 9.7 Biological activity of food-derived peptides in body.......................................... 232 9.8 Conclusion and future prospects ........................................................................ 233 References ................................................................................................................. 234

Chapter 10: Methodologies for studying the structure
function relationship of food-derived peptides with biological activities ............239 Advaita Ganguly, Kumakshi Sharma and Kaustav Majumder Introduction ..................................................................................................... 239 Bioactivity prediction of peptides .................................................................... 240 Mapping methods to predict structure
function of bioactive peptides ............. 241 In silico methods predicting bioactivity in food-derived peptides .................... 242 Methods to analyze the physicochemical feature of bioactive peptide ............. 243 Quantitative structure
activity relationship methods to assess food-derived peptide functions ......................................................................... 244 10.7 Artificial neural networking and quantitative structure
activity relationship integrative approach to assess bioactive of peptides ..................... 246 10.8 Limitations of classical bioinformatics and computational biology approach for peptide analysis ........................................................................... 247 10.9 Conclusion and future directions ...................................................................... 248 References ................................................................................................................. 249 10.1 10.2 10.3 10.4 10.5 10.6

Contents xi

Chapter 11: Methodologies for investigating the vasorelaxation action of peptides............................................................................255 Mitsuru Tanaka and Toshiro Matsui 11.1 Introduction.................................................................................................... 255 11.2 Principles ....................................................................................................... 257 11.2.1 Measurement of vascular tension ...................................................... 257 11.2.2 Measurement of [Ca21]i .................................................................... 258 11.2.3 Assay for Ca21
CaM complex formation ........................................ 258 11.3 Materials, equipments, and reagents ............................................................... 258 11.3.1 Measurement of vascular tension ...................................................... 259 11.3.2 Measurement of intracellular Ca21 concentration [Ca21]i ................. 260 11.3.3 Assay for Ca21
CaM complex formation ........................................ 260 11.4 Protocols ........................................................................................................ 261 11.4.1 Measurement of vascular tension ...................................................... 261 11.4.2 Measurement of [Ca21]i .................................................................... 263 11.4.3 Assay for Ca21
CaM complex formation ........................................ 266 11.5 Analysis and statistics .................................................................................... 266 11.5.1 Measurement of vascular tension ...................................................... 266 11.5.2 Measurement of [Ca21]i .................................................................... 267 11.5.3 Percentage of Ca21
CaM complex formation .................................. 267 11.5.4 The Hill-plot analysis ........................................................................ 268 11.6 Safety considerations and standards ............................................................... 268 11.6.1 Animal ethics .................................................................................... 268 11.7 Pros and cons ................................................................................................. 270 11.7.1 Measurement of vascular tension ...................................................... 270 11.7.2 Measurement of [Ca21]i .................................................................... 270 11.7.3 Assay for Ca21
CaM complex formation ........................................ 270 11.8 Alternative methods/procedures ..................................................................... 270 11.8.1 Measurement of vascular tension using rat mesenteric arteries ............................................................................ 270 11.8.2 The patch clamp test ......................................................................... 271 11.9 Troubleshooting and optimization .................................................................. 271 11.9.1 Measurement of vascular tension ...................................................... 271 11.9.2 Measurement of [Ca21]i .................................................................... 272 11.10 Summary........................................................................................................ 272 References ................................................................................................................. 273

xii Contents

Chapter 12: Methodologies for studying mechanisms of action of bioactive peptides: a multiomic approach ..........................................275 Hua Zhang and Yoshinori Mine 12.1 Introduction ..................................................................................................... 275 12.2 Investigation of the regulatory properties of dietary peptides in cellular signaling events ............................................................................................... 276 12.2.1 In silico approach for characterizing bioactive peptides ...................... 277 12.2.2 In silico approach for investigation of the interaction between bioactive peptides and molecular target .............................................. 277 12.2.3 Exploration of the molecular basis of the dietary peptide modulating cellular signaling transduction via an integrated approach ............................................................................. 279 12.3 Conclusion ....................................................................................................... 282 References ................................................................................................................. 283

Chapter 13: CRISPR
Cas systems in bioactive peptide research ..........................285 Khushwant S. Bhullar, Nan Shang and Jianping Wu Introduction.................................................................................................... 285 Timeline and development of CRISPR
Cas system ...................................... 286 Beyond Cas9 .................................................................................................. 288 Advancing biological research ....................................................................... 289 Bioactive peptides and CRISPR
Cas9 .......................................................... 292 13.5.1 Generating CRISPR-guided targets for peptide-based studies in mammalian cells ............................................................... 292 13.6 Materials, equipment, and reagents ................................................................ 293 13.7 Protocols ........................................................................................................ 294 13.8 Analysis and quality control........................................................................... 295 13.9 Ethical reflections .......................................................................................... 296 13.10 Future directions ............................................................................................ 297 13.11 Conclusions.................................................................................................... 300 References ................................................................................................................. 300 13.1 13.2 13.3 13.4 13.5

Chapter 14: Databases of bioactive peptides .......................................................309 Anna Iwaniak, Małgorzata Darewicz and Piotr Minkiewicz 14.1 14.2 14.3 14.4

Introduction ..................................................................................................... 309 General overview of databases and their classification .................................... 310 Biological and chemical information on peptides in brief ................................ 312 Some databases of bioactive peptide sequences ............................................... 316

Contents xiii 14.5 Using bioinformatic databases for the analysis of food proteins and peptides ....................................................................................... 320 14.6 Conclusion ....................................................................................................... 325 Acknowledgments ...................................................................................................... 325 References ................................................................................................................. 325

Chapter 15: Encapsulation technology for protection and delivery of bioactive peptides ...........................................................................331 Xiaohong Sun, Ogadimma D. Okagu and Chibuike C. Udenigwe 15.1 Introduction ..................................................................................................... 331 15.2 Microparticulate delivery systems .................................................................... 332 15.2.1 Food-grade microparticulate carrier materials ..................................... 333 15.2.2 Techniques for fabricating microparticles ........................................... 335 15.2.3 Bitter taste and hygroscopicity of microencapsulated peptides ............ 336 15.2.4 Release characteristics, gastric stability, and bioavailability of microencapsulated peptides ............................................................ 338 15.3 Hydrogel delivery systems ............................................................................... 339 15.3.1 Fabrication of bioactive peptide-loaded microgels .............................. 339 15.3.2 Encapsulation efficiency of bioactive peptides in microgels ............... 342 15.3.3 Release behavior and bioactive properties of encapsulated peptides in microgels .......................................................................... 343 15.4 Nanoparticulate delivery systems for bioactive peptides .................................. 344 15.4.1 Liposome-based nanoencapsulation system for bioactive peptides ............................................................................... 345 15.4.2 Polyelectrolyte-based nanoencapsulation system for bioactive peptide delivery ................................................................... 347 15.4.3 Nanoemulsion-based delivery system for bioactive peptides delivery ................................................................................. 349 15.4.4 Solid lipid nanoparticles for bioactive peptide delivery ....................... 350 15.5 Conclusion and future perspectives .................................................................. 351 References ................................................................................................................. 352

Chapter 16: Plant sources of bioactive peptides ...................................................357 Vermont P. Dia 16.1 Introduction ..................................................................................................... 357 16.2 Plant proteins classification and isolation and extraction methods ................... 358 16.3 Sources and production of bioactive plant peptides.......................................... 364 16.3.1 Naturally occurring bioactive peptides in plants.................................. 364 16.3.2 Plant-derived bioactive peptides through enzymatic hydrolysis ........... 369

xiv

Contents 16.3.3 Plant-derived bioactive peptides through fermentation ........................ 373 16.3.4 Unique aspects of plant proteins and preparing bioactive peptides from plant sources ................................................................. 375 16.4 Mechanistic insights on the biological activities of bioactive peptides from plants....................................................................................................... 377 16.4.1 The role of plant-derived peptides in inflammation and immunomodulation ............................................................................. 377 16.4.2 The anticancer effect of plant-derived peptides: prevention, initiation, and progression ................................................................... 379 16.4.3 The role of plant-derived peptides in metabolic syndrome .................. 381 16.5 Challenges and opportunities in studying the health benefits of plant-derived peptides ...................................................................................... 383 16.6 Conclusion ....................................................................................................... 386 Acknowledgements .................................................................................................... 386 References ................................................................................................................. 386

Chapter 17: Generation of bioactivities from proteins of animal sources by enzymatic hydrolysis and the Maillard reaction ............................403 Keizo Arihara, Issei Yokoyama and Motoko Ohata 17.1 Introduction ..................................................................................................... 403 17.2 Bioactive peptides from milk ........................................................................... 405 17.2.1 Generation of peptides from milk ....................................................... 405 17.2.2 Utilization of cheese whey for producing peptides .............................. 406 17.2.3 Evaluation of milk proteins for bioactive peptides .............................. 407 17.3 Bioactive peptides from meat........................................................................... 407 17.3.1 Generation of peptides by gastrointestinal digestion ........................... 407 17.3.2 Generation of peptides during aging ................................................... 408 17.3.3 Generation of peptides during fermentation ........................................ 408 17.3.4 Generation of peptides by protease treatments .................................... 409 17.4 Bioactive peptides from animal by-products .................................................... 410 17.4.1 Generation of peptides from blood ...................................................... 410 17.4.2 Generation of peptides from collagen ................................................. 410 17.5 Bioactive peptides from marine sources ........................................................... 411 17.5.1 Generation of peptides from seafood and its by-products .................... 411 17.5.2 Commercial development of marine-derived peptides ......................... 412 17.6 Bioactive peptides and the Maillard reaction ................................................... 413 17.6.1 The Maillard reaction .......................................................................... 413 17.6.2 The Maillard reaction and meat .......................................................... 414 17.6.3 Bioactivities of Maillard reaction products from peptides ................... 415

Contents xv 17.6.4 Bioactivities of volatile Maillard reaction products from peptides ...................................................................................... 416 17.7 Conclusion ....................................................................................................... 418 References ................................................................................................................. 418

Chapter 18: Sustainable, alternative sources of bioactive peptides ........................427 J.E. Aguilar-Toala´, F.G. Hall, U. Urbizo-Reyes and A.M. Liceaga 18.1 Introduction ..................................................................................................... 427 18.2 Fungi ............................................................................................................... 427 18.2.1 Major fungi protein and mechanisms of extraction ............................. 428 18.2.2 Bioactive properties of peptides derived from fungi............................ 429 18.3 Edible insects ................................................................................................... 432 18.3.1 Extraction of bioactive peptides from insects ...................................... 432 18.3.2 Bioactivity of peptides derived from insects ....................................... 433 18.4 Marine macroalgae .......................................................................................... 435 18.4.1 Mechanisms of extraction of bioactive peptides from marine macroalgae .............................................................................. 436 18.4.2 Bioactive properties of peptides from macroalgae proteins ................. 437 18.5 Underutilized agricultural by-products ............................................................. 438 18.5.1 Mechanisms for extraction of bioactive peptides from underutilized agricultural by-products ................................................. 439 18.5.2 Bioactivity of peptides derived from underutilized agricultural by-products ...................................................................... 440 18.6 Conclusion ....................................................................................................... 444 References ................................................................................................................. 445

Chapter 19: Application in nutrition: mineral binding ..........................................455 Sarah El Hajj, Tatiana Sepulveda-Rincon, Ce´dric Paris, Tristan Giraud, Gizella Csire, Loic Stefan, Katalin Selmeczi, Jean-Michel Girardet, Ste´phane Desobry, Said Bouhallab, Laurence Muhr, Caroline Gaucher and Laetitia Canabady-Rochelle 19.1 Introduction ..................................................................................................... 455 19.2 Importance of minerals for nutrition ................................................................ 456 19.2.1 Main mineral involved in nutrition and their needs in human ............. 456 19.2.2 Safety considerations and standards/regulation.................................... 457 19.2.3 Bioavailability and metabolism of minerals ........................................ 459 19.3 Evidence of health effects of mineral-binding peptide ..................................... 460

xvi

Contents 19.4 Mineral-binding peptides: potential applications, sources, production, and commercialization ..................................................................................... 462 19.4.1 Application of mineral-binding peptides in nutrition ........................... 462 19.4.2 Sources of mineral-binding peptides ................................................... 464 19.5 Selective extraction of mineral-binding peptides from complex hydrolyzates ........468 19.5.1 Peptides
metal ion interactions .......................................................... 468 19.5.2 Mineral-binding peptide screening techniques..................................... 469 19.5.3 Immobilized metal-ion affinity chromatography separation ................ 480 19.6 Summary ......................................................................................................... 482 Acknowledgment ....................................................................................................... 483 References ................................................................................................................. 483

Chapter 20: Applications in nutrition: clinical nutrition ........................................495 Wen-Ying Liu, Liang Chen, Ying Wei, Guo-Ming Li, Yan Liu, Yu-Chen Wang, Yu-Qing Wang, Xiu-Yuan Qin, Xin-Yue Cui, Rui-Zeng Gu and Jun Lu 20.1 Introduction ..................................................................................................... 495 20.1.1 Overview of clinical nutritional support and clinical nutrition therapy .................................................................................. 495 20.1.2 Application of biologically active peptides in clinical nutritional support and therapy............................................................ 497 20.2 Application of biologically active peptides in disease treatment ...................... 498 20.2.1 Application of biologically active peptides in the clinical treatment of cardiovascular diseases ................................................... 500 20.2.2 Application of biologically active peptides in the clinical treatment of cancer ............................................................................. 501 20.2.3 Application of biologically active peptides in the clinical treatment of liver injury ...................................................................... 503 20.2.4 Application of biologically active peptides in the clinical treatment of diabetes mellitus ............................................................. 505 20.2.5 Application of biologically active peptides in the clinical treatment of other diseases .................................................................. 507 20.3 Application of biologically active peptides in clinical nutritional foods ........... 510 20.3.1 Determination of proportions of biologically active peptides in products with specific nutritional requirements ................................... 510 20.3.2 Source selection of biologically active peptides in products for patients with specific health needs ...................................................... 511 20.3.3 Product forms...................................................................................... 514 20.4 Summary and prospects ................................................................................... 516 References ................................................................................................................. 518

Contents xvii

Chapter 21: Applications in nutrition: sport nutrition ..........................................525 J. Kohl, S. Jerger, D Ko¨nig and C. Centner 21.1 Introduction ..................................................................................................... 525 21.2 Rationale .......................................................................................................... 526 21.3 Application in sports nutrition.......................................................................... 527 21.3.1 Bioactive peptides, body composition, and muscular performance .................................................................. 527 21.3.2 Bioactive peptides and muscle damage ............................................... 531 21.3.3 Bioactive peptides and connective tissue............................................. 535 21.4 Limitations ....................................................................................................... 538 21.5 Practical applications ....................................................................................... 539 21.6 Summary ......................................................................................................... 541 References ................................................................................................................. 542

Chapter 22: Application in nutrition: cholesterol-lowering activity.........................551 Carmen Lammi, Carlotta Bollati, Gilda Aiello and Anna Arnoldi 22.1 Introduction ..................................................................................................... 551 22.2 Rationale: peptides activity and characterization.............................................. 552 22.3 Peptides from plant proteins ............................................................................ 552 22.3.1 Soybean peptides ................................................................................ 552 22.3.2 Lupin peptides .................................................................................... 555 22.3.3 Hempseed peptides ............................................................................. 556 22.4 Hypocholesterolemic peptide from other seeds: amaranth, cowpea, and rice ............................................................................................................ 557 22.5 Peptides from animal sources........................................................................... 558 22.5.1 Milk peptides ...................................................................................... 558 22.5.2 Meat peptides ...................................................................................... 559 22.5.3 Fish peptides ....................................................................................... 560 22.5.4 Egg peptides ....................................................................................... 560 22.5.5 Royal jelly peptides ............................................................................ 561 22.6 Structure
activity relationship of hypocholesterolemic peptides ..................... 562 22.7 Summary ......................................................................................................... 563 References ................................................................................................................. 563

Chapter 23: Applications in nutrition: Peptides as taste enhancers .......................569 Yu Fu, Mohammad Sadiq Amin, Qian Li, Kathrine H. Bak and Rene´ Lametsch 23.1 Introduction ..................................................................................................... 569

xviii

Contents 23.2 Umami and umami-enhancing peptides ........................................................... 570 23.2.1 Umami taste ........................................................................................ 570 23.2.2 Umami taste receptors ......................................................................... 571 23.2.3 Structural characteristics of umami and umami-enhancing peptides .... 571 23.3 Bitter and bitter inhibitory peptides ................................................................. 572 23.3.1 Bitter taste........................................................................................... 572 23.3.2 Bitter taste receptor ............................................................................. 572 23.3.3 Bitter taste inhibitory peptides ............................................................ 573 23.4 Salt taste-enhancing peptides ........................................................................... 573 23.4.1 Salt taste ............................................................................................. 573 23.4.2 Salty taste receptors ............................................................................ 574 23.4.3 Structural characteristics of salty taste-enhancing peptides ................. 574 23.5 Kokumi peptides .............................................................................................. 575 23.5.1 Kokumi taste ....................................................................................... 575 23.5.2 Kokumi taste receptors........................................................................ 575 23.5.3 The characteristics of kokumi peptides ............................................... 575 23.6 Summary ......................................................................................................... 576 Acknowledgments ...................................................................................................... 577 References ................................................................................................................. 577

Chapter 24: Cardiovascular benefits of food protein-derived bioactive peptides......581 Rotimi E. Aluko 24.1 Introduction ..................................................................................................... 581 24.2 Inhibition of the renin
angiotensin
aldosterone system: antihypertensive peptides ................................................................................. 583 24.2.1 ACE- and renin-inhibitory peptides..................................................... 584 24.2.2 Foods formulated with antihypertensive protein hydrolysates and peptides ........................................................................................ 598 24.3 Conclusions ..................................................................................................... 601 24.4 Future trends .................................................................................................... 602 References ................................................................................................................. 602

Chapter 25: Applications in medicine: hypoglycemic peptides ................................607 Forough Jahandideh and Jianping Wu 25.1 Introduction ..................................................................................................... 607 25.2 Carbohydrate digestion and glucose homeostasis ............................................. 608 25.3 Pathophysiology of type 2 diabetes .................................................................. 609 25.4 Clinical diagnosis of diabetes........................................................................... 610

Contents xix 25.5 Diverse physiological properties of protein hydrolysates and bioactive peptides ...................................................................................... 611 25.6 Antidiabetic properties of protein hydrolysates/peptides (in vivo studies) ........ 612 25.7 Antidiabetic properties of protein hydrolysates/peptides (clinical studies) ....... 612 25.8 Conclusions ..................................................................................................... 623 References ................................................................................................................. 623

Chapter 26: Application in medicine: obesity and satiety control ...........................629 Alina Kondrashina, Shauna Heffernan, Nora O’Brien and Linda Giblin Abbreviations ............................................................................................................. 629 26.1 Introduction ..................................................................................................... 630 26.2 Synthetic peptides ............................................................................................ 631 26.2.1 Synthetic peptides: glucagon-like peptide-1 mimetics ......................... 631 26.2.2 Synthetic peptides: multiple actions mimetics ..................................... 637 26.2.3 Safety considerations and limitations for synthetic peptides ................ 638 26.2.4 Other synthetic peptides in preclinical trials and in vitro development ........................................................................... 639 26.3 Food-derived peptides ...................................................................................... 640 26.3.1 Food-derived peptides targeting CCK and GI enzymes with proven in vivo efficacy ....................................................................... 644 26.3.2 Food-derived peptides targeting ghrelin, opioid receptor, and GI transit with proven in vivo efficacy ......................................... 646 26.3.3 Food-derived peptides targeting lipid metabolism with proven in vivo efficacy ....................................................................... 647 26.3.4 Food-derived peptides inhibiting protease dipeptidyl peptidase-4 ....... 648 26.3.5 In vitro evidence of food-derived peptides .......................................... 649 26.3.6 Limitations: survival of food-derived peptides during gut transit ........ 649 26.4 Commercial dietary protein hydrolyzates with antiobesity potential ................ 652 26.5 Summary ......................................................................................................... 655 Acknowledgments ...................................................................................................... 656 References ................................................................................................................. 656

Chapter 27: Food-derived osteogenic peptides towards osteoporosis ......................665 Ming Du, Zhe Xu, Hui Chen, Fengjiao Fan, Pujie Shi and Di Wu 27.1 Introduction ..................................................................................................... 665 27.2 Evaluation and diagnosis of osteoporosis ......................................................... 667 27.2.1 Bone formation and resorption biomarkers ......................................... 667 27.2.2 Computed tomography diagnosis ........................................................ 669

xx Contents 27.3 Osteogenic agents ............................................................................................ 670 27.3.1 Drugs for osteoporosis ........................................................................ 670 27.3.2 Osteogenic peptides ............................................................................ 672 27.4 Characterization of osteogenic peptides ........................................................... 679 27.4.1 Preparation of osteogenic peptides ...................................................... 679 27.4.2 Identification of osteogenic peptides ................................................... 679 27.5 Bioavailability of osteogenic peptides .............................................................. 681 27.5.1 Absorption analysis ............................................................................. 681 27.5.2 Pharmacokinetic analysis .................................................................... 681 27.6 Conclusions ..................................................................................................... 682 Acknowledgments ...................................................................................................... 682 Reference ................................................................................................................... 682

Chapter 28: Applications in medicine: mental health ............................................689 Yorick Janssens, Evelien Wynendaele, Kurt Audenaert and Bart De Spiegeleer 28.1 Introduction ..................................................................................................... 689 28.1.1 Peptide transport across the blood
brain barrier and use as shuttles ..................................................................................... 691 28.2 Peptides as diagnostic tools in brain tumors and CNS disorders ...................... 694 28.2.1 Peptide-based imaging tracers ............................................................. 694 28.2.2 Peptides as biomarkers ........................................................................ 695 28.3 Therapeutic applications of peptides for mental health .................................... 697 28.3.1 Neurodevelopmental disorders ............................................................ 698 28.3.2 Psychotic disorders ............................................................................. 703 28.3.3 Depressive, bipolar, and anxiety disorders .......................................... 705 28.3.4 Neurocognitive and neurodegenerative disorders ................................ 708 28.3.5 Others ................................................................................................. 711 28.4 Conclusion ....................................................................................................... 711 References ................................................................................................................. 712

Chapter 29: Applications in medicine: joint health ...............................................723 Ezequiel R. Coscueta, Marı´a Emilia Brassesco, Patrı´cia Batista, Sandra Borges and Manuela Pintado 29.1 Introduction ..................................................................................................... 723 29.2 Overview of joint diseases ............................................................................... 724 29.2.1 Osteoarthritis ....................................................................................... 724 29.2.2 Rheumatoid arthritis ............................................................................ 725

Contents xxi 29.3 Peptides activity and characterization .............................................................. 725 29.3.1 Natural bioactive peptide sources ........................................................ 725 29.3.2 Peptidome analysis .............................................................................. 727 29.4 Mechanisms of action ...................................................................................... 728 29.4.1 Cartilage proliferation ......................................................................... 728 29.4.2 Antioxidant, antimicrobial, and antiinflammatory activities ................ 731 29.4.3 Neuroactivity ...................................................................................... 733 29.5 Evidence in joint health benefits ...................................................................... 733 29.6 Potential applications, production, and commercialization ............................... 736 29.6.1 Diagnostic ........................................................................................... 736 29.6.2 Prophylaxis/therapeutic ....................................................................... 738 29.6.3 Production and commercialization ...................................................... 739 29.7 Summary ......................................................................................................... 740 Acknowledgments ...................................................................................................... 740 References ................................................................................................................. 741

Chapter 30: Applications in food technology: antimicrobial peptides .....................745 En Huang, Walaa E. Hussein, Emily P. Campbell and Ahmed E. Yousef 30.1 Introduction ..................................................................................................... 745 30.2 Classification ................................................................................................... 746 30.3 Current and potential food applications............................................................ 748 30.3.1 Commercial application of nisin ......................................................... 751 30.3.2 Commercial application of pediocin.................................................... 752 30.3.3 Commercial application of MicroGARD ............................................. 753 30.3.4 Commercial application of ε-polylysine .............................................. 753

30.4 30.5

30.6

30.7

30.3.5 Other antimicrobial peptide preparations received regulatory approval ............................................................................. 753 Hurdle approach............................................................................................... 754 Application of antimicrobial peptides for improving human health.................. 754 30.5.1 Antimicrobial peptides production by probiotic strains ....................... 754 30.5.2 Antiinfective activity of antimicrobial peptides ................................... 755 30.5.3 Antiviral effect of antimicrobial peptides ............................................ 756 30.5.4 Bioavailability and metabolism ........................................................... 756 Mechanisms of action ...................................................................................... 758 30.6.1 Mechanisms of action against bacteria and fungi ................................ 758 30.6.2 Mechanisms of action against viruses ................................................. 759 Safety considerations and regulations .............................................................. 759 30.7.1 Safety of antimicrobial peptides .......................................................... 759 30.7.2 Regulatory aspects of using AMPs or AMP producers in food ........... 760

xxii

Contents 30.8 Limitations ....................................................................................................... 761 30.9 Summary ......................................................................................................... 763 References ................................................................................................................. 764

Index ..................................................................................................................771

List of contributors J.E. Aguilar-Toala´ Protein Chemistry and Bioactive Peptides Laboratory, Department of Food Science, Purdue University, West Lafayette, IN, United States Gilda Aiello Department of Human Science and Quality of Life Promotion, Telematic University San Raffaele, Rome, Italy Sara Elsa Aita Department of Chemistry, Sapienza University of Rome, Rome, Italy Rotimi E. Aluko The Richardson Centre for Functional Foods and Nutraceuticals, Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada Miryam Amigo-Benavent Department of Biological Sciences, University of Limerick, Limerick, Ireland Mohammad Sadiq Amin College of Food Science, Southwest University, Chongqing, China Keizo Arihara School of Veterinary Medicine, Kitasato University, Towada, Japan Anna Arnoldi Department of Pharmaceutical Sciences, University of Milan, Milan, Italy Kurt Audenaert Department of Psychiatry and Medical Psychology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Kathrine H. Bak Institute of Food Safety, Food Technology and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria Ana Paulina Barba de la Rosa IPICYT, Potosino Institute of Scientific and Technological Research A.C., San Luis Potosi, Mexico Patrı´cia Batista Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal Khushwant S. Bhullar Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada; Department of Pharmacology, University of Alberta, Edmonton, AB, Canada; 4–10 Agricultural/Forestry Centre, Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Carlotta Bollati Department of Pharmaceutical Sciences, University of Milan, Milan, Italy Sandra Borges Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal Said Bouhallab STLO, INRAE, Institut Agro, Rennes, France Marı´a Emilia Brassesco Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal Emily P. Campbell Department of Food Science and Technology, The Ohio State University, Columbus, OH, United States Laetitia Canabady-Rochelle Universite´ de Lorraine, CNRS, LRGP, Nancy, France

xxiii

xxiv List of contributors Anna Laura Capriotti Department of Chemistry, Sapienza University of Rome, Rome, Italy C. Centner Department of Sport and Sport Science, University of Freiburg, Freiburg, Germany Andrea Cerrato Department of Chemistry, Sapienza University of Rome, Rome, Italy Hui Chen College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, P.R. China Liang Chen Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Ezequiel R. Coscueta Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal Gizella Csire Universite´ de Lorraine, CNRS, LCPM, Nancy, France; Universite´ de Lorraine, CNRS, L2CM, Nancy, France Xin-Yue Cui Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Małgorzata Darewicz Department of Food Biochemistry, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland Antonio De Leo´n-Rodrı´guez IPICYT, Potosino Institute of Scientific and Technological Research A.C., San Luis Potosi, Mexico Bart De Spiegeleer Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Ste´phane Desobry Universite´ de Lorraine, LIBio, Nancy, France Vermont P. Dia Department of Food Science, The University of Tennessee, Knoxville, TN, United States Ming Du School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, P.R. China Sarah El Hajj Universite´ de Lorraine, CNRS, LRGP, Nancy, France; Universite´ de Lorraine, CITHEFOR, Nancy, France Fengjiao Fan College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, P.R. China Richard J. FitzGerald Department of Biological Sciences, University of Limerick, Limerick, Ireland Yu Fu College of Food Science, Southwest University, Chongqing, China Advaita Ganguly Comprehensive Tissue Centre, UAH Transplant Services, Alberta Health Services, Edmonton, AB, Canada Caroline Gaucher Universite´ de Lorraine, CITHEFOR, Nancy, France Linda Giblin Food Biosciences Department, Teagasc Food Research Centre, Cork, Ireland Jean-Michel Girardet Universite´ de Lorraine, INRAE, IAM, Nancy, France Tristan Giraud Universite´ de Lorraine, CNRS, LCPM, Nancy, France Elvira Gonzalez de Mejia Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Champaign, IL, United States; Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Champaign, IL, United States Rui-Zeng Gu Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China

List of contributors xxv F.G. Hall Protein Chemistry and Bioactive Peptides Laboratory, Department of Food Science, Purdue University, West Lafayette, IN, United States Shauna Heffernan School of Food and Nutritional Sciences, University College Cork, Cork, Ireland En Huang Department of Environmental and Occupational Health, College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR, United States Walaa E. Hussein Department of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, OH, United States; Department of Microbiology and Immunology, National Research Center, Giza, Egypt Anna Iwaniak Department of Food Biochemistry, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland Forough Jahandideh Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada Yorick Janssens Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium S. Jerger Department of Sport and Sport Science, University of Freiburg, Freiburg, Germany Mohammadreza Khalesi Department of Biological Sciences, University of Limerick, Limerick, Ireland J. Kohl Department of Sport and Sport Science, University of Freiburg, Freiburg, Germany Alina Kondrashina Food Biosciences Department, Teagasc Food Research Centre, Cork, Ireland D Ko¨nig Department of Sport and Sport Science, University of Freiburg, Freiburg, Germany Aldo Lagana` Department of Chemistry, Sapienza University of Rome, Rome, Italy; CNR NANOTEC, Campus Ecotekne, University of Salento, Lecce, Italy Rene´ Lametsch Department of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark Carmen Lammi Department of Pharmaceutical Sciences, University of Milan, Milan, Italy Guo-Ming Li Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Qian Li Department of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark A.M. Liceaga Protein Chemistry and Bioactive Peptides Laboratory, Department of Food Science, Purdue University, West Lafayette, IN, United States Wen-Ying Liu Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Yan Liu Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Jun Lu Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Kaustav Majumder Department of Food Science and Technology, University of NebraskaLincoln, Lincoln, NE, United States

xxvi List of contributors Toshiro Matsui Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka, Japan; Division of Taste Sensor/Odor Sensor, Research and Development Center for Five-Sense Devices, Kyushu University, Fukuoka, Japan Yoshinori Mine Department of Food Science, University of Guelph, Guelph, ON, Canada Piotr Minkiewicz Department of Food Biochemistry, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland Carmela Maria Montone Department of Chemistry, Sapienza University of Rome, Rome, Italy Leticia Mora Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), Valencia, Spain Laurence Muhr Universite´ de Lorraine, CNRS, LRGP, Nancy, France Motoko Ohata College of Bioresource Sciences, Nihon University, Fujisawa, Japan Ogadimma D. Okagu Department of Chemistry and Biomolecular Sciences, Faculty of Science, University of Ottawa, Ottawa, ON, Canada Nora O’Brien School of Food and Nutritional Sciences, University College Cork, Cork, Ireland Ce´dric Paris Universite´ de Lorraine, CNRS, LRGP, Nancy, France; Universite´ de Lorraine, LIBio, Nancy, France Manuela Pintado Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal Susy Piovesana Department of Chemistry, Sapienza University of Rome, Rome, Italy Xiu-Yuan Qin Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Kenji Sato Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan Katalin Selmeczi Universite´ de Lorraine, CNRS, L2CM, Nancy, France Tatiana Sepulveda-Rincon Universite´ de Lorraine, CNRS, LRGP, Nancy, France; Faculty of Pharmaceutical and Food Sciences, University of Antioquia, Medellin, Colombia Fereidoon Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada Nan Shang Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada; 4–10 Agricultural/Forestry Centre, Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Kumakshi Sharma Health, Safety and Environment Branch, National Research Council Canada, Edmonton, AB, Canada Pujie Shi School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, P.R. China Loic Stefan Universite´ de Lorraine, CNRS, LCPM, Nancy, France Xiaohong Sun School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada; College of Food and Biological Engineering, Qiqihar University, Qiqihar, P.R. China Mitsuru Tanaka Division of Integrated Research for Five-sense Devices, Research and Development Center for Five-Sense Devices, Kyushu University, Fukuoka, Japan Ganesh Thapa Department of Biological Sciences, University of Limerick, Limerick, Ireland Fidel Toldra´ Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), Valencia, Spain

List of contributors xxvii Chibuike C. Udenigwe School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada; Department of Chemistry and Biomolecular Sciences, Faculty of Science, University of Ottawa, Ottawa, ON, Canada U. Urbizo-Reyes Protein Chemistry and Bioactive Peptides Laboratory, Department of Food Science, Purdue University, West Lafayette, IN, United States Gabriela Va´zquez-Rodrı´guez IPICYT, Potosino Institute of Scientific and Technological Research A.C., San Luis Potosi, Mexico; Department of Biomedical and Clinical Sciences, Linko¨ping University, Sweden Aı´da Jimena Velarde-Salcedo Faculty of Chemical Sciences, Autonomous University of San Luis Potosı´, Mexico Yu-Chen Wang Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Yu-Qing Wang Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Ying Wei Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China Di Wu School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, P.R. China Jianping Wu Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada; 4–10 Agricultural/Forestry Centre, Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Evelien Wynendaele Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium Feiran Xu Department of Food Science and Human Nutrition, University of Illinois at UrbanaChampaign, Champaign, IL, United States; School of Food Science and Technology, Jiangnan University, Wuxi, P.R. China Zhe Xu School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, P.R. China; College of Life Sciences, Key Laboratory of Biotechnology and Bioresources Utilization, Dalian Minzu University, Ministry of Education, Dalian, P.R. China JuDong Yeo Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada Issei Yokoyama School of Veterinary Medicine, Kitasato University, Towada, Japan Ahmed E. Yousef Department of Food Science and Technology, The Ohio State University, Columbus, OH, United States; Department of Microbiology, The Ohio State University, Columbus, OH, United States Hua Zhang Department of Food Nutrition and Safety, College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, P.R. China

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Preface Bioactive peptides are derived from food proteins and are involved in metabolic regulation with a variety of benefits for human health and therefore have been proposed as functional food ingredients for controlling, or even reducing, the risk of chronic diseases. The scientific knowledge on bioactive peptides is a very dynamic field, developing and changing very rapidly in the recent years. This book is providing readers with an updated and comprehensive overview of bioactive peptides with special emphasis on the recent advances and state of the art on bioactive peptides’ fundamental concepts, methodology, production, structure
activity relationships, and benefits for human health. It covers a variety of disciplines such as chemistry, biochemistry, cell and molecular biology, physics, and medicine. Such multidisciplinary approach, intrinsic to the field of bioactive peptides, fills the gap between peptide chemistry and physiological functionality, while reviewing recent methodological progress in peptides analysis and bioactivity, and applications in the medical, pharmaceutical, nutritional, and food fields. This book can be used as a key reference for the readers in the field of food science, nutrition, functional foods/ nutraceuticals, biochemistry, biotechnology, industry and marketing sectors, or as a textbook/reference for undergraduate and graduate students. The book contains 30 chapters divided into three parts. The first part compiles recent methodologies with a detailed description on protocols and analysis for the generation and production of bioactive peptides, their extraction from different source materials, analysis, and identification; in vitro and in vivo bioactivity assays; and mechanisms of action and databases of bioactive peptides sequences. The second part discusses the plant and animal sources as well as new alternative sources such as insects, and the latest developed technologies for bioactive peptides’ protection for its appropriate delivery to the target organs. The third and last part is reporting the applications of bioactive peptides in clinical and sport nutrition and how sensory aspects are considered; medical applications with evidence of beneficial effects in animals and humans for cardiovascular benefits, diabetes prevention, obesity and satiety, osteoporosis, mental and joints health; and finally the applications of antimicrobial peptides in foods.

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Preface

We wish to thank all the contributing authors for sharing their time, knowledge, and expertise for making this book possible. We also want to thank the production team at Elsevier with special recognition to Sam Young, Editorial Project Manager, and Peter Linsley, Senior Acquisitions Editor.

´ and Jianping Wu Fidel Toldra

CHAPTER 1

Bioactive peptides in health and disease: an overview JuDong Yeo and Fereidoon Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada

1.1 Introduction Human body is constantly exposed to extrinsic toxic substances that disturb its normal functions, thus causing severe health conditions and diseases that lead to extensive use of health-promoting agents to prevent chronic diseases. Ames, Shigena, and Hegen (1993) Recently, much interest has been paid to explore structural, compositional, and sequential properties of bioactive peptides due to their variety of health benefits such as anticancer, opioid activity, anti-inflammation, antihypertensive activity, antibacterial effect, and enhancement of intestinal activity, among others (Amarowicz & Shahidi, 1997; Ambigaipalan & Shahidi, 2017; Ariyoshi, 1993; Clare & Swaisgood, 2000; Cumby, Zhong, Naczk, & Shahidi, 2008; Kim et al., 2001; Nwachukwu & Aluko, 2019; Park, Jung, Nam, Shahidi, & Kim, 2001; Shahidi, Han, & Synowiecki, 1995; Shahidi, Synowiecki, & Balejko, 1994; Wu & Ding, 2002; Yi, Zhao, & Wu, 2017). Moreover it is necessary to minimize the side effects including nephrotoxic, neurotoxic, cardiotoxic, and gonadotoxic implications which are of major concern for synthetic products (Gutierrez et al., 2016; Oun, Plumb, Rowan, & Wheate, 2013; Van Acker et al., 2016). Bioactive peptides consist of a diverse combination of amino acids and are specific fragments of food proteins that provide certain health benefits to the human body (Wu, Liao, & Udenigwe, 2017). Bioactive food-derived peptides were first reported by Mellander (1950), in which casein-derived phosphorylated peptides significantly improved vitamin D-independent calcification in rachitic neonates (Mellander, 1950). Since then, more than 1250 peptides with various bioactivities have been found from natural sources. Inactive amino acid sequences that are encrypted in the primary structure of plant and animal proteins are converted into active molecules upon liberation from parental protein, leading to the physiological modulators with hormone-like activity. In the body, signaling and modulation processes are primarily governed by the chemical interaction of specific segments of amino

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00007-8 © 2021 Elsevier Inc. All rights reserved.

1

2

Chapter 1

acid sequences, either in the form of peptides or parts of proteins, thus bioactive peptides hold future promise for diverse therapeutic applications (Fields, Falla, Rodan, & Bush, 2009). Bioactive peptides are liberated from proteins through enzymatic proteolysis (i.e., gastrointestinal digestion, in vitro hydrolysis by proteolytic enzymes) as well as food processing (i.e., cooking, fermentation, ripening, etc.) (Toldra´, Reig, Aristoy, & Mora, 2018). The enzymatic hydrolysis of food proteins leads to three major structural changes, namely reducing average molecular mass/size, producing higher availability of hydrophobic regions, and generating ionizable groups, and this structural alteration results in their wide spectrum of bioactivities. Indeed, the effectiveness of bioactive peptides depends on their structural properties such as amino acid sequence, the electronic charge of peptides, length and weight of the peptide, and hydrophobic/hydrophilic properties. These structural effects are deeply involved in their wide spectrum of biological functions such as anticancer, antimicrobial, immunomodulatory, antihypertension, opioid agonists or antagonists, antithrombotic, antioxidant activities, and nutrient utilization (Clare & Swaisgood, 2000; Elias, Kellerby, & Decker, 2008). This contribution provides a critical overview on bioactive peptides derived from diverse food sources by focusing on their health-promoting effects and inhibitory activities against chronic diseases.

1.2 Preparation of bioactive peptides A variety of plant- and animal-based foods containing a high level of proteins are excellent sources for the production of bioactive peptides in which several factors such as availability and affordability of raw material and their compositional characteristics are most important. Numerous animal and plant food proteins have been used for the production of bioactive peptides, namely animal proteins (i.e., milk proteins, casein, whey, egg, meat muscle proteins, etc.), marine proteins (i.e., salmon, macroalgae, oyster sea urchin, squid, snow crab, shrimp, seahorse, etc.), and plant food proteins (i.e., lentil, chickpea, pea, beans, oat, wheat, hemp seed, canola, flaxseed, etc.). Generally, peptides released from food proteins upon hydrolysis have displayed better bioactivity compared to their parent proteins, indicating the significance of the hydrolysis of peptide bonds for producing bioactive peptides (Aluko, 2008; Hartmann & Meisel, 2007). Three common methods such as the enzymatic hydrolysis, microbial fermentation, and chemical synthesis (which is mainly conducted for their characterization) have so far been widely used to obtain bioactive peptides (Lee et al., 2017), and among them, enzymatic hydrolysis has provided the most popular means in isolating bioactive peptides from natural protein sources. Enzymatic hydrolysis to release peptides of interest is carried out using single or multiple, specific or nonspecific, proteases after the selection of raw material (protein sources) for the production of

Bioactive peptides in health and disease: an overview 3 bioactive peptides (Udenigwe & Aluko, 2012). Several common enzymes are widely used to hydrolyze food-derived proteins such as Alcalase, trypsin, pepsin, Flavourzyme, and simulated gastrointestinal digestion enzymes (trypsin and pancreatin). In the hydrolysis process, reaction conditions such as hydrolysis time and temperature, degree of hydrolysis of the protein, enzyme-substrate ratio, and pretreatment of the protein before hydrolysis are significant factors affecting the yield and the composition of liberated bioactive peptides (Udenigwe & Aluko, 2012). To induce microbial fermentation, a variety of species of microbes including Bacillus subtilis, Aspergillus oryzae, Lactobacillus plantarum, Mucor michei ex fries have been utilized to acquire bioactive peptides. The released peptides are then fractionated by using advanced techniques based on the physical and chemical properties of bioactive peptides such as size, net charge, and hydrophobicity. Membrane ultrafiltration, ultracentrifugation, and size-exclusion chromatography have been widely used to separate peptides depending on defined molecular weight ranges, in particular for obtaining low-molecular-weight peptides. Reverse-phase high performance liquid chromatography (HPLC) is also used to fractionate peptides based on the discrepancy in their hydrophobic/hydrophilic properties in which the physical separation of individual peptides is carried out depending on their affinity to the stationary phase in the column (Pownall, Udenigwe, & Aluko, 2010). In addition, particular net charges can be exploited to separate bioactive peptides using selective ion-exchange columns (Pownall et al., 2010), and electrodialysis-ultrafiltration to fractionate cationic, anionic, and neutral peptides of defined molecular sizes (Firdaous et al., 2009). Thus, an appropriate approach should be used to acquire desired bioactive peptides by considering their structural properties.

1.3 Absorption of peptides in the small intestine 1.3.1 Paracellular transport In the intestinal surface, there are water-filled pores or channels between cells, referred to as paracellular space, and this route plays a significant role in the absorption of micro- and macromolecules (Fig. 1.1) (Renukuntla, Vadlapudi, Patel, Bodduc, & Mitra, 2013). This paracellular route accounts for 0.01%
0.1% of the total intestinal surface area: Given that the surface area of the intestinal epithelium is approximately 2 x 106 cm2, the water-filled pore (paracellular route) corresponds to 200
2000 cm2 (Fasano, 1998). This route is an efficient pathway for the absorption of peptides produced from the hydrolysis of proteins, in particular hydrophilic peptides and low-molecular-weight molecules (Renukuntla et al., 2013). However, there is a limitation in the penetration of polar macromolecules due to the presence of tight junctions or zonula occludens, which are multiprotein junctional complexes and play an important role in preventing leakage of transported solutes and water at the paracellular pathway (Stella, 2007). Nevertheless, the paracellular route is an efficient pathway for the penetration of polypeptides in which their physicochemical properties such as molecular dimension and the overall ionic charge of peptides decide their

4

Chapter 1

Figure 1.1 Intestinal drug transport mechanisms.

permeability through paracellular route (Salamat-Miller & Johnston, 2005). Chittchang et al. (2002) examined the effect of the secondary structure of peptides on their diffusion rate in the microporous membrane. Interestingly, the structural changes from random coil to the α-helix did not show the significant alteration in permeability of peptides, however, β-sheet conformer showed the decreased rate in permeability to the microporous membrane. Dodoo et al. (2000) investigated the permeability of 14 synthetic model peptides on rat alveolar cell monolayers in which most peptides penetrated the cells via the paracellular route, and apparent permeability (Papp) values were inversely proportional to the molecular size. In addition, the structural modification of drug molecules and modulation of tight junctions were investigated to enhance the penetration of bioactive molecules (Lane & Corrigan, 2006).

1.3.2 Transcellular transport Transcellular transport occurs through the diffusion of molecules at the apical and basolateral membranes in the digestive tract (Fig. 1.1). This pathway of transport includes transcellular diffusion, active carrier-mediated transport, and transcytosis. The transcellular diffusion involved in the transport of molecules includes moving of molecules from an area of high concentration to an area of low concentration. The rate of molecular transfer is followed by Fick’s law of diffusion (Gibaldi, 1991) (Eq. 1.1). dQ DKAðC1 2 C2 Þ 5 dt h

(1.1)

Bioactive peptides in health and disease: an overview 5 The rate of diffusion (dQ/dt) is affected by several factors such as diffusion coefficient (D), oil/water partition coefficient of drug or bioactive molecules (K), surface area of the membrane across which transfer occurs (A), thickness of the membrane through which diffusion occurs (h), difference in concentrations in areas 1 and 2 (C1 2 C2), respectively. This pathway is preferred to the lipophilic compounds possessing a relatively high affinity to the lipid membrane (Renukuntla et al., 2013). Carrier-mediated transport involves the movement of molecules via membrane protein transporters, referred to as active transport. The representative membrane transporters for the absorption of peptides in the digestive tract are peptide transporter 1 (PEPT1) and PEPT2. Di- and tripeptides are absorbed into the small intestinal epithelial cell by a transporter called (PEPT1) with H1 ions followed by an introduction into the bloodstream in the intact form from the enterocyte (Rubio-Aliaga & Daniel, 2002). Subsequently, these peptides circulate in plasma and enter the target cells through PEPT1 or PEPT2, depending on the type of cells. Another pathway is transcytosis referred to as cytopempsis. In this pathway, a variety of molecules, in particular macromolecules such as peptides, are transferred across the interior of the epithelial cells in which lipid membrane at one side of the cell captures molecules by forming vesicles, then they transport captured molecules across the cell inside, followed by ejection on the other side.

1.3.3 Absorption of peptides in the large intestine (colon) The large intestine consists of three segments, cecum, colon, and rectum, and their wall has simple columnar epithelium and mucus-secreting goblet cells (Renukuntla et al., 2013). More than 400 species of gut microbiota reside in the large intestine in which they support the digestion of foods that are not absorbed in the small intestine by secreting extracellular enzymes such as carbohydrases and proteases, among others. Some compounds formed from fermentation are in turn absorbed by passive diffusion through the large intestinal wall (Rafii, Franklin, & Cerniglia, 1990). The gut microbiota also produces vitamins including vitamin K, thiamine, riboflavin, and biotin, which are then absorbed and circulated, followed by transfer to different organ cells. A large intestine is also a suitable place for the absorption of peptides due to the presence of proteases produced by gut microbiota and relatively long residence time (Sinha & Kumria, 2003). Vitamin influx receptors also play a significant role in delivering peptides using surface-modified particulate systems (Renukuntla et al., 2013). The epithelial junctions of the colon do not allow the permeation ˚ , however, some polypeptides having excellent of molecules larger than 8
9 A conformational flexibility can diffuse through the tight epithelial junction in the colon (Tomita, Shiga, Hayashi, & Awazu, 1988).

6

Chapter 1

1.3.4 Approaches for enhancing the absorption of peptides Two major factors, physical and biochemical barriers, interfere with the absorption of bioactive peptides in the gastrointestinal epithelium. The physical barrier is mainly caused by poor penetration rate across the intestinal membrane, while the biochemical barrier stems from the enzymatic degradation of peptides by peptidases (Hamman, Enslin, & Kotze, 2005). Thus careful consideration regarding physicochemical properties (i.e., molecular weight, ionization constant, pH stability, molecular size, hydrophobicity, etc.) and biological barriers (i.e., poor permeation and membrane efflux, variable pH, enzymatic hydrolysis, etc.) should be taken into consideration when developing efficient bioactive peptides having high bioavailability (Mahato, Narang, Thoma, & Miller, 2003). Until now, many formulation strategies including absorption enhancers, mucoadhesive polymers, liposomes, nanoparticles, enzyme inhibitors, and hydrogels, among others, have been developed to enhance the bioavailability of peptides. Absorption enhancers are molecules that improve/promote the absorption of peptides in the digestive tract (Aungst, 2012). The majority of bioactive peptides are hydrophilic in nature along with high-molecular-weight, in particular peptides with long-chain amino acids, in which their absorption rate via transcellular and paracellular routes in the gastrointestinal epithelium is severely restricted (Shaji & Patole, 2008). There are several underlying mechanisms of absorption enhancers that temporarily interrupt the structural integrity of the intestinal barrier, reducing the mucus viscosity, improving the space in the tight junctions, and enhancing the membrane fluidity (Liu, LeCluyse, & Thakker, 1999). A variety of absorption enhancers have been investigated to improve peptide absorption through the intestinal membrane, namely cationic and anionic polymers, chelating agents, acylcarnitines, surfactants, bile salts, fatty acids and their derivatives (Renukuntla et al., 2013). The absorption enhancers help therapeutic agents (peptides) to penetrate across biological membranes, followed by an introduction into the systemic circulation and reaching to the target sites to exert their health-beneficial effects (Shaji & Patole, 2008). The effectiveness/efficiency of absorption enhancers depends on the physical and chemical properties of peptides, regional differences in the intestinal membrane, and other excipients. Some properties including safe and non-toxic, pharmacologically and chemically inert, non-irritant, and non-allergenic are also required to be an appropriate absorption enhancer (Sagar, Sharma, Bansal, & Banik, 2011). Mucoadhesive systems consist of synthetic or natural polymers that bind to mucosal membranes, which allows a higher level of the bioactive peptide to be absorbed in the epithelium cells, thus their capacity to adhere to mucin layer on the mucosal epithelium dictates their ability as the mucoadhesive polymers (Renukuntla et al., 2013). Mucoadhesive polymers decrease the rate of clearance of drug or bioactive peptide molecules from the absorption site, which leads to the extension of time for absorption. They also suppress the

Bioactive peptides in health and disease: an overview 7 activity of proteolytic enzymes and involve modulating the permeability of tight epithelial tissue barriers (Lehr, 1996). There are two types of mucoadhesive polymers such as synthetic or natural polymers. Synthetic bioadhesive polymers include polyacrylic acids (i.e., carbopol, polycarbophil, polyacrylic acid, polyacrylate, poly(methylvinylether-co-methacrylic) acid) and cellulose derivatives (i.e., carboxymethyl cellulose, methylhydroxyethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, and methyl cellulose), while chitosan and a variety of gums including xanthan, guar, crylamide-acrylate polymer, poly (vinylpyrrolidone), and poly (vinyl alcohol) are representative (semi-) natural bioadhesive polymers (Renukuntla et al., 2013). Aside from absorption enhancers and mucoadhesive polymers, several delivery systems such as liposomes, nanoparticles, microparticles, and cyclodextrins have been developed to protect bioactive peptides from pH, oxidation, bile salts and various enzymes present in the digestive tract (Sprott et al., 2003). Liposomes are referred to as microscopic vesicles consisting of one or multiple phospholipid bilayer membranes with a diameter ranging from 0.0 to 10 μm. They are lipid-based delivery systems that can protect bioactive peptides in the GI tract (Li, Chen, Sun, & Xu, 2010). Liposomes have been successfully used for the delivery of a diverse array of therapeutics such as nucleotides, proteins, peptides and plasmids (Kurz & Ciulla, 2002). In the structure of liposome, hydrophilic molecules are encapsulated in the inner aqueous core, while the hydrophobic ones are localized in the lipid bilayer (Kaur, Garg, Singla, & Aggarwal, 2004). In some cases, liposomes are utilized for the site-specific delivery of peptides by decorating the surface with targeting moieties such as antibodies. A variety of parameters including liposome size, surface charge, the composition of the liposomes, and encapsulation efficiency are significant factors affecting the efficiency in the delivery of peptides by liposomes (Renukuntla et al., 2013). Recently, accumulating evidence has indicated the application of nanoparticles as an effective delivery system. Nanoparticles are known as colloidal carriers with size ranging between 1 and 100 nm. They are divided into two groups of nanospheres that are matrix systems in which bioactive molecules are uniformly physically dispersed and nanocapsules that are encapsulated by a polymeric membrane (Yun, Cho, & Park, 2012). Nanoparticles are relatively stable in the harsh GI environment compared to other delivery systems such as liposomes and micelles, and their optimized polymer features and surface chemistry enable the controlled release and targeting specific tissue (Panyam & Labhasetwar, 2003). Tailor-made pH sensitive nanoparticles allow delivery of bioactive peptides to different sections of the intestine by using the different pH in the GI tract; polyanionic or polycationic polymers and their mixtures are widely used to make such nanoparticles. The central mechanism of bioactive release from nanoparticles is primarily based on the dissolution of the bioactive and the swelling of polymers (or both of these) at a specific pH. Enteric coating materials have commonly been utilized in most pH-sensitive carriers to extend a period of time as well as to improve their safety in biological systems.

8

Chapter 1

For instance, diethylenetriaminepentaacetic acid has been used to interrupt intestinal tight junctions in order to enhance the absorption of nanoparticles in the gastrointestinal epithelium and to inhibit the activity of intestinal proteases by chelating divalent metal ions (Su et al., 2012). In addition to the above approaches, hydrogels, microparticles, and cyclodextrins have also been widely used to enhance the bioavailability of peptides. The detailed explanations of those approaches are not included in this chapter due to space limitations.

1.3.5 Structure-activity relationship of bioactive peptides The specific health-beneficial activity of peptides originates from a diverse combination of amino acid residues with specific sequences. In other words, the discrepancy in the chemical structure of bioactive peptides including the amino acid composition, the type of amino acid in C- and N-terminal, spatial structure, the hydrophobic/hydrophilic property, the length and weight of the peptide chain, the charge character of amino acid, and other factors dictate their chemical and physical properties (Ji, Li, He, & Qian, 2011). For instance, in angiotensin-converting enzyme (ACE; EC 3.4. 15.1), the inhibitory activity of bioactive peptides show a strong relationship between amino acid composition and their inhibitory activity. Many studies have reported that the chemical interaction of bioactive peptides with ACE is greatly governed by the sequences of three amino acids in C-terminal section; in particular aromatic or alkaline amino acids in the N-terminal of bioactive peptides remarkably enhances the ACE inhibitory activity of such peptides (Aleman, Gimenez, Perez-Santin, Gomez-Guillen, & Montero, 2011; Pan, Cao, & Guo, 2012). Wu et al. (2016) reported that aromatic amino acids were preferred for the carboxyl terminus, while positively charged amino acids were preferred for the middle position, as well as hydrophobic amino acids were preferred for the amino terminus in antihypertensive tripeptides. The presence of leucine, isoleucine, and valine in the N-terminal of bioactive peptides enhances their antihypertensive effect. On the other hand, the presence of proline in the N-terminal reduces their ACE inhibitory activity (Li & Yu, 2014). A high proportion of ACE inhibitory peptides contained amino acids tyrosine (Tvr), proline (Pro), tryptophan (Trp), phenylalanine (Phe), and leucine (Leu) in the C-terminal, while arginine (Arg), Tvr (Gly), glycine, valine (Va1), alanine (Ala), and isoleucine (Ile) were present in the N-terminal of the ACE inhibitory peptides. Thus ACE inhibitory activities of bioactive peptides are strongly related to their terminal amino acid compositions. The spatial structure also greatly affects the bioactivities of peptides. Thus the high amphipathic and stable helical section of bioactive peptides play a central role in their anticancer activity. The hydrophilic and hydrophobic surfaces are formed by the arrangement of hydrophilic and hydrophobic amino acid side chains in the two sides of

Bioactive peptides in health and disease: an overview 9 α-helix structure or by the concentration of hydrophilic and hydrophobic sides in the N-terminal and C-terminal (Li & Yu, 2014). Dennison, Whittaker, Harris, and Phoenix (2006) reported that the architectural features of α-ACPs, including amphilicity levels and hydrophobic arc size are responsible for their excellent penetration capacity to the cancer cell membranes. The potent hemolytic activity of peptides displayed a high correlation with high helicity, whereas the reduced helicity caused a decrease in the anti-HeLa activity of peptides (Huang, He, Jiang, & Chen, 2012). The β-sheet peptides also showed strong anticancer activity, and their structure was generally stabilized by the disulfide bonds. The representative β-sheet anticancer peptides are defensins (small cysteine-rich cationic proteins), lactoferricin (isolated by the pepsin-mediated digestion of lactoferrin), and tachyplesin (isolated from the horseshoe crab). Tachyplesin I, which possesses β-sheet (residues 3
8 and 11
16) associated by a type I β-turn (residues 8
11) along with two disulfide bonds, significantly suppressed human gastric adenocarcinoma, a type of cancerous tumor, and human hepatocarcinoma cells (Kawano et al., 1990; Li, Ouyang, Peng, & Hong, 2003; Shi, Wang, Liang, & Li, 2006). Buforin IIb exhibited anticancer activity against 62 cancer cell lines by binding with cell surface gangliosides (Vitor, Maria, & Margarida, 2012). Bovine lactoferricin (LfcinB) exhibited strong anticancer capacity in the different cancer cell lines such as leukemia cells, fibrosarcoma cells, various carcinomas, and neuroblastoma cells in which it inhibited the basic fibroblast growth factors, that is, bFGF- and VEGF- driven proliferation and the movement of human endothelial cells (Eliassen, Haug, Berge, & Rekdal, 2003). The electronic charge is another significant factor affecting the bioactivities of peptides. The interaction of bioactive peptides with the cancer cell membrane occurs via electrostatic interactions in which the cationic portion of peptides binds to the anionic section of the lipopolysaccharide of cancer cells at the surface of the membrane, leading to membrane perturbation (Schweizer, 2009). The net charge and the number of positive charges from functional groups of peptides also affect their bioactivity. Aside from the above properties of peptides, molecular shape and structural folding, the hydrophobic/hydrophilic property, the length and weight of the peptide chain are also important factors influencing their bioactivities.

1.4 Bioactivities of food-derived bioactive peptides focusing on inhibiting chronic diseases 1.4.1 Anticancer activity The occurrence of a malignant tumor (cancer) is a physiological process due to the growth/ proliferation of the abnormal cells in body, characterized by their uncontrollable growth and fast spread into surrounding tissues. Thus, it is extremely important to inhibit the deregulated

10

Chapter 1

cell proliferation to prevent the growth of tumors in the body (Chi, Hu, Wang, Li, & Ding, 2015). Chemoprevention is one of the most effective anticancer approaches among other treatments, and it has widely been used to weaken the morbidity and mortality of cancer by decelerating the progression of carcinogenesis (Pan, Zhao, Hu, Chi, & Wang, 2016; Sheih, Fang, Wu, & Lin, 2010). However, increasing evidence has reported severe side effects of synthetic anticancer agents such as nephrotoxic, neurotoxic, cardiotoxic and gonadotoxic effects (Gutierrez et al., 2016; Oun et al., 2013; Van Acker et al., 2016). The above disadvantages of synthetic agents have led to the increasing attraction of natural bioactive peptides by consumers and researchers. Epidemiological evidence has demonstrated the remarkable anticancer effect of protein hydrolysates by retarding the initiation and progression of cancers in which they constrain the transformation of normal cells, the growth of tumors, angiogenesis, and metastasis. A large number of studies has been conducted on the anticancer activity of bioactive peptides from foods (Table 1.1). For instance, two peptides KPEGMDPPLSEPEDRRDGAAGPK and KLPPLLLAKLLMSGKLLAEPCTGR released from tuna cooking juice exhibited potent antiproliferative activity in breast cancer cell line MCF-7 (Hung, Yang, Kuo, & Hsu, 2014): These two peptides induced cell arrest in the S phase by elevating p21 and p27 expression while reducing cyclin A expression and downregulating Bcl-2, PARP, and caspase 9 expression as well as upregulating p53 and Bax expression. A peptide QPK isolated from sepia ink protein hydrolysates significantly suppressed the proliferation of DU-145, PC-3 and LNCaP cells in which this peptide reduced Table 1.1: Anticancer activities of bioactive peptides from different food sources. Source Tuna cooking juice Sepia ink Oyster hydrolysates Chickpea hydrolysates Soybean hydrolysates Fermented rapeseed S. Platensis hydrolysates Blood clam muscle hydrolysates

Isolated peptide

Anticancer effect (model)

Reference

KPEGMDPPLSEPEDRRDGAAGPK KLPPLLLAKLLMSGKLLAEPCTGR

Breast cancer cell line MCF-7

Hung et al. (2014)

QPK

DU-145, PC-3 and lncap cells

LANAK

Human colon carcinoma (HT-29) cell lines Breast cancer cell lines

Huang, Yang, et al. (2012) Umayaparvathi et al. (2014) Xue et al. (2015)

RQSHFANAQP RKQLQGVN, GLTSK, LSGNK, GEGSGA, MPACGSS, MTEEY

Colorectal cancer HT-29 cells

Peptide mixture

Human hepg2 liver cancer, human MCF-7 breast cancer and human MCF-7 breast cancer cell lines HT-29 cancer cell

HVLSRAPR WPP

PC-3, DU-145, H-1299 and hela cell lines

Ferna´ndez-Tome´ et al. (2018), Vital et al. (2014) Xie et al. (2015)

Wang and Zhang (2017) Chi, Hu, et al. (2015)

Bioactive peptides in health and disease: an overview 11 the expression of the anti-apoptotic protein Bcl-2 and enhanced the expression of apoptogenic protein Bax (Huang, Yang, et al., 2012). A peptide LANAK prepared from oyster hydrolysate also displayed potent anticancer activity against human colon carcinoma (HT-29) cell lines (Umayaparvathi et al., 2014). In addition, peptides from plant-based food hydrolysates also showed an effective anticancer activity. A peptide RQSHFANAQP, isolated from chickpea hydrolysates, remarkably increased the level of p53 in breast cancer cell lines, leading to the efficient inhibition of their proliferation (Xue et al., 2015). Several protein hydrolysates from soybean including Lunasin, RKQLQGVN, GLTSK, LSGNK, GEGSGA, MPACGSS, and MTEEY exerted excellent antiproliferative effects on colorectal cancer HT-29 cells (Ferna´ndez-Tome´, Sancho´n, Recio, & Herna´ndez-Ledesma, 2018; Vital, de Mejı´a, Dia, & Loarca-Pin˜a, 2014). Peptides isolated from rapeseed protein upon fermentation also showed strong inhibitory activity against the proliferation of human HepG2 liver cancer, human MCF-7 breast cancer and human MCF-7 breast cancer cell lines (Xie et al., 2015). Moreover, a peptide HVLSRAPR prepared from S. platensis hydrolysates displayed potent inhibitory activity against HT-29 cancer cell proliferation, while it showed relatively mild inhibition against normal liver cells (Wang & Zhang, 2017). Chi, Hu, et al. (2015) reported that the tripeptide WPP prepared from blood clam muscle exhibited excellent cytotoxicity toward PC-3, DU-145, H-1299 and HeLa cell lines.

1.4.2 Anti-inflammatory effect Inflammation is a critical biological response to tissue damage in which the immune system secretes pro-inflammatory cytokines to respond against stimuli such as infection, injury, or irritation (Wang, Huang, Lu, & Chang, 2013). The overproduction of pro-inflammatory cytokines such as interleukin (IL)-1b, IL-6, and tumor necrosis factor alpha (TNF-α) triggers the incidence of chronic diseases including arthritis, allergy, atherosclerosis, and cancer, thus preventing the overproduction of pro-inflammatory cytokines is extremely critical to suppress the occurrence of aforementioned diseases (Devi et al., 2015). The anti-inflammatory effect of food-derived peptides has become an active area of research due to the aforementioned negative effects of inflammation. Many food-derived peptides have shown anti-inflammatory potential by suppressing the production of cytokines (i.e., IL-1β, IL-6, IL-8, and TNF-α), NO, and prostaglandin E2 (PGE2) (Ahn, Cho, & Je, 2015; Karnjanapratum, O’Callaghan, Benjakul, & O’Brien, 2016; Masotti, Buckley, Champagne, & Green-Johnson, 2011; Meram & Wu, 2017). Lee et al. (2012) reported that the isolated peptide, QCQQAVQSAV, from short-necked clam (Ruditapes philippinarum) hydrolysate showed a potent inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells. Kim et al. (2013) identified a novel anti-inflammatory peptide GVSLLQQFFL from Mytilus coruscus that significantly inhibited NO production (62.16%) in LPSstimulated RAW 264.7 cells. Moreover, Vo, Ryu, and Kim (2013) found two effective

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anti-inflammatory peptides, LDAVNR (686 Da) and MMLDF (655 Da) from edible microalgae (Spirulina maxima) hydrolysate after enzymatic hydrolysis by gastrointestinal endopeptidases (trypsin, α-chymotrypsin and pepsin). In another study, Ndiaye, Vuong, Duarte, Aluko, and Matar (2011) reported potent anti-inflammatory activity of protein hydrolysates from yellow pea seed that repressed NO production, TNF-α and IL-6 in activated macrophages. Protein hydrolysates isolated from soybean and amaranth remarkably inhibited the production of NO, PGE2, COX-2, iNOS, and TNF-α in LPS-induced RAW 264.7 macrophages (Barba de la Rosa et al., 2010; Vernaza, Dia, Mejia, & Chang, 2012). The effective anti-inflammatory activity of hydrolysates from lupine protein was found by inhibiting the expression of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and NO production in THP-1 macrophages (Millan-Linares, Bermudez, Yust, Millan, & Pedroche, 2014). Moreover, Chalamaiah, Yu, and Wu (2018) reported that enzymatically prepared hydrolysates (livetins fraction) of hen egg yolk attenuated the expression of the proinflammatory markers (i.e., IL-1β, IL-6, and TNF-α) and NO production in LPS-stimulated RAW 264.7 macrophages.

1.4.3 Antimicrobial activity Antimicrobial agents kill or slow down the action of bacteria and viruses without inflicting any damage to the surrounding cells and tissues (Shahidi & Yeo, 2018). Antimicrobial peptides (AMP) exert direct effects on bacteria, yeast and viruses. Individual AMPs show a discrepancy in the length of amino acid chains, amino acid composition, electronic charge and position of disulfide bonds (Pane et al., 2017). In the structure of AMP, the presence of a positive charge or amphipathic character due to the presence of both hydrophilic and hydrophobic amino acids at the terminals primarily contributes to their interaction with microbes and the subsequent action. Pane et al. (2017) reported that the antimicrobial potential of a cationic AMP is highly related to its electronic charge and the length of the peptide chain. There are two main mechanisms by which peptides kill bacteria; these are either by penetrating the bacteria cell membrane and making pores or by directly interacting with macromolecules present in the intracellular space of the microbial cells (Farkas, Maro´ti, Kereszt, & Kondorosi, 2017; Shah, Hsiao, Ho, & Chen, 2016; Taniguchi et al., 2016; Zhang et al., 2017). AMPs that are abundant in positively charged amino acids (i.e., arginine and lysine) directly pass through the microbial cell membrane by inducing an energy-dependent endocytic process such as micropinocytosis (Guterstam et al., 2009). Until now, many peptides possessing potent antimicrobial effects have been found in the hydrolysate of a wide range of foods. Zhang et al. (2017) found that the peptide ELLLNPTHQIYPVTQPLAPV isolated from human colostrum showed potent antimicrobial activity by disintegrating the cytoplasmic membrane and cell wall of microbes. A peptide IKHQGLPQE prepared from casein hydrolysates effectively reduced the number of

Bioactive peptides in health and disease: an overview 13 pathogenic bacteria spiked in infant formula (Kamali Alamdari & Ehsani, 2017). A variety of antimicrobial peptides have also been found from hydrolysates of fish and fish products. Ennaas, Hammami, Beaulieu, and Fliss (2015) obtained SIFIQRFTT from the hydrolysates of mackerel by-products, and this peptide exhibited powerful antimicrobial activity against Listeria innocua and Escherichia coli. Tang, Zhang, Wang, Qian, and Qi (2015) reported that the AMPGLSRLFTALK from cooked anchovy effectively inhibited the growth of S. aureus, B. subtilis, S. pneumoniae, E. coli, S. dysenteriae, P. aeruginosa and S. typhimurium.

1.4.4 Antihypertensive effect Hypertension, referred to as high blood pressure, is a long-term medical condition in which the arterial systolic and/or diastolic blood pressure is persistently increased ($ 140/90 mmHg) in the resting status. The average incidence rate of hypertension is globally about 15% (Patricia, Megan, & Kristi, 2005). Blood pressure is primarily regulated by the balance of boost system (renin-angiotensin system, RAS) and depressurization system (kallikrein-kinin system) in which the ACE (EC 3.4.15.1) plays a key role in controlling the boost and depressurization system as a rate-limiting enzyme. In the RAS system, renin activates angiotensinogen to secrete a nonactive peptide-angiotensin I in which ACE catalyzes the structural change of angiotensin I (10 peptides) into angiotensin II (8 peptides). The produced angiotensin II, which is a strong vasoconstrictor and boost effect, leads to the elevation of blood volume and blood pressure (Wilson, Hayes, & Carney, 2011). Thus inhibition of ACE activity can reduce the biosynthesis of angiotensin II followed by inactivation of the bradykinin, and this fact enables the use of ACE as an ideal target for the treatment of hypertension. Up until now, many synthetic ACE inhibitors have been developed to control hypertension, however, some undesired side effects such as dizziness, dysgeusia, headache, angioedema, and cough have been found after the use of synthetic antihypertensive drugs (Daliri, Lee, & Oh, 2016). This led to the search for natural source-derived ACE inhibitors. Food-derived antihypertensive peptides possess higher tissue affinities than those of synthetic drugs, resulting in slow elimination from tissues (Koyama, Hattori, Amano, Watanabe, & Nakamura, 2014). Many antihypertensive peptides have been found from natural sources (Table 1.2). Olagunju, Omoba, Enujiugha, Alashi, and Aluko (2018) reported that pepsin-pancreatinhydrolyzed pea protein (PPHPp) showed a remarkable systolic blood pressure-lowering effect (226.12 mmHg) in hypertensive rats. A peptide AEKTK was isolated from fermented whey by Lactobacillus brevis, and it showed a strong angiotensin 1-converting enzyme inhibition activity (Ahn, Park, Atwal, Gibbs, & Lee, 2009). IRW (Ile-Arg-Trp) prepared from egg-white hydrolysates exhibited excellent performance in hypertensive rats (Liao, Fan, Davidge, & Wu, 2019). Chakrabarti, Liao, Davidge, and Wu (2017) reported the antihypertensive effects of milk-derived bioactive peptides, IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro), through RAS modulation. Some experimental trials investigated the antihypertensive effect of milk

14

Chapter 1 Table 1.2: Antihypertensive activities of bioactive peptides from different food sources.

Source

Isolated peptide

Egg-white hydrolysates Fermented whey Milk

IRW

IPP, VPP

Milk

IPP, VPP

Fermented lactoferrin Bamboo shoots

DPYKLRP

Palmaria palmata hydrolysates Fermented milk

IRLIIVLMPILMA

AEKTK

DY

IPP, VPP

Antihypertensive effect

Reference

Reducing blood pressure in spontaneously hypertensive rats Angiotensin 1-converting enzyme inhibition activity

Liao et al. (2019) Ahn et al. (2009) Chakrabarti et al. (2017) Sipola et al. (2002) Garcı´a-Tejedor et al. (2015) Liu et al. (2013)

Antihypertensive effects in cultured vascular smooth muscle cells Reducing high blood pressure in spontaneously hypertensive rats Suppressing systolic blood pressure in spontaneously hypertensive rats Reducing systolic blood pressure by 18 mmhg at the administration level of 10 mg/kg body weight/day in spontaneously hypertensive rats Decreasing systolic blood pressure by 33 mmhg in spontaneously hypertensive rats Inhibiting the arterial stiffness and blood pressure of 89 hypertensive patients

Fitzgerald et al. (2014) Jauhiainen et al. (2010)

peptides, IPP and VPP, in animal models in which they found that these peptides significantly reduced high blood pressure after a single dose and after long-term administrations as well (Jauhiainen, Collin, Narva, Paussa, & Korpela, 2005; Sipola, Finckenberg, Korpela, Vapaatalo, & Nurminen, 2002). An ACE inhibitory peptide DPYKLRP, isolated from fermented lactoferrin using Kluyveromyces marxianus, significantly suppressed systolic blood pressure in spontaneously hypertensive rats (Garcı´a-Tejedor, Castello´-Ruiz, Gimeno-Alcan˜´ız, Manzanares, & Salom, 2015). In addition, dipeptide DY extracted from bamboo shoots remarkably decreased systolic blood pressure by 18 mmHg at the administration level of 10 mg/kg body weight/day in spontaneously hypertensive rats (Liu, Liu, Lu, Chen, & Zhang, 2013). Fitzgerald, Aluko, Hossain, Rai, and Hayes (2014) studied the antihypertensive effect of peptide IRLIIVLMPILMA from hydrolyzed Palmaria palmata with papain, and found that IRLIIVLMPILMA reduced systolic blood pressure by 33 mmHg in spontaneously hypertensive rats. Jauhiainen et al. (2010) reported that the daily consumption of fermented milk containing 5 mg of IPP and VPP for 12 weeks significantly reduced the arterial stiffness and blood pressure of 89 hypertensive patients in a double-blind parallel-group intervention study. The position of certain amino acid residues in the structure of peptides plays an important role in their antihypertensive activity, however, it has not yet been fully understood as to which specific amino acids are responsible for the antihypertensive effect of peptides. Meanwhile, Lin et al. (2017) reported the transport of antihypertensive peptide, LSW (Leu-Ser-Trp), across Caco-2 monolayers with permeability coefficient of 11.53 6 1.82 3 1028 cm/s as well as the improvement of LSW transport by cytochalasin D

Bioactive peptides in health and disease: an overview 15 (a tight junction expander) and reduction by theaflavin-30 -O-gallate (a tight junction enhancer). Moreover, Xu, Fan, Yu, Hong, and Wu (2017) confirmed a high permeability of antihypertensive peptides (LKP and IQW) in Caco-2 and ht29 coculture monolayers models.

1.4.5 Immunomodulatory peptides The immune system is a complex network of cells, tissues, and organs that defend the body against infection caused by outer harmful substances such as bacteria, viruses, fungi, and protozoans. The immune system can be influenced by many factors including stress, unhealthy lifestyle, and antigens (Segerstrom & Miller, 2004). Immunomodulators involve controlling/ modifying the immune responses by intervening in both innate and adaptive functional categories of the immune system. A variety of immunomodulators including levamisole, glucocorticoids, phytol, tacrolimus, aristolochic acid, plumbagin and cyclosporine has been successfully used to improve the immune response in humans (Gertsch, Viveros-paredes, & Taylor, 2011), however, some drawbacks of the above immunomodulators, including severe side effects and high cost of the allopathic drugs has prompted the search for new immunomodulators which can address these concerns (Wang et al., 2010). Recently, a large number of studies has been reported on exploring novel immunemodulating peptides derived from natural sources (Chalamaiah et al., 2014). These peptides have shown a variety of immunomodulatory effects including improvement of natural killer cell capacity, stimulation of lymphocytes to proliferate, modulating the production of cytokine and antibody, as well as the enhancement of the defensive ability from invading pathogens and the suppression of pro-inflammatory responses of host cells to bacterial components (Chalamaiah et al., 2014; Duarte, Vinderola, Ritz, Perdigon, & Matar, 2006; Hou, Fan, Li, Xue, & Yu, 2012; Wenjia et al., 2016; Yang et al., 2009). The immunomodulatory activity of peptides prepared from food hydrolysates was highly related to their structural properties including length, amino acid composition, sequence, electronic charge, and hydrophobicity of the peptide molecules (Berthou et al., 1987; Jacquot, Gauthier, Drouin, & Boutin, 2010). The various immunomodulatory effects of food-derived peptides might be due to the direct association of peptides to the receptors on the surface of immune cells. However, food-derived immunomodulatory peptides may not interact with the pathogen directly, but they are involved in the host’s defense response (Maestri, Marmiroli, & Marmiroli, 2016). A wide range of immunomodulatory peptides has been found from various food protein sources. Milk is an excellent source of immunomodulatory peptides. Peptide isolated from enzymatic digestion of milk proteins showed an excellent immunomodulatory activity, which was reported as early as 1984 (Parker et al., 1984). Berthou et al. (1987) isolated two tripeptides LLY (Leu-Leu-Tyr) from bovine casein and GLF (Gly-Leu-Phe) from human casein, and they found that these two tripeptides significantly improved the phagocytosis

16

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activity of red blood cells by mouse peritoneal macrophages. Saint-Sauveur, Gauthier, Boutin, and Montoni (2008) prepared whey protein hydrolysate after combined digestion of trypsin and chymotrypsin and reported its remarkable immunostimulatory effects on the proliferation of splenocytes in both the presence or absence of concanavalin and the secretion of IL-2 and IFN-γ. Aside from milk proteins, immunomodulatory peptides have also been found from protein hydrolysates of soybean, rice, fish and other marine species. Yoshikawa et al. (1993) isolated a peptide HCQRPR from tryptic digests of soy protein. This hexapeptide strongly stimulated the activity of phagocytes (white blood cells) in mice model. Chen, Suetsuna, and Yamauchi (1995) also reported the potent immunostimulatory effect of peptides isolated from soybean protein in which they prepared three immunostimulatory peptides, namely AEINMPDT, IQQGN, and SGFAP after enzymatic hydrolysis of soybean protein using pepsin. Subsequently, they confirmed the enhanced proliferation of mice splenocytes by these three peptides. Takahashi, Moriguchi, Yoshikawa, and Sasaki (1994) found an immunomodulatory peptide, GYPMYPLPR, from rice soluble protein hydrolysate after the tryptic digest, and reported the elevation of phagocytosis activity for human polymorphonuclear leukocytes by the isolated peptide. Hou et al. (2012) isolated three immunomodulating peptides, NGMTY, NGLAP, and WT, from Alaska pollock trypsin hydrolysate, and these three peptides showed potent lymphocyte proliferation activities. Moreover, He, Cao, Pan, Yang, and Zhang (2015) reported six effective immunomodulatory peptides including PHTC, VGTT, EF, LF, EGAK, and WI or WL from Paphia undulate clam.

1.4.6 Antidiabetic effect There are two types of diabetes mellitus, type I and type II. Type I accounts for approximately 5%
10% of diabetes patients and is insulin-dependent; this type of diabetes is caused by the inability of the pancreas in secreting insulin due to the destruction of beta cells (Lauritano & Ianora, 2016). On the other hand, type II diabetes shows a higher prevalence (about 90%
95%) compared to type I diabetes and is noninsulin-dependent. Type II diabetes occurs mainly by the low efficiency of insulin production or in making use of insulin secreted. Environmental conditions such as high body mass index, sedentary lifestyle, ageing and hereditary have been suggested as the major factors elevating the incidence of type II diabetes (Anguizola et al., 2013). A variety of medications for the management of diabetes has so far been developed such as DPP-IV inhibitors, GLP-1 receptor agonists, α-glucosidase inhibitors, SGLT-2 inhibitors, and α-amylase inhibitors (Deacon, 2018; Kalita, Holm, LaBarbera, Petrash, & Jayanty, 2018). The DPP-IV suppression and GLP-1 receptor agonism enhance the synthesis of incretin hormones such as glucose-dependent insulinotropic polypeptide (known as a gastric inhibitory peptide) and glucagon-like peptide-1 (GLP-1), which are hormonal peptides

Bioactive peptides in health and disease: an overview 17 secreted to regulate blood glucose levels and the suppression of glucagon release from pancreatic cells upon ingestion of foods (Amaya-Farfan, Moura, Morato, & Lollo, 2016). α-Glucosidase, an enzyme (EC 3.2.1.20) present in the intestinal brush border, is responsible for hydrolyzing 1,4-α-glycosidic linkages present in oligosaccharides, assisting the production of monosaccharides, that are absorbable form, in the intestine. Thus α-glucosidase inhibitors play a key role in reducing blood glucose levels. However, there are some drawbacks to the use of such medications, despite their potent effects on diabetic patients. The drawbacks include causing clinical symptoms and side effects such as nausea, weight gain, vomiting, and increased risk of cardiovascular as well as infection of the pancreas and cancer of the bladder (Chaudhury et al., 2017). These disadvantages of antidiabetic medications have prompted the search for novel antidiabetic compounds with less/low side effects. Bioactive peptides have been suggested as excellent alternatives to replace the aforementioned medications as the pharmaceutical therapy of diabetes, administered orally or intravenously (Shaji & Patole, 2008). Even though bioactive peptides are somewhat less effective, food-derived peptides and hydrolysates have attracted great attention from consumers and researchers due to their minimal side effects based on their natural sources and mechanisms of action (Li-Chan, 2015). The inhibition of enzymes such as DPP-IV, α-glucosidase, and α-amylase is a major mechanism for the antidiabetic potential of hydrolysates and peptides from natural sources. Therefore many antidiabetic peptides from food-derived hydrolysates following these mechanisms have been reported. Proteins from dairy sources are among the excellent sources of bioactive peptides (Ricci-Cabello, Herrera, & Artacho, 2012). El-Sayed et al. (2016) studied the antidiabetic effect of milk protein and its hydrolysate in a diabetic rat model. The hydrolysates and milk protein were orally administered at 800 mg/kg of body weight daily for 6 weeks and the result showed that the administration of milk protein and its hydrolysate remarkably reduced the blood glucose level in the diabetic rat as well as significantly decreasing the level of low-density lipoproteins, very-low-density lipoproteins, triacylglycerols and total cholesterol in rat plasma, thus demonstrating their potential as effective antidiabetic agents. Lacroix and LiChan (2013) investigated the antidiabetic activity of hydrolysates from pepsin-treated whey proteins by focusing on their enzyme inhibitory potential against DPP-IV and α-glucosidase in which α-lactalbumin hydrolysate exerted the highest antidiabetic activity among other whey protein hydrolysates with an IC50 value of 0.036 mg/mL. In addition to dairy foods, many antidiabetic peptides have been found from a wide range of foods such as egg (egg white and egg yolk), marine sources, and plant-based foods (i.e., cereals, seeds, legumes, vegetables, etc.). Aside from the above health-beneficial effects of bioactive peptides, a wide range of peptides having antioxidant activity has been found in food protein hydrolysates, and those are summarized in Table 1.3.

18

Chapter 1 Table 1.3: Antioxidant peptides from different food sources.

Source Rice Rice endosperm protein Algae protein waste Peptide from frog skin Egg white protein Rice residue protein Oyster Bluefin leatherjacket heads. Tilapia Gelatin Blue mussel Sweet potato protein Skate cartilage Sandfish Blood clam muscle

Isolated peptide

Reference

IP, MP, VP, LP

Hatanaka, Uraji, Fujita, and Kawakami (2015) Zhang et al. (2010) Sheih, Wu, and Fang (2009) Qian, Jung, and Kim (2008) Liu, Jin, Lin, Jones, and Chen (2015) Yan, Huang, Sun, Jiang, and Wu (2015)

FRDEHKK, KHDRGDEF VECYGPNRPQF LEELEEELEGCE DHTKE, MPDAHL, FFGFN RPNYTDA, TSQLLSDQ, TRTGDPFF, NFHPQ LANAK, PSLVGRPPVGKLTL, GPP, GVPLT LSGYGP

Umayaparvathi et al. (2014) Chi, Wang, Wang, Zhang, and Deng (2015) Sun, Zhang, and Zhuang (2013)

PIIVYWK, TTANIEDRR, FSVVPSPK YYIVS FIMGPY, GPAGDY, IVAGPQ ATSHH TPP

Park, Kim, Ahn, and Je (2016) Zhang and Mu (2016) Pan, Zhao, Hu, and Wang (2016) Jang, Liceaga, and Yoon (2016) Chi, Hu, et al. (2015)

1.5 Conclusion In this contribution, various health beneficial effects of bioactive peptides derived from a wide range of plant- and animal-based food protein hydrolysates were discussed. In recent years, the use of bioactive peptides as functional foods and nutraceuticals has been dramatically increased along with the discovery of diverse bioactive peptides from food protein hydrolysates. However, further research including testing their bioavailability, measuring pharmacokinetics in human subjects, exploring mechanisms of their bioactivities is required to facilitate their use. In particular, the safety of these peptide-based products should be thoroughly verified before their commercial production and consumer use.

References Ahn, C. B., Cho, Y. S., & Je, J. Y. (2015). Purification and anti-inflammatory action of tripeptide from salmon pectoral fin by-product protein hydrolysate. Food Chemistry, 168, 151
156. Ahn, J., Park, S., Atwal, A., Gibbs, B., & Lee, B. (2009). Angiotensin I-converting enzyme (ACE) inhibitory peptides from whey fermented by Lactobacillus species. Journal of Food Biochemistry, 33, 587
602. Aleman, A., Gimenez, B., Perez-Santin, E., Gomez-Guillen, M. C., & Montero, P. (2011). Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chemistry, 125, 334
341. Aluko, R. E. (2008). Antihypertensive properties of plant-derived inhibitors of angiotensin Iconverting enzyme activity: a review. In J. N. Govil, & V. K. Singh (Eds.), Recent Progress in Medicinal Plants— Phytopharmacology and Therapeutic Values (Vol. IV, pp. 541
561). Houston: Stadium Press.

Bioactive peptides in health and disease: an overview 19 Amarowicz, R., & Shahidi, F. (1997). Antioxidant activity of peptide fractions of capelin protein hydrolysates. Food Chemistry, 58, 355
359. Amaya-Farfan, J., Moura, C., Morato, P., & Lollo, P. (2016). Chapter 17
Dietary whey protein and type 2 diabetes: Molecular aspects. In D. Mauricio (Ed.), Molecular nutrition and diabetes (pp. 211
220). Cambridge, Massachusetts: Academic press. Ambigaipalan, P., & Shahidi, F. (2017). Bioactive peptides from shrimp shell processing discards: Antioxidant and biological activities. Journal of Functional Foods, 34, 7
17. Ames, B. N., Shigena, M. K., & Hegen, T. M. (1993). Oxidants, antioxidants and the degenerative diseases of aging. Proceedings of the National Academy of Sciences of the United States of America, 90, 7915
7922. Anguizola, J., Matsuda, R., Barnaby, O. S., Hoy, K. S., Wa, C., DeBolt, E., . . . Hage, D. S. (2013). Review: Glycation of human serum albumin. Clinica Chimica Acta, 425, 64
76. Ariyoshi, Y. (1993). Angiotensin converting enzyme inhibitors derived from food proteins. Trends in Food Science & Technology, 4, 139
144. Aungst, B. J. (2012). Absorption enhancers: Applications and advances. The AAPS Journal, 14, 10
18. Barba de la Rosa, A. P., Barba Montoya, A., Martinez-Cuevas, P., Hernandez-Ledesma, B., Le´on Galva´n, M. F., De Le´on-Rodrı´guez, A., & Gonza´lez, C. (2010). Tryptic amaranth glutelin digests induce endothelial nitric oxide production through inhibition of ACE: Antihypertensive role of amaranth peptides. Nitric Oxide: Biology and Chemistry/Official Journal of the Nitric Oxide Society, 23, 106
111. Berthou, J., Migliore-Samour, D., Lifchitz, A., Delettre´, J., Floc’h, F., & Jolle`s, P. (1987). Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins. Federation of European Biochemical Societies Letters, 218, 55
58. Chakrabarti, S., Liao, W., Davidge, S. T., & Wu, J. (2017). Milk-derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) differentially modulate angiotensin II effects on vascular smooth muscle cells. Journal of Functional Foods, 30, 151
158. Chalamaiah, M., Hemalatha, R., Jyothirmayi, T., Diwan, P. V., Uday Kumar, P., Chetan, N., & Dinesh Kumar, B. (2014). Immunomodulatory effects of protein hydrolysates from rohu (Labeo rohita) egg in BALB/c mice. Food Research International, 62, 1054
1061. Chalamaiah, M., Yu, W., & Wu, J. (2018). Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins: A review. Food Chemistry, 245, 205
222. Chaudhury, A., Duvoor, C., Dendi, R. V. S., Kraleti, S., Chada, A., Ravilla, R., . . . Mirza, W. (2017). Clinical review of antidiabetic drugs: Implications for type-2 diabetes mellitus management. Frontiers in Endocrinology, 8, 6. Chen, J., Suetsuna, K., & Yamauchi, F. (1995). Isolation and characterization of immunostimulative peptides from soybean. The Journal of Nutritional Biochemistry, 6, 310
313. Chi, C. F., Hu, F. Y., Wang, B., Li, T., & Ding, G. F. (2015). Antioxidant and anticancer peptides from the protein hydrolysate of blood clam (Tegillarca granosa) muscle. Journal of Functional Foods, 15, 301
313. Chi, C. F., Wang, B., Wang, Y. M., Zhang, B., & Deng, S. G. (2015). Isolation and characterization of three antioxidant peptides from protein hydrolysate of Bluefin leatherjacket (Navodon septentrionalis) heads. Journal of Functional Foods, 12, 1
10. Chittchang, M., Salamat-Miller, N., Alur, H. H., Velde, D. G. V., Mitra, A. K., & Johnston, T. P. (2002). Poly(llysine) as a model drug macromolecule with which to investigate secondary structure and microporous membrane transport, part 2: Diffusion studies. Journal of Pharmacy and Pharmacology, 54, 1497
1505. Clare, D. A., & Swaisgood, H. E. (2000). Bioactive milk peptides: A prospectus. Journal of Dairy Science, 83, 1187
1195. Cumby, N., Zhong, Y., Naczk, M., & Shahidi, F. (2008). Antioxidant activity and water-holding capacity of canola protein hydrolysates. Food Chemistry, 109, 144
148. Daliri, E. B. M., Lee, B. H., & Oh, D. H. (2016). Current perspectives on antihypertensive probiotics. Probiotics Antimicrob Proteins, 9, 91
101. Deacon, C. F. (2018). A review of dipeptidyl peptidase-4 inhibitors. Hot topics from randomized controlled trials. Diabetes, Obesity and Metabolism, 20, 34
46.

20

Chapter 1

Dennison, S. R., Whittaker, M., Harris, F., & Phoenix, D. A. (2006). Anticancer alpha-helical peptides and structure/function relationships underpinning their interactions with tumour cell membranes. Current Protein & Peptide Science, 7, 487
499. Devi, K. P., Malar, D. S., Nabavi, S. F., Sureda, A., Xiao, J., & Nabavi, S. M. (2015). Kaempferol and inflammation: From chemistry to medicine. Pharmacological Research, 99, 1
10. Dodoo, A. N., Bansal, S., Barlow, D. J., Bennet, F. C., Hider, R. C., Lansley, A. B., . . . Marriott, C. (2000). Systematic investigations of the influence of molecular structure on the transport of peptides across cultured alveolar cell monolayers. Pharmaceutical Research, 17, 7
14. Duarte, J., Vinderola, G., Ritz, B., Perdigon, G., & Matar, C. (2006). Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation. Immunobiology, 211, 341
350. Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and peptides. Critical Reviews in Food Science and Nutrition, 48, 430
441. Eliassen, L. T., Haug, B. E., Berge, G., & Rekdal, O. (2003). Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications. Journal of Peptide Science, 9, 510
517. El-Sayed, M. I., Awad, S., Wahba, A., El Attar, A., Yousef, M. I., & Zedan, M. (2016). In vivo anti-diabetic and biological activities of milk protein and milk protein hydrolysate. Advance D Research, 4, 154. Ennaas, N., Hammami, R., Beaulieu, L., & Fliss, I. (2015). Purification and characterization of four antibacterial peptides from protamex hydrolysate of Atlantic mackerel (Scomber scombrus) by-products. Biochemical and Biophysical Research Communications, 462, 195
200. Farkas, A., Maro´ ti, G., Kereszt, A., & Kondorosi, E´ . (2017). Comparative analysis of the bacterial membrane disruption effect of two natural plant antimicrobial peptides. Frontiers in Microbiology, 8, 51. Fasano, A. (1998). Modulation of intestinal permeability: An innovative method of oral drug delivery for the treatment of inherited and acquired human diseases. Molecular Genetics and Metabolism, 64, 12
18. Ferna´ndez-Tome´, S., Sancho´n, J., Recio, I., & Herna´ndez-Ledesma, B. (2018). Transepithelial transport of lunasin and derived peptides: Inhibitory effects on the gastrointestinal cancer cells viability. Journal of Food Composition and Analysis, 68, 101
110. Fields, F., Falla, T. J., Rodan, K., & Bush, L. (2009). Bioactive peptides: signaling the future. Journal of Cosmetic Dermatology, 8, 8
13. Firdaous, L., Dhulster, P., Amiot, J., Gaudreau, A., Lecouturier, D., Kapel, R., . . . Bazinet, L. (2009). Concentration and selective separation of bioactive peptides from an alfalfa white protein hydrolysate by electrodialysis with ultrafiltration membranes. Journal of Membrane Science, 329, 60
67. Fitzgerald, C., Aluko, R. E., Hossain, M., Rai, D. K., & Hayes, M. (2014). Potential of a renin inhibitory peptide from the red seaweed Palmaria palmata as a functional food ingredient following confirmation and characterization of a hypotensive effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 62, 8352
8356. Garcı´a-Tejedor, A., Castello´-Ruiz, M., Gimeno-Alcan˜´ız, J. V., Manzanares, P., & Salom, J. B. (2015). In vivo antihypertensive mechanism of Lactoferrin-derived peptides: Reversion of Angiotensin I- and Angiotensin II-induced hypertension in wistar rats. Journal of Functional Foods, 15, 294
300. Gertsch, J., Viveros-paredes, J. M., & Taylor, P. (2011). Plant immunostimulants—Scientific paradigm or myth? Journal of Ethnopharmacology, 136, 385
391. Gibaldi, M. (1991). Biopharmaceutics and Clinical Pharmacokinetics (fourth ed). Philadelphia: Lea & Febiger. ¨ . (2009). Guterstam, P., Madani, F., Hirose, H., Takeuchi, T., Futaki, S., Andaloussi, S. E., . . . Langel, U Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate. Biochimica Biophysica Acta (BBA)Biomembranes, 1788, 2509
2517. Gutierrez, K., Glanzner, W. G., Chemeris, R. O., Rigo, M. L., Comim, F. V., Bordignon, V., & Gonc¸alves, P. B. (2016). Gonadotoxic effects of busulfan in two strains of mice. Reproductive Toxicology, 59, 31
39.

Bioactive peptides in health and disease: an overview 21 Hamman, J. H., Enslin, G. M., & Kotze, A. F. (2005). Oral delivery of peptide drugs: Barriers and developments. BioDrugs: Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy, 19, 165
177. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: From research to food applications. Current Opinion in Biotechnology, 18, 163
169. Hatanaka, T., Uraji, M., Fujita, A., & Kawakami, K. (2015). Anti-oxidation activities of rice-derived peptides and their inhibitory effects on dipeptidylpeptidase-IV. International Journal of Peptide Research and Therapeutics, 21, 479
485. He, X. Q., Cao, W. H., Pan, G. K., Yang, L., & Zhang, C. H. (2015). Enzymatic hydrolysis optimization of Paphia undulata and lymphocyte proliferation activity of the isolated peptide fractions. Journal of the Science of Food and Agriculture, 95, 1544
1553. Hou, H., Fan, Y., Li, B., Xue, C., & Yu, G. (2012). Preparation of immunomodulatory hydrolysates from Alaska pollock frame. Journal of the Science of Food and Agriculture, 92, 3029
3038. Huang, F., Yang, Z., Yu, D., Wang, J., Li, R., & Ding, G. (2012). Sepia ink oligopeptide induces apoptosis in prostate cancer cell lines via caspase-3 activation and elevation of Bax/Bcl-2 ratio. Marine Drugs, 10, 2153
2165. Huang, Y. B., He, L. Y., Jiang, H. Y., & Chen, Y. X. (2012). Role of helicity on the anticancer mechanism of action of cationic-helical peptides. International Journal of Molecular Sciences, 13, 6849
6862. Hung, C. C., Yang, Y. H., Kuo, P. F., & Hsu, K. C. (2014). Protein hydrolysates from tuna cooking juice inhibit cell growth and induce apoptosis of human breast cancer cell line MCF-7. Journal of Functional Foods, 11, 563
570. Jacquot, A., Gauthier, S. F., Drouin, R., & Boutin, Y. (2010). Proliferative effects of synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes. International Dairy Journal, 20, 514
521. Jang, H. L., Liceaga, A. M., & Yoon, K. Y. (2016). Purification, characterisation and stability of an antioxidant peptide derived from sandfish (Arctoscopus japonicus) protein hydrolysates. Journal of Functional Foods, 20, 433
442. Jauhiainen, T., Collin, M., Narva, M., Paussa, T., & Korpela, R. (2005). Effect of long-term intake of milk peptides and minerals on blood pressure and arterial function in spontaneously hypertensive rats. Milchwissenschaft, 60, 358
362. Jauhiainen, T., Ro¨nnback, M., Vapaatalo, H., Wuolle, K., Kautiainen, H., Groop, P. H., & Korpela, R. (2010). Long-term intervention with Lactobacillus helveticus fermented milk reduces augmentation index in hypertensive subjects. European Journal of Clinical Nutrition, 64, 424
431. Ji, Y. W., Li, B., He, J. G., & Qian, P. (2011). Quantitative structure-activity relationship study of antioxidative peptide by using different sets of amino acids descriptors. Journal of Molecular Structure, 998, 53
61. Kalita, D., Holm, D. G., LaBarbera, D. V., Petrash, J. M., & Jayanty, S. S. (2018). Inhibition of α-glucosidase, a-amylase, and aldose reductase by potato polyphenolic compounds. PLoS One, 13, e0191025. Kamali Alamdari, E., & Ehsani, M. (2017). Antimicrobial peptides derived from milk: A review. Journal of Food Biosciences and Technology, 7, 49
56. Karnjanapratum, S., O’Callaghan, Y. C., Benjakul, S., & O’Brien, N. (2016). Antioxidant, immunomodulatory and antiproliferative effects of gelatin hydrolysate from Unicorn leatherjacket skin. Journal of the Science of Food and Agriculture, 96, 3220
3226. Kaur, I. P., Garg, A., Singla, A. K., & Aggarwal, D. (2004). Vesicular systems in ocular drug delivery: An overview. International Journal of Pharmaceutics, 269, 1
14. Kawano, K., Yoneya, T., Miyata, T., Yoshikawa, K., Tokunaga, F., & Terada, Y. (1990). Antimicrobial peptide, tachyplesin I, isolated from hemocytes of the horseshoe crab (Tachypleus tridentatus). NMR determination of the beta-sheet structure. Journal of Biological Chemistry, 265, 15365
15367. Kim, E. K., Kim, Y. S., Hwang, J. W., Kang, S. H., Choi, D. K., Lee, K. H., . . . Park, P. J. (2013). Purification of a novel nitric oxide inhibitory peptide derived from enzymatic hydrolysates of Mytilus coruscus. Fish & Shellfish Immunology, 34, 1416
1420.

22

Chapter 1

Kim, S. K., Kim, Y. T., Byun, H. G., Nam, K. S., Joo, D. S., & Shahidi, F. (2001). Isolation and characterization of antioxidative peptides from gelatin hydrolysate of alaska pollack skin. Journal of Agricultural and Food Chemistry, 49, 1984
1989. Koyama, M., Hattori, S., Amano, Y., Watanabe, M., & Nakamura, K. (2014). Blood pressure-lowering peptides from neo-fermented buckwheat sprouts: A new approach to estimating ACE-inhibitory activity. PLoS One, 9, e105802. Kurz, D., & Ciulla, T. A. (2002). Novel approaches for retinal drug delivery. Ophthalmology Clinics of North America, 15, 405
410. Lacroix, I. M. E., & Li-Chan, E. C. Y. (2013). Inhibition of dipeptidyl peptidase (DPP)-IV and α-glucosidase activities by pepsin-treated whey proteins. Journal of Agricultural and Food Chemistry, 61, 7500
7506. Lane, M. E., & Corrigan, O. I. (2006). Paracellular and transcellular pathways facilitate insulin permeability in rat gut. Journal of Pharmacy and Pharmacology, 58, 271
275. Lauritano, C., & Ianora, A. (2016). Marine organisms with anti-diabetes properties. Marine Drugs, 14, 220. Lee, J. H., Moon, S. H., Kim, H. S., Park, E., Ahn, D. U., & Paik, H.-D. (2017). Antioxidant and anticancer effects of functional peptides from ovotransferrin hydrolysates. Journal of the Science of Food and Agriculture, 97, 4857
4864. Lee, S. J., Kim, E. K., Kim, Y. S., Hwang, J. W., Lee, K. H., Choi, D. K., . . . Park, P. J. (2012). Purification and characterization of a nitric oxide inhibitory peptide from Ruditapes philippinarum. Food and Chemical Toxicology, 50, 1660
1666. Lehr, C. M. (1996). From sticky stuff to sweet receptors
achievements, limits and novel approaches to bioadhesion. European Journal of Drug Metabolism and Pharmacokinetics, 21, 139
148. Li, Q., Ouyang, G., Peng, X., & Hong, S. (2003). Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells. World Journal of Gastroenterology, 9, 454
458. Li, Y., & Yu, J. (2014). Research progress in structure-activity relationship of bioactive peptides. Journal of Medicinal Food, 18, 147
156. Li, Z., Chen, J., Sun, W., & Xu, Y. (2010). Investigation of archaeosomes as carriers for oral delivery of peptides. Biochemical and Biophysical Research Communications, 394, 412
417. Liao, W., Fan, H., Davidge, S. T., & Wu, J. (2019). Egg white
derived antihypertensive peptide IRW (Ile-ArgTrp) reduces blood pressure in spontaneously hypertensive rats via the ACE2/Ang (1-7)/Mas receptor axis. Molecular Nutrition & Food Research, 63, 1900063. Li-Chan, E. C. Y. (2015). Bioactive peptides and protein hydrolysates: Research trends and challenges for application as nutraceuticals and functional food ingredients. Current Opinion in Food Science, 1, 28
37. Lin, Q., Xu, Q., Bai, J., Wu, W., Hong, H., & Wu, J. (2017). Transport of soybean protein-derived antihypertensive peptide LSW across Caco-2 monolayers. Journal of Functional Foods, 39, 96
102. Liu, D. Z., LeCluyse, E. L., & Thakker, D. R. (1999). Dodecylphosphocholine-mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers. Journal of Pharmaceutical Sciences, 88, 1161
1168. Liu, J., Jin, Y., Lin, S., Jones, G. S., & Chen, F. (2015). Purification and identification of novel antioxidant peptides from egg white protein and their antioxidant activities. Food Chemistry, 175, 258
266. Liu, L., Liu, L., Lu, B., Chen, M., & Zhang, Y. (2013). Evaluation of bamboo shoot peptide preparation with Angiotensin converting enzyme inhibitory and antioxidant abilities from byproducts of canned bamboo shoots. Journal of Agricultural and Food Chemistry, 61, 5526
5533. Maestri, E., Marmiroli, M., & Marmiroli, N. (2016). Bioactive peptides in plant-derived foodstuffs. Journal of Proteomics, 147, 140
155. Mahato, R. I., Narang, A. S., Thoma, L., & Miller, D. D. (2003). Emerging trends in oral delivery of peptide and protein drugs. Critical Reviews in Therapeutic Drug Carrier Systems, 20, 153
214. Masotti, A. I., Buckley, N., Champagne, C. P., & Green-Johnson, J. (2011). Immunomodulatory bioactivity of soy and milk ferments on monocyte and macrophage models. Food Research International, 44, 2475
2481. Mellander, O. (1950). The physiological importance of the casein phosphopeptide calcium salts. II. Per oral calcium dosage of infants. Acta Soc Med Uppsala, 55, 247
255.

Bioactive peptides in health and disease: an overview 23 Meram, C., & Wu, J. (2017). Anti-inflammatory effects of egg yolk livetins (α, β, and γ-livetin) fraction and its enzymatic hydrolysates in lipopolysaccharide-induced RAW 264.7 macrophages. Food Research International, 100, 449
459. Millan-Linares, M. C., Bermudez, B., Yust, M. M., Millan, F., & Pedroche, J. (2014). Antiinflammatory activity of lupine (Lupinus angustifolius L.) protein hydrolysates in THP-1-derived macrophages. Journal of Functional Foods, 8, 224
233. Ndiaye, F., Vuong, T., Duarte, J., Aluko, R. E., & Matar, C. (2011). Antioxidant, anti-inflammatory and immunomodulatory properties of an enzymatic protein hydrolysate from yellow field pea seeds. European Journal of Nutrition, 51, 29
37. Nwachukwu, I. D., & Aluko, R. E. (2019). Structural and functional properties of food protein-derived antioxidant peptides. Journal of Food Biochemistry, 43, e12761. Olagunju, A. I., Omoba, O. S., Enujiugha, V. N., Alashi, A. M., & Aluko, R. E. (2018). Antioxidant properties, ACE/renin inhibitory activities of pigeon pea hydrolysates and effects on systolic blood pressure of spontaneously hypertensive rats. Food Science & Nutrition, 7, 1879
1889. Oun, R., Plumb, J., Rowan, E., & Wheate, N. (2013). Encapsulation of cisplatin by cucurbiuril decreases the neurotoxic and cardiotoxic side effects of cisplatin. Toxicology Letters, 221, S92. Pan, D. D., Cao, J. X., & Guo, H. Q. (2012). Studies on purification and the molecular mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate. Food Chemistry, 130, 121
126. Pan, X., Zhao, Y., Hu, F., Chi, C., & Wang, B. (2016). Anticancer activity of a hexapeptide from skate (Raja porosa) cartilage protein hydrolysate in HeLa Cells. Marine Drugs, 14, 153
164. Pan, X., Zhao, Y. Q., Hu, F. Y., & Wang, B. (2016). Preparation and identification of antioxidant peptides from protein hydrolysate of skate (Raja porosa) cartilage. Journal of Functional Foods, 25, 220
230. Pane, K., Durante, L., Crescenzi, O., Cafaro, V., Pizzo, E., Varcamonti, M., . . . Notomista, E. (2017). Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: Application to the detection of “cryptic” antimicrobial peptides. Journal of Theoretical Biology, 419, 254
265. Panyam, J., & Labhasetwar, V. (2003). Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Advanced Drug Delivery Reviews, 55, 329
347. Park, P. J., Jung, W. K., Nam, K. S., Shahidi, F., & Kim, S. K. (2001). Purification and characterization of antioxidative peptides from protein hydrolysate of lecithin-free egg yolk. Journal of the American Oil Chemists’ Society, 78, 651
656. Park, S. Y., Kim, Y. S., Ahn, C. B., & Je, J. Y. (2016). Partial purification and identification of three antioxidant peptides with hepatoprotective effects from blue mussel (Mytilus edulis) hydrolysate by peptic hydrolysis. Journal of Function Foods, 20, 88
95. Parker, F., Migliore-Samour, D., Floc’h, F., Zerial, A., Werner, G. H., Jolle`s, J., . . . Jolle`s, P. (1984). Immunostimulating hexapeptide from human casein: amino acid sequence, synthesis and biological properties. European Journal of Biochemistry, 45, 677
682. Patricia, M. K., Megan, W., & Kristi, R. (2005). Global burden of hypertension: Analysis of worldwide data. Lancet, 365, 217
223. Pownall, T. L., Udenigwe, C. C., & Aluko, R. E. (2010). Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. Journal of Agriccultural and Food Chemistry, 58, 4712
4718. Qian, Z. J., Jung, W. K., & Kim, S. K. (2008). Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw. Bioresour Technology, 99, 1690
1698. Rafii, F., Franklin, W., & Cerniglia, C. E. (1990). Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora. Applied Environmental Microbiology, 56, 2146
2151. Renukuntla, J., Vadlapudi, A. D., Patel, A., Bodduc, S. H. S., & Mitra, A. K. (2013). Approaches for enhancing oral bioavailability of peptides and proteins. International Journal of Pharmaceutics, 447, 75
93. Ricci-Cabello, I., Herrera, M. O., & Artacho, R. (2012). Possible role of milk-derived bioactive peptides in the treatment and prevention of metabolic syndrome. Nutrition Reviews, 70, 241
255.

24

Chapter 1

Rubio-Aliaga, I., & Daniel, H. (2002). Mammalian peptide transporters as targets for drug delivery. Trends in Pharmacological Sciences, 23, 434
440. Sagar, J., Sharma, P. K., Bansal, S., & Banik, A. (2011). Noninvasive routes of proteins and peptides drug delivery. Indian Journal of Pharmaceutical Sciences, 73, 367
375. Saint-Sauveur, D., Gauthier, S. F., Boutin, Y., & Montoni, A. (2008). Immunomodulating properties of a whey protein isolate, its enzymatic digest and peptide fractions. International Dairy Journal, 18, 260
270. Salamat-Miller, N., & Johnston, T. P. (2005). Current strategies used to enhance the paracellular transport of therapeutic polypeptides across the intestinal epithelium. International Journal of Pharmaceutics, 294, 201
216. Schweizer, F. (2009). Cationic amphiphilic peptides with cancer-selective toxicity. European Journal of Pharmacology, 625, 190
194. Segerstrom, S. C., & Miller, G. E. (2004). Psycological stress and the human immune system: a meta analytic study of 30 years of inquiry. Psychology Bulletin, 130, 601
630. Shah, P., Hsiao, F. S. H., Ho, Y. H., & Chen, C. S. (2016). The proteome targets of intracellular targeting antimicrobial peptides. Proteomics, 16, 1225
1237. Shahidi, F., Han, X. Q., & Synowiecki, J. (1995). Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chemistry, 53, 285
293. Shahidi, F., Synowiecki, J., & Balejko, J. (1994). Proteolytic hydrolysis of muscle proteins of harp seal (Phoca groenlandica). Journal of Agricultural and Food Chemistry, 42, 2634
2638. Shahidi, F., & Yeo, J. D. (2018). Bioactivities of phenolics by focusing on suppression of chronic diseases: A review. International Journal of Molecular Sciences, 19, 1573. Shaji, J., & Patole, V. (2008). Protein and peptide drug delivery: Oral approaches. Indian Journal of Pharmaceutical Sciences, 70, 269
277. Sheih, I. C., Fang, T. J., Wu, T., & Lin, P. (2010). Anticancer and antioxidant activities of the peptide fraction from algae protein waste. Journal of Agricultural and Food Chemistry, 58, 1202
1207. Sheih, I. C., Wu, T. K., & Fang, T. J. (2009). Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresource Technology, 100, 3419
3425. Shi, S., Wang, Y., Liang, Y., & Li, Q. (2006). Effects of tachyplesin and nsodium butyrate on proliferation and gene expression of human gastric adenocarcinoma cell line BGC-823. World Journal of Gastroenterology, 12, 1694
1698. Sinha, V. R., & Kumria, R. (2003). Microbially triggered drug delivery to the colon. European Journal of Pharmaceutical Sciences, 18, 3
18. Sipola, M., Finckenberg, P., Korpela, R., Vapaatalo, H., & Nurminen, M. (2002). Effect of long-term intake of milk products on blood pressure in hypertensive rats. Journal of Dairy Research, 6, 103
111. Sprott, G. D., Sad, S., Fleming, L. P., Dicaire, C. J., Patel, G. B., & Krishnan, L. (2003). Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen. Archaea (Vancouver, B.C.), 1, 151
164. Stella, V. J. (2007). Prodrug approaches to enhancing the oral delivery of poorly permeable drugs. In V. J. Stella, R. T. Borchardt, M. J. Hageman, H. Maag, R. Oliyai, & J. W. Tilley (Eds.), Prodrugs Challenges and Rewards Part 1 (pp. 37
82). New York, NY: American Association of Pharmaceutical Scientists, SpringerLink (Online service). Su, F. Y., Lin, K. J., Sonaje, K., Wey, S. P., Yen, T. C., Ho, Y. C., . . . Sung, H. W. (2012). Protease inhibition and absorption enhancement by functional nanoparticles for effective oral insulin delivery. Biomaterials, 33, 2801
2811. Sun, L., Zhang, Y., & Zhuang, Y. (2013). Antiphotoaging effect and purification of an antioxidant peptide from Tilapia (Oreochromis niloticus) gelatin peptides. Journal of Functional Foods, 5, 154
162. Takahashi, M., Moriguchi, S., Yoshikawa, M., & Sasaki, R. (1994). Isolation and characterization of oryzatensin: A novel bioactive peptide with ileumcontracting and immunomodulating activities derived from rice albumin. Biochemistry and Molecular Biology International, 33, 1151
1158. Tang, W., Zhang, H., Wang, L., Qian, H., & Qi, X. (2015). Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis. Food Chemistry, 168, 115
123.

Bioactive peptides in health and disease: an overview 25 Taniguchi, M., Ochiai, A., Kondo, H., Fukuda, S., Ishiyama, Y., Saitoh, E., . . . Tanaka, T. (2016). Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system. Journal of Bioscience and Bioengineering, 121, 591
598. Toldra´, F., Reig, M., Aristoy, M. C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404. Tomita, M., Shiga, M., Hayashi, M., & Awazu, S. (1988). Enhancement of colonic drug absorption by the paracellular permeation route. Pharmaceutical Research, 5, 341
346. Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides: Production, processing, and potential health benefits. Journal of Food Science, 71, R11
R24. Umayaparvathi, S., Meenakshi, S., Vimalraj, V., Arumugam, M., Sivagami, G., & Balasubramanian, T. (2014). Antioxidant activity and anticancer effect of bioactive peptide from enzymatic hydrolysate of oyster (Saccostrea cucullata). Biomedicine & Preventive Nutrition, 4, 343
353. Van Acker, T., Van Malderen, S. J., Van Heerden, M., McDuffie, J. E., Cuyckens, F., & Vanhaecke, F. (2016). High-resolution laser ablation-inductively coupled plasma-mass spectrometry imaging of Cisplatin-induced nephrotoxic side effects. Analytica Chimica Acta, 945, 23
30. Vernaza, M. G., Dia, V. P., Mejia, E. G., & Chang, Y. K. (2012). Antioxidant and anti-inflammatory properties of germinated and hydrolysed Brazilian soybean flours. Food Chemistry, 134, 2217
2225. Vital, D. A. L., de Mejı´a, E. G., Dia, V. P., & Loarca-Pin˜a, G. (2014). Peptides in common bean fractions inhibit human colorectal cancer cells. Food Chemistry, 157, 347
355. Vitor, T., Maria, J. F., & Margarida, B. (2012). Role of lipids in the interaction of antimicrobial peptides with membranes. Progress in Lipid Research, 51, 149
177. Vo, T., Ryu, B., & Kim, S. (2013). Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maxima. Journal of Functional Foods, 5, 1336
1346. Wang, B. S., Huang, G. J., Lu, Y. H., & Chang, L. W. (2013). Anti-inflammatory effects of an aqueous extract of Welsh onion green leaves in mice. Food Chemistry, 138, 751
756. Wang, Y., He, H., Wang, G., Wu, H., Zhou, B., Chen, X., & Zhang, Y. Z. (2010). Oyster (Crassostrea gigas) hydrolysates produced on a plant scale have antitumor activity and immunostimulating effects in BALB/c mice. Marine Drugs, 8, 255
268. Wang, Z., & Zhang, X. (2017). Isolation and identification of anti-proliferative peptides from spirulina platensis using three-step hydrolysis. Journal of the Science of Food Agriculture, 97, 918
922. Wenjia, W., Mengmeng, Z., Chongzhen, S., Margaret, B., Huixian, L., Guang, W., & Hui, W. (2016). Enzymatic preparation of immunomodulatory hydrolysates from defatted wheat germ (Triticum vulgare) globulin. International Journal of Food Science and Technology, 51, 2556
2566. Wilson, J., Hayes, M., & Carney, B. (2011). Angiotensin-I-converting enzyme and prolyl endopeptidase inhibitory peptides from natural sources with a focus on marine processing by-products. Food Chemistry, 129, 235
244. Wu, J., & Ding, X. (2002). Characterization of inhibition and stability of soy-protein-derived angiotensin Iconverting enzyme inhibitory peptides. Food Research International, 35, 367
375. Wu, J., Liao, W., & Udenigwe, C. C. (2017). Revisiting the mechanisms of ACE inhibitory peptides from food proteins. Trends in Food Science & Technology, 69, 214
219. Wu, W., Zhang, M., Sun, C., Brennan, M., Li, H., Wang, G., . . . Wu, H. (2016). Enzymatic preparation of immunomodulatory hydrolysates from defatted wheat germ (Triticum vulgare) globulin. International Journal of Food Science and Technology, 51, 2556
2566. Xie, H., Wang, Y., Zhang, J., Chen, J., Wu, D., & Wang, L. (2015). Study of the fermentation conditions and the antiproliferative activity of rapeseed peptides by bacterial and enzymatic cooperation. International Journal of Food Science & Technology, 50, 619
625. Xu, Q., Fan, H., Yu, W., Hong, H., & Wu, J. (2017). Transport study of egg-derived antihypertensive peptides (LKP and IQW) using caco-2 and ht29 coculture monolayers. Journal of Agricultural and Food Chemistry, 65, 7406
7414.

26

Chapter 1

Xue, Z., Wen, H., Zhai, L., Yu, Y., Li, Y., Yu, W., . . . Kou, X. (2015). Antioxidant activity and antiproliferative effect of a bioactive peptide from chickpea (Cicer arietinum L.). Food Research International, 77, 75
81. Yan, Q. J., Huang, L. H., Sun, Q., Jiang, Z. Q., & Wu, X. (2015). Isolation, identification and synthesis of four novel antioxidant peptides from rice residue protein hydrolyzed by multiple proteases. Food Chemistry, 179, 290
295. Yang, R., Zhang, Z., Pei, X., Han, X., Wang, J., Wang, L., . . . Li, Y. (2009). Immunomodulatory effects of marine oligopeptide preparation from chum Salmon (Oncorhynchus keta) in mice. Food Chemistry, 113, 464
470. Yi, J., Zhao, J., & Wu, J. (2017). Egg ovotransferrin derived IRW exerts protective effect against H2O2-induced oxidative stress in Caco-2 cells. Journal of Functional Foods, 39, 160
167. Yoshikawa, M., Kishi, K., Takahashi, M., Watanabe, A., Miyamura, T., Yamazaki, M., & Chiba, H. (1993). Immunostimulating peptide derived from soybean protein. Annals of the New York Academy of Sciences, 685, 375
376. Yun, Y., Cho, Y. W., & Park, K. (2012). Nanoparticles for oral delivery: Targeted nanoparticles with peptidic ligands for oral protein delivery. Advanced Drug Delivery Reviews, 65, 822
832. Zhang, F., Cui, X., Fu, Y., Zhang, J., Zhou, Y., Sun, Y., . . . Chen, T. (2017). Antimicrobial activity and mechanism of the human milk-sourced peptide casein201. Biochemical Biophysical Research Communications, 485, 698
704. Zhang, J., Zhang, H., Wang, L., Guo, X., Wang, X., & Yao, H. (2010). Isolation and identification of antioxidative peptide from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS. Food Chemistry, 119, 226
234. Zhang, M., & Mu, T. H. (2016). Optimisation of antioxidant hydrolysate production from sweet potato protein and effect of in vitro gastrointestinal digestion. International Journal of Food Science & Technology, 51, 1844
1850.

CHAPTER 2

Enzymatic mechanisms for the generation of bioactive peptides ´ and Leticia Mora Fidel Toldra Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), Valencia, Spain

2.1 Introduction Proteins in foods are very important due to their nutritional relevance as a source of essential amino acids in the diet. However, proteins may experience some hydrolysis during food processing and generate peptides and free amino acids 1
3, especially in relatively long processes such as fermentation and/or ripening that are typical in the manufacture of dairy and meat products and alcoholic beverages (i.e., fermented sausages, dry-cured meats, cheese, yogurt, wine, etc.). In such products, numerous bioactive peptides have been reported (Correˆa, et al., 2014; Mohanty, Mohapatra, Misra, & Sahu, 2016; Mora, Escudero, & Toldra´, 2016; Mora, Gallego, & Toldra´, 2018; Toldra´, Reig, Aristoy, & Mora, 2018). On the other hand, the most important production of bioactive peptides at industrial scale is obtained in reactors using commercial proteolytic enzymes or microorganisms for protein hydrolysis under controlled conditions (Moayedi, et al., 2017; Toldra´, et al., 2018). Proteins extracted from food by-products constitute the raw material. Typical sources are byproducts from slaughterhouses, fisheries, olivemill wastewater, cheese whey, winery sludge, citrus peel, or any other agricultural practice (Ferraro, et al., 2013; Mora, et al., 2015; Reyes-Dı´az, Gonza´lez-Co´rdova, Herna´ndez-Mendoza, & Vallejo-Co´rdoba, 2016; Toldra´, Mora, & Reig, 2016). The scheme showing the hydrolysis of proteins either by endogenous or microbial peptidases in foods or by commercial peptidases or microorganisms with proteolytic activity is shown in Fig. 2.1. Both routes of formation of bioactive peptides and the mechanisms involved in protein hydrolysis are described below.

2.1.1 Enzymatic mechanisms in the hydrolysis of food proteins Protein hydrolysis starts with the cleavage of internal linkages by endopeptidases. This releases numerous polypeptides and peptides that act as substrate for exopeptidases (Toldra´, et al., 2018). A scheme of the mode of action of major types of peptidases is shown in Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00031-5 © 2021 Elsevier Inc. All rights reserved.

27

28

Chapter 2 Proteins in foods

Isolated food proteins

Endogenous peptidases

Microbial peptidases

Peptidases

Microbial peptidases

Ripening

Fermentation

Reactor

Fermenter

Large amounts of bioactive peptides as extracts

Small amounts of bioactive peptides within the food

Gastrointestinal digestion

Absorption in the organism Resistance to serum peptidases

Bioactive peptides Physiological functions

Figure 2.1 Scheme of the generation of bioactive peptides from protein hydrolysis in foods and/or the hydrolysis of isolated proteins. Source: Reproduced from Toldra´, F., Reig, M., Aristoy, M.C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404 with permission from Elsevier.

Fig. 2.2 for endopeptidases and Fig. 2.3 for exopeptidases. There are many types of exopeptidases that may act either on N- or C-terminal and with a varied specificity for specific terminal amino acids that will affect its hydrolysis rate. Tripeptidylpeptidases release tripeptides and dipeptidylpeptidases (DPP) release dipeptides from the N-terminal. In this way, dipeptide Ala-Arg is preferred by DPP I, whereas Gly-Pro and Arg-Pro are preferently cleaved by DPP II and IV, and dipeptides Ala-Arg and Arg-Arg by DPP III (Sentandreu & Toldra´, 2007; Toldra´, Gallego, Reig, Aristoy, & Mora, 2020a). Other dipeptides such as Lys-Ile and Phe-Asp may be released from the C terminal by peptidyldipeptidase (Mora, et al., 2018). The final step in the proteolysis chain is the release of free amino acids either from the N-terminal by aminopeptidases (Pep N, Pep A, Pep C, Pep P, among others) or the C terminal by carboxypeptidases A or B. In addition, some of the generated tripeptides can be further hydrolyzed by tripeptidases into a dipeptide and a single amino acid, and dipeptides can also be hydrolyzed by dipeptidases into the two single constituent amino acids (Mora, et al., 2015; Sanz, Mulholland, & Toldra´, 1998).

Enzymatic mechanisms for the generation of bioactive peptides 29 P0 Sequence 3' FVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTL I FVKTLTGKTITL L EVEPSDTIENVKAKIQDK K EGIPPDQQRL L IFAGKQLEDGRTL L SDYNIQKESTL

Pf 67' E E I S H

Figure 2.2 Peptides identified by nanoESI-LC-MS/MS derived from ubiquitin 60S ribosomal protein (UniProtKB/TrEMBL protein database accession number P63053). Endopeptidase activity is shown in black arrows. Source: Reprinted with permission from Toldra´, F., Gallego, M., Reig, M., Aristoy, M.-C., & Mora, L. (2020a). Recent progress in enzymatic release of food-derived peptides and assessment of bioactivity. Journal of Agricultural & Food Chemistry, 68, 12842
12855. doi:10.1021/acs. jafc.9b08297. 2020 American Chemical Society.

A Thr

Val

Lys

C

E

D Glu

Asp

Gln

Val

Phe

Pro

Met

Asn

Pro

Pro

Lys

C Phe

C

P Asp

Lys

Ile

C Glu

Asp

TVKEDQVFPMNPPKFDKIED PPKFDKIED TVKEDQVFPMNPPKFDKIED VKEDQVFPMNPPKFDKIED EDQVFPMNPPKFDKIED VKEDQVFPMNPPKFDKIED VKEDQVFPMNPPKFDKIE VKEDQVFPMNPPKFDKI TVKEDQVFPMNPPKFD TVKEDQVFPMNPPK TVKEDQVFPMNPP

Figure 2.3 Scheme of food protein hydrolysis and enzymes involved. The amino acids sequence is a fragment belonging to myosin heavy chain. Aminopeptidase (A), Dipeptidylpeptidase (D), Endopeptidase (E), Carboxypeptidase (C), and Peptidyldipeptidase (P). Source: Adapted from Mora et al. Mora, L., Gallego, M., Aristoy, M.C., Fraser, P.D., & Toldra´, F. (2015). Peptides naturally generated from ubiquitin60S ribosomal protein as potential biomarkers of dry-cured ham processing time. Food Control, 48, 102
107 with permission from Elsevier.

The final result of such proteolysis is the accumulation of small peptides and free amino acids that are then subjected to gastrointestinal (GI) digestion once the food is consumed. So, salivary, stomachal, intestinal, and pancreatic enzymes are engaged in further hydrolysis

30

Chapter 2

of the polypeptides and peptides present in foods generating smaller peptides that may be bioactive depending on the length and sequence of residues (Capriotti, et al., 2015; Mora, et al., 2018). In vitro and in vivo assays have to be performed with laboratory animals to confirm physiological effects, and final trials with humans are requested by health agencies to corroborate such effects.

2.1.2 Bioactive peptides generated during food processing There are several enzymes usually involved in the hydrolysis of proteins during food fermentation and/or ripening. On one side, the endogenous enzymes like the muscle peptidases found in meat or fish, and on the other hand, the enzymes provided by microorganisms involved in the fermentation [i.e., lactic acid bacteria (LAB) peptidases]. The proteolysis chain follows an initial step of protein breakdown by endopeptidases at internal linkages, followed by the action of exopeptidases (Mora, et al., 2015). Muscle foods are rich in exopeptidases that are active at the pH found in meat and meat products. For instance, DPP I and II that release dipeptides from the N-terminal, aminopeptidases that release amino acids from the N-terminal and carboxypeptidases that release amino acids from the C terminal (Toldra´, et al., 2020a). Some of the generated dipeptides are bioactive, whereas others contribute to the taste (Gallego, Mora, & Toldra´, 2019a). An example of sequential release of dipeptide Pro-Ala from the N-terminal of myosin light chain I (Mora, Sentandreu, & Toldra´, 2011) is shown in Fig. 2.4. Dry-cured ham, that has a very long ripening/drying process, has been reported as a potential source of antioxidant (Escudero, Aristoy, Nishimura, Arihara, & Toldra´, 2012), angiotensin-converting enzyme (ACE) inhibitory peptides (Mora, Escudero, Arihara, Toldra´ et al., 2015; Toldra´, Gallego, Reig, Aristoy, & Mora, 2020b), cardioprotective peptides (Gallego, Mora, & Toldra´, 2019b) and α-glucosidase-inhibitory peptides (Mora, Gonza´lez-Rogel, Heres, & Toldra´, 2020). LAB are typical microorganisms used in food fermentation where they exert proteolytic activity due to their extracellular endopeptidases and intracellular peptidases with a wide range of specificity. Numerous bioactive peptides were reported in dry-fermented sausages with Lactobacillus pentosus and Staphylococcus carnosus (Mora, et al., 2015). ACE-inhibitory peptides were reported in milk fermented with the yeast Kluyveromyces marxianus Z17 (Li, Sadiq, Liu, Chen, & He, 2015). A couple of antioxidant hexapeptides released from β-casein after simulated GI digestion were reported in Italian Stracchino cheese (Pepe, et al., 2016).

2.1.3 Bioactive peptides generated through the hydrolysis of proteins with commercial peptidases The usual production of bioactive peptides is through the hydrolysis of proteins with specific commercial peptidases or microorganisms (Ryder, Bekhit, McConnell, & Carne,

Enzymatic mechanisms for the generation of bioactive peptides 31

A

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Figure 2.4 Intense degradation of myosin light chain 1 (accession number A1XQT6_PIG in UniProtKB/ TrEMBL database), evidencing the action of aminopeptidases (in dark black) and dipeptidylpeptidases (in light black). Source: Reproduced from Toldra´, F., Reig, M., Aristoy, M.C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404 with permission from Elsevier.

2016; Toldra´, et al., 2018). There are many types of enzymes as shown in Table 2.1. Those produced from microorganisms are cheaper than those from plant or animal origin. Each enzyme has its own activity and specificity and therefore may cleave the protein at different sites. The enzymatic hydrolysis is performed in reactors with controlled pH and temperature followed by downstream processing for the separation of the bioactive peptides fraction. The sources of proteins are typically by-products from the food industry like dairy, fish, meat, eggs (Abdelhedi, et al., 2016; Lassoued, et al., 2015a; Oseguera-Toledo, Gonza´lez de Mejı´a, Reynoso-Camacho, Cardador-Martı´nez, & Amaya-Llano, 2014; Ryder, et al., 2016; Tanzadehpanah, Asoodeh, & Chamani, 2012), and vegetable sources like soybean, peanuts, among other (De Oliveira, et al., 2015; Ji, Sun, Zhao, Xiong, & Sun, 2014). In view of the large variety of peptidases available for protein hydrolysis, as reported in Table 2.1, and the type of bioactive peptides obtained depending on the hydrolysis reaction conditions, as reported in Table 2.2, it is very important to consider very carefully the most appropriate enzyme for each type of protein and target bioactive peptides (Toldra´, et al.,

Table 2.1: Commercial enzyme preparations with specific characteristics and some relevant applications. Commercial preparation Flavourzyme 1000M

Prolidase

Bioprase SP20FG

Origin

Manufacturers

Activity

Aspergillus oryzae

3 endopeptidases 2 aminopeptidases 2 dipeptidylpeptidases 1 α-amylase

L-lactis cremoris Other many sources

Dipeptidase

Bacillus sp.

Subtilisin Endo metalloprotease Aminopeptidase Metalloprotease

Neutrase

Bacillus subtilis B. amyloliquefaciens

Novozymes

Alcalase 2.4L

Bacillus licheniformis

Novozymes

Savinase

Bacillus lentus

Novozymes

Esperase

Bacillus lentus

Novozymes

Subtilisin Alkaline serin endopeptidase Extracellular neutral metalloprotease Aminopeptidase SubtilisinAlkaline serin endopeptidase Subtilisin Alkaline serin endopeptidase

Cleavage sites

Bonds including proline or hydroxiproline

Application

References

Cereals Calcium-chelating peptides, Soy

Huang, et al. (2015), Meinlschmidt, Sussmann, SchweiggertWeisz, and Eisner (2016), Merz, et al. (2015) Kitchener and Grunden (2012)

Cheese making

Merz, Claaßena et al. (2016)

Nonespecific

Higher affinity by Phe and Leu Broad especificity

Collagen Calcium- and ironchelating peptides Soy Calcium-chelating peptides

Lentil proteins

Meinlschmidt, et al. (2016), Ou, et al. (2010) Charoenphun, Cheirsilp, Sirinupong, and Youravong (2013), Choi, Lee, Chun, and Song (2012) Garcı´a-Mora, et al. (2014) Georgieva, Stoeva, Voelter, Genov, and Betzel (2001)

Protamex

Bacillus licheniformis Bacillus amyloliquefaciens

Novozymes

Subtilisin Serin endopeptidase, Metallo endopeptidase Neutral protease

Protex 6L

Bacillus licheniformis

Genencor

Alcaline serine endopeptidase Endo and Exo

Protease M Promod 439L

Bacillus licheniformis

Biocatalysts

Subtilisin

Pronase Corolase 7089

Streptomyces griseus Bacillus subtilis

Sigma-Aldrich AB Enzymes GmbH

Endo and Exo Neutral endopeptidase

Porcine pancreatic gland

AB enzymes GmbH

Bacillus thermoproteolyticusBacillus stearothermophylus Aspergillus niger

AB enzymes

Genencor Biotech

Porcine pancreatic glands

Novozymes

Bovine pancreas

Sigma-Aldrich Fluka

Corolase PP

Corolase 2TS GC106

Pancreatic trypsin Novo Trypsin

Meinlschmidt, et al. (2016), Slizyte, et al. (2016), Sung, et al. (2014)

Calcium-chelating peptides in soybean Generation of flavor in animal, vegetable, and fish proteins.

Lv, Bao, Liu, Ren, and Guo (2013) ´, Vejlupkova´, Mihulova Hanuˇsova´, ˇ Stˇetina, and Panovska´ (2013) Yoshida, et al. (1988) Garcı´a-Mora, et al. (2014), Slizyte, et al. (2016) Slizyte, et al. (2016)

Bioactivity in fish

Bioactivity in fish

Endopeptidase, Amino- and carboxypeptidase Endopeptidase

Cereals, soy

Acid proteinase@@Aspartictype peptidase Endopeptidase Endopeptidase

Calcium-chelating peptides Bioactivity in fish, antiallergenic in cereals Soy Bioactivity in fish

Antiallergenic in cereals

Broad specificity Arg-|-Xaa, Lys-|-Xaa

Calcium- chelating peptides from shrimp byproducts, Tilapia, or Alaska Pollock skin

Slizyte, et al. (2016)

Meinlschmidt, et al. (2016), Merz, et al. (2015) Sung, et al. (2014)

Meinlschmidt, et al. (2016) Guo, et al. (2015)

(Continued)

Table 2.1: (Continued) Commercial preparation

Origin

Manufacturers

Activity

Cleavage sites

Application

References

Bacillus licheniformis

Genencor

Serine-type peptidase

Antiallergenic in cereals

Sung, et al. (2014)

Bromelain

Pineapple stem

Great food (Biochem)

Cysteine-type peptidase

Aromatic or hydrophobic residues Broad especificity

Antiallergenic in cereals

Sung et al. (2014)

Collupulin

Carica papaya

Gist-brocades

Cysteine-type peptidase

Antiallergenic in cereals

Sung, et al. (2014)

Papain

Carica papaya

Sigma-Aldrich Fluka Sigma-Aldrich Fluka

Cysteine-type peptidase

Collagen antiallergenic in cereals

Sung, et al. (2014)

Alkaline protease

Ficain (ficin)

Figs latex

Cysteine-type peptidase

Aromatic or hydrophobic residues Broad especificity Broad especificity

Bekhit, Hopkins, Geesink, Bekhit, and Franks (2014)

Source: Reprinted from Toldra´, F., Reig, M., Aristoy, M.C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404 with permission from Elsevier.

Enzymatic mechanisms for the generation of bioactive peptides 35 Table 2.2: Examples of hydrolysis treatments of different types of foods and bioactive peptides identified in the hydrolyzates. Origin of proteins

Hydrolysis treatment 

Peptidase

Peptide sequence

Reported activity valuesa

References Harnedy, O’Keeffe, and FitzGerald (2017) Admassu, Gasmalla, Yang, and Zhao (2018) Neves, Harnedy, O’Keeffe, and FitzGerald (2017)

Algae Palmaria palmata

2%, 50 C, pH 7, 4 h

Corolase PP

SDITRPGGQM

ORAC: 152.43 nmol TE/ μmol RP: 21.23 nmol TE/μmol

Red seeweed (Porphyra spp.) Atlantic salmon (Salmo salar)

1%, 37 C, pH 2, 3 h

Pepsin

GGSK ELS

α-Amylase inhibition: IC50 5 2.58 mM IC50 5 2.62 mM

1%, 50 C, pH 7, 1 h

Corolase PP

GPAV

ACE inhibition: IC50 5 415.91 μM DPP-IV inhibition: IC50 5 245.58 μM ORAC: 9.51 μmol TE/ μmol ACE inhibition: IC50 5 59.151 μM DPP-IV inhibition: IC50 5 546.84 μM ORAC: 8.47 μmol TE/ μmol ACE inhibition: IC50 5 0.23 mM β-CBA: IC50 5 0.64 mM ACE inhibition: IC50 5 0.41 mM DPPH: IC50 5 1.32 mMACE inhibition: IC50 5 0.21 mMDPPH: IC50 5 1.41 mM Alcohol dehydrogenase stabilization

FF

Sardinelle (Sardinella aurita)

4%, 37 C, Bacillus 24 h amyloliquefaciens

Chicken breast

0.5%, 55 C, 8 h

Alcalase 2.4L

Soy

6 U/kg, 50 C, pH 9

Alkaline proteinase

Pork loin

0.008%, 5 C, 24 h

Thermolysin

LVGRPRHGQ VFPS

Goat (Capra hircus) milk

3%, 37 C, pH 8, 3 h

Trypsin

INNQFLPYPY MHQPPQPL SPTVMFPPQSVL

ITALAPSTM SLEAQAEKY GTEDELDKY NVPVYEGY

DPQYPPGPPAF KPC APGH LLPLPVLK SWLRL WLRL

α-Glusosidase inhibition: IC50 5 237.43 μM IC50 5 182.05 μM IC50 5 162.29 μM ACE inhibition: IC50 5 15.69 μM IC50 5 3.60 μM DPP-IV inhibition: IC50 5 40.08 μM IC50 5 350.41 μM IC50 5 376.31 μM

Jemil, et al. (2016)

Xiao, et al. (2020) Wang, et al. (2019)

Choe, et al. (2019) Zhang, Chen, Ma, and Chen (2015)

(Continued)

36

Chapter 2 Table 2.2: (Continued)

Origin of proteins

Hydrolysis treatment

Peptidase

Chicken combs and wattles

5%, 4 h

Alcalase

APGLPGPR FPGPPGP Piro-GPPGPT

Oil palm (Elaeis guineensis Jacq) kernel expeller

0.5%, 45 C, pH 8.5, 2h1 0.5%, 50 C, pH 7, 2h1 0.3%, 37 C, pH 2, 1 h1 0.3%, 37 C, pH 7, 1h 2%, 37 C, 24 h

Alcalase 1 Flavourzyme 1 pepsin 1 trypsin

ADVFNPR LPILR VIEPR VVLYK

Bacillus subtilis

DGVVYY GQVPP

Tomato seeds

Peptide sequence

Reported activity valuesa ACE inhibition: IC50 5 53 μM IC50 5 38 μM IC50 5 88 μM ACE inhibition: IC50 5 485.7 μM IC50 5 779.8 μM IC50 5 632.0 μM IC50 5 533.9 μM

ACE inhibition: IC50 5 2 μM DPPH: 97% at 0.4 mM RP: 0.95 UA at 0.5 mM

References Bezerra, et al. (2019)

Zheng, Li, Zhang, Ruan, and Zhang (2017)

Moayedi, et al. (2018)

a Activity values: IC50 value is the peptide concentration that inhibits 50% of activity. Angiotensin-converting enzyme (ACE), Antioxidant activity: ABTS radical-scavenging activity (ABTS), DPPH radical scavenging assay (DPPH), hydroxyl radical scavenging activity (OH
), reducing power (RP), β-carotene bleaching (β-CBA), and oxygen radical absorbance capacity assay (ORAC).

2018). However, the choice of the most adequate peptidase is not always easy due to its specificity that may vary depending on the particular enzyme and the lack of enough information in the manufacturers specifications. This is the case of some peptidases that may present some minor side reactions due to other enzymes present in small content (see Table 2.1). Activity based on the release of amino acids at N or C terminal was assayed in eight different commercial enzyme preparations. It was reported that Flavourzyme 1000L (Novozymes) and Protease AN (Amano Enzyme Inc.) hydrolyzed lupine with a high degree of hydrolysis (DH) and large amounts of free amino acids, whereas Alcalase 2.4L (Novozymes) and Maxazyme NNP DS (DSM) resulted in intermediate DH and release of free amino acids (Merz, Claaßena et al., 2016). Flavourzyme (Novozymes) showed a

Enzymatic mechanisms for the generation of bioactive peptides 37 variety of three endopeptidases but also other enzymes like two aminopeptidases, two DPP, and one amylase (Merz, et al., 2015). Commercial enzymes, like Flavourzyme, may also have batch to batch variability in the activity that may affect its efficacy, reproducibility, and stability during the storage time (Merz, Appel, et al., 2016). Neutrase was able to generate smaller peptides from collagen than those obtained using papain (Damrongsakkul, Ratanathammapan, Komolpis, & Tanthapanichakoon, 2008). The DH constitutes a good parameter for monitoring the extension of protein hydrolysis. The functional properties of the protein hydrolyzate depends on the DH and type of peptidase preparation used (Mune, 2015). Although the DH is a very important control parameter, the final target is the number and amount of bioactive peptides able to exert the desired activity. So, thornback ray gelatin hydrolyzate treated with Alcalase was the most effective to prevent DNA oxidation, even though the DH was not so high (Lassoued, et al., 2015b). Also lentil protein hydrolyzate treated with Savinase was reported to generate more bioactive peptides than Alcalase that gave a higher amount of peptides (Garcı´a-Mora, Pen˜as, Frias, & Martı´nezVillaluenga, 2014). Examples of hydrolysis conditions for different types of food proteins and the reported major peptides and its bioactivities are shown in Table 2.2. A good alternative to produce bioactive peptides of interest is to proceed with the sequential hydrolysis using different peptidases. So, a higher antioxidant and antimicrobial activity of peptides was obtained from hen egg white lysozyme using trypsin and papain enzymes, instead of using them alone (Memarpoor-Yazdi, Asoodeh, & Chamani, 2012). Also more antioxidant peptides were obtained from Brassica carinata using trypsin, chymotrypsin, and carboxypeptidase A (Pedroche, et al., 2007); more antioxidant, ACEinhibitory, and antibacterial peptides were obtained in Smooth hound viscera of M. mustelus using Purafeet, Neutrase, and Esperase (Abdelhedi, et al., 2016); more ACE-inhibitory peptides were obtained from casein protein using Neutrase and plastein enzymes (Xu, Kong, & Zhao, 2014), as well as a higher antioxidant and ACE-inhibitory activity was observed in defatted salmon backbone hydrolyzed using trypsin, bromelain, papain, and protamex enzymes together (Slizyte, et al., 2016).

2.2 Degree of hydrolysis 2.2.1 Definition The DH constitutes a good parameter for monitoring the extension of protein hydrolysis so that the reaction may be stopped once the desired DH is reached. DH is defined as the percentage of cleaved peptide bonds through the following equation: DH 5 h=htot 3 100

38

Chapter 2

where h is the number of hydrolyzed bonds and htot the total number of peptide bonds per protein equivalent (Adler-Nissen, 1976).

2.2.2 Precursor techniques and alternative methods/procedures There are different available methods for the determination of DH. The pH stat, that only works at neutral or alkaline pH, is based on the net release of protons when the pH value approaches the pK value of the α-amino group, and therefore the base consumption is proportional to the number of cleaved peptide bonds (Adler-Nissen, 1984). The osmometer technique is a fast method that measures the freezing point depression which is converted to mosmol so that DH may be determined from the increase in osmolality (Adler-Nissen, 1984). However, this technique cannot be applied to highly viscous solutions (Nielsen, Petersen, & Dambmann, 2001). The soluble nitrogen (SN)-trichloroacetic acid (TCA) method analyses the amount of SN in aqueous TCA obtaining the SN-TCA index, also known as nonprotein nitrogen, that gives good correlation with the pH stat (Margot, Flaschel, & Renken, 1994) but it majorly works for endopeptidase activity since the correlation is worse (less protein solubility) when a major exopeptidase activity is present (Nielsen, et al., 2001). Some authors determine the percent DH as the ratio of SN-TCA to the total nitrogen (Kaewka, Therakulkait, & Cadwallader, 2009). The trinitrobenzenesulfonic acid (TNBS) method is a widely used spectrophotometric method that determines the absorbance at 340 nm of the chromophore formed from the reaction of primary amino groups in the hydrolyzate with TNBS under slightly alkaline conditions. A L-leucine solution is used as standard (Adler-Nissen, 1979). An improved method was based on the reaction of o-phtaldialdehyde with primary amino groups in the presence of mercaptoethanol giving a colored compound determined spectrophotometrically at 340 nm (Church, Swaisgood, Porter, & Catignani, 1983). Such method was improved by replacing mercaptoethanol by dithiothreitol and using serine as standard (Nielsen, et al., 2001).

2.3 Assay of endopeptidase activity 2.3.1 Definition Initially, endopeptidase activity was determined using proteins as substrates to measure product peptides as general proteolysis without considering any specificity. The current assays of endopeptidase activity are usually based on the hydrolysis of synthetic substrates conjugated to a chromophore compound like p-nitroanilide (pNA) or containing a highly fluorescent substituent such as fluorescein isothiocyanate (FITC) or 7-amino-4methylcoumarin. In this way, the assays are based on the determination of the increase in absorbance when the pNA molecule is cleaved and released, or the increase of fluorescence due to the release of the fluorescein substituent when a fluorescent substrate is used. The

Enzymatic mechanisms for the generation of bioactive peptides 39 endopeptidase activity of microorganisms used for protein hydrolysis is usually determined with whole cells through the use of labeled substrates. The protocol described herein was developed by Twining (Twining, 1984) and modified by Sanz and Toldra´ (Sanz & Toldra´, 1997) and has been applied to determine the endopeptidase activity of numerous microorganisms like L. curvatus (Fadda, et al., 1999a), L. sakei (Sanz & Toldra´, 1997), L. plantarum (Fadda, et al., 1999b), L. casei (Sanz, et al., 1999), or Staphylococci (Casaburi, Villani, Toldra´, & Sanz, 2006). 2.3.1.1 Materials, equipment, and reagents • • • •

• • • •

Assay buffer: 50 mM Tris-HCl buffer (pH 6.5) containing 20 mM CaCl2 Standard: FITC (Sigma, St. Louis, MO). Substrate: Casein-FITC type II (Sigma, St. Louis, MO). Enzyme solution: Peptidase at appropriate dilution or whole-cell suspension of the tested microorganism having the peptidase that, after harvesting and centrifugation, is resuspended at 2% in 50 mM Tris-HCl buffer (pH 6.5). Temperature-controlled water bath Centrifuge Plate-reader for fluorescence detection equipped with temperature-controlled plate housing. Multichannel pipettes, tips, and black 96-well plates.

2.3.1.2 Protocol 1. Place all solutions (buffer, substrate, and enzyme) in a constant-temperature water bath set at 37 C. 2. Background: Pipette 170 μL of assay buffer into the background wells. 3. Control: Pipette 70 μL of assay buffer containing 0.4% of substrate 1 100 μL of 50 mM Tris-HCl buffer (pH 6.5) into the control wells. 4. Enzyme sample: Pipette 70 μL of assay buffer containing 0.4% of substrate into the assay wells. 5. Initiate the reaction by pipetting 100 μL of enzyme solution into the assay wells. 6. Place the multiwall plate in the plate-reader, mix to start the reaction and incubate at 37 C for 60 min. 7. Measure the fluorescence at 0 and 30 min of incubation at excitation and emission wavelengths of 485 and 538 nm, respectively. 2.3.1.3 Analysis One unit (U) of endopeptidase activity is defined as the amount of required enzyme to release 1 μmol of FITC from the substrate per hour at 37 C. A standard curve is prepared with known concentrations of FITC for the equivalence relating fluorescence intensity to FITC concentration.

40

Chapter 2

2.3.1.4 Alternative methods/procedures In some cases, the protein is chosen depending on the peptidase to be assayed. Other assays are based on the use of chromogenic derivatives of proteins like azocasein. Such substrate is incubated with the endopeptidase, the reaction is stopped by adding TCA, and the absorbance of the supernatant is measured at 450 nm (Mune, 2015). Other methods are based on the use of substrates with pNA and the spectrophotometric determination at 405 nm of the released pNA after the enzymatic action. However, the sensitivity of colorimetric substrates is lower than when using fluorescent substrates. An alternative fluorescent substrate is N-succinyl-leucine-tyrosine-7-amido-4-methylcoumarin that releases the fluorescent molecule 7-amido-4-methylcoumarin (AMC) that can be measured at excitation and emission wavelengths of 355 and 460 nm, respectively (Bolumar, Sanz, Aristoy, & Toldra´, 2006). The protocols may be adapted to a one single sampling time by stopping the reaction if the rate at such time is found within the straight part of the curve of product concentration versus time of reaction.

2.4 Assay of exopeptidase activity 2.4.1 Definition The exopeptidase activity of microorganisms used for protein hydrolysis is usually determined with whole cells or cell-free extract of the microorganisms (Sanz & Toldra´, 1997) through the use of labeled substrates. The assays are usually based on the hydrolysis of synthetic substrates conjugated to a chromophore compound like pNA or containing a highly fluorescent substituent like AMC. Such assays determine the increase in absorbance when the pNA molecule is cleaved and released, or the increase of fluorescence due to the cleavage of AMC when a fluorescent substrate is used.

2.4.2 Materials, equipment, and reagents • • • •

• • • •

Assay buffer: 50 mM Tris-HCl buffer (pH 7.0) Standard: 7-amino-4-methylcoumarin (Sigma, St. Louis, MO). Substrate: L-Ala-AMC trifluoroacetate salt (Sigma, St. Louis, MO). Enzyme solution: Exopeptidase at appropriate dilution, or whole-cell suspension, or cell-free extract of the tested microorganism that, after harvesting and centrifugation, is resuspended at 1.5% in 50 mM Tris-HCl buffer (pH 7.0) Temperature-controlled water bath Centrifuge Plate-reader with incubation and fluorescence detection. Multichannel pipettes and disposable black 96-well plates.

Enzymatic mechanisms for the generation of bioactive peptides 41

2.4.3 Protocol 1. Place all solutions (buffer, substrate, and enzyme) in a constant-temperature water bath set at 37 C. 2. Background: Pipette 300 μL of assay buffer into the background wells. 3. Control: Pipette 250 μL of assay buffer containing 0.1 mM substrate 1 50 μL of 50 mM Tris-HCl buffer (pH 7.0) into the control wells. 4. Enzyme sample: Pipette 250 μL of assay buffer containing 0.4% of substrate into the assay wells. 5. Initiate the reaction by pipetting 50 μL of enzyme solution into the assay wells. 6. Place the multiwell plate in the plate-reader, mix to start the reaction and incubate at 37 C for 15 min 7. Measure the fluorescence at 0 and 15 min of incubation at excitation and emission wavelengths of 360 and 440 nm, respectively. 2.4.3.1 Analysis One unit (U) of exopeptidase activity is defined to be the amount of enzyme required to release 1 μmol of AMC from the substrate per hour at 37 C. A standard curve is prepared with known concentrations of AMC for the equivalence relating fluorescence intensity to AMC concentration. 2.4.3.2 Alternative methods/procedures There is a wide range of substrates containing 7-amino-4-methylcoumarin that depend on the terminal amino acid (Ala, Met, Leu, Pro, etc.) and the choice depends on the specificity of the enzyme to be assayed. Such substrates are soluble in water and can be stored with low rates of autolysis. The substrate may be as much specific as possible to get the best activity determination. It can also be marked (i.e., isotope) for follow up more specific action. Other assays are available for the determination of exopeptidase activity. Some of them are based on the use of substrates with pNA and the spectrophotometric determination at 405 nm of the released pNA after the enzymatic action (Merz, et al., 2015). It must be said that the sensitivity of fluorescent substrates is much higher than colorimetric substrates.

2.4.4 Pros and cons Pros

Cons

The protocol is easy to be performed

Commercial enzymes usually contain other enzymes even though with minor activity. So, the activity only represents the enzyme if it is really pure (Continued)

42

Chapter 2 (Continued)

The materials and plate-reader spectrophotometers are usually available in any laboratory Results may be obtained in a short period of time due to the reduced time needed (usually less than 2 hours)

Other enzymes in the fermentation media or in the food may be also extracted and interfere in the activity assay Plate-readers with fluorescence detection are not so usual in laboratories Interferences in product measurement, especially if using spectroscopy tools

2.4.5 Summary This chapter is reporting the main mechanisms involved in the endo and exopeptidases action on proteins and how bioactive peptides are generated either in the food itself by endogenous or microbial peptidases, or in reactors with commercial peptidases. Most relevant methods used for the determination of the DH are briefly summarized as well as examples of protocols for the assay of activity of endopeptidases and aminopeptidases.

References Abdelhedi, O., Jridi, M., Jemil, I., Mora, L., Toldra´, F., Aristoy, M. C., et al. (2016). Combined biocatalytic conversion of smooth hound viscera: Protein hydrolysates elaboration and assessment of their antioxidant, anti-ACE and antibacterial activities. Food Research International, 86, 9
23. Adler-Nissen, J. (1976). Enzymatic hydrolysis of proteins for increased solubility. Journal of Agricultural & Food Chemistry, 24, 1090
1093. Adler-Nissen, J. (1979). Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. Journal of Agricultural & Food Chemistry, 27, 1256
1262. Adler-Nissen, J. (1984). Control of the proteolytic reaction and of the level of bitterness in protein hydrolysis processes. Journal of Chemical Techmology & Biotechnology, 34B, 215
222. Admassu, H., Gasmalla, M. A., Yang, R., & Zhao, W. (2018). Identification of bioactive peptides with α-amylase inhibitory potential from enzymatic protein hydrolysates of red seaweed (Porphyra spp). Journal of Agriculture & Food Chemistry, 66, 4872
4882. Bekhit, A. A., Hopkins, D. L., Geesink, G., Bekhit, A. A., & Franks, P. (2014). Exogenous proteases for meat tenderization. Critical Reviews in Food Science and Nutrition, 54, 1012
1031. Bezerra, T. K. A., de Lacerda, J. T. J. G., Salu, B. R., Oliva, M. L. V., Juliano, M. A., Pacheco, M. T. B., & Madruga, M. S. (2019). Identification of angiotensin I-converting enzyme-inhibitory and anticoagulant peptides from enzymatic hydrolysates of chicken combs and wattles. Journal of Medicinal Food, 22, 12. Bolumar, T., Sanz, Y., Aristoy, M. C., & Toldra´, F. (2006). Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources. International Journal of Food Microbiology, 107, 20
26. Capriotti, A. L., Caruso, G., Cavaliere, C., Samperi, R., Ventura, S., Chiozzi, R. Z., et al. (2015). Identification of potential bioactive peptides generated by simulated gastrointestinal digestion of soybean seeds and soy milk proteins. Journal of Food Composition and Analysis, 44, 205
213. Casaburi, A., Villani, F., Toldra´, F., & Sanz, Y. (2006). Protease and esterase activity of Staphylococci. International Journal of Food Microbiology, 112, 223
229.

Enzymatic mechanisms for the generation of bioactive peptides 43 Charoenphun, N., Cheirsilp, B., Sirinupong, N., & Youravong, W. (2013). Calcium-binding peptides derived from tilapia (Oreochromisniloticus) protein hydrolysate. European Food Research and Technology, 236, 57
63. Choe, J., Seol, K. H., Son, D. I., Lee, H. J., Lee, M., & Jo, C. (2019). Identification of angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates of pork loin. International Journal of Food Properties, 22, 1112
1121. Choi, D. W., Lee, J. H., Chun, H. H., & Song, K. B. (2012). Isolation of a calcium-binding peptide from bovine serum protein hydrolysates. Food science and biotechnology, 21, 1663
1667. Church, F. C., Swaisgood, H. E., Porter, D. H., & Catignani, G. L. (1983). Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science, 66, 1219
1227. Correˆa, A. P. F., Daroit, D. J., Fontoura, R., Meira, S. M. M., Segalin, J., & Brandelli, A. (2014). Hydrolysates of sheep cheese whey as a source of bioactive peptides with antioxidant and angiotensin-converting enzyme inhibitory activities. Peptides, 6, 48
55. Damrongsakkul, S., Ratanathammapan, K., Komolpis, K., & Tanthapanichakoon, W. (2008). Enzymatic hydrolysis of raw hide using papain and neutrase. Journal of Industrial and Engineering Chemistry, 14, 202
206. Escudero, E., Aristoy, M. C., Nishimura, H., Arihara, K., & Toldra´, F. (2012). Antihypertensive effect and antioxidant activity of peptide fractions extracted from dry-cured ham. Meat Science, 91, 306
311. Fadda, S., Sanz, Y., Vignolo, G., Aristoy, M.-C., Oliver, G., & Toldra´, F. (1999a). Hydrolysis of pork muscle sarcoplasmic proteins by Lactobacillus curvatus and Lactobacillus sake. Applied and Environmental Microbiology, 65, 578
584. Fadda, S., Sanz, Y., Vignolo, G., Aristoy, M.-C., Oliver, G., & Toldra´, F. (1999b). Characterization of muscle sarcoplasmic and myofibrillar protein hydrolysis caused by Lactobacillus plantarum. Applied and Environmental Microbiology, 65, 3540
3546. Ferraro, V., Carvalho, A. P., Piccirillo, C., Santos, M. M., Castro, P. M. L., & Pintado, M. E. (2013). Extraction of high added value biological compounds from sardine, sardine-type fish and mackerel canning residues. Materials Science and Engineering C, 33, 3111
3120. Gallego, M., Mora, L., & Toldra´, F. (2019a). The relevance of dipeptides and tripeptides in the bioactivity and taste of dry-cured ham. Food Production, Processing & Nutrition, 1, 2. Gallego, M., Mora, L., & Toldra´, F. (2019b). Potential cardioprotective peptides generated in Spanish dry-cured ham. Journal of Food Bioactives, 6, 110
117. Garcı´a-Mora, P., Pen˜as, E., Frias, J., & Martı´nez-Villaluenga, C. (2014). Savinase, the most suitable enzyme for releasing peptides from Lentil (Lens culinaris var. Castellana) protein concentrates with multifunctional properties. Journal of Agricultural and Food Chemistry, 62, 4166
4174. Georgieva, D. N., Stoeva, S., Voelter, W., Genov, N., & Betzel, C. (2001). Substrate specificity of the highly alkalophilic bacterial proteinase Esperase: Relation to the X-ray structure. Current Microbiology, 42, 368
371. Guo, L., Harnedy, P. A., O’Keeffe, M. B., Zhang, L., Li, B., Hou, H., & Fitzgerald, R. J. (2015). Fractionation and identification of Alaska pollock skin collagen-derived mineral chelating peptides. Food Chemistry, 173, 536
542. Harnedy, P. A., O’Keeffe, M. B., & FitzGerald, R. J. (2017). Fractionation and identification of antioxidant peptides from an enzymatically hydrolysed Palmaria palmata protein isolate. Food Research Intermational, 100, 416
422. Huang, S. L., Zhao, L. N., Cai, X., Wang, S. Y., Huang, Y. F., Hong, J., & Rao, P. F. (2015). Purification and characterisation of a glutamic acid-containing peptide with calcium-binding capacity from whey protein hydrolysate. Journal of Dairy Research, 82, 29
35. Jemil, I., Mora, L., Nasri, R., Abdelhedi, O., Aristoy, M. C., Hajji, M., . . . Toldra´, F. (2016). A peptidomic approach for the identification of antioxidant and ACE-inhibitory peptides in sardinelle protein

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Chapter 2

hydrolysates fermented by Bacillus subtilis A26 and Bacillus amyloliquefaciens An6. Food Research International, 89, 347
358. Ji, N., Sun, C. X., Zhao, Y. X., Xiong, L., & Sun, Q. J. (2014). Purification and identification of antioxidant peptides from peanut protein isolate hydrolysates using UHR-Q-TOF mass spectrometer. Food Chemistry, 161, 148
154. Kaewka, K., Therakulkait, C., & Cadwallader, K. R. (2009). Effect of preparation conditions on composition and sensory aroma characteristics of acid hydrolyzed rice bran protein concentrate. Journal of Cereal Science, 50, 56
60. Kitchener, R. L., & Grunden, A. M. (2012). Prolidase function in proline metabolism and its medical and biotechnological applications. Journal of Applied Microbiology, 113, 233
247. Lassoued, I., Mora, L., Nasri, R., Aydi, M., Toldra´, F., Aristoy, M. C., . . . Nasri, M. (2015a). Characterization, antioxidative and ACE inhibitory properties of hydrolysates obtained from Thornback Ray (Raja clavata) muscle. Journal of proteomics, 128, 458
468. Lassoued, I., Mora, L., Nasri, R., Jridi, M., Toldra´, F., Aristoy, M. C., . . . Nasri, M. (2015b). Characterization and comparative assessment of antioxidant and ACE inhibitory activities of thornback ray gelatin hydrolysates. Journal of Functional Foods, 13, 225
238. Li, Y., Sadiq, F. A., Liu, T., Chen, J., & He, G. (2015). Purification and identification of novel peptides with inhibitory effect against angiotensin I-converting enzyme and optimization of process conditions in milk fermented with the yeast Kluyveromyces marxianus. Journal of Functional Foods, 16, 278
288. Lv, Y., Bao, X., Liu, H., Ren, J., & Guo, S. (2013). Purification and characterization of calcium-binding soybean protein hydrolysates by Ca2 1 /Fe3 1 immobilized metal affinity chromatography (IMAC). Food Chemistry, 141, 1645
1650. Margot, A., Flaschel, E., & Renken, A. (1994). Continuous monitoring of enzymatic whey protein hydrolysis. Correlation of base consumption with soluble nitrogen content. Process Biochemistry, 29, 257
262. Meinlschmidt, P., Sussmann, D., Schweiggert-Weisz, U., & Eisner, P. (2016). Enzymatic treatment of soy protein isolates: Effects on the potential allergenicity, technofunctionality, and sensory properties. Food Science & Nutrition, 4, 11
23. Memarpoor-Yazdi, M., Asoodeh, A., & Chamani, J. (2012). A novel antioxidant and antimicrobial peptide from hen egg white lyzozyme hydrolysates. Journal of Functional Foods, 4, 278
286. Merz, M., Appel, D., Berends, P., Rabe, S., Blank, I., Stressler, T., et al. (2016). Batch
to
batch variation and storage stability of the commercial peptidase preparation Flavourzyme in respect of key enzyme activities and its influence on process reproducibility. European Food Research and Technology, 242, 1005
1012. Merz, M., Claaßena, W., Appel, D., Berends, P., Rabe, S., Blank, I., . . . Fischer, L. (2016). Characterization of commercially available peptidases in respect of the production of protein hydrolysates with defined compositions using a three-step methodology. Journal of Molecular Catalysis B: Enzymatic, 127, 1
10. Merz, M., Eisele, T., Berends, P., Appel, D., Rabe, S., Blank, I., et al. (2015). Flavourzyme, an enzyme preparation with industrial relevance: Automated nine-step purification and partial characterization of eight enzymes. Journal of Agricultural and Food Chemistry, 63, 5682
5693. ˇ etina, J., & Panovska´, Z. (2013). Effect of modified whey Mihulova´, M., Vejlupkova´, M., Hanuˇsova´, J., Stˇ proteins on texture and sensory quality of processed cheese. Czech Journal of Food Science, 31, 55
558. Moayedi, A., Mora, L., Aristoy, M.-C., Hashemi, M., Safari, M., & Toldra´, F. (2017). ACE-inhibitory and antioxidant activities obtained in fermented tomato seed by-products using Bacillus subtilis: Effect of amino Acid composition and peptides molecular weight distribution. Applied Biochemistry & Biotechnology, 181, 48
64. Moayedi, A., Mora, L., Aristoy, M. C., Safari, M., Hashemi, M., & Toldra´, F. (2018). Peptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteins fermented using Bacillus subtilis. Food Chemistry, 250, 180
187. Mohanty, D. P., Mohapatra, S., Misra, S., & Sahu, P. S. (2016). Milk derived bioactive peptides and their impact on human health—A review. Saudi Journal of Biological Sciences, 23, 577
583.

Enzymatic mechanisms for the generation of bioactive peptides 45 Mora, L., Escudero, E., Arihara, K., & Toldra´, F. (2015). Antihypertensive effect of peptides naturally generated during Iberian dry-cured ham processing. Food Research International, 78, 71
78. Mora, L., Escudero, E., & Toldra´, F. (2016). Characterisation of the peptide profile of Spanish Teruel, Italian Parma and Belgian dry-cured hams and its potential bioactivity. Food Research International, 89, 638
646. Mora, L., Gallego, M., Aristoy, M. C., Fraser, P. D., & Toldra´, F. (2015). Peptides naturally generated from ubiquitin-60S ribosomal protein as potential biomarkers of dry-cured ham processing time. Food Control, 48, 102
107. Mora, L., Gallego, M., Escudero, E., Reig, M., Aristoy, M.-C., & Toldra´, F. (2015). Small peptides hydrolysis in dry-cured meats. International Journal of Food Microbiology, 212, 9
15. Mora, L., Gallego, M., & Toldra´, F. (2018). ACE-inhibitory peptides naturally generated in meat and meat products and their health relevance. Nutrients, 10(1259), 1
12. Mora, L., Gonza´lez-Rogel, D., Heres, A., & Toldra´, F. (2020). Iberian dry-cured ham as a potential source of α-glucosidase-inhibitory peptides. Journal of Functional Foods, 67, 103840. Mora, L., Sentandreu, M. A., & Toldra´, F. (2011). Intense degradation of myosin light chain isoforms after drycured ham processing. Journal of Agricultural & Food Chemistry, 59, 3884
3892. Mune, M. A. M. (2015). Influence of degree of hydrolysis on the functional properties of cowpea protein hydrolysates. Journal of Food Processing and Preservation, 39, 2386
2392. Neves, A. C., Harnedy, P. A., O’Keeffe, M. B., & FitzGerald, R. J. (2017). Bioactive peptides from Atlantic salmon (Salmo salar) with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory, and antioxidant activities. Food Chemistry, 218, 396
405. Nielsen, P. M., Petersen, D., & Dambmann, C. (2001). Improved method for determining food protein degree of hydrolysis. Journal of Food Science, 66, 642
646. De Oliveira, C. F., Correˆa, A. P., Coletto, D., Daroit, D. J., Cladera-Olivera, F., & Brandelli, A. (2015). Soy protein hydrolysis with microbial protease to improve antioxidant and functional properties. Journal of Food Science & Technology, 52, 2668
2678. Oseguera-Toledo, M. E., Gonza´lez de Mejı´a, E., Reynoso-Camacho, R., Cardador-Martı´nez, A., & AmayaLlano, S. L. (2014). Proteins and bioactive peptides: Mechanisms of action on diabetes management. Nutrafoods, 13, 147
157. Ou, K., Liu, Y., Zhang, L., Yang, X., Huang, Z., Nout, M. J. R., & Liang, J. (2010). Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by Caco-2 cells. Journal of Agricultural and Food Chemistry, 58, 4894
4900. Pedroche, J., Yust, M. M., Lqari, H., Megias, C., Giro´n-Calle, J., Alaiz, M., . . . Milla´n, F. (2007). Obtaining of Brassica carinata protein hydrolysates enriched in bioactive peptides using immobilized digestive proteases. Food Research International, 40, 931
938. Pepe, G., Sommella, E., Ventre, G., Scala, M. C., Adesso, S., Ostacolo, C., . . . Campiglia, P. (2016). Antioxidant peptides released from gastrointestinal digestion of “Stracchino” soft cheese: Characterization, in vitro intestinal protection and bioavailability. Journal of Functional Foods, 26, 494
505. Reyes-Dı´az, A., Gonza´lez-Co´rdova, A. F., Herna´ndez-Mendoza, A., & Vallejo-Co´rdoba, B. (2016). Immunomodulating peptides obtained from milk proteins. Interciencia, 41, 84
91. Ryder, K., Bekhit, A. E. D., McConnell, M., & Carne, A. (2016). Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases. Food Chemistry, 208, 42
50. Sanz, Y., Fadda, S., Vignolo, G., Aristoy, M.-C., Oliver, G., & Toldra´, F. (1999). Hydrolytic action of Lactobacillus casei CRL 705 on pork muscle sarcoplasmic and myofibrillar proteins. Journal of Agricultural and Food Chemistry, 47, 3441
3448. Sanz, Y., Mulholland, F., & Toldra´, F. (1998). Purification and characterization of a tripeptidase from Lactobacillus sake. Journal of Agricultural and Food Chemistry, 46, 349
353. Sanz, Y., & Toldra´, F. (1997). Purification and characterization of an aminopeptidase from Lactobacillus sake. Journal of Agricultural and Food Chemistry, 45, 1552
1558.

46

Chapter 2

Sentandreu, M. A., & Toldra´, F. (2007). Evaluation of ACE inhibitory activity of dipeptides generated by the action of porcine muscle dipeptidyl peptidases. Food Chemistry, 102, 511
515. Slizyte, R., Rommi, K., Mozuraityte, R., Eck, P., Five, K., & Rustad, T. (2016). Bioactivities of fish protein hydrolysates from defatted salmon backbones. Biotechnology Reports, 11, 99
109. Sung, D. E., Lee, J., Han, Y., Shon, D.-W., Ahn, K., Oh, S., et al. (2014). Effects of enzymatic hydrolysis of buckwheat protein on antigenicity and allergenicity. Nutrition Research and Practice, 8, 278
283. Tanzadehpanah, H., Asoodeh, A., & Chamani, J. (2012). An antioxidant peptide derived from Ostrich (Struthio camelus) egg white protein hydrolysates. Food Research International, 49, 105
111. Toldra´, F., Gallego, M., Reig, M., Aristoy, M.-C., & Mora, L. (2020a). Recent progress in enzymatic release of food-derived peptides and assessment of bioactivity. Journal of Agricultural & Food Chemistry, 68, 12842
12855. Available from https://doi.org/10.1021/acs.jafc.9b08297. Toldra´, F., Gallego, M., Reig, M., Aristoy, M.-C., & Mora, L. (2020b). Generated bioactive peptides in the processing of dry-cured ham. Food chemistry, 321, 126689. Toldra´, F., Mora, L., & Reig, M. (2016). New insights into meat by-products utilization. Meat Science, 120, 54
59. Toldra´, F., Reig, M., Aristoy, M. C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404. Twining, S. S. (1984). Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Analytical Biochemistry, 143, 30
34. Wang, R., Zhao, H., Pan, X., Orfila, C., Lu, W., & Ma, Y. (2019). Preparation of bioactive peptides with antidiabetic, antihypertensive, and antioxidant activities and identification of α-glucosidase inhibitory peptides from soy protein. Food Science & Nutrition, 7, 1848
1856. Xiao, C., Zhao, M., Zhou, F., Gallego, M., Gao, J., Toldra´, F., & Mora, L. (2020). Isolation and identification of alcohol dehydrogenase stabilizing peptides from Alcalase digested chicken breast hydrolysates. Journal of Functional Foods, 64, 103617. Xu, W., Kong, B. H., & Zhao, X. H. (2014). Optimization of some conditions of Neutrase-catalyzed plastein reaction to mediate ACE-inhibitory activity in vitro of casein hydrolysate prepared by Neutrase. Journal of Food Science and Technology Mysore, 51, 276
284. Yoshida, N., Tsuruyama, S., Nagata, K., Hirayama, K., Noda, K., & Makisumi, S. (1988). Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase). Journal of Biochemistry, 104, 451
456. Zhang, Y., Chen, R., Ma, H., & Chen, S. (2015). Isolation and identification of dipeptidyl peptidase IVinhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates by 2D-TLC and LC
MS/MS. Journal of Agriculture & Food Chemistry, 63, 8819
8828. Zheng, Y., Li, Y., Zhang, Y., Ruan, X., & Zhang, R. (2017). Purification, characterization, synthesis, in vitro ACE inhibition and in vivo antihypertensive activity of bioactive peptides derived from oil palm kernel glutelin-2 hydrolysates. Journal of Functional Foods, 28, 48
58.

CHAPTER 3

Novel technologies in bioactive peptides production and stability ´zquez-Rodrı´guez2,3, Aı´da Jimena Velarde-Salcedo1, Gabriela Va 2 ´n-Rodrı´guez and Ana Paulina Barba de la Rosa2 Antonio De Leo 1

Faculty of Chemical Sciences, Autonomous University of San Luis Potosı´, Mexico, 2IPICYT, Potosino Institute of Scientific and Technological Research A.C., San Luis Potosi, Mexico, 3Department of Biomedical and Clinical Sciences, Linko¨ping University, Sweden

3.1 Introduction Bioactive peptides (BAPs) are small amino acid sequences, usually among 2
20 residues, that are natively encrypted in proteins and when they are released (by a physical treatment, enzymatic hydrolysis, or gastrointestinal digestion) exert a biological effect in the body (Bhandari et al., 2019). BAPs can be obtained from several sources such as endogenous peptides, animal and vegetable-derived foods, and fermented products; they participate in different homeostatic mechanisms resulting in a positive health effect making them useful for the prevention or treatment of diseases. Discovery and applications of BAPs have been widely studied in the last 20 years, which include peptides with immunomodulatory, antioxidant, antimicrobial, antithrombotic, antihypertensive, hypoglycemic, antiproliferative activity, amongst others. Furthermore, BAPs have several advantages in medicine against their pharmaceutical counterpart (chemical-based drugs); they are diverse in number, activity, specificity with low toxicity and immunogenicity. Even synthetic peptides allow de novo approaches to explore new therapeutic applications (Kang et al., 2019). At least four different methodologies for the generation of BAPs have been described: (1) chemical synthesis, (2) fermentation by microorganisms, (3) enzymatic hydrolysis, and (4) design and biosynthesis through recombinant DNA technology (Agyei, Ahmed, Akram, Iqbal, & Danquah, 2017; Kang et al., 2019). Chemical synthesis is usually carried out by polymerization methods coupled to a liquid or solid phase, which allows a simpler purification, bigger selectivity by generating one peptide at a time, and the implementation of automated systems; however, this is achieved with low yields and at high production costs (Fields, 2002; Kang et al., 2019). Microbial fermentation has been used throughout human history for generation of foods; it was observed that microorganisms such as lactic Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00021-2 © 2021 Elsevier Inc. All rights reserved.

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48

Chapter 3

acid bacteria in fermentation of dairy foods (cheese and yogurt) and soy-derived products were also able to release BAPs (Kang et al., 2019). Enzymatic digestion is the most common method for BAP release, it uses one or different combinations of enzymes like alcalase, pepsin, trypsin, and papain on specific proteins from a food source. This approach offers an important advantage: from one protein, several sequences can be released and each sequence has a particular biological activity. However, some enzymes are expensive and their yields are sometimes difficult for their profitability in the commercial level. These hydrolyzates can be selectively purified (or not) and be used for the preparation of new fortified foods, for the elaboration of nutraceutics or food supplements. Moreover, if BAPs come from a food source, this is usually prepared through a cooking process like boiling, roasting, popping, and baking. These physical treatments change the release pattern of BAPs, due to protein denaturalization, facilitating their enzymatic digestion and changing their physicochemical properties by interacting with the food matrix and increasing their solubility. Hence, it is important to consider that the methods to obtain BAPs may have a significant impact on their biological activity and if the intention is to commercialize and handle them on a large scale, then it is necessary to implement affordable and consistent methods that guarantee the efficacy of these novel products. In this sense, recombinant DNA technology can be a viable tool; this technology has been widely used for the production of biopharmaceuticals, particularly bacterial culture systems, and can be applied for the production of BAPs to an industrial scale (Kamerzell, Esfandiary, Joshi, Middaugh, & Volkin, 2011; McClements, 2018). Furthermore, different administration routes besides oral can be tested, excipients that guarantee the peptides stability can be used as well as several encapsulation strategies can be applied for the peptides delivery in particular tissues or cell types.

3.2 Expression of recombinant peptides Recombinant proteins, from viral to human sources, have been produced for more than 30 years (Palomares, Kuri-Bren˜a, & Ramı´rez, 2009). Proteins that are scarce and difficult to obtain can now be easily produced as recombinant proteins, increasing their uses and applications in pharmaceutical, agriculture, and food industries (Terpe, 2006). With the increase in chronic diseases worldwide, the interest in BAPs has been renewed, which have shown to be important actors in the cure and control of those illnesses (Korhonen, 2009; Udenigwe & Aluko, 2012). Hence, recombinant BAPs production has opened a new era in opportunities for innovation to produce a wide range of novel formulations for therapeutic uses. Currently, there are a wide variety of available expression systems for large-scale production of recombinant proteins. Within the prokaryotic systems the Gram-negative bacterium Escherichia coli is currently the most used organism. E. coli is a widely studied

Novel technologies in bioactive peptides production and stability 49 and established microorganism, its biology is well understood, and there are several tools for its genetic manipulation (Schmidt, 2004; Terpe, 2006). For this reason, it is one of the most important organisms used on an industrial scale (Gonza´lez & Fillat, 2018). A disadvantage for therapeutic use of produced recombinant proteins in typical E. coli strains is the presence of lipopolysaccharides, generally referred as endotoxins, which are pyrogenic in humans and other animals; therefore recombinant proteins produced in this system should be purified (Terpe, 2006). Nevertheless, there are detoxifying E. coli strains for endotoxin-free recombinant proteins (Mamat et al., 2015). Although E. coli is very easy to handle at relatively low costs, sometimes it presents serious limitations in the production of eukaryotic proteins, which sometimes requires posttranslational modifications such as the formation of disulfide bridges, glycosylation, and phosphorylation that are necessary for their correct folding, stability, and biological activity. Furthermore, in bacteria, it is difficult to extract large amounts of the recombinant protein into the extracellular medium, and when high levels of expression are achieved, aggregates of recombinant proteins known as inclusion bodies are frequently formed. For inclusion bodies purification, it is necessary to use chaotropic agents that denature the protein with a subsequent renaturation step, which often results in a considerable loss of yields and increased production costs (Gonza´lez & Fillat, 2018). Gram-positive bacteria usually possess only a single membrane (cytoplasmic membrane) and export of a target protein across this major permeability barrier can directly result in its release into the culture supernatant. Due to this fact, members of this class of microorganisms are considered especially useful as potentially host organism for the industrial recombinant proteins. In fact, since many years, various Gram-positive bacteria (Bacillus species) are extensively used in the industry for the secretory production of a variety of enzymes such as lipases, amylases, and proteases (Mamat et al., 2015). Many pharmaceutically relevant proteins have been successfully expressed in different strains, such as Bacillus megaterium (Biedendieck, 2016) and Bacillus subtilis (Rojas Contreras et al., 2010). Losurdo et al. (2013) reported the production of recombinant antihypertensive peptides using a novel Bifidobacterium pseudocatenulatum system. In terms of downstream processing and efficiency, yeasts, like Saccharomyces cerevisiae, can be grown cheaply and rapidly and are amenable to high-cell-density fermentations. Yeast systems offer efficient secretory expression systems to the culture medium with advantages for the production of recombinant proteins, compared with inclusion body forming cytosolic systems (Schmidt, 2004). Besides, yeast possesses complex posttranslational modification pathways, offering the advantage of being neither pyrogenic nor pathogenic to humans. Species established in industrial production are S. cerevisiae, Kluyveromyces lactis, Pichia pastoris, and Hansenula polymorpha (Ogataea polymorpha). P. pastoris is considered to be superior to any other known yeast species with respect to its

50

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secretion efficiency, allowing recombinant protein production without excessive recovery and purification processes (Cregg et al., 2009; Damasceno et al., 2012; Macauley-Patrick, Fazenda, McNeil, & Harvey, 2005). In addition, there are several commercial plasmids for P. pastoris recombinant protein production as well as different yeast strains (Baghban et al., 2019).

3.2.1 Escherichia coli expression vectors and strains for recombinant protein production There are many E. coli commercial and noncommercial expression vectors (plasmids) available; however, several points need to be considered before choosing a vector, such as (1) the type of bacterial strain that will be used, (2) if there will be added a tag or not, (3) the desired level of protein accumulation, (4) the toxicity of the protein/peptide for E. coli, and (5) the use of rare codons if working with eukaryotic protein/peptides (Langlais & Korn, 2006). Some of these vectors are listed in Table 3.1. All expression vectors contain a selectable antibiotic-resistance gene, which facilitates the selection of positive transformant colonies. The levels of expression of mRNA are mainly determined by the promoter properties, which in general are strong promoters to achieve 10%
30% of recombinant protein in the total protein content. The origin (ori) of replication determines the approximate number of copies of the plasmid in a single bacterial cell. High copy number plasmids will yield more DNA and therefore more recombinant protein. However, if the desired protein is harmful to the bacterial metabolism, then a low Table 3.1: Some commercial vectors used for the recombinant protein expression in Escherichia coli. Vector

Promoter/ repressor Resistance

Fusion

Host strain

pET-28a (1)

T7/LacI

Kan

N-His C-His

BL21 (DE3)

pET-20b (1)

T7

Amp

BL21 (DE3)

pET-32a (1)

T7/LacI

Amp

C-His Signal peptide N-Trx N-His C-His

pET-12a-c pGEX-4T-3 pETDuet-1

T7 tac 2x (T7/ LacI)

Amp Amp Amp

N-GST N-His C-S

Origami (DE3)

Application Cytoplasm expression IPTG-inducible IMAC purification Periplasm expression IPTG-inducible IMAC purification Expression of proteins with disulfide bonds IMAC purification

BL21-SI Salt induction BL21 Purification through glutathione affinity BL21 Coexpression of two proteins, with (DE3) independent purification (IMAC and protein-S)

Amp, Ampicillin; C-His, tag of histidine at C-terminal; IMAC, immobilized metal affinity chromatography; IPTG, isopropylβ-D-thiogalactoside; Kan, kanamycin; N-His, tag of histidine at N-terminal; N-Trx, thioredoxin cleavage site at N-terminal

Novel technologies in bioactive peptides production and stability 51

Figure 3.1 Diagram of a simple expression vector (plasmid) in bacteria. The vector contains an origin of replication (ORI), and an antibiotic-resistance gene (here ampr is been shown, which encodes β-lactamase, an enzyme that inactivates ampicillin), a promoter, RBS, MCS containing specific restriction sites, optional tags that could be located at N- or C-terminal of both, and the stop codon. MCS, Multiple cloning site; RBS, ribosome binding site.

copy vector is more appropriate. The region where fragment DNA can be inserted, known as the multiple cloning site, is also present as well as different tags (His-tag/6xHis, GST, amongst others) that could be in fusion to the N- or C-terminal of the recombinant protein. Tags considerably facilitate the identification and purification of recombinant proteins. A simple schematic of the main components in an expression plasmid is shown in Fig. 3.1. The most common promoter is the lac promoter, which initiates transcription only when lactose, or the nonhydrolysable lactose analog isopropyl-β-D-thiogalactoside (IPTG) is added to the culture (Fig. 3.2). However, the use of IPTG for therapeutic protein production is inadequate due to its toxicity for humans; in this sense, new promoters induced by salt are frequently used. Following induction with IPTG, the gene under control of the lac promoter is transcribed into mRNA, which is then translated into a protein (Fig. 3.2). Most E. coli strains used for protein expression are depleted of proteases that could degrade the recombinant protein (Novagen Inc.). In E. coli systems, most of recombinant proteins are accumulated in the cytoplasm, but translocation of these proteins into the periplasm provides a favorable environment for oxidative folding via disulfide bond (Balderas Herna´ndez et al., 2008; Baumgarten, Ytterberg, Zubarev, & de Gier, 2018; Malik, 2016). For routine protein expression, E. coli BL21 and their derivatives are most frequently used, and in general, overexpressed recombinant proteins accumulate either in the cytoplasm or in the periplasmic space (Terpe, 2006). Other bacterial hosts become more and more attractive for heterologous protein production. One reason is that the increasing genomics knowledge

52

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Figure 3.2 Schematic representation of recombinant protein expression in bacteria. The two-step mechanism of protein expression with the T7 Lac promoter. A section of Escherichia coli genome shows the gene for T7 RNA polymerase under control of the lac promoter. The expression vector (pET) is also shown which contains an ORF cloned into the vector under control of the T7 late promoter. When IPTG is added to the culture medium, the lac promoter initiates transcription of the T7 RNA polymerase gene, and the mRNA is translated into protein. T7 RNA polymerase then initiates transcription at the T7 late promoter on the expression vector and the ORF is translated into the desired protein. Protease genes (lon, Ompt) are depleted in the E. coli strain. IPTG, Isopropyl-β-Dthiogalactoside. Source: Modified from pET Manual System.

enables us to compare the codon usage of host and original organisms. Codons AGG, AGA, CUA, AUA, CCC, CGA, CGG, and UCG are very rarely used in E. coli, their presence causes a decrease in expression, and some specialized protein expression hosts have been designed to provide enhanced protein yield (BL32 Star), or expression hosts containing rare tRNA genes (Rosetta strain). For toxic proteins, BL32-A1 vectors are used to tight regulation and strong expression or for reduction of the basal expression of target genes BL32(DE3)pLysE or pLysS are used. Thus the decision of which particular plasmid and host bacteria to be used depends on the individual application and the characteristics of the protein of interest (Table 3.2).

3.3 Stability of proteins and peptides Peptides as proteins are polymeric amino acid chains joined by peptide bonds, and they are subject to physical (mechanic stress, pH changes) and chemical (digestive enzymes) factors

Novel technologies in bioactive peptides production and stability 53 Table 3.2: Some Escherichia coli strains used for recombinant protein expression. Strain

Key features

Recommended uses

BL21

Deficient in lon and ompT proteases. Lack of T7 RNA polymerase.

Gene expression under the control of promoters recognized by Escherichia coli polymerase: lac, tac, trc, T5. Expression of recombinant genes under control of promoters T7, T7-lac, and promoters recognized by E. coli polymerase. Expression where is required a control of basal expression.

BL21(DE3)

BL21(DE3)pLysS

BL21(DE3)pLysE

BL21 trxB BL21-SI and BL21-AI

Derived from BL21. Contains ˆI»DE3 which transport T7RNA polymerase gene under the control of promoter lacUV5, inducible by IPTG. Derived from BL21(DE3) contains the plasmid pLysS which transport the T7 lysozyme (LysS) gene, inhibitor of T7RNA polymerase that prevents basal expression in cultures no induced with IPTG. Derived from BL21(DE3). Contains the plasmid pLysE that express higher levels of T7 lysozyme. Derived from BL21, trxB mutant; facilitates disulfide bond formation. Contains the T7 RNA polymerase under the control of promoter proU, inducible by salt concentration in the culture media.

BL21star (DE3) BL21-CodonPlusRP Origami (DE3)

Rosetta-gammi

Lemo21 (DE3)

Where higher control of basal expression. Especially for toxic proteins. Where proteins contains an internal disulfide bond. For toxic proteins.

General expression, not recommend for toxic proteins. General protein expression.

Enhances expression of eukaryotes containing codons rarely used in E. coli: AGG, AGA, CCC. trxb/gor (tioredoxin reductase/glutathione Expression of recombinant proteins that reductase) mutant, which facilitates require the formation of disulfide bond to bisulfide bond format in cytoplasm. reach its native structure. Derived from BL21. Contains the plasmid Enhances expression of eukaryotic proteins that contain codons rarely used in E. coli. pRARE or pRARE2, that transport gene for tRNA for AGG, AGA, AUA, CUA, CCC, GGA (pRARE) and AGA, AGG, AUA, CUA, GGA, CCC, CGG (PRARE2). Expression of toxic, insoluble, or Deficient in lon and ompT proteases. membrane proteins.

IPTG, Isopropyl-β-D-thiogalactoside.

that could result in a complete breakdown into their basic units (amino acids) and loss of their activity. All food proteins are subjected to some processing and heat is commonly applied to kill microorganisms, improve organoleptic properties, and increase food shelf life. Heat may have a positive or negative effect on proteins; the positive effects are the denaturation of the polypeptide chain increasing its digestibility and peptides release. Negative effects are oxidation and hydrolysis reactions which could cause some modifications of reactive groups that could result in nonpredicted complex formation

54

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between amino, carboxyl, or sulfhydryl groups of BAPs and other food components, so it is necessary to avoid aggressive treatments that could alter BAPs quality and therefore their efficacy (Dullius et al., 2020; Sun, Acquah, Aluko, & Udenigwe, 2020). For example, heating can cause Maillard (glycation) reactions, which create a crosslinking between the carbohydrates and proteins present in food systems. Maillard reactions can have favorable effects on foods in terms of handling, flavor, and digestibility by altering their ´ lvarez, & Mullen, 2020; Bhandari et al., 2019). physicochemical properties (Anzani, A Proteins glycation can be accomplished in an unspecific but controlled way to generate more soluble and easier to absorb peptides (by addition of hydrophilic groups). Protein glycation can be done previous the generation of BAPs, after adding them into a food; this is an important point to consider because it has been reported that a previous heating or cooking process may change the functional behavior of BAPs (Korczek, Tkaczewska, Duda, & Migdał, 2020; Velarde-Salcedo et al., 2013). On the other hand, when heating (and possible glycation of peptides) is carried out after BAPs releasing, their functional properties are not significantly altered (Ontiveros et al., 2020). Furthermore, it is even interesting that within the same food product, the properties of a particular group of BAPs can be changed while others do not. Korczek et al. (2020) observed that different thermic treatments like boiling, roasting, and sterilization decrease the antioxidant capacity of hydrolyzates obtained from mackerel, whereas the angiotensin-converting enzyme (ACE) inhibitory activity was merely affected. Evidence may suggest that the temperature and time of exposure are important to determine the stability of BAPs during food processing. Therefore oral administration of therapeutic BAPs is not a viable route for delivery (bioavailability less than 1%) and the elaboration of parenteral formulations is important to assure that the majority of BAPs reaches their site of action (Brandelli, 2012; Tiwari, Gajbhiye, Sharma, & Jain, 2010). Although biopharmaceutics and BAPs have differences in terms of production methods, cellular targets, and mechanisms of action, they share some similarities. A brief comparison between biopharmaceutics and BAPs is shown in Table 3.3. Despite the reasons mentioned earlier, oral route is the least invasive and easiest administration route for a patient (because it does not require assistance of a healthcare professional) and still is the delivery route of choice for the administration of pharmaceutical and nutraceutical formulations. Studies in cell lines have suggested that the intestinal absorption of BAPs (up to 26 amino acids) could be achieved through several mechanisms: passive diffusion, PepT-mediated active transport, tight junction-mediated para-cellular transport, and vesicle-mediated transcytosis (Grootaert et al., 2017; Sun et al., 2020). Moreover, during the production, purification, and handling of proteins and BAPs, these substances are prone to alterations that can compromise their biological activity, which include oxidation, deamidation, hydrolysis, denaturation, aggregation, isomerization, amongst others (Bjeloˇsevi´c, Zvonar Pobirk, Planinˇsek, & Ahlin Grabnar, 2020; Geraldes

Novel technologies in bioactive peptides production and stability 55 Table 3.3: Characteristics of classic biopharmaceutics and bioactive peptides. Attribute Structural basis Size (length)

Biopharmaceuticsa

Amino acids ˆh”2000 amino acids (antibodies 30 aaa and coagulation factors) PTM Frequent and commonly related to biological activity, stability, and time of action Order of structure Up to tertiary or quaternary structure Administration route Parenteral (i.v., s.c., i.m., etc.) Half life time Minutes to weeks (antibodies) Distribution after Bloodstream, lymphatic vessels, administration coupled to transport proteins (serum albumin) Metabolism/ Hydrolysis by intra or extracellular elimination proteases mechanism Synthesis Recombinant DNA technology, hybridomas Purificationb Therapeutic classification Therapeutic target

Industrial production Immunogenicity

Bioactive peptides Amino acids 2
26 amino acids, although there can be longer Not usually described

Usually primary and secondary structure Oral administration is suggested Hours Bloodstream

Hydrolysis by intra or extracellular proteases Enzymatic digestion, microbial fermentation, chemical synthesis, recombinant DNA technology SEC, IEX, RP-HPLC

Precipitation, diafiltration, SEC, IEX, AC, RP-HPLC, etc. Antihypertensive, osteoprotectants, Hormones, antibodies, cytokines, antioxidants, immunomodulators, coagulation factors, thrombolytic antidislypidemic, anticancer, etc. agents, growth factors, etc. The same peptide can have different Usually specific for each targets and different peptides can have the biopharmaceutic, cellular, and same target noncellular targets Well established, automatized with Early stages and nonprofitable standardized and optimized procedures Relevant for large and complex proteins Not described to date

a

Biopharmaceutics are defined as a therapeutic protein obtained by biotechnological methods. AC, Affinity chromatography; IEX, ionic-exchange chromatography; RP-HPLC, reverse phase HPLC; SEC, size-exclusion chromatography.

b

et al., 2020). Thus BAPs stability is considered to be low and measures must be taken to increase their stability and absorption. For these purposes, the following approaches have been suggested: (1) chemical modification of peptides to increase their stability; (2) use of protease inhibitors to protect BAPs enzymatic degradation; (3) increase the permeation of BAPs through the enterocyte, circulation, and target cells using biliary salts, surfactants, or chelating agents; and (4) design delivery systems that allow to direct BAPs to a specific cell or tissue like nanoencapsulation (Gleeson, Ryan, & Brayden, 2016; Sharma, Agrawal, & Vyas, 2014; Sun et al., 2020). To accomplish some of these aims, excipients can be added (Table 3.4). However, it is important to consider if excipients used in the pharmaceutical industry could be also used in

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Table 3.4: Some excipients used for drugs delivery that could be used for bioactive formulation. Category Buffering agents Surfactants

Inorganic salts Antioxidants Bulking agents and lyoprotectants

Osmolytes

Function

Examples

Maintain solution pH in the appropriate range of activity/stability of the bioactive principle Reduces superficial tension and adsorption of hydrophobic interphase regions between drug-drug and drug-recipient

Histidine, phosphate salts, citrate salts, glycine, tris, succinate Polysorbate 80, polysorbate 20, poloxamer 188, SDS, Carbopol, Tween, clyclodextrins, serum albumin NaCl, CaCl2, ZnCO3, Na2SO4, Mg (OH)2, MgCO3 Glutathione, methionine

Maintain isotonicity of proteins, and therefore their stability and function Prevent reactive oxygen species formation in aqueous solutions Stabilize proteins during the drying process in freeze-dried formulations

Replace water during the drying process in freezedried formulations, maintain protein conformation and facilitate reconstitution

Mannitol, glycine, leucine, methionine, arginine, histidine, sucrose, glucose, polyethylene glycol, dextrans, Ficoll, modified starch Sucrose, trehalose, clyclodextrins, mannitol

food products. Therefore studies should be designed to determine if there are changes in food stability, shelf life, taste, and other desirable properties for consumers, besides the BAPs
excipient
food matrix interaction mandatory studies to establish the efficacy of the product. It is worth to mention that some peptides present dual activity when used in pharmaceutical preparations; for example, it has been observed that branched chain amino acid
derived peptides (valine, leucine, isoleucine) used as antimicrobial excipients also present anticancer, antihypertensive, and antioxidant activity, which could allow them to be used as excipients in other BAP formulations or in the generation of new foods with additional value (Dullius et al., 2020). The fact that BAPs have been obtained most of the time as an heterogeneous mixture, where each peptide has its own physicochemical characteristics and where all peptides can interact with each other, is a challenge to establish which would be the ideal method to develop a stable formulation of BAPs (Dullius et al., 2020). Solid formulations are usually associated with prolonged shelf life. Lyophilization, milling, grinding, spray-drying, spray-freezing, extraction, and drying by supercritical fluids, among others, are the most common methods used for the generation of solid formulations (Molavi, Barzegar-Jalali, & Hamishehkar, 2020; Yu, Rogers, Hu, Johnston, & Williams, 2002). However, some of these methods use abrupt temperature changes that can alter the integrity and thus the stability of bioactive principles, especially BAPs. Moreover, because of the properties of most hydrolyzates and BAPs, such as high hydrophilicity, hygroscopicity, or bitter taste, additional studies should be carried out when these formulations are pretended to be added in food products, so it is also relevant to

Novel technologies in bioactive peptides production and stability 57 test encapsulation of BAPs, in addition to improve their stability (Sarabandi, Gharehbeglou, & Jafari, 2020). These issues will be addressed further in this book.

3.4 Definition: production of recombinant bioactive peptides in Escherichia coli BAPs are short amino acid sequences that are released during the digestion of food proteins proving health benefits. Yields of peptides release and their variability in composition are usually the limiting factor in exploiting their full biological potential; hence, the use of molecular biology tools to guarantee a more homogeneous peptides production is a viable alternative that is also easy to scale to an industrial level. In this chapter a methodology for the production of recombinant peptides using the E. coli bacterial system is presented. The basic steps to obtain the recombinant peptides with antihypertensive activity as well as the vasoinhibin, a peptide with antiangiogenic activity, are described. Because antihypertensive peptides are very short sequences (3
4 amino acids), the strategy is to link several of them joined by a protease site to obtain a larger peptide sequence (expression cassette), individual peptides will be released after hydrolysis giving higher yields and peptide homogeneity. After cassette design and gene amplification the processes of cell transformation, recombinant protein expression, and protein purification are described. Furthermore, the nanoencapsulation of the peptides obtained using a chitosan
alginate matrix by emulsification processes is also described. The production of therapeutic recombinant proteins/peptides can be broadly divided into six general steps (Fig. 3.3). 1. Design of the synthetic peptide or cloning from a natural source. 2. Cloning the DNA of interest in a suitable vector under an adequate promoter. 3. Transformation of the host cells: cells are cultured in presence of a transformer marker (antibiotic). 4. Induction of the expression of the desired protein under controlled conditions. 5. Recovery and purification of the recombinant product: after induction, cells are harvested by centrifugation and disrupted. Soluble and insoluble fractions are analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) or by immunoblotting. Also, the identity of enzymatic proteins can be measured by their specific activity. 6. Preparation for delivery. Preformulation studies must be performed to determine the appropriate combination of active ingredient (BAP), excipients, and final formulation.

3.4.1 Antihypertensive peptides Hypertension is a major public health problem worldwide; hence, the discovery and design of antihypertensive substances (such as peptides) are of great interest. To date, most antihypertensive peptides function as inhibitors of ACE, which is a dipeptidyl peptidase that plays an important physiological role in both regulation of blood pressure and

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Figure 3.3 Schematic representation of the main steps for recombinant peptide expression. The synthetic peptide is amplified by PCR and ligated in a transient vector. Plasmid is recovered and construction is restricted with specific enzymes as well as the expression vector. Escherichia coli is transformed, cultured in selection media (antibiotic), positive clones are selected for plasmid recuperation and induction of protein expression. PCR, Polymerase chain reaction.

cardiovascular function (Fleming, 2006). ACE inhibitory peptides have been reported from different sources such as milk and dairy products (Herna´ndez-Ledesma, del Mar Contreras, & Recio, 2011; Herna´ndez-Ledesma, Recio, Ramos, & Amigo, 2002; Pihlanto-Leppa¨la¨, Koskinen, Phlola, Tupasela, & Korhonen, 2000; Saito, Nakamura, Kitazawa, Kawai, Itoh, 2000), amaranth (Barba de la Rosa et al., 2010), wheat (Motoi and Kodama, 2003), rice (Li, Qu, Wan, & You, 2007), and fish muscle proteins (Yathisha, Bhat, Karunasagar, & Mamatha, 2019; Yi, Lv, Zhang, Yang, & Shi, 2018). Some of the characterized peptides are di, tri, and tetrapeptides, which are too small to be produced by recombinant technology, then, a cassette of repetitive sequences joined by protease cleave sites should be constructed; in this way, antihypertensive peptides have been produced successfully in E. coli recombinant systems (Huang, Ma, Li, & Li, 2012; Lv, Huo, & Fu, 2003).

3.4.2 Antiangiogenic peptides Cancer is the second cause of death worldwide, where a third of the cases are a consequence of behavioral and dietary risks, such as high body mass index, low fruit and

Novel technologies in bioactive peptides production and stability 59 vegetable intake, lack of physical activity, and tobacco and alcohol abuse (World Health Organization, 2018). The most studied cancer-preventive peptide derived from cereals is the well-known lunasin. This peptide has been expressed in E. coli (Liu and Pan, 2010) as well as in P. pastoris systems (Zhu, Nadia, Yao, Shi, & Ren, 2018). Angiogenesis is the physiological process, which involves the formation of new capillaries from preexisting vasculature; it is considered a key process for local invasion and the development of primary and metastatic tumors (Clapp, Martial, Guzman, Rentier-Delure, & Weiner, 1993; World Health Organization, 2018). Efforts have been concentrated in discovering pro- and antiangiogenic molecules to treat cancer and other diseases (Carmeliet and Jain, 2000). Some groups of antiangiogenic molecules are vasoinhibins, which belong to a family of antiangiogenic peptides that are encrypted in the N-terminal of prolactin (PRL). Cleavage of PRL by cathepsin-D or by matrix metalloproteases generates Nterminal fragments that act on endothelial cells to suppress vasodilation and angiogenesis and promote vascular regression (Clapp et al., 2006). Vasoinbins have also been produced in both E. coli (Vazquez Rodriguez, Gonzalez, & de Leon Rodriguez, 2013) and P. pastoris systems (Calderon-Salais, Velazquez-Bernardino, Balderas-Herna´ndez, Barba de la Rosa, & de Leo´n-Rodrı´guez, 2018).

3.5 Protocol 3.5.1 Antihypertensive cassette design The decision to choose which particular antihypertensive peptide is going to be produced should be based on reports about its activity in vivo and in vitro, its efficient absorption through the intestine in an active form without degradation, and the ease to be released from the antihypertensive cassette with gastrointestinal proteases. Once the peptides are selected, they will be arranged in tandem linked by amino acid sequence of specific proteases, where the most common one is trypsin that cleaves at the peptide bond between the carboxyl group or arginine (R) or lysine (K). The cassette design solves the technical difficulty of unstable expression of small molecule peptides in bacteria, overcoming the shortcomings of low yield and reducing the purification cost. The expressed multimer peptides linked with gastrointestinal proteases as the final product could be used as the final product (Rao, Su, Li, Xu, & Yang, 2009). Here, we have selected the peptides LKPNM, LKP, IPP, IKP, ACEIP, KVLPVP, IYPR because of their low IC50 (Barba de la Rosa et al., 2010; Huang et al., 2012; Liang, Zhang, & Lin, 2014; Rao et al., 2009). As shown in Fig. 3.4A, the designed peptide sequence was reverse transcribed using one of the several free platforms located at Bioinofrmatics Resource Portal (expasy.org) or at

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Figure 3.4 (A) Antihypertensive multimer fusion peptide. The antihypertensive peptide sequence is shown in bold capital letters below the nucleotide sequence. Trypsin digestion is carried out at carboxyl-end of each lysine (K) or arginine (R). Nucleotide sequences for BamHI and XhoI restriction enzymes at 50 - and 30 -end, respectively, are shown in red lower case letters. (B) Oligonucleotide design for the antihypertensive peptide cassette amplification by PCR. Sequences for BamHI and XhoI restriction enzymes are shown in lowercase red letters. The initiation and termination codons ATG and TTA, respectively, iare shown in bold capital letters. PCR, Polymease chain reaction.

http://www.bioinformatics.org/sms2/rev_trans.html) site. Resulting nucleotide sequence is shown at the top of the amino acid sequence. Nucleotide sequences for BamHI and XhoI restriction enzymes (lowercase letters) were added at each end, as well as the first methionine codon (ATG) and the last stop codon or terminator (TAA). The gene sequence is then synthesized; several companies offer these services and delivery is linked into a transient plasmid or as free peptide. A pair of oligonucleotides should be also synthesized for amplification of synthetic gene. The forward primer is designed in the sense of 50 to 30 of nucleotide sequence; the reverse primer is designed in the complementary sequence and in 50 to 30 direction (Fig. 3.4B). For primers design, computer programs such as Primer3 (http://frodo.wi.mit.edu/primer3) could be used.

3.5.2 Amplification of the encrypted vasoinhibin peptide The prolactin (PRL) gene with number of access in GenBank M29386.1 was retrieved from databases at https://www.ncbi.nlm.nih.gov/nucleotide/ (Fig. 3.5A). Vasoinhibin region was selected and design of specific oligonucleotides for PCR amplification was carried out. At the N-terminal and C-terminal the sequences GGATCC and GTCGACG for BamHI and Sal1 cleavage, respectively, were added (Fig. 3.5B). Amplification was carried out with high fidelity Taq Pfu (Promega) that generates blunt ends (Table 3.5).

Novel technologies in bioactive peptides production and stability 61

Figure 3.5 (A) Human prolactin sequence retrieved from GenBank (M29386.1). The sequence that codifies for a vasoinhibin is shown in red flanked by arrows. Arrows indicate the direction of forward and reverse oligonucleotides.(B) Specific oligonucleotides designed for vasoinhibin peptide amplification. The restriction sequences for BamH1 and Sal1 at 50 and 30 end, respectively, are shown in red lowercase letters. ATG for the initial methionine and TTA for the stop codon are shown in capital bold letters. Table 3.5: Comparison of polymerase chain reaction product properties for thermostable DNA polymerases. Taq DNA polymerase Tfl, Tth Vent (Tli), Deep Vent Pfu Pfx Phusion Accuzyme

Resulting end 0

3 -A-tailed Blunt Blunt Blunt Blunt Blunt

Brand Promega Promega Promega Invitrogen Thermo Bioline

3.5.3 DNA cloning into a suitable vector 3.5.3.1 Fragment amplification by PCR and purification of PCR product For gene amplification, it is necessary to work with a high-fidelity DNA polymerase, there are polymerases that could generate 30 -A-tailed ends, which added several residues of adenine (A) or polymerases that generates blunt ends (Table 3.5).

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A standard PCR consists of four steps: 1. initial denaturation: 95 C for 30 seconds; 2. 30 cycles: denaturation: 95 C for 15
30 seconds, annealing: 45 C
68 C for 15
60 seconds, is based on the Tm of the primer pair, and extension: 72 C for 1 minute/kb; 3. extension: 72 C for 5
10 minutes; and 4. hold at 4 C
10 C. For 50 μL reaction, the amount of template is recommended from 1 ng to 1 μg if genomic DNA is used or 1 pg to 1 ng if plasmid is used. The final concentration of primers in a reaction may be 0.005
1 μM, typically 0.1
0.5 μM. Mg21 concentration of 1.5
2.0 mM is optimal for most PCR products generated with Taq DNA polymerase. Amplification of some difficult targets, like GC-rich sequences, may be improved with additives such as DMSO or formamide. The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Taq polymerase concentration is used at 25 units/mL (1.25 units/50 μL reaction). However, optimal Taq concentration should be reviewed in the manufacturer’s protocol. All reactants are assembled on ice and quickly transferred to a thermocycler preheated to the denaturation temperature (95 C) as shown in Table 3.6. Mix the reaction gently; collect all liquid to the bottom of the tube by a quick spin if necessary. Once the PCR cycle is finished, fragments can be observed in 0.8%
1.0% agarose gel, depending on the fragment size, also a nucleotide pair base ladder is used to corroborate the fragment size. The loading buffer is added to the tube, mixed and sample is loaded onto gel. Efficient separation is usually achieved at 70 V. Gel can be stained with nontoxic dye such as SYBR green (Invitrogen), Gel Green (Biotium), amongst other commercial dyes. Once the fragment is observed, it is excised from the gel and purified using commercial kits (QIAquick PCR purification, Quiagen) or home-made filters. Table 3.6: Setup reaction mixture for DNA amplification. Component 10 3 standard Taq buffer 10 mM dNTPs 10 μM Forward primer 10 μM Reverse primer Template DNA Taq DNA Polymerase Nuclease-free water

25 μL reaction

Final concentration

2.5 0.5 0.5 0.5 Variable 0.125 To 25

13 200 μM 0.2 μM (0.05
1 μM) 0.2 μM (0.05
1 μM) Less 1000 ng 1.25 units

Novel technologies in bioactive peptides production and stability 63 3.5.3.2 Ligation of amplified fragments by PCR into transient vectors Amplified fragments by PCR are ligated into a transient plasmid; the most common vectors are pCR-TOPO cloning systems, ClonJET (Thermo), and pGEM (Promega), which can accept blunt or sticky ends (Fig. 3.6). Once selected the transient plasmid, ligation should be carried out following the manufacturer’s protocols.

3.5.4 Transformation of the host cells 3.5.4.1 Competent cells preparation Once the cDNA has been cloned into the appropriate vector, then it is ready for plasmid insertion into the selected bacterial cell, in a process called transformation. To achieve a successful transformation, first E. coli cells need to be made competent or permeable to foreign DNA insertion. A classical method of making bacterial cells competent is to expose them to high concentration of calcium chloride to carry out the heat-shock transformation method. E. coli cells such as Top10, DH5α 6 , or JM109 are used for transformation. Competent cells can be commercial or prepared in the lab, following the protocol 1.82

Figure 3.6 pJET vector showing the unique restriction enzymes that are used for the released fragment. PCR blunt fragments are ligated between XhoI and XbaI sites, where the blunt ends are located. PCR, Polymerase chain reaction. Source: Modified from cloneJET PCR cloning manual, Thermo Sci.

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reported in Maniatis, Fritsch, and Sambrook (1982), which yields 5 3 106 to 2 3 107 transformed colonies per microgram of plasmid DNA. Briefly: 1. Pick a single fresh colony (Top10, DH5α 6 , or JM109) from a plate freshly grown for 16
20 hours at 37 C and transfer it into 100 mL LB (Luria Bertani) broth. Incubate the culture for approximately 3 hours at 37 C with vigorous shaking (300 cycles/min). Culture should be carefully monitored by continually measuring optical density at 600 nm (OD600). To obtain high transformation efficiency, it is crucial that the cell growth is in the mid-log phase at the time of harvest, which means when OD600 is between 0.4 and 0.9, with optimal value depending on the culture volume and strain. 2. Aseptically transfer the cells to sterile, disposable, ice-cold 50 mL polypropylene tube. Cool down the cultures to 0 C on ice for 10 minutes. All subsequent steps should be carried out aseptically at 4 C. 3. Recover the cells by centrifugation at 4000 rpm for 10 minutes at 4 C (Sorvall GS3 rotor or equivalent) and decant the media from the pellets. Stand the tube in an inverted position for 1 minute to allow the last traces of media to drain away. 4. Resuspend the pellet in 10 mL of ice-cold 0.1 M CaCl2 and keep on ice, recover the cells by centrifugation as mentioned earlier. Decant the fluid and stand the tubes in an inverted position for 1 minute. 5. Resuspend the pellet in 2 mL of ice-cold 0.1 M CaCl2 for each 50 mL of original culture. 6. Using a chilled, sterile pipette tip, transfer 200 μL of cell suspension to a sterile microtube. Tubes can be transferred to a 280 C freezer or used immediately for transformation. 3.5.4.2 Transformation 1. Take 200 μL of competent cell suspension on ice and add plasmidic DNA (no more than 50 ng in a volume of 10 μL or less). 2. Transfer the tubes in a circulating water bath preheated at 42 C and kept for exactly 90 seconds. 3. Rapidly transfer the tubes to an ice bath and allow the cell to chill for 1
2 minutes. 4. Add 800 μL of SOC (super optimal broth with catabolite repression) or LB medium and incubate for 45 minutes in a water bath set at 37 C to allow the bacteria to recover and to express the antibiotic-resistance marker. 5. Transfer up 200 μL per 90 mm plate of transformed competent cells onto agar SOB (super optimal broth) or LB medium containing the appropriate antibiotic. Using sterile bent glass rod, gently spread the transformed cells over the surface of the agar plate. 6. Leave the plates at room temperature until the liquid has been absorbed and incubate at 37 C. Colonies should appear in 12
16 hours.

Novel technologies in bioactive peptides production and stability 65 3.5.4.3 Preparation of plasmid DNA Minipreparations of plasmid DNA can be obtained either by the alkaline lysis method of obliging methods both described step by step in Maniatis et al. (1982), protocol 1.25. In addition, several commercial kits are available which facilitate the procedure. Minipreparation diagram of plasmid DNA is shown in Fig. 3.7. Selected colony is picked in LB containing the selection marker (Amp, Kan) and grown overnight at 37 C with constant agitation. Next day, the cell pellet is recovered by centrifugation following the manufacturer’s instructions for plasmid DNA extraction. 3.5.4.4 Fragment restriction and ligation into expression vector Restriction of both plasmid containing the DNA of interest and the expression vector (pET) should be carried out with the same enzymes, which were added to the 50 and 30 ends of the peptide (Fig. 3.5), so fragment and vector should have compatible ends. Each restriction enzyme arrives with its own buffer and digestion will be carried out following the manufacturers’ protocol. After digestion, the released fragment is separated from the transient vector in an agarose gel (Fig. 3.8A) and excised from gel and purified using protocols described at Maniatis et al. (1982) or using one commercial kit for gel DNA purification (Qiagen, Promega). The purified fragment (Fig. 3.8B) is then ligated with the restricted pET expression vector (Fig. 3.9A). Ligation pET-peptide is transformed in

Figure 3.7 Recuperation of plasmid DNA using a miniprep commercial kit protocol.

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Figure 3.8 (A) Restriction analysis of transient plasmid with peptide gene. Once plasmid was purified, the fragment is released by BamHI and SalI restriction generating cohesive ends. (B) Vasoinhibin, fragment of 394 pb in part A was purified and now is ready to be ligated on pET expression vector.

competent cells and transformed cells are recovered in LB containing the antibiotic for plasmid selection. Recombinant cells are used for plasmid recovery for sequencing and for protein expression. As shown in Fig. 3.9B, peptide is ligated in BamHI and SalI restriction sites and peptide sequences and peptide will be expressed as a fusion protein with His-Tag, which could be released after purification with the use of the specific protease that cleavage it, releasing the recombinant protein.

3.5.5 Induction of the expression of the desired protein under controlled conditions A single colony of transformed selected E. coli strain is inoculated into 5 mL LB medium containing 50 μg/mL kanamycin and cultured overnight at 37 C with constant shaking at 200 rpm. An aliquot (1.5 mL) of the overnight culture is transferred into 150 mL fresh LB medium and incubated at 37 C with constant shaking at 200 rpm until culture reaches an OD600 around 0.8
1.0. At this time, induction of the recombinant gene is carried out by addition of IPTG. At this point, several strategies are used to increase or optimize the recombinant peptide expression: temperature could be set as low as 20 C and IPTG concentrations could range from 0.1 to 1.2 mM. An aliquot of culture medium is taken at 3, 5, 7, 9, and 12 hours after induction. Cells in each aliquot sample are pelleted and resuspended in 100 μL of lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 5 8.0), and then mixed by vortex or by sonication at 200 W for 2 minutes at 4 C (on-time of ultrasonic pulse of 2 seconds and off-time of 2 seconds). Sometimes, lysozyme could be added to a concentration of 100 μg/mL.

Novel technologies in bioactive peptides production and stability 67

Figure 3.9 (A) Amplified fragment, which was cut with BamHI and SalI restriction enzymes, with cohesive ends is ligated into BamHI and SalI restricted pET plasmid. (B) pET nucleotide sequences of the MCS (Multiple cloning site) region. Dotted lines indicate where the fragment is inserted at BamHI and SalI sies. Blue boxes indicates de His-tag at N- and C-terminal. The specific protease cleavage sequences is underlined with dotted lines. T7 promoter, Lac operator and ribosome binding site (rbs) are shown.

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Figure 3.10 (A) Coomassie stained gel of vasoinhibin expression in E. coli. Samples were taken prior to induction of protein expression with IPTG and after induction (T3
12). When cells are fractionated as soluble (FS) and insoluble proteins (FI) is observed that recombinant protein was found at inclusion bodies. (B) Western-blot analysis confirming the identity of the peptide. The molecular weight marker is indicated at the left of the figure. IPTG, Isopropyl-β-D-thiogalactoside.

After 20 minutes incubation on ice or at 220 C the mix is centrifuged at 12,000 g for 10 minutes at 4 C. The soluble and insoluble fraction are analyzed by SDS-PAGE (Fig. 3.10A) and Western blot (Fig. 3.10B). If the recombinant protein is observed in the insoluble fraction, the pellet or inclusion bodies can be washed with 2 mL of washing buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5% Triton X-100, pH 5 8.0) and collected by centrifugation as mentioned earlier. The washed inclusion bodies should be solubilized in 2 ml buffer (8 M urea, 50 mM Tris-HCl, 100 mM NaCl, pH 5 8.0) at 4 C for 4 hours. After centrifugation at 12,000 g for 10 minutes at 4 C, the soluble recombinant peptides will be ready for purification.

3.5.6 Recovery and purification of the recombinant product Expression vectors generally contain the His-tag that is very useful for recombinant protein purification using immobilized metal affinity chromatography. The commercial resin, Ni21NTA His-Bind resin, could be purchased from several brands (Sigma, Novagen, Merck). The column is preequilibrated with 10 column volumes of binding buffer (50 mM Tris-HCl, 300 mM NaCl, pH 5 7.4). After the soluble recombinant peptide is loaded onto the column and washed with binding buffer, recombinant peptide will be eluted with several washes of elution buffer (binding buffer containing different concentrations of imidazole: 1, 20, 50, 100, 200 mM). Each fraction at different imidazole concentration will be collected separately and analyzed by SDS-PAGE (Fig. 3.11).

Novel technologies in bioactive peptides production and stability 69

Figure 3.11 Schematic process for recombinant protein purification using IMAC affinity columns. IMAC, Immobilized metal affinity chromatography.

3.5.7 Preparation and encapsulation of recombinant peptides Polymeric microgels are colloidal nanoencapsulates comprising one or more biopolymers (McClements, 2018); here, it is described a modified protocol for the encapsulation of BAPs, using a chitosan
alginate mixture by the membrane emulsification method (Wang, Ma, & Su, 2005; Zhang, Wei, Lv, Wang, & Ma, 2011). This is a relatively simple and lowcost technique that has also been used for the encapsulation of BAPs. Briefly, a solution of 1.0 wt/alginate in acetic acid buffer solution (pH 5 4.2) is pressed under nitrogen pressure through a premodified Shirasu Porous glass membrane (7 μm pore size) into a paraffin: petroleum ether (7:5) emulsifier. A mini-emulsion of CaCl2 is prepared by dispersing 1.5 mL of CaCl2 solution into the emulsifier by ultrasonication. The alginate and CaCl2 emulsions are mixed to form a gel for 5 hours under stirring. The solidified alginate gels are collected and washed twice with petroleum ether and four times with distilled water and then centrifuged for 5 minutes at 1000 g and 25 C. A second step of solidification is carried out by dispersing the alginate gel with 1.6 wt.% chitosan solution in acetic acid (pH 5 4.2) for 1 hour under stirring. Then, the alginate-chitosan microspheres are washed twice with 1 wt.% aqueous acetic acid to remove residual chitosan and dried by lyophilization. Vasoinhibin or antihypertensive peptide can be loaded into the alginate-chitosan

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Chapter 3

Figure 3.12 Schematic process for BAPsaˆht encapsulation. BAP, Bioactive peptide.

microspheres by dissolving the protein in the 1.6% chitosan solution during the second solidification step (Fig. 3.12).

3.6 Summary In this chapter the use of recombinant methodology for BAPs production has been reviewed. A review of vectors (plasmids) currently used for protein recombinant expression as well as different E. coli strains was described. A cassette containing several antihypertensive peptides was designed and an encrypted peptide was selected and

Novel technologies in bioactive peptides production and stability 71 amplified for production as recombinant protein. A step-by-step procedure was described for peptide amplification, cloning into an expression vector, transformation into cells for induction of expression and purification of the recombinant protein. Different methodologies for proteins/peptide delivery were reviewed and a simple method for encapsulation of recombinant peptides was described.

References Agyei, D., Ahmed, I., Akram, Z., Iqbal, H. M. N., & Danquah, M. K. (2017). Protein and peptide biopharmaceuticals: An overview. Protein & Peptide Letters, 24(2), 94
101. ´ lvarez, C., & Mullen, A. M. (2020). Assessing the effect of Maillard reaction with dextran on the Anzani, C., A techno-functional properties of collagen-based peptides obtained from bovine hides. LWT, 118, 108800. Baghban, R., Farajnia, S., Rajabibazl, M., Ghasemi, Y., Mafi, A. A., Hoseinpoor, R., et al. (2019). Yeast expression systems: Overview and recent advances. Molecular Biotechnology, 61(5), 365
384. Balderas Herna´ndez, V. E., Paz Maldonado, L. M. T., Medina Rivero, E., Barba de la Rosa, A. P., Jime´nez-Bremont, J. F., Ordon˜ez Acevedo, L. G., et al. (2008). Periplasmic expression and recovery of human interferon gamma in Escherichia coli. Protein Expression and Purification, 59(1), 169
174. Barba de la Rosa, A. P., Barba Montoya, A., Martı´nez-Cuevas, P., Herna´ndez-Ledesma, B., Leo´n-Galva´n, M. F., de Leo´n-Rodrı´guez, A., et al. (2010). Tryptic amaranth glutelin digests induce endothelial nitric oxide production through inhibition of ACE: Antihypertensive role of amaranth peptides. Nitric Oxide, 23(2), 106
111. Baumgarten, T., Ytterberg, A. J., Zubarev, R. A., & de Gier, J. W. (2018). Optimizing recombinant protein production in the Escherichia coli periplasm alleviates stress. Applied and Environmental Microbiology, 84(12). Bhandari, D., Rafiq, S., Gat, Y., Gat, P., Waghmare, R., & Kumar, V. (2019). A review on bioactive peptides: Physiological functions, bioavailability and safety. International Journal of Peptide Research and Therapeutics, 26(1), 139
150. Biedendieck, R. (2016). A Bacillus megaterium system for the production of recombinant proteins and protein complexes. Advances in experimental medicine and biology (pp. 97
113). New York LLC: Springer. Bjeloˇsevi´c, M., Zvonar Pobirk, A., Planinˇsek, O., & Ahlin Grabnar, P. (2020). Excipients in freeze-dried biopharmaceuticals: Contributions toward formulation stability and lyophilisation cycle optimisation. International Journal of Pharmaceutics, 576, 119029. Brandelli, A. (2012). Nanostructures as promising tools for delivery of antimicrobial peptides. Mini-Reviews in Medicinal Chemistry, 12(8), 731
741. Calderon-Salais, S., Velazquez-Bernardino, P., Balderas-Herna´ndez, V. E., Barba de la Rosa, A. P., & de Leo´nRodrı´guez, A. (2018). Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H. Protein Expression and Purification, 144, 40
45. Carmeliet, P., & Jain, R. K. (2000). Angiogenesis in cancer and other diseases. Nature, 407(6801), 249
257. Clapp, C., Gonza´lez, C., Macotela, Y., Aranda, J., Rivera, J. C., Garcı´a, C., et al. (2006). Vasoinhibins: A family of N-terminal prolactin fragments that inhibit angiogenesis and vascular function. Pituitary today: Molecular, physiological and clinical aspects (pp. 64
73). Basel: KARGER. Clapp, C., Martial, J. A., Guzman, R. C., Rentier-Delure, F., & Weiner, R. I. (1993). The 16-kilodalton Nterminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology, 133(3), 1292
1299. Cregg, J. M., Tolstorukov, I., Kusari, A., Sunga, J., Madden, K., & Chappell, T. (2009). Chapter 13— Expression in the yeast Pichia pastoris. Methods in enzymology (pp. 169
189). Academic Press Inc. Damasceno, L. M., Huang, C. J., & Batt, C. A. (2012). Protein secretion in Pichia pastoris and advances in protein production. Applied Microbiology and Biotechnology, 93(1), 31
39.

72

Chapter 3

Dullius, A., Fassina, P., Giroldi, M., Goettert, M. I., Volken., & de Souza, C. F. (2020). A biotechnological approach for the production of branched chain amino acid containing bioactive peptides to improve human health: A review. Food Research International, 131, 109002. Fields, G. B. (2002). Introduction to peptide synthesis. Current Protocols in Immunology, 47(1), 9.1.1
9.1.9. Fleming, I. (2006). Signaling by the angiotensin-converting enzyme. Circulation Research, 98(7), 887
896. ˜ ojo, V. L., Pardo, B. O. P., Junior, A. P., Stephano, M. A., & de OliveiraGeraldes, D. C., Beraldo-de-AraA % drug delivery: Current dosage form profile and formulation strategies. Nascimento, L. (2020). Protein Journal of Drug Targeting, 28(4), 339
355. Gleeson, J. P., Ryan, S. M., & Brayden, D. J. (2016). Oral delivery strategies for nutraceuticals: Delivery vehicles and absorption enhancers. Trends in Food Science and Technology, 53, 90
101. Gonza´lez, A., & Fillat, M. F. (2018). Aspectos metodolo´gicos de la expresio´n de proteı´nas recombinantes en Escherichia coli. Revista de Educacio´n Bioquı´mica (REB), 37, 14
27. Grootaert, C., Matthijs, B., Voorspoels, S., Possemiers, S., Smagghe, G., & van Camp, J. (2017). Egg-derived bioactive peptides with ACE-inhibitory properties: A literature update. Food & Function, 8, 3847
3855. Herna´ndez-Ledesma, B., del Mar Contreras, M., & Recio, I. (2011). Antihypertensive peptides: Production, bioavailability and incorporation into foods. Advances in colloid and interface science (pp. 23
35). Elsevier. Herna´ndez-Ledesma, B., Recio, I., Ramos, M., & Amigo, L. (2002). Preparation of ovine and caprine β-lactoglobulin hydrolysates with ACE-inhibitory activity. Identification of active peptides from caprine β-lactoglobulin hydrolysed with thermolysin. International Dairy Journal, 12(10), 805
812. Huang, L., Ma, H., Li, Y., & Li, S. (2012). Antihypertensive activity of recombinant peptide IYPR expressed in Escherichia coli as inclusion bodies. Protein Expression and Purification, 83(1), 15
20. Kamerzell, T. J., Esfandiary, R., Joshi, S. B., Middaugh, C. R., & Volkin, D. B. (2011). Protein-excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development. Advanced Drug Delivery Reviews, 63(13), 1118
1159. Kang, N. J., Jin, H. S., Lee, S. E., Kim, H. J., Koh, H., & Lee, D. W. (2019). New approaches towards the discovery and evaluation of bioactive peptides from natural resources. Critical Reviews in Environmental Science and Technology, 50(1), 72
103. Korczek, K. R., Tkaczewska, J., Duda, I., & Migdał, W. (2020). Effect of heat treatment on the antioxidant and antihypertensive activity as well as in vitro digestion stability of Mackerel (Scomber scombrus) protein hydrolysates. Journal of Aquatic Food Product Technology, 29(1), 73
89. Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications. Journal of Functional Foods, 1(2), 177
187. Langlais, C., & Korn, B. (2006). Recombinant protein expression in bacteria. Encyclopedic reference of genomics and proteomics in molecular medicine (pp. 1609
1616). Berlin, Heidelberg: Springer. Li, G.-H., Qu, M.-R., Wan, J.-Z., & You, J.-M. (2007). Antihypertensive effect of rice protein hydrolysate with in vitro angiotensin I-converting enzyme inhibitory activity in spontaneously hypertensive rats. Asia-Pacific Journal of Clinical Nutrition, 16(1), 275
280. Liang, R., Zhang, M., & Lin, S. (2014). Recombinant expression of bioactive peptides: A review. Advanced Materials Research, 881
883, 331
334. Liu, C. F., & Pan, T. M. (2010). Recombinant expression of bioactive peptide lunasin in Escherichia coli. Applied Microbiology and Biotechnology, 88(1), 177
186. Losurdo, L., Quintieri, L., Caputo, L., Gallerani, R., Mayo, B., & de Leo, F. (2013). Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum. FEMS Microbiology Letters, 340(1), 24
32. Lv, G. S., Huo, G. C., & Fu, X. Y. (2003). Expression of milk-derived antihypertensive peptide in Escherichia coli. Journal of Dairy Science, 86(6), 1927
1931. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast, 22(4), 249
270. Malik, A. (2016). Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli. 3 Biotech, 6(1), 44.

Novel technologies in bioactive peptides production and stability 73 Mamat, U., Wilke, K., Bramhill, D., Schromm, A. B., Lindner, B., Kohl, T. A., et al. (2015). Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins. Microbial Cell Factories, 14(1), 57. (Cited 27 April 2020) ,http://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-015-0241-5.. Maniatis, T., Fritsch, E. F., & Sambrook, J. (1982). Molecular cloning: A laboratory manual (p. 545) Cold Spring Harbor Laboratory. McClements, D. J. (2018). Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems: A review. Advances in Colloid and Interface Science, 253, 1
22. Molavi, F., Barzegar-Jalali, M., & Hamishehkar, H. (2020). Polyester based polymeric nano and microparticles for pharmaceutical purposes: A review on formulation approaches. Journal of Controlled Release, 320, 265
282. Motoi, H., & Kodama, T. (2003). Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides from wheat gliadin hydrolysate. Nahrung—Food, 47(5), 354
358. Novagen Inc. (2021) pET system manual (11th ed.). Novagen Inc. ,https://www.merckmillipore.com/MX/es/ product/pET-Expression-System-28-Novagen,EMD_BIO-70777#anchor_USP.. Reviewed May 2021. Ontiveros, N., Lo´pez-Teros, V., Vergara-Jime´nez, M. d J., Islas-Rubio, A. R., Ca´rdenas-Torres, F. I., CuevasRodrı´guez, E. O., et al. (2020). Amaranth-hydrolyzate enriched cookies reduce the systolic blood pressure in spontaneously hypertensive rats. Journal of Functional Foods, 64. Palomares, L. A., Kuri-Bren˜a, F., & Ramı´rez, O. T. (2009). Industrial recombinant protein production. In H. Werner Doelle, S. Roken, & M. Berovic (Eds.), Encyclopedia of life support systems: Biotechnology fundamentals (Vol V, pp. 203
245). EOLSS Publishers/UNESCO. Pihlanto-Leppa¨la¨, A., Koskinen, P., Phlola, K., Tupasela, T., & Korhonen, H. (2000). Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides. Journal of Dairy Research, 67(1), 53
64. Rao, S., Su, Y., Li, J., Xu, Z., & Yang, Y. (2009). Design and expression of recombinant antihypertensive peptide multimer gene in Escherichia coli BL21. Journal of Microbiology and Biotechnology, 19(12), 1620
1627. Rojas Contreras, J. A., Pedraza-Reyes, M., Ordon˜ez, L. G., Estrada, N. U., Barba de la Rosa, A. P., & de Leo´nRodrı´guez, A. (2010). Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis. Plasmid, 64(3), 170
176. Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y., & Itoh, T. (2000). Isolation and structural analysis of antihypertensive peptides that exist naturally in gouda cheese. Journal of Dairy Science, 83. Sarabandi, K., Gharehbeglou, P., & Jafari, S. M. (2020). Spray-drying encapsulation of protein hydrolysates and bioactive peptides: Opportunities and challenges. Drying Technology, 38(5
6), 577
595. Schmidt, F. R. (2004). Recombinant expression systems in the pharmaceutical industry. Applied Microbiology and Biotechnology, 65(4), 363
372. Sharma, R., Agrawal, U., & Vyas, S. (2014). Polymeric nanocarriers for the oral delivery of bioactives. Current Drug Therapy, 9(1), 21
34. Sun, X., Acquah, C., Aluko, R. E., & Udenigwe, C. C. (2020). Considering food matrix and gastrointestinal effects in enhancing bioactive peptide absorption and bioavailability. Journal of Functional Foods, 64, 103680. Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 72(2), 211
222. Tiwari, A. K., Gajbhiye, V., Sharma, R., & Jain, N. K. (2010). Carrier mediated protein and peptide stabilization. Drug Delivery, 17(8), 605
616. Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides: Production, processing, and potential health benefits. Journal of Food Science, 77(1), R11
R24. Vazquez Rodriguez, G., Gonzalez, C., & de Leon Rodriguez, A. (2013). Novel fusion protein derived from vasostatin 30 and vasoinhibin II-14.1 potently inhibits coronary endothelial cell proliferation. Molecular Biotechnology, 54(3), 920
929.

74

Chapter 3

Velarde-Salcedo, A. J., Barrera-Pacheco, A., Lara-Gonza´lez, S., Montero-Mora´n, G. M., Dı´az-Gois, A., Gonza´lez De Mejia, E., et al. (2013). In vitro inhibition of dipeptidyl peptidase IV by peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins. Food chemistry, 136(2). Wang, L.-Y., Ma, G.-H., & Su, Z.-G. (2005). Preparation of uniform sized chitosan microspheres by membrane emulsification technique and application as a carrier of protein drug. Journal of Controlled Resease, 106(1
2), 62
75. World Health Organization. (2018). Cancer. Available from https://www.who.int/news-room/fact-sheets/detail/ cancer. (Cited 9 April 2020). Yathisha, U. G., Bhat, I., Karunasagar, I., & Mamatha, B. S. (2019). Antihypertensive activity of fish protein hydrolysates and its peptides. Critical Reviews in Food Science and Nutrition, 59(15), 2363
2374. Yi, Y., Lv, Y., Zhang, L., Yang, J., & Shi, Q. (2018). High throughput identification of antihypertensive peptides from fish proteome datasets. Marine Drugs, 16(10), 365. Yu, Z., Rogers, T. L., Hu, J., Johnston, K. P., & Williams, R. O. (2002). Preparation and characterization of microparticles containing peptide produced by a novel process: Spray freezing into liquid. European Journal of Pharmaceutics and Biopharmaceutics, 54(2), 221
228. Zhang, Y., Wei, W., Lv, P., Wang, L., & Ma, G. (2011). Preparation and evaluation of alginate-chitosan microspheres for oral delivery of insulin. European Journal of Pharmaceutics and Biopharmaceutics, 77(1), 11
19. Zhu, Y., Nadia, E., Yao, Y., Shi, Z., & Ren, G. (2018). Tandem repeated expression of lunasin gene in Pichia pastoris and its anti-inflammatory activity in vitro. Journal of Bioscience and Bioengineering, 126(1), 1
8.

CHAPTER 4

Methodologies for extraction and separation of short-chain bioactive peptides Andrea Cerrato1, Sara Elsa Aita1, Carmela Maria Montone1, Anna Laura Capriotti1, Susy Piovesana1 and Aldo Lagana`1,2 1

Department of Chemistry, Sapienza University of Rome, Rome, Italy, 2CNR NANOTEC, Campus Ecotekne, University of Salento, Lecce, Italy

4.1 Introduction Bioactive peptides (BPs) refer to various amino acids (AAs) sequences, normally comprising 2
20 residues. Several routes can lead to the release of BPs. For instance, they can be naturally present in food matrices due to common food processing such as ripening, fermentation or cooking (Tu, Cheng, Lu, & Du, 2018), or they can be formed by protein degradation (enzymatic degradation and physicochemical non-enzymatic degradation such as radicals) or gene-encoding (BPs) and gene-independent enzymatic formation (e.g. glutathione) in vivo in biological matrices (Peng, Zhang, Niu, & Wu, 2020). Mediumsized endogenous peptides have been characterized in several food matrices, such as aged duck meat (Liu, Chen, Huang, & Zhou, 2019), fermented meat source, chicken breast and ham (Liu, Xing, Fu, Zhou, & Zhang, 2016), milk (Capriotti, Cavaliere, Piovesana, Samperi, & Lagana`, 2016; Nongonierma & FitzGerald, 2016; Piovesana et al., 2015; Zenezini Chiozzi et al., 2016), and vegetables (Piovesana et al., 2018) and have already been demonstrated to be valuable bioactive compounds. Alternatively, protein digestion by trypsin or mixtures of enzymes, which can simulate gastrointestinal digestion, can actively produce BPs. The use of trypsin leads to the generation of medium-sized peptides (6
20 AAs) due to its specific cleavage mechanism (Capriotti et al., 2015), while the use of enzyme cocktails can also produce shorter sequences (2
5 AAs) (Gallego, Mauri, Aristoy, Toldra´, & Mora, 2020; Le Maux, Nongonierma, Murray, Kelly, & FitzGerald, 2015). BPs length is of extreme importance from several points of view, both because the analytical techniques for their extraction, separation and identification are quite different and because they possess different physico-chemical properties, biological functions and activities. Karami and Akbari-adergani (2019) Several recent reviews have been focused on the progress of analytical techniques for the enrichment, purification and identification of

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00002-9 © 2021 Elsevier Inc. All rights reserved.

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medium-sized peptides, (Aydo˘gan, 2020; Nongonierma & FitzGerald, 2017; Piovesana et al., 2018). However, the enrichment, separation and identification of short-chain peptides has long been an analytical challenge. In fact, even though well-established analytical methodologies have been developed for the study of medium-sized BPs, few papers have specifically dealt with short-chain BPs, although it is well known that the most bioactive sequences are those possessing five or less AAs in their chain Chakrabarti, Guha, and Majumder (2018). At present, the specific issues of short-chain BPs isolation and identification has been faced for the investigation of milk-derived short-chain BPs (Montone et al., 2019; O’Keeffe & Fitzgerald, 2015) but these compounds have been emerging as fundamental in other matrices as well, like dry-cured ham, where dipeptides and tripeptides are not only abundant, but also display multiple bioactivities and important contribution to taste (Gallego, Mora, & Toldra´, 2019). The restricted present literature could be attributed to three main problems in the analysis of short-chain peptide sequences. The first issue is related to the wide range of physicochemical properties of such peptides, such as molecular weight, polarity and acid-base properties, which makes not perfectly suitable the typical C18 reverse phase (RP) separation, standardly employed in proteomics and medium-sized peptidomics. Moreover, very hydrophilic peptides can be lost during the common desalting step or during analysis if the chromatographic method comprises an on-line precolumn enrichment prior to peptide separation and MS. The use of different separation strategies can help in retaining such short sequences and efficiently separate them. For example, short-chain angiotensinconverting-enzyme inhibitor peptides were recently identified in Tilapia using column Acquity HSS T3, a particular column which still use a RP separation mechanism but is designed for more polar compounds (Yesmine et al., 2017); HILIC has also been used for the separation of short-chain peptide sequences (Le Maux, Nongonierma, & Fitzgerald, 2015) and combined with RP for separation of isobaric short-chain peptides (Le Maux, Nongonierma, Murray, et al., 2015). Recently, our research group has introduced an effective separation of small hydrophilic peptides by porous graphitic carbon (PGC) stationary phase in milk and serum samples (Montone et al., 2019; Piovesana, Montone, et al., 2019) due to the PGC behaviour as reverse phase as well as electronic ion-exchanger. The second problem is associated with the poor ionization efficiency of short-chain peptides when the common electrospray ionization is used (Meo, Pasic, & Yousef, 2016). In fact, short-chain peptides generate low-abundance and singly charged precursor ions, hindering the use of common nano-HPLC-MS approaches which are mainly based to multi-charged ions. This problem has been partially overcome by adding a supercharging reagent to the electrospray solution or to the mobile phase in HPLC-MS which is able to increase the ionization efficiency of short-chain peptides, leading to improved peptide identification in large-scale applications (Miladinovi´c et al., 2012; Piovesana, Montone, et al., 2019). The third problem is related to short-chain BPs identification, since unknown short-chain

Methodologies for extraction and separation of short-chain bioactive peptides 77 peptide sequences cannot be confidently identified by common proteomics software. Such short-chain sequences, in fact, cannot be uniquely attributed to proteins present in the databases. Different solutions were proposed and introduced over the past years including, targeted analysis (Takahashi et al., 2012), de novo sequence identification (Nongonierma & FitzGerald, 2017), retention time validation by retention time prediction models (Le Maux, Nongonierma, & Fitzgerald, 2015) and use of metabolomics approaches (Guijas et al., 2018). Evidently, each one of these approaches has not allowed a comprehensive identification of all possible short-chain peptides present in a matrix for different limitations that were already described and discussed in a recent review article (Peng et al., 2020). Recently, an untargeted approach by suspect screening was developed in our research group leading to a broad increase in the number of identifications (Cerrato et al., 2020). Essentially, this chapter aims to provide the reader with an overview of the most relevant and recent approaches for the enrichment, clean-up and separation of short-chain bioactive by solidphase extraction and liquid chromatography techniques. A brief description and explanation of the mass-spectrometric workflow for short-chain BPs identification will also be provided in the last part of the chapter for a complete description of the whole analytical platform.

4.2 Definition: Short-chain peptide enrichment Short-chain peptides are present in food and biological samples in very low concentrations and in most cases their detection is vitiated by the presence of other higher abundance compounds such as metabolites, lipids, proteins and medium-sized peptides. For these reasons, when untargeted short-chain peptidomics studies are carried out, an enrichment and clean-up step is mandatory. Moreover, due to the wide range of physico-chemical properties of short-chain peptides, an enrichment method based on a peculiar material suitable both for hydrophobic and hydrophilic sequences is also necessary.

4.3 Materials, equipment and reagents Carbograph 4 (Lara S.r.l., Formello, RM, Italy); polypropylene tubes; polypropylene frit (Sigma); analytical balance (E50S by Gibertini Elettronica, Milan, Italy); a mixture of the following synthetic peptides Gly-Asp-Leu-Glu (GDLE), Leu-Pro-Leu (LPL), Ile-Pro-ProLeu (IPPL), Lys-His (KH), Pro-Ile (PI), Arg-Phe (RF), Ser-His (SH), Val-Glu-Pro (VEP), Val-Arg-Gly-Pro (VRGP), Pro-Leu (PL), Ile-Pro-Ile (IPI), Leu-Pro (LP), and Lys-His- Lys (KHK), all were purchased from Thermo Fisher Scientific (Ulm, Germany). Optima LC 2 MS grade water, acetonitrile (ACN) and methanol (MeOH) were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Trifluoroacetic acid (TFA), hydrochloric acid, formic acid (FA), ammonium formate and dichloromethane (DCM) were supplied by Romil Ltd. (Cambridge). Speed-Vac SC250 Express (Thermo Savant,

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Holbrook, NY, USA). Kinetex XB-C18 (100 3 2.1 mm, 2.6 μm particle size, Phenomenex, Torrance, USA) and iHILIC-Fusion UHPLC Column, SS (100 3 2.1 mm, 1.8 μm particle size, Hilicon, Umea, Sweden). Ultra-high performance liquid chromatography system Vanquish binary pump H, equipped with a thermostated auto-sampler and column compartment, coupled to a hybrid quadrupole orbitrap (Q Exactive) mass-spectrometer (Thermo Fisher Scientific, Bremen, Germany).

4.4 Protocols 1. 500 mg of Carbograph 4 were manually packed into a 6 mL polypropylene tube. A polypropylene frit was placed at the bottom and on top of the cartridge in order to avoid dispersion of the sorbent during all operations. 2. The cartridge was washed with 5 mL of CH2Cl2/MeOH, 80:20 (v/v) with 20 mmol L21 TFA and 5 mL of MeOH with 20 mmol L21 TFA. 3. The material was activated by flushing with 10 mL H2O of 0.1 mol L21 HCl. 4. The material was conditioned with 10 mL H2O of 20 mmol L21 TFA. 5. Sample was loaded (in the case of urine sample: 2 mL urine diluted to 10 mL with 20 mmol L21 TFA; in the case of plasma sample: 100 μL plasma diluted to 10 mL with 20 mmol L21 TFA; in the case of fish sample: 40 mg of fish muscle were extracted with 2 mL H2O and diluted to 10 mL with H2O with 20 mmol L21 TFA). 6. After sample loading, the cartridge was washed with 2 mL of 20 mmol L 2 1 TFA and 0.5 mL MeOH (used to remove the cartridge dead volume). 7. Short-chain peptides were eluted by back flushing with 10 mL of CH2Cl2/MeOH and 80:20 (v/v) with 20 mmol L21 TFA. 8. The eluate was evaporated at room temperature in a Speed-Vac and the residue reconstituted either in 200 μL H2O for RP separation or 200 μL of ACN/H2O, 75:25 (v/v) for HILIC separation.

4.5 Pros and cons Pros

Cons

The GCB can work as a RP adsorbentand retain analytes by strong hydrophobic interaction, which is suitable for non-polar and polar compounds.

Need to elute some compounds (basic peptides, for instance) in backflushing mode. This is quite laborious and time-consuming since in most cases two elution steps must be carried out. The GCB is produced at high temperature and some reproducibility problems could occur between two different preparation batches or between two different manufacturers.

Graphitic structure can contribute to retention of both hydrophilic and hydrophobic peptides by a wide range of chemical interactions. The main mechanisms are hydrophobic interactions between the aromatic graphene-like structures and the hydrophobic sections of the peptides. Moreover, electrostatic interaction to

(Continued)

Methodologies for extraction and separation of short-chain bioactive peptides 79 (Continued) Pros

Cons

anions with chromene-like heterogeneities on the GCB surface, and to cations with H-π interactions help retaining hydrophilic, acid and basic compounds. Finally, also π stacking interaction with aromatic moieties of some AAs occur. Peptide clean-up on GCB is suitable to maximize weak hydrophobic interactions with short-chain peptides as well as interactions with charged side chains, charged C- and N-terminus, and aromatic residues, therefore making it a suitable strategy to enrich the entire shortchain peptidome and to simultaneously remove salts as well as compounds with strong acid moieties, such as sulfate and glucoronate conjugated metabolites, which are often highly abundant in biological samples.

4.6 Alternative methods/procedures An alternative enrichment method could be porous graphitic carbon (PGC) cartridges. Commercially, Hypercarb SPE Cartridges feature a unique graphite carbon-based material that retains highly polar compounds. The interaction behaviour is quite similar to the GCB material with some differences due to their peculiar structure. PGC cartridges are stable over a wide range of pH values (0
14), temperatures and salt concentrations and they are effective for the purification and enrichment of highly polar compounds such as short-chain peptides (Desportes, Charpentier, Duteurtre, Maujean, & Duchiron, 2000). The disadvantage in the use of PGC cartridges is essentially due to their long and expensive manufacturing process which hinders a routine use when many biological samples must be analysed (West, Elfakir, & Lafosse, 2010).

4.7 Troubleshooting & Optimization Problem

Solution

Poor solubility of some peptides in pure water. GCB possesses heterogeneities on its structure with potentially reactive oxygen species, such as epoxides, which can lead to an irreversible chemical bond by nucleophilic addition of amine moieties with these oxygen groups. Low retentions of the lowest molecular weight peptides as well as the most polar ones.

Acid loading buffer should be used. Acid loading buffers avoid this problem.

TFA allows to reach higher retention due to its capability as ion acidic pairing agent to generate heavier, neutral, and less hydrophilic species, thus enhancing their interactions with GCB. (Continued)

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Problem

Solution

The use of acidic condition in the elution buffer can lead to the formation of positively charged residues for peptides and consequently a strong interaction between graphene and this charge state. In fact, positively charged residues, along with aromatic amino acids, represent the best residues for the interaction with graphene due to the right combination of long side positively charged chains of lysine and arginine, and aromatic positively charged residues of histidine. The use of positively charged ion pairing agents, such as tetramehtylamonium chloride (TMAC) in the elution buffer leads to several issues due to its ion suppressing nature in ESI sources and a noticeable loss in reproducibility.

TFA as acidic modifier is the best compromise in order to have higher recovery and overcome the issue related to the strong interaction. TFA has a double nature, it works as acidic modifier and an ion pairing agent. TFA together with back-flushing elution helps improving short-chain peptide elution and recovery.

TFA at 20 mmol L-1 in the elution buffer is the best ion pairing agent since it does not contribute to massive ion suppressing phenomena in positive polarity. TFA is also used in RP separation phases for improving hydrophilic sequences retention.

Definition: Short-chain peptide chromatographic separation by porous graphitic carbon stationary phase Several chromatographic separation strategies were employed over the past years for the separation of short-chain hydrophilic peptides, such as RP, HILIC and PGC (Adoubel, Guenu, Elfakir, & Dreux, 2000; Nova´kova´, Havlı´kova´, & Vlˇckova´, 2014; Peng et al., 2020) but none of them is capable to separate all peptides. With the purpose of achieving a more comprehensive characterization of short-chain peptides in biological matrices, two columns with different selectivity mechanisms should be employed. For instance, the use of HILIC and PGC separation is suitable for the separation of small polar and charged compounds, which are poorly retained in RP separation, a better choice for more hydrophobic molecules. PGC exploits, in fact, multiple retention mechanisms. Its ability in retaining hydrophilic and ionic molecules (Ne`meth-Kiss, Forga´cs, & Cserha´ti, 1997; Pereira, 2008; Zhu, Huang, Zhao, Zhong, & Mechref, 2020) was previously applied to the fractionation of short-chain BPs and phosphopeptides in wine (Desportes et al., 2000), for desalting flow-through fractions from a C18 column (Chin & Papac, 1999) and to separate underivatized di-, tri-, tetrapeptides using nonafluoropentanoicacid as an ion pair reagent (Adoubel et al., 2000), but never in direct coupling with MS detection. Here, we describe the first use and optimization, highlighting the advantages and challenges, when PGC was coupled to an Orbitrap mass spectrometric system for applications to serum or milk samples (Montone et al. 2019; Piovesana, Cerrato, et al., 2019).

4.8 Materials, equipment and reagents Materials Optima® LC
MS grade water, acetonitrile (ACN) and methanol (MeOH) were purchased from Thermo Fisher Scientific (Bremen, Germany); trifluoroacetic acid (TFA) and tetrahydrofuran (THF) were supplied by Romil Ltd (Cambridge). Formic acid and 3-nitrobenzyl alcohol (3-NBA) were purchased from Sigma-Aldrich (Germany). Hypercarbt Porous Graphitic Carbon LC Column (50 3 2.1 mm,3 μm particle size).

Methodologies for extraction and separation of short-chain bioactive peptides 81

Figure 4.1 Extracted ion chromatogram of the short-chain peptide standard mixture under the final chromatographic conditions (10 μL injected volume, 1 μg μL 2 1 concentration). Analyte order: SH (1), KH (2), PL (3), LP and PI (4), KHK (5), VEP (6), GDLE (7), VRGP (8), RF (9), LPL (10), RKKH (11), IPI (12), IPPL (13).

Ultra-high performance liquid chromatography system Vanquish binary pump H, equipped with a thermostated auto-sampler and column compartment, coupled to a hybrid quadrupole orbitrap (Q Exactive) mass-spectrometer (Thermo Fisher Scientific, Bremen, Germany). Synthetic peptides Gly-Asp-Leu-Glu (GDLE), Leu-Pro-Leu (LPL), Ile-Pro-Pro-Leu (IPPL), Lys-His (KH), Pro-Ile (PI), Arg-Phe (RF), Ser-His (SH), Val-Glu-Pro (VEP), Val-Arg-Gly-Pro (VRGP), Pro-Leu (PL), Ile-Pro-Ile (IPI), Leu-Pro (LP), and Lys-His- Lys (KHK) were purchased from Thermo Fisher Scientific (Ulm, Germany). {{{Fig. 4.1}}}

4.9 Protocols Peptide separation was carried out at 0.5 mL min21 flow rate. Ten μL of each sample were injected onto a Hypercarbt PGC column.  The column was maintained at 50 C using the still air option. Peptides were eluted using H2O (phase A) and ACN/THF 99:1 (v/v) (phase B) both with 0.15% of TFA, with post-column addition of ACN with 1.2% 3-NBA at 0.1 mL min21. 5. The chromatographic gradient was the following: 0% B for 2 min, 0
35% B in 15 min, 35
90% B in 8 min; at the end of the gradient, a washing step was carried out at 90% B for 3 min and a re-equilibration step at 0% B for 7 min.

1. 2. 3. 4.

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6. The chromatographic system was coupled to a hybrid quadrupole-Orbitrap mass spectrometer Q Exactive (Thermo Fisher Scientific) using a heated electrospray ionization (ESI) source.

4.10 Pros and cons Pros

Cons

PGB is stable over the entire pH range of 0 2 14. PGC is stable under high temperature (over 200  C).

PGB can withstand pressures of maximum 400 bar. PGC columns are only available as 3, 5 and 7 μm particles (UHPLC columns possess particles , 2.1 μm). PGC columns suffer from scarce reproducibility over time or frequent usage, especially the 3 μm particles.

PGC possess unique retention mechanisms; besides hydrophobic interactions, strong interactions between negatively charged analytes and aromatic moieties of molecules and the delocalized electron cloud on the PGC occur.

PGC needs long equilibration times in order to obtain good reproducibility.

4.11 Alternative methods/procedures An alternative separation strategy to PGC for small hydrophilic compounds is HILIC (Nova´kova´ et al., 2014). In order to obtain larger information on hydrophilic short-chain BPs, a retention time prediction model using an Acquity BEH Amide column ˚ ) has been described (Le Maux, Nongonierma, & Fitzgerald, (2.1 x 150 mm, 1.7 μm, 130 A 2015). In fact, the retention time observed in RP-HPLC is directly related to the apparent hydrophobic character of the analyzed molecule, while HILIC elutes compounds in order of increasing polarity. Moreover, HILIC can enhance MS sensitivity since highly organic mobile phases are easily desolvated, resulting in an improved ionization efficiency. Differently from PGC, which still shows a RP mechanism, HILIC presents a completely orthogonal retention selectivity and therefore, it is the most suitable chromatographic system for the most polar and charged BPs. However, in complex matrices, retention time prediction models can often perform poorly. Since BPs, and especially endogenous peptides, are low-abundant compounds in matrices in which other higher abundant isobaric compounds could also be present, a large number of false positives could occur. Moreover, HILIC columns cause usually broader peaks than RP ones, resulting in high-abundance metabolites suppression when common data dependent acquisition (DDA) mode MS analyses are performed. Recently, zwitterionic iHILIC-Fusion UHPLC Column, SS (100 3 2.1 mm, 1.8 μm particle size, Hilicon, Umea, Sweden) has been employed for the characterization of short-chain peptide sequences. (Cerrato et al., 2020; Piovesana, Capriotti, et al., 2019; Piovesana, Montone, et al., 2019). The iHILIC-Fusion is a

Methodologies for extraction and separation of short-chain bioactive peptides 83 charge-modulated hydroxyethyl amide HILIC, in which the cationic ammonium site is at the terminal position separated from mixed sulfates and phosphates anionic sites by a linker with hydroxyethyl amide side chains. The column was previously employed for separation in metabolomics analyses (Cuykx, Claes, Rodrigues, Vanhaecke, & Covaci, 2018; Sa´nchezSobero´n et al., 2018); the iHILIC Fusion column proved better than other HILIC columns, with a good coverage of metabolite classes like amino acids (Cuykx et al., 2018; Sa´nchez-Sobero´n et al., 2018).

4.12 Troubleshooting & Optimization Problem

Solution

Oxidation of the PGC stationary phase could potentially occur.

Properly connect the PGC column to the grounding system, typically included in in standard modern ESI interfaces for safety reasons. Rodriquez et al. (2015) The use of TFA as an ion-pairing agent (for retention) and organic modifier (for elution) is strongly recommended. The use of post column addition of propionic acid and isopropanol is strongly recommended. Kuhlmann, Apffel, Fischer, Goldberg, and Goodley (1995) PGC must be regenerated as follows every 50
60 runs: (i) 70 volumes of isopropanol, (ii) 70 volumes of THF and (iii) 70 volumes of isopropanol. Since PGC is stable up to 200  C, pression issues can be solved by increasing the column temperature.

The various retention mechanisms of PGC do not allow to use only the eluotropic solvent series for eluting all compounds. TFA could cause ion suppression phenomena in ESI sources. Garcı́a, Hogenboom, Zappey, and Irth (2002) The use of TFA could cause permanent absorption on the surface of PGC, causing the modification of the stationary phase. PGC column cannot bear pression higher than 400 bar.

4.13 Summary Carbonaceous material, with their unique retention mechanisms, are promising sorbents both for sample preparation and chromatographic separation of short-chain BPs. Since such materials present peculiar characteristics, attention to details and care of the columns shall be taken and a deep knowledge on the troubleshooting is highly recommended. Preconcentration strategies are a crucial step for low-abundance compounds, like shortchain endogenous peptides, and further efforts must be taken for proper separation of the most hydrophilic sequences. Finally, particular attention must also be paid on the MS analysis (Cerrato et al., 2020). When orbitrap instruments are employed, DDA methods, in which top n ranked precursor ions are sequentially isolated and fragmented, would repeatedly cause high-abundance

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species to suppress less concentrated coeluting compounds, which would not be fragmented and, eventually, identified. On the other hand, data independent acquisition (DIA) strategies, which would assure all ion fragmentation, cannot be usually performed on such slow instruments. In order to overcome DDA limits, a suspect screening approach, in which an inclusion list with the combination of amino acids in di-, tri- and tetrapeptides is implemented in the MS method, can be chosen. Thus, many low-abundance species that would have normally been neglected, can be fragmented and validated Cerrato et al. (2020).

References Adoubel, A. A., Guenu, S., Elfakir, C., & Dreux, M. (2000). SEPARATION OF UNDERIVATIZED SMALL PEPTIDES ON A POROUS GRAPHITIC CARBON COLUMN BY ION-PAIR CHROMATOGRAPHY AND EVAPORATIVE LIGHT SCATTERING DETECTION. J Liq Chromatogr Relat Technol, 23, 2433
2446. Available from https://doi.org/10.1081/JLC-100100499. Aydo˘gan, C. (2020). Recent advances and applications in LC-HRMS for food and plant natural products: a critical review. Analytical and bioanalytical chemistry, 412, 1973
1991. Available from https://doi.org/ 10.1007/s00216-019-02328-6. Capriotti, A. L., Cavaliere, C., Foglia, P., Piovesana, S., Samperi, R., Zenezini Chiozzi, R., et al. (2015). Development of an analytical strategy for the identification of potential bioactive peptides generated by in vitro tryptic digestion of fish muscle proteins. Analytical and bioanalytical chemistry, 407, 845
854. Available from https://doi.org/10.1007/s00216-014-8094-z. Capriotti, A. L., Cavaliere, C., Piovesana, S., Samperi, R., & Lagana`, A. (2016). Recent trends in the analysis of bioactive peptides in milk and dairy products. Analytical and bioanalytical chemistry, 408, 2677
2685. Available from https://doi.org/10.1007/s00216-016-9303-8. Cerrato, A., Aita, S. E., Capriotti, A. L., Cavaliere, C., Montone, C. M., Lagana`, A., et al. (2020). A new opening for the tricky untargeted investigation of natural and modified short peptides. Talanta, 219121262. Available from https://doi.org/10.1016/j.talanta.2020.121262. Chakrabarti, S., Guha, S., & Majumder, K. (2018). Food-Derived Bioactive Peptides in Human Health: Challenges and Opportunities. Nutrients, 10, 1738. Available from https://doi.org/10.3390/nu10111738. Chin, E. T., & Papac, D. I. (1999). The Use of a Porous Graphitic Carbon Column for Desalting Hydrophilic Peptides prior to Matrix-Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry. Analytical Biochemistry, 273, 179
185. Available from https://doi.org/10.1006/abio.1999.4242. Cuykx, M., Claes, L., Rodrigues, R. M., Vanhaecke, T., & Covaci, A. (2018). Metabolomics profiling of steatosis progression in HepaRG® cells using sodium valproate. Toxicology Letters, 286, 22
30. Available from https://doi.org/10.1016/j.toxlet.2017.12.015. Desportes, C., Charpentier, M., Duteurtre, B., Maujean, A., & Duchiron, F. (2000). Liquid chromatographic fractionation of small peptides from wine. Journal of Chromatography. A, 893, 281
291. Available from https://doi.org/10.1016/S0021-9673(00)00698-1. Gallego, M., Mauri, L., Aristoy, M. C., Toldra´, F., & Mora, L. (2020). Antioxidant peptides profile in dry-cured ham as affected by gastrointestinal digestion. J Funct Foods, 69103956. Available from https://doi.org/ 10.1016/j.jff.2020.103956. Gallego, M., Mora, L., & Toldra´, F. (2019). The relevance of dipeptides and tripeptides in the bioactivity and taste of dry-cured ham. Food Prod Process Nutr, 1, 2. Available from https://doi.org/10.1186/s43014-019-0002-7. Garcı́a, M. C., Hogenboom, A. C., Zappey, H., & Irth, H. (2002). Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography
electrospray mass spectrometry. Journal of Chromatography. A, 957, 187
199. Available from https://doi.org/10.1016/ S0021-9673(02)00345-X.

Methodologies for extraction and separation of short-chain bioactive peptides 85 Guijas, C., Montenegro-Burke, J. R., Domingo-Almenara, X., Palermo, A., Warth, B., Hermann, G., et al. (2018). METLIN: A Technology Platform for Identifying Knowns and Unknowns. Analytical Chemistry, 90, 3156
3164. Available from https://doi.org/10.1021/acs.analchem.7b04424. Karami, Z., & Akbari-adergani, B. (2019). Bioactive food derived peptides: a review on correlation between structure of bioactive peptides and their functional properties. Journal of Food Science and Technology, 56, 535
547. Available from https://doi.org/10.1007/s13197-018-3549-4. Kuhlmann, F. E., Apffel, A., Fischer, S. M., Goldberg, G., & Goodley, P. C. (1995). Signal enhancement for gradient reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol. Journal of the American Society for Mass Spectrometry, 6, 1221
1225. Available from https://doi.org/10.1016/1044-0305(95)00571-4. Le Maux, S., Nongonierma, A. B., & Fitzgerald, R. J. (2015). Improved short peptide identification using HILIC-MS/MS: Retention time prediction model based on the impact of amino acid position in the peptide sequence. Food chemistry, 173, 847
854. Available from https://doi.org/10.1016/j.foodchem.2014.10.104. Le Maux, S., Nongonierma, A. B., Murray, B., Kelly, P. M., & FitzGerald, R. J. (2015). Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate. Food Res Int, 77, 534
539. Available from https://doi.org/10.1016/j.foodres.2015.09.012. Liu, D., Chen, X., Huang, M., & Zhou, G. (2019). Antioxidant activity of peptides in postmortem aged duck meat as affected by cooking and in vitro digestion. Int J Food Prop, 22, 727
736. Available from https:// doi.org/10.1080/10942912.2019.1605374. Liu, R., Xing, L., Fu, Q., Zhou, G., & Zhang, W. (2016). A Review of Antioxidant Peptides Derived from Meat Muscle and By-Products. Antioxidants, 5, 32. Available from https://doi.org/10.3390/antiox5030032. Meo, A. D., Pasic, M. D., & Yousef, G. M. (2016). Proteomics and peptidomics: moving toward precision medicine in urological malignancies. Oncotarget, 7, 52460
52474. Available from https://doi.org/ 10.18632/oncotarget.8931. Miladinovi´c, S. M., Fornelli, L., Lu, Y., Piech, K. M., Girault, H. H., & Tsybin, Y. O. (2012). In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry. Analytical Chemistry, 84, 4647
4651. Available from https://doi.org/10.1021/ac300845n. Montone, C. M., Capriotti, A. L., Cerrato, A., Antonelli, M., La Barbera, G., Piovesana, S., et al. (2019). Identification of bioactive short peptides in cow milk by high-performance liquid chromatography on C18 and porous graphitic carbon coupled to high-resolution mass spectrometry. Analytical and bioanalytical chemistry, 411, 3395
3404. Available from https://doi.org/10.1007/s00216-019-01815-0. Ne`meth-Kiss, V., Forga´cs, E., & Cserha´ti, T. (1997). Anomalous retention behaviour of peptides on porous graphitized carbon column. Journal of Chromatography. A, 776, 147
152. Available from https://doi.org/ 10.1016/S0021-9673(97)00269-0. Nongonierma, A. B., & FitzGerald, R. J. (2016). Strategies for the discovery, identification and validation of milk protein-derived bioactive peptides. Trends in Food Science & Technology, 50, 26
43. Available from https://doi.org/10.1016/j.tifs.2016.01.022. Nongonierma, A. B., & FitzGerald, R. J. (2017). Strategies for the discovery and identification of food proteinderived biologically active peptides. Trends in Food Science & Technology, 69, 289
305. Available from https://doi.org/10.1016/j.tifs.2017.03.003. Nova´kova´, L., Havlı´kova´, L., & Vlˇckova´, H. (2014). Hydrophilic interaction chromatography of polar and ionizable compounds by UHPLC. TrAC Trends Anal Chem, 63, 55
64. Available from https://doi.org/ 10.1016/j.trac.2014.08.004. O’Keeffe, M. B., & Fitzgerald, R. J. (2015). Identification of short peptide sequences in complex milk protein hydrolysates. Food chemistry, 184, 140
146. Available from https://doi.org/10.1016/j. foodchem.2015.03.077. Peng, J., Zhang, H., Niu, H., & Wu, R. (2020). Peptidomic analyses: The progress in enrichment and identification of endogenous peptides. TrAC Trends Anal Chem, 125115835. Available from https://doi.org/ 10.1016/j.trac.2020.115835.

86

Chapter 4

Pereira, L. (2008). Porous Graphitic Carbon as a Stationary Phase in HPLC: Theory and Applications. J Liq Chromatogr Relat Technol, 31, 1687
1731. Available from https://doi.org/10.1080/10826070802126429. Piovesana, S., Capriotti, A. L., Cavaliere, C., La Barbera, G., Montone, C. M., Zenezini Chiozzi, R., et al. (2018). Recent trends and analytical challenges in plant bioactive peptide separation, identification and validation. Analytical and bioanalytical chemistry, 410, 3425
3444. Available from https://doi.org/10.1007/s00216-018-0852-x. Piovesana, S., Capriotti, A. L., Cavaliere, C., La Barbera, G., Samperi, R., Zenezini Chiozzi, R., et al. (2015). Peptidome characterization and bioactivity analysis of donkey milk. Journal of proteomics, 119, 21
29. Available from https://doi.org/10.1016/j.jprot.2015.01.020. Piovesana, S., Capriotti, A. L., Cerrato, A., Crescenzi, C., La Barbera, G., Lagana`, A., et al. (2019). Graphitized Carbon Black Enrichment and UHPLC-MS/MS Allow to Meet the Challenge of Small Chain Peptidomics in Urine. Analytical Chemistry. Available from https://doi.org/10.1021/acs.analchem.9b03034. Piovesana, S., Cerrato, A., Antonelli, M., Benedetti, B., Capriotti, A. L., Cavaliere, C., et al. (2019). A clean-up strategy for identification of circulating endogenous short peptides in human plasma by zwitterionic hydrophilic liquid chromatography and untargeted peptidomics identification. Journal of Chromatography. A460699. Available from https://doi.org/10.1016/j.chroma.2019.460699. Piovesana, S., Montone, C. M., Cavaliere, C., Crescenzi, C., La Barbera, G., Lagana`, A., et al. (2019). Sensitive untargeted identification of short hydrophilic peptides by high performance liquid chromatography on porous graphitic carbon coupled to high resolution mass spectrometry. Journal of Chromatography. A, 1590, 73
79. Available from https://doi.org/10.1016/j.chroma.2018.12.066. Rodriquez, M., Cretoso, D. S., Euterpio, M. A., Russo, P., Crescenzi, C., & Aquino, R. P. (2015). Fast determination of underivatized gentamicin C components and impurities by LC-MS using a porous graphitic carbon stationary phase. Analytical and bioanalytical chemistry, 407, 7691
7701. Available from https://doi.org/10.1007/s00216-015-8933-6. Sa´nchez-Sobero´n, F., Cuykx, M., Serra, N., Linares, V., Belle´s, M., Covaci, A., et al. (2018). In-vitro metabolomics to evaluate toxicity of particulate matter under environmentally realistic conditions. Chemosphere, 209, 137
146. Available from https://doi.org/10.1016/j.chemosphere.2018.06.065. Takahashi, K., Tokuoka, M., Kohno, H., Sawamura, N., Myoken, Y., & Mizuno, A. (2012). Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/ electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry. Journal of Chromatography. A, 1242, 17
25. Available from https://doi.org/10.1016/j.chroma.2012.03.076. Tu, M., Cheng, S., Lu, W., & Du, M. (2018). Advancement and prospects of bioinformatics analysis for studying bioactive peptides from food-derived protein: Sequence, structure, and functions. TrAC Trends Anal Chem, 105, 7
17. Available from https://doi.org/10.1016/j.trac.2018.04.005. West, C., Elfakir, C., & Lafosse, M. (2010). Porous graphitic carbon: A versatile stationary phase for liquid chromatography. Journal of Chromatography. A, 1217:, 3201
3216. Available from https://doi.org/ 10.1016/j.chroma.2009.09.052. Yesmine, B. H., Antoine, B., da Silva Orteˆncia Leoca´dia, N. G., Roge´rio, B. W., Ingrid, A., Nicolas, B., et al. (2017). Identification of ace inhibitory cryptides in Tilapia protein hydrolysate by UPLC
MS/MS coupled to database analysis. J Chromatogr B, 1052, 43
50. Available from https://doi.org/10.1016/j.jchromb.2017.02.015. Zenezini Chiozzi, R., Capriotti, A. L., Cavaliere, C., La Barbera, G., Piovesana, S., Samperi, R., et al. (2016). Purification and identification of endogenous antioxidant and ACE-inhibitory peptides from donkey milk by multidimensional liquid chromatography and nanoHPLC-high resolution mass spectrometry. Analytical and bioanalytical chemistry, 408, 5657
5666. Available from https://doi.org/10.1007/s00216-016-9672-z. Zhu, R., Huang, Y., Zhao, J., Zhong, J., & Mechref, Y. (2020). Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS. Analytical Chemistry, acs. analchem.0c00668. Available from https://doi.org/10.1021/acs.analchem.0c00668.

CHAPTER 5

Methodologies for peptidomics: Identification and quantification Leticia Mora and Fidel Toldra´ Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), Valencia, Spain

5.1 Introduction Traditionally, proteomics has been used to identify and quantify proteins from a mix or an extract, obtaining the characterization of the protein profile of the sample. The most frequently used strategy has been the separation of proteins using gel electrophoresis and the subsequent digestion of the isolated proteins with well-known proteases like trypsin, which specifically cleaves peptides on the C-terminal side of lysine and arginine amino acid residues. Once the proteins are hydrolyzed, the resulting peptides are analyzed by mass spectrometry (MS) approaches, obtaining a list of peaks to be processed by data analysis. This experimental mass profile is matched against the theoretical masses obtained from the in silico digestion of the protein that are included in the databases. This strategy is named protein mass fingerprint, and the results are given as a report where identified proteins are ordered according to the number of coincidences. To identify a protein, it is necessary that the masses of a certain number of peptides show a coincidence with the theoretical masses contained in the databases. The result is statistically analyzed to get the best match (Saraswathy & Ramalingam, 2011). Despite these approaches that have been very useful for a better understanding of protein changes, when the objective is to analyze naturally generated peptides such as those obtained from natural processes as fermentation, curing, or gastrointestinal digestion, the strategy needs to be adapted as traditional proteomics procedures are not adequate. In this sense, peptidomics is the area of science focused on the comprehensive characterization of peptides present in a biological sample by studying their structure, composition, interactions, and properties. Peptidomics has become an essential tool in food science, combined with innovative MS approaches and bioinformatics, to study quality and safety parameters, and it is fundamental for the detection and identification of naturally generated peptides (Peng, Zhang, Niu, & Wu, 2020). In fact, peptidomics has been used to study the proteolysis occurred in natural and complex food processes such as curing or fermentation, to characterize the influence of processing on the generated peptides, to

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00010-8 © 2021 Elsevier Inc. All rights reserved.

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follow the gastrointestinal digestion under in vitro conditions, to identify potential peptide biomarkers, or to detect distinctive peptides that allow the differentiation between species, muscles, or types of products. However, in the analysis of natural peptides, there are numerous complications to be considered due to the complexity of the samples, the changes occurred on peptides in the course of food processing and/or gastrointestinal digestion, and the intrinsic characteristics of short sequence and low amounts of these peptides. The technical evolution of mass spectrometers arisen during the last years and the advance in bioinformatics data analysis have led to an increasing development in quantitative proteomic techniques (Feng, Cappelletti, & Picotti, 2017). Peptidomics methods can be developed for the absolute or relative quantification of peptides, using label-free or labeling approaches. The relative quantification of peptides is focused on the assessment of peptide amounts in two or more samples, and the results are given by the comparison of these amounts between samples. These methodologies provide a simple, reliable, versatile, and cost-effective quantification that can be based on peak intensity measurements or spectral counting (Bantscheff, Schirle, Sweetman, Rick, & Kuster, 2007; Zhu, Smith, & Huang, 2010). On the other hand, the labeled approaches provide the most accurate quantitative values, but they require complex experimental protocols and frequently expensive isotope labels. Among them, multiple reaction monitoring (MRM) is the most appropriate methodology for the analysis of peptides due to its specificity and extensive linear range. MRM is a highly sensitive and selective method of directed MS that uses three quadrupoles for the analysis of short sequence and low concentration peptides in complex mixtures (Picotti & Aebersold, 2012; Sherwood et al., 2009).

5.2 Identification of naturally generated peptides The major difficulty in the identification of naturally generated peptides is just the small size of these fragments which are not previously hydrolyzed using trypsin enzyme. This fact gives several difficulties because the generated peptides are very small and frequently in the limit of some MS instruments range and they are also a complex mixture generated from different proteins of origin and unspecific cleavage sites, that is, in the sample. In this sense, it is critical to use modern proteomic techniques such as MS in tandem to elucidate the sequence of these small peptides, as it allows faster and more reliable identification. In proteomics, the identification of peptides is mostly supported using database search methodologies. This approach has been frequently used in the identification of peptides generated through the action of endogenous enzymes where the main challenge is the correct choice of searching parameters in data analysis (Fricker, Lim, Pan, & Che, 2006; Geho, Liotta, Petricoin, Zhao, & Araujo, 2006; Hardt, Thomas et al., 2005; Hardt, Witkowska, & Webb, 2005; Villanueva, Martorella, & Lawlor, 2006). In this regard, database searching of peptides that have not been trypsin digested is less effective due to

Methodologies for peptidomics: Identification and quantification 89 the absence of the specific cleavage sites in naturally generated peptides. Also, the charge of these peptides can be from 11 to 17, wider than trypsin digested peptides. The main parameters to consider in the data analysis for the identification of naturally generated peptides are the search on the protein databases using nonspecific enzymes as well as the error-tolerant option to search the unmatched spectra considering potential modifications. The most widely used search engines are Mascot, SEQUEST, Phenyx, MassWiz, Hydra, and the free software OMSSA and X!Tandem, although there are other software packages aimed to identify peptide sequences from MS/MS spectra. Between these search engines, Mascot is the most commonly used software, while UniProt and NCBInr are the most commonly used protein databases.

5.3 Materials, equipment, and reagents Reagents: Trifluoroacetic acid (TFA) from Sigma-Aldrich (St. Louis, MO, United States), acetonitrile (ACN), formic acid (FA), and water of MS grade from Scharlab (Sentmenat, Barcelona, Spain). Trypsin protein mixture digest used to optimize source and spray parameters was from LC Packings (P/N 161088): alcohol dehydrogenase, apo-transferrin, bovine serum albumin, beta-galactosidase, cytochrome C, lysozyme. Equipment: Liquid chromatography Ultimate Plus/Famos nano-LC system from LC Packings (Amsterdam, The Netherlands) and mass spectrometer instrument QSTAR XL hybrid quadrupole-time-of-flight (TOF) from AB Sciex (Redwood City, CA, United States) with a nanoelectrospray ion source (ESI). Materials: C18 PepMap trap column (0.3 3 5 mm, 3 μm; LC Packings), Dionex C18 PepMap column (0.075 3 150 mm, 3 μm; LC Packings).

5.3.1 Protocol 1. Dissolve fractions of peptides in loading buffer (0.1% formic acid (FA) and 2% acetonitrile (ACN) and injected. 2. Clean samples and concentrate them on a trap column at a flow rate of 40 μL/min with 0.1% trifluoroacetic acid (TFA) as the mobile phase for 3 minutes. 3. Switch in-line the trap column with a separation column. Mobile phases are solvent A, with 0.1% FA, and solvent B, with 0.1% FA in 95% ACN. 4. Perform the liquid chromatography separation of peptides at a flow rate of 0.2 μL/min for 30 minutes using a linear gradient from 95% to 50% of solvent A. Keep the column outlet straight attached to the nano-electrospray ionization (ESI) source. 5. Use the MS instrument in positive mode. Record the time-of-flight (ToF) MS survey scans for mass range m/z from 350 to 1800 followed by MS/MS in tandem scans of the three most intense peaks.

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5.3.2 Analysis and statistics The analysis of the data to get the identification of peptides is done using Mascot search engine together with Mascot Daemon interface from Matrix Science (Boston, MA, United States), and the ProteinPilot software (AB Sciex, Redwood City, CA, United States) in parallel. The Paragon algorithm is used to search in Uniprot database using the search parameters no enzyme and no taxonomy control. The NCBInr protein database could also be used with no enzyme specificity.

5.3.3 Pros and cons The following are pros and cons: Pros Potential contaminants or interferences are removed using the C18 PepMap trap column step so a better ionization of peptides is obtained when analyzing complex matrices. Peptides are retained in the trap column and later eluted using FA mobile phase. The identification of the peptides can be done using protein databases combined with search engines.

Cons The most polar peptides could be discarded to waste using the C18 PepMap trap column step as they might not be retained in C18. The use of TFA as sample eluent can cause ionization suppression. Complex matrices containing salts, proteins, etc., can cause interferences in the ionization and a decrease in the reproducibility. Some species are not included in the databases so it is necessary to search for the protein of origin using BLAST tool, for example.

5.3.4 Alternative methods/procedures Other authors have identified naturally generated peptides using Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI)-quadrupole (Q)/ToF approaches such as Zhu, Tian, Li, Liu, and Zhao (2017), that uses the liquid chromatography system ACQUITY Ultra Performance Liquid Chromatography (UPLC) attached to a photodiode array detector (PDA) ACQUITY both from Waters (Milford, MA, United States) with a range of 200
700 nm at 45 C. The separation of the peptides was done using a BEH C18 column (100 3 2.1 mm; 1.7 μm particle size, Waters). The chromatographic conditions were solvent A, 0.9% TFA, and solvent B, 0.1% TFA in water/ACN (10:90), using a 5 minute isocratic gradient with solvent A, and a linear gradient from 0% to 60% of solvent B within 40 minutes at a flow rate of 0.3 mL/min. The analysis of MS in tandem was done using a MALDI SYNAPT Q-TOF mass spectrometer instrument from Waters (Milford, MA, United States). The main parameters of the analysis were 200
4000 Da of scanning range, nitrogen as collision gas, capillary

Methodologies for peptidomics: Identification and quantification 91 voltage at 3.5 kV, and cone voltage 20 V. The temperature of the block was 100 C, and the desolvation temperature was 250 C. The collision energy was 15 V, the detector voltage was 1600 V, the desolvation gas flow 500 L/h, and the cone gas flow 50 L/h. On the other hand, the use of MALDI-TOF/TOF could be an alternative as it is a faster and less expensive technique. In fact, the main benefit of MALDI-TOF is its fast turnaround time (,10 minutes) and its low cost in regents and materials and low personnel processing time. On the other hand, sample preparation is also very simple and the type of ionization is very “slight” with little to no fragmentation due to the fact that formed ions have low internal energy (monocharged ions). The highest limitation of MALDI-TOF includes low analytical sensitivity and low reproducibility especially in very complex samples with many peptides to identify without previous isolation.

5.3.5 Troubleshooting and optimization The following are the problems and their solutions: Problem Small size peptides are not identified probably due to trap column does not retain them. Natural peptides showing different polarities and characteristics difficult to isolate using a C18 column. Unstable baseline values with normal pressure of the column. The sequences of the peptides might not have been previously sequenced in the studied species so unknown protein of origin is determined.

Solution Pumping the sample to be analyzed directly into the mass spectrometer using direct infusion MS. Combine the use of C18 column with HILIC stationary phase column to cover the maximum polarity range. Insufficient eluent replacement in the HPLC before ESI so increase the flow to wash the line of HPLC to the ESI. Use of BLAST tool to identify all protein of origin possibilities considering all sequences included in the databases.

HILIC, hydrophilic interaction liquid chromatography; HPLC, High performance liquid chromatography.

5.4 Label-free relative quantitation of naturally generated peptides In peptidomics, the study of peptides often includes their relative quantitation with labeled or label-free methods. There are different approaches for label-free quantitation and the methodology based on peak intensity has been chosen to be described in this chapter. This methodology is based on the measurement of the peak intensity through the integration of the extracted ion chromatogram (EIC) obtained in the MS1, whereas the MS2 will be used for the identification of the sequence (Gallego, Mora, Hayes, Reig, & Toldra´, 2017). It has been established that the EIC values correlate to the amount of the peptide so accurate and precise relative quantitation measurements can be done to evaluate variations in abundance between samples.

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5.4.1 Materials, equipment, and reagents Reagents: TFA from Sigma-Aldrich (St. Louis, MO, United States), ACN, FA, and water were of MS grade from Scharlab (Sentmenat, Barcelona, Spain). A known protein hydrolyzate such as Bovine Serum Albumin (BSA) protein, lactoferrin protein, or beta-hemoglobin protein from Aldrich (St. Louis, MO, United States) hydrolyzated with trypsin enzyme should be included in the sample before MS/MS analysis to normalize the results. Working solutions, at concentrations of 50, 20, 10, 5, 2.5, and 1 fmol/μL, were prepared and subsequently digested with trypsin, to test the linearity of the generated peptides as well as their reproducibility and repeatability under the experimental conditions (see Fig. 5.1 as an example). Tips of Zip-Tip C18 with standard bed format (Millipore Corporation, Bedford, MA) were used before the MS/MS analysis to clean and concentrate the samples. Equipment: Liquid chromatography system Eksigent Nano-LC Ultra 1D Plus system from AB Sciex (CA, United States) coupled to a tandem mass spectrometer with a Q/TOF TripleTOF 5600 1 system from AB Sciex Instruments (MA, United States) with a nanoelectrospray ionization source. Materials: Eksigent C18 trap column (3 μm, 350 μm, 0.5 mm; Eksigent of AB Sciex, CA, United States); nano-HPLC capillary column (3 mm, 75 mm, 12.3 cm, C18; Nikkyo Technos Co., Ltd., Japan).

5.4.2 Protocols 1. Resuspend lyophilized extracts in 100 μL of 0.1% (v/v) TFA. 2. Clean and concentrate 10 μL of each sample using tip cartridges Zip-Tip C18 with standard bed format from Millipore Corporation (Bedford, MA). 3. Concentrate 4 μL of the supernatant using the C18 trap column at a flow rate of 3 mL/min for 5 minutes and using 0.1% TFA as mobile phase. 4. The trap column is automatically switched in-line onto the nano-HPLC capillary column. Mobile phases are 0.1% (v/v) FA in H2O as solvent A, and 0.1% (v/v) FA in ACN as solvent B. HPLC conditions are a linear gradient from 5% to 35% of solvent B over 90 minutes, and 10 minutes from 35% to 65% of solvent B, at a flow rate of 0.3 μL/min at 30 C. 5. Couple the column outlet to the electrospray ionization source, and set the mass spectrometer in tandem to run in positive polarity and information-dependent acquisition mode, with 250 ms TOF MS scan from 300 to 1250 m/z, followed by 50 ms product ion scans from 100 to 1500 m/z on the 50 most intense 1
5 charged ions.

Methodologies for peptidomics: Identification and quantification 93 1.2 1 0.8

Ratio

GLDIQK

0.6

IPAVFK ALPMHIR

0.4

IDALNENK TPEVDDEALEK

0.2 0 0

20

40

60

80

100

-0.2

Concentration (fmol/µl)

Peptide number

Mass (Da)

Peptide sequence

Equation

1

673.388

GLDIQK

y = 0.0104x + 0.0121

2

674.423

IPAVFK

y = 0.0112x - 0.0315

R-squared LOD value (fmol/µl) R2 = 0.99

5

2

5

2

R = 0.99

3

837.476

ALPMHIR

y = 0.0114x - 0.1076

R = 0.97

5

4

916.473

IDALNENK

y = 0.0109x + 0.0091

R2 = 0.99

5

5

1245.584

TPEVDDEALEK

y = 0.0106x + 0.0304

2

R = 0.98

5

Figure 5.1 Representation of the linearity range and regression coefficients of LACB protein (n 5 3), showing the five peptides obtained after trypsin digestion of this protein which were used to normalize the datasets. A digested control sample at a concentration of 100 fmol/L was used to calculate the ratio. The LOD ´, 2015). LACB, β-lactoglobulin; were determined for the five peptides (Gallego, Mora, Aristoy, & Toldra LOD, Limits of detection. Source: Reproduced from Gallego, M., Mora, L., Aristoy, M. C., & Toldra´, F. (2015). Optimisation of a simple and reliable label-free methodology for the relative quantitation of raw pork meat proteins. Food Chemistry, 182, 74
80. https://doi.org/10.1016/j.foodchem.2015.02.114, with the permission of Elsevier.

5.4.3 Analysis and statistics Automated spectral processing, peak list generation, database search, normalization, and quantitative comparisons are performed using Mascot Distiller software (Matrix Science, Inc., Boston, MA, United States). The optimized approach in this study is based on the development of label-free quantitation methodologies through the study of different

(A)

412.6 619

(B)

90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5

(102)

412.0

412.5

413.0

413.5

414.0

414.5

m/z

(Continued)

Methodologies for peptidomics: Identification and quantification 95 replicates of the integration of relative intensities of EICs for the peaks corresponding to the peptides aligned using mass and elution time (Silva et al., 2005; Wang, Wu, Zeng, Chou, & Shen, 2006). When the objective is the identification and relative quantitation at peptide level the robustness of the methodology is very important as any outlier peak indicates one less peptide that is quantified. In this respect, data required for the identification of the peptides can be adequate whereas similar data obtained for the quantitation of same peptides could not reach the quality parameters needed for an adequate quantitation, this is why many peptides are identified but only a few are finally quantified. The identification of the peptides can be done using UniProt or NCBInr databases. The search parameters are specified in the previous section on “Identification of peptides.” A threshold P , 0.5, False Discovery Rate (FDR) of 0.5, and a tolerance of 50 ppm in MS and 0.3 Da in MS/MS are specified in the search parameters of Mascot search engine. The label-free option provided in the Mascot search engine that allows the comparison between peak intensities is also selected. Different quality principles are established to define the peptide ratios used in the quantification and those peaks that are not in the range are eliminated. To obtain the different ratios, peaks are integrated according to the following parameters: standard error of 0.2, correlation coefficient of 0.95, and fraction threshold value of 0.5.

5.4.4 Pros and cons The following are pros and cons (see Fig. 5.2): Pros

L

The accuracy of this protocol is determined by the robustness of the mass accuracy and a reproducible chromatography. The protocol has proved its linearity, reproducibility, and repeatability for plant and animal samples. Simplicity of sample preparation once it is optimized.

Cons It requires careful and reproducible sample preparation techniques. A high resolution of survey scans is necessary to obtain a high number of data points that allow the accurate integration and alignment of the peaks (see Fig. 5.2). The data analysis requires the accurate alignment of retention times permitting the comparison of profiles between samples, noise suppression, best peak picking, and normalization.

Figure 5.2 (A) TICs of extracts of sarcoplasmic proteins at different times of the dry-cured ham processing obtained from nLC
MS/MS analysis. (B) EIC of the peptide YNQLMR used to quantify the ENO protein (Gallego, Mora, Aristoy, & Toldra´, 2016). EIC, Extracted ion chromatogram; TICs, total ion chromatograms. Source: Reproduced from Gallego, M., Mora, L., Aristoy, M. C., & Toldra´, F. (2016). The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham. Food Chemistry, 196, 437
444. https://doi.org/10.1016/j. foodchem.2015.09.062, with permission from Elsevier.

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5.4.5 Alternative methods/procedures There are currently two different strategies for label-free approaches: the spectral counting, and the measurement of relative intensities. In essence, spectral counting is based on counting the total number of spectra identified for a protein in an MS/MS experiment and some examples are emPAI methodology (Exponentially Modified Protein Abundance Index to estimate protein abundances) or APEX methodology (Absolute Protein Expression, improving the spectral counting method including a correction factor for each protein). In this respect the measurement of relative intensities shows certain gains in comparison with other label-free methods where the aim is to evaluate changes in proteins from sample to sample. In conclusion, MS1 quantitation is more precise and accurate than spectral counting, which is very convenient in the assessment of the relative concentration of proteins in a single sample.

5.4.6 Troubleshooting and optimization The following are the problems and their solutions: Problem The quantification of the previously identified peptides using MS2 can be difficult and always there are a higher number of peptides identified than quantified. This is because the spectra quality can be good for identification but not good enough for quantification. The chromatograms must be aligned in MS1 for the relative quantitation of the peptides.

Presence of different outlier points after the statistical analysis of the MS results.

Solution Use of high-resolution equipment with exact mass.

If this not occurs, a peptide or group of known peptides should be used as normalizers to correct the retention times of the chromatogram and align the peak intensities. Main outliers should be removed.

5.5 Absolute quantitation of naturally generated peptides MRM is the most used method for the quantification of peptides in complex mixtures (Hu et al., 2011; Nakashima et al., 2011; Priyanto et al., 2015; Rawendra et al., 2014). This methodology is a significantly sensitive method of targeted MS that can selectively identify and quantify small and low abundance peptides based on the screening of specific transitions generated from the precursor peptides (Lange, Picotti, Domon, & Aebersold, 2008; Picotti & Aebersold, 2012). However, frequently there are some problems in the analysis when peptides have been generated with nonspecific enzymes during processing or gastrointestinal digestion. Another important challenge is the optimization of the MRM parameters of low abundant peptides or small size peptides to get a final accurate and sensitive quantification without

Methodologies for peptidomics: Identification and quantification 97 interferences from other peptides or signal suppression (Capriotti, Cavaliere, Piovesana, Samperi, & Lagana`, 2016; Mora, Gallego, Reig, & Toldra´, 2017).

5.5.1 Materials, equipment, and reagents Reagents: Water and ACN were of LC
MS grade from Sharlab, S.L. (Barcelona, Spain), FA of LC
MS grade from Sigma-Aldrich (St. Louis, MO). Equipment: Liquid chromatography system Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA); triple quadrupole mass spectrometer (QQQ) 6420 Triple Quad LC/MS (Agilent Technologies, Santa Clara, CA) with an electrospray ionization source. Materials: ZORBAX StableBond guard column (5 μm particle size, 1 mm 3 17 mm) from Agilent Technologies (Santa Clara, CA); ZORBAX 300SB-C18 capillary column (5 μm particle size, 250 mm 3 0.3 mm) from Agilent Technologies (Santa Clara, CA).

5.5.2 Protocols 1. Inject 5 μL of each sample on the guard column at a flow rate of 0.1 mL/min for 5 minutes, using 5% (v/v) ACN in water as mobile phase. 2. The guard column is automatically switched in-line onto the chromatographic column. Mobile phases are solvent A 0.1% (v/v) FA and solvent B 100% ACN. Gradient elution is set at a flow rate of 6 μL/min at 30 C as follows: 0
2 minutes, 5% B; 2
20 minutes, linear from 5% to 45% B; 20
25 minutes, linear from 45% to 65% B; 25
28.5 minutes, linear from 65% to 98% B; and 28.5
30 minutes, 98% B. 3. Attach the column outlet to an ESI, and set the triple quadrupole to run in positive polarity to acquire full scan mass spectra from m/z 80 to 1200. The optimized mass spectrometer parameters are nitrogen gas temperature, 350 C; gas flow, 6 L/min; nebulizer pressure, 15 psi; capillary, 3500 V; fragmentor, 100 V; scan time, 500 ms; cell accelerator, 4 V. 4. The synthesized peptides of interest (1000 fmol/μL) are previously analyzed using the QQQ to know and optimize the different parameters such as their m/z and retention time. From each peptide, a standard curve is prepared (100
3000 fmol/μL) using the integrated peak areas extracted from the EIC of the total ion chromatograms (see Fig. 5.3).

5.5.3 Analysis and statistics Samples are processed and data evaluated using MassHunter LC/MS Data Acquisition and MassHunter Qualitative Analysis software (Agilent Technologies), respectively. Quantitative analysis is done in the tripe quadrupole MRM mode after optimization of the acquisition parameters using Skyline software. Peptides to be quantified by MRM are synthesized to prepare a standard curve from 50 to 1000 fmol/μL to study the linearity of

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Figure 5.3 (A) TIC, (B) mass spectrum, and (C) EIC of the peptide AEEEYPDL after MS analysis (Gallego, Mora, ´, 2018). EIC, Extracted ion chromatogram; MS, mass spectrometry; TICs, total ion Aristoy, & Toldra chromatograms. Source: Reproduced from Gallego, M., Mora, L., Aristoy, M. C., & Toldra´, F. (2018). Characterisation of the antioxidant peptide AEEEYPDL and its quantification in Spanish dry-cured ham. Food Chemistry, 258, 8
15. https://doi.org/10.1016/j.foodchem.2018.03.035, with permission from Elsevier.

Methodologies for peptidomics: Identification and quantification 99 the peptides and to calculate the peak areas. These peptides are used as standards to optimize the QQQ instrument parameters. Statistical analysis is done using XLSTAT software (Addinsoft, Barcelona, Spain).

5.5.4 Pros and cons The following are the pros and cons: Pros Accurate and sensitive quantification of peptides. It is possible to detect peptide compounds that cannot be seen by a typical MS approach. Useful in very complex mixtures of peptides as the method is very selective when it is optimized.

Cons Need to synthesize the peptide to study its retention time and establish the calibration curves.

The optimization of MRM parameters is difficult and requires expertise as it is based on the screening of specific transitions obtained from the precursor peptides. The generation of MRM transitions of small peptides is very difficult. Each analysis is specific for one or certain peptides and needs a long time for optimization of the MS parameters and data analysis.

5.5.5 Alternative methods/procedures The absolute quantification gives the accurate concentration of the peptide of interest in the sample. There are different procedures based on metabolic (SILAC) (Han et al., 2019), chemical (iTRAQ, ICAT) (Wu, Liu, Li, & Chi, 2019), and enzymatic (16O/18O) (Barnaby, Wa, Cerny, Clarke, & Hage, 2010) methodologies (see Fig. 5.4). The absolute quantitation using labeled approaches offers the most accurate results although require more complex experimental protocols and the use of expensive labels.

5.5.6 Troubleshooting and optimization The following are the problems and their solutions: Problem Very complex samples can cause interferences and ion signal suppression. No positive MRMs found in the Q1 and Q3 Bad quality in MS/MS spectra

Solution For complex mixtures, it is necessary a clean-up before the chromatographic separation. Probably this is due to incorrect masses were introduced or the collision energy is inappropriate. Inappropriate collision energy.

100 Chapter 5

Figure 5.4 Scheme with the different types of absolute and relative quantitation.

5.6 Summary In the study of naturally generated peptides, there is an increasing interest to improve the understanding of their generation, stability, and effects on different systems, and therefore a better knowledge of the identification and quantitation methodologies based on peptidomics approaches is required. In this sense, a main imperative task to overcome still exists when analyzing complex matrices with naturally generated peptides that have been generated after nonspecific enzymatic action. The identification and quantification of naturally generated peptides require the analytical and experimental variation of previously proteomics strategies using MS in tandem and modern bioinformatics tools. The main adaptation in the identification is the absence of previous trypsin digestion and the use of certain search parameters in the data analysis. Regarding the quantification methodologies, the label-free approaches permit a simple, reliable, versatile, and cost-effective quantification, whereas labeled approaches offer the most accurate quantitative results. In this respect, MRM is a very sensitive and selective methodology that is developed using triple quadrupole instruments for the identification and quantification of small and low abundant peptides in complex mixtures. The main problem of this methodology is the difficulty of the optimization of MRM parameters to perform a sensitive and accurate quantification without interferences or signal suppression, which requires specialized personnel. During the last years, there has been an increase in the use of empirical approaches for the identification and quantification of peptides, which has allowed an important advance in the field of peptidomics. The use of bioinformatics and computational tools reduces the economic cost and time required to develop conventional methodologies used to study the properties or characteristics of the peptides, to predict both the products of the proteolysis and biological activities, and to evaluate potential biomarkers (Agyei, Ongkudon, Wei, Chan, & Danquah, 2018; Sanchez-Rivera, Martinez-Maqueda, Cruz-Huerta, Miralles, & Recio, 2014).

Methodologies for peptidomics: Identification and quantification 101

References Agyei, D., Ongkudon, C. M., Wei, C. Y., Chan, A. S., & Danquah, M. K. (2018). Bioinformatics and peptidomics approaches to the discovery and analysis of food-derived bioactive peptides. Analytical and Bioanalytical Chemistry, 15, 3463. Available from https://doi.org/10.1007/s00216-018-0974-1. Bantscheff, M., Schirle, M., Sweetman, G., Rick, J., & Kuster, B. (2007). Quantitative mass spectrometry in proteomics: A critical review. Analytical and Bioanalytical Chemistry, 389(4), 1017
1031. Available from https://doi.org/10.1007/s00216-007-1486-6. Barnaby, O. S., Wa, C., Cerny, R. L., Clarke, W., & Hage, D. S. (2010). Quantitative analysis of glycation sites on human serum albumin using 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Clinica Chimica Acta, 411(15
16), 1102
1110. Available from https:// doi.org/10.1016/j.cca.2010.04.007. Capriotti, A. L., Cavaliere, C., Piovesana, S., Samperi, R., & Lagana`, A. (2016). Recent trends in the analysis of bioactive peptides in milk and dairy products. Analytical and Bioanalytical Chemistry, 408(11), 2677
2685. Available from https://doi.org/10.1007/s00216-016-9303-8. Feng, Y., Cappelletti, V., & Picotti, P. (2017). Quantitative proteomics of model organisms. Current Opinion in Systems Biology, 6, 58
66. Available from https://doi.org/10.1016/j.coisb.2017.09.004. Fricker, L. D., Lim, J., Pan, H., & Che, F. Y. (2006). Peptidomics: Identification and quantification of endogenous peptides in neuroendocrine tissues. Mass Spectrometry Reviews, 25(2), 327
344. Available from https://doi.org/10.1002/mas.20079. Gallego, M., Mora, L., Aristoy, M. C., & Toldra´, F. (2015). Optimisation of a simple and reliable label-free methodology for the relative quantitation of raw pork meat proteins. Food Chemistry, 182, 74
80. Available from https://doi.org/10.1016/j.foodchem.2015.02.114. Gallego, M., Mora, L., Aristoy, M. C., & Toldra´, F. (2016). The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham. Food Chemistry, 196, 437
444. Available from https://doi.org/10.1016/j.foodchem.2015.09.062. Gallego, M., Mora, L., Aristoy, M. C., & Toldra´, F. (2018). Characterisation of the antioxidant peptide AEEEYPDL and its quantification in Spanish dry-cured ham. Food Chemistry, 258, 8
15. Available from https://doi.org/10.1016/j.foodchem.2018.03.035. Gallego, M., Mora, L., Hayes, M., Reig, M., & Toldra´, F. (2017). Effect of cooking and in vitro digestion on the antioxidant activity of dry-cured ham by-products. Food Research International, 97, 296
306. Available from https://doi.org/10.1016/j.foodres.2017.04.027. Geho, D. H., Liotta, L. A., Petricoin, E. F., Zhao, W., & Araujo, R. P. (2006). The amplified peptidome: The new treasure chest of candidate biomarkers. Current Opinion in Chemical Biology, 10(1), 50
55. Available from https://doi.org/10.1016/j.cbpa.2006.01.008. Han, J., Yi, S., Zhao, X., Zheng, Y., Yang, D., Du, G., . . . Sun, X. (2019). Improved SILAC method for double labeling of bacterial proteome. Journal of Proteomics, 194, 89
98. Available from https://doi.org/10.1016/ j.jprot.2018.12.011. Hardt, M., Thomas, L. R., Dixon, S. E., Newport, G., Agabian, N., Prakobphol, A., . . . Fisher, S. J. (2005). Toward defining the human parotid gland salivary proteome and peptidome: Identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry. Biochemistry, 44(8), 2885
2899. Available from https://doi.org/10.1021/bi048176r. Hardt, M., Witkowska, H. E., Webb, S., et al. (2005). Assessing the effects of diurnal variation on the composition of human parotid saliva: Quantitative analysis of native peptides using iTRAQ reagents. Analytical Chemistry, 77(15), 4947
4954. Available from https://doi.org/10.1021/ac050161r. Hu, Y., Stromeck, A., Loponen, J., Lopes-Lutz, D., Schieber, A., & Ga¨nzle, M. G. (2011). LC-MS/MS quantification of bioactive angiotensin I-converting enzyme inhibitory peptides in rye malt sourdoughs. Journal of Agricultural and Food Chemistry, 59(22), 11983
11989. Available from https://doi.org/10.1021/jf2033329. Lange, V., Picotti, P., Domon, B., & Aebersold, R. (2008). Selected reaction monitoring for quantitative proteomics: A tutorial. Molecular Systems Biology, 4(1), 222. Available from https://doi.org/10.1038/ msb.2008.61.

102 Chapter 5 Mora, L., Gallego, M., Reig, M., & Toldra´, F. (2017). Challenges in the quantitation of naturally generated bioactive peptides in processed meats. Trends in Food Science and Technology, 69, 306
314. Available from https://doi.org/10.1016/j.tifs.2017.04.011. Nakashima, E. M., Kudo, A., Iwaihara, Y., Tanaka, M., Matsumoto, K., & Matsui, T. (2011). Application of 13C stable isotope labeling liquid chromatography
multiple reaction monitoring
tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats. Analytical Biochemistry, 414(1), 109
116. Available from https://doi.org/10.1016/j.ab.2011.02.037. Peng, J., Zhang, H., Niu, H., & Wu, R. (2020). Peptidomic analyses: The progress in enrichment and identification of endogenous peptides. TrAC Trends in Analytical Chemistry, 125, 115835. Available from https://doi.org/10.1016/j.trac.2020.115835. Picotti, P., & Aebersold, R. (2012). Selected reaction monitoring-based proteomics: Workflows, potential, pitfalls and future directions. Nature Methods, 9(6), 555
566. Available from https://doi.org/10.1038/nmeth.2015. Priyanto, A. D., Doerksen, R. J., Chang, C. I., Sung, W. C., Widjanarko, S. B., Kusnadi, J., et al. (2015). Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. Journal of Proteomics, 128, 424
435. Available from https://doi.org/10.1016/j.jprot.2015.08.018. Rawendra, R. D., Chen, S. H., Chang, C. I., Shih, W. L., Huang, T. C., Liao, M. H., et al. (2014). Isolation and characterization of a novel angiotensin-converting enzyme-inhibitory tripeptide from enzymatic hydrolysis of soft-shelled turtle (Pelodiscus sinensis) egg white: In vitro, in vivo, and in silico study. Journal of Agricultural and Food Chemistry, 62(50), 12178
12185. Available from https://doi.org/10.1021/jf504734g. Sanchez-Rivera, L., Martinez-Maqueda, D., Cruz-Huerta, E., Miralles, B., & Recio, I. (2014). Peptidomics for discovery, bioavailability and monitoring of dairy bioactive peptides. Food Research International, 63(Part B), 170
181. Available from https://doi.org/10.1016/j.foodres.2014.01.069. Saraswathy, N., & Ramalingam, P. (2011). Chapter 13—Protein identification by peptide mass fingerprinting (PMF). In N. Saraswathy, & P. Ramalingam (Eds.), Woodhead publishing series in biomedicine, concepts and techniques in genomics and proteomics (pp. 185
192). Woodhead Publishing, ISBN 9781907568107. Sherwood, C. A., Eastham, A., Lee, L. W., Risler, J., Mirzaei, H., Falkner, J. A., & Martin, D. B. (2009). Rapid optimization of MRM-MS instrument parameters by subtle alteration of precursor and product m/z targets. Journal of Proteome Research, 8(7), 3746
3751. Available from https://doi.org/10.1021/pr801122b. Silva, J. C., Denny, R., Dorschel, C. A., Gorenstein, M., Kass, I. J., Li, G. Z., . . . Geromanos, S. (2005). Quantitative proteomic analysis by accurate mass retention time pairs. Analytical Chemistry, 77(7), 2187
2200. Available from https://doi.org/10.1021/ac048455k. Villanueva, J., Martorella, A. J., Lawlor, K., et al. (2006). Serum peptidome patterns that distinguish metastatic thyroid carcinoma from cancer-free controls are unbiased by gender and age. Molecular and Cellular Proteomics, 5(10), 1840
1852. Available from https://doi.org/10.1074/mcp.M600229-MCP200. Wang, G., Wu, W. W., Zeng, W., Chou, C. L., & Shen, R. F. (2006). Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes. Journal of Proteome Research, 5(5), 1214
1223. Available from https://doi.org/10.1021/pr050406g. Wu, X., Liu, L., Li, J., & Chi, F. (2019). Proteome analysis using iTRAQ reveals the differentiation between Tibetan and ordinary ovalbumin peptides. International Journal of Biological Macromolecules, 132, 722
728. Available from https://doi.org/10.1016/j.ijbiomac.2019.03.075. Zhu, C. Z., Tian, W., Li, M. Y., Liu, Y. X., & Zhao, G. M. (2017). Separation and identification of peptides from dry-cured Jinhua ham. International Journal of Food Properties, 20(Suppl. 3), S2980
S2989. Available from https://doi.org/10.1080/10942912.2017.1389954. Zhu, W., Smith, J. W., & Huang, C. M. (2010). Mass spectrometry-based label-free quantitative proteomics. Journal of Biomedicine & Biotechnology, 840518. Available from https://doi.org/10.1155/2010/840518.

CHAPTER 6

Methodologies for bioactivity assay: biochemical study Miryam Amigo-Benavent, Mohammadreza Khalesi, Ganesh Thapa and Richard J. FitzGerald Department of Biological Sciences, University of Limerick, Limerick, Ireland

6.1 Introduction Consumer awareness and demand for disease-preventing and health-promoting food ingredients, known as functional food ingredients, are growing rapidly (Karami & Akbari-adergani, 2019). Bioactive peptides (BAPs) derived from food proteins represent a large group of molecules with biological activities such as antioxidant (Cermen˜o, Connolly et al., 2019; Cermen˜o, Stack et al., 2019; Power-Grant et al., 2016; Wong, Xiao, Wang, Ee, & Chai, 2020), antidiabetic (Harnedy-Rothwell et al., 2020; Kehinde & Sharma, 2020), antiobesity (Kumar, 2019), and antimicrobial activity (Ahmed & Hammami, 2019) along with the potential to act as regulators of satiety (Kondrashina, Brodkorb, & Giblin, 2020). The validation of BAPs with demonstrated biological activity ultimately relies on the assessment of specific in vivo markers. However, due to the time requirement, high cost, and availability of suitable animal models and human volunteers, BAPs are routinely initially screened using a range of in vitro, or biochemical, assays. The in vitro assays are more rapid, less expensive, and less laborious compared to in vivo assessments. Furthermore, these assays may provide valuable information during the screening process in the discovery of novel BAPs. In addition, in vitro assays can play a major role in elucidating the mechanism(s) by which specific BAPs may mediate their physiological effects in vivo. A diverse number of biochemical assays exist for assessment of the biological activity of BAPs. A range of colorimetric, fluorescent, and chemiluminescent approaches are available, for example, for characterization of the ability of specific peptides or crude hydrolyzates (having large numbers of peptides) to display antioxidant activity and to inhibit or activate specific enzyme activities in vitro. Once the specificity and accuracy of these assays are

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00030-3 © 2021 Elsevier Inc. All rights reserved.

103

104 Chapter 6 established, it is possible to adapt assay sensitivity by developing specific substrates, for example, containing fluorescent labels. This in turn can allow miniaturization and adaption for high-throughput screening of large numbers of BAPs. This chapter deals with some in vitro/biochemical assays that are currently routinely utilized in screening of BAPs derived from food protein sources. These include assays that determine antioxidant activity as well as a range of assays based on screening the ability of BAPs to inhibit specific enzymes which can play a role in metabolic control.

6.2 Antioxidant activity assays All living cells are continuously exposed to a variety of challenges exerting oxidative stress; this may be due to exposure to pollutants, ionizing radiation, and other hostile environmental factors. Oxidative stress is either associated with the generation of reactive oxygen species (ROS) or leads to the generation of ROS, including free radicals (Nita & Grzybowski, 2016). The amount of ROS present in cells at any given time depends not only on the generation rate but also on the antioxidant defense system(s) of the organism (Schieber & Chandel, 2014). ROS are linked with the pathophysiology of diseases such as cancer, heart disease, atherosclerosis, aging, diabetes mellitus, along with renal, inflammatory, infectious, and neurologic diseases (Peng, 2009). To survive, cells possess different antioxidant mechanisms including reducing the level of active products by scavenging molecular oxygen, removal of transition metals (Fe, Cu) by binding with proteins and elimination of ROS via interaction with antioxidant compounds (SantosSa´nchez, Salas-Coronado, Villanueva-Can˜ongo, & Herna´ndez-Carlos, 2019). The vast majority of the currently known BAPs is encrypted within the primary structure of proteins and is released mainly by enzymatic processes (Daliri, Lee, & Oh, 2018). Several reviews have dealt with the antioxidative activity of BAPs (Lorenzo et al., 2018; Power, Jakeman, & FitzGerald, 2013). Exogenously generated antioxidative BAPs are considered as a new generation of biologically active regulators: they can prevent oxidation in foods and may contribute to the management of various diseases and disorders, thus improving the quality of life. Just as there are several antioxidant mechanisms, a large variety of corresponding antioxidant assays also exist. There are two main mechanisms of deactivation of radicals: hydrogen atom transfer (HAT) and single electron transfer (SET), and they may occur in parallel. These mechanisms provide a classification of the antioxidant activity assays in HAT-based methods, in which the antioxidant deactivates the free radical by hydrogen donation, and SET-based methods, in which the ability of a potential antioxidant to transfer one electron to reduce any compound from its oxidation state is detected (Prior et al., 2005). In this chapter, the details of commonly used in vitro antioxidant assays employed for the assessment of BAPs derived from food proteins are outlined.

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6.2.1 Ferric-reducing antioxidant power assay The ferric-reducing antioxidant power (FRAP) assay is a relatively simple, rapid, and inexpensive direct method of measuring the total antioxidant activity of reductive (electron-donating) antioxidants in a test sample (Benzie, Chung, & Strain, 1999; Benzie & Strain, 1996). The assay exploits the reduction of ferric (Fe31) to ferrous (Fe21) ions which in turn can be assessed spectrophotometrically. A wide range of sample types can be tested in the FRAP assay. It is a straightforward assay that requires little in the way of specialized equipment and can be semiautomated using a microplate reader. The assay can also be adapted to run in fully automated mode by employing a user-defined program in a biochemical analyzer. The reagents are stable and of low toxicity, the sensitivity and precision of the method are high and the stoichiometric factors of reacting antioxidants are constant over varied concentrations. Furthermore, the test is robust, in that small differences in reaction conditions do not markedly affect the results. In a modified version known as the ferric-reducing activity and ascorbic acid concentration assay, both the nonenzymatic antioxidant capacity and the ascorbic acid (vitamin C) concentration of the test sample can be measured virtually simultaneously (Benzie & Choi, 2014; Benzie et al., 1999; Benzie & Strain, 1996). 6.2.1.1 Definition The FRAP assay does not involve radical generation or the scavenging of added radicals. It uses a simple redox reaction, performed under defined conditions, in which a signal oxidant molecule changes color when it is reduced by the combined action of the redox active, electron-donating reductants (antioxidants) in the test sample. The FRAP assay is classified as a SET reaction mechanism assay (Prior et al., 2005). The FRAP assay uses the following redox reaction: Fe 31salt 1 electron-Fe 21-salt (Benzie & Strain, 1996; Benzie & Strain, 1999; Halliwell & Gutteridge, 2015). It measures the reduction of Fe 31 in the form of an aqueous ferric tripyridyltriazine (TPTZ) salt solution (pale yellow) to the intensively blue colored ferrous form by antioxidants in an acidic medium. Antioxidant activity is determined colorimetrically by monitoring the absorbance at 593 nm and the results are transformed into total antioxidant activity by comparing the change in absorbance obtained in the presence of antioxidants. The results are expressed as a FRAP value, that is, in μmol Fe 21 equivalents or 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox) equivalents (TE) per unit volume or unit weight of test sample. The FRAP assay has been used, for example, to determine the antioxidant activity of protein hydrolyzates from seaweed (Harnedy & FitzGerald, 2013; Stack et al., 2017), fish (Franco et al., 2020; Guo et al., 2015), and brewer’s spent grain (Connolly et al., 2019).

106 Chapter 6 6.2.1.2 Materials, equipment, and reagents • •

• • •

•

• • •

40 mM HCl. Solution A: 300 mM sodium acetate buffer, pH 3.6. Add 1.55 g sodium acetate trihydrate to B450 mL distilled water, adjust to pH 3.6 with acetic acid. Adjust final volume to 500 mL with distilled water. Solution B: 10 mM TPTZ. Dissolve 15.6 mg in 5 mL of 40 mM HCl. Solution C: 20 mM FeCl3  6H2O. Dissolved 27 mg in 5 mL distilled water. FRAP reagent: Prepared by mixing solutions A, B, and C in the following proportion 10:1:1 (v/v/v), protect from light, and use within 3 hours. The FRAP reagent should be pale yellow-orange in color. Preheat at 37 C before use. Standard: Trolox methanolic stock solution. Weigh out 20.64 mg Trolox and dissolve in 40 mL MeOH. Prepare a Trolox standard calibration curve by dissolving the stock solution in a concentration range between 10 and 200 μM with methanol and keep on ice until use. Alternatively, ferrous sulfate can be used as a standard. Dissolve and appropriately dilute the test samples in acetate buffer. Microplate reader. Pipettes, plastic vials, multichannel pipette, tips, and clear 96-well plates.

6.2.1.3 Protocols Microplate set-up: design the microplate layout including triplicates for standards, quadruplicates for test samples, and a sample blank for each sample concentration. It is recommended to use two clear 96-well plates: a sample preparation plate and a reaction plate, to avoid delay and evaporation of methanol. 1. Pipette 120 μL of each sample, blank and standard into the sample preparation plate and cover with parafilm to avoid methanol evaporation. 2. Add 150 μL of prewarmed FRAP reagent to each well in the reaction plate, except for the sample blank to which 150 μL of sodium acetate buffer is added instead. 3. Place the reaction plate in a preheated (37 C) plate reader and determine the absorbance at 590 nm (zero FRAP). 4. Add 20 μL sample/standard to reaction plate directly from sample preparation plate. 5. Mix and incubate the reaction plate at 37 C for 30 minutes. 6. Determine the absorbance 590 nm after 30 minutes. 6.2.1.4 Analysis and statistics Calculate the mean of every triplicate/quadruplicate reading for each standard, control, and sample. Subtract the mean value of the blank (150 μL of FRAP reagent 1 20 μL of methanol). If significant, subtract the sample background (sample blank) from sample readings. Calculate delta values using the formula: ΔAbs 5 (Abs30 min 2 Abszero). Plot the delta values for each standard as a function of the final concentration of Trolox. The change

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in absorbance for the sample is interpolated to calculate the concentration in TE (μM) of the samples. The results are expressed in μmol TE/g sample or μmol TE/mol of peptide. Alternatively, if ferrous sulfate is used as a standard, the results are expressed as μmol Fe21 equivalents /g sample. 6.2.1.5 Safety considerations and standards Avoid contamination: if a visible blue color is seen in the working reagent before the addition of sample, discard the reagent and prepare a new batch as a visible blue color without added antioxidants (sample) which indicates potential contamination by ferrous iron (Fe21) in one or more of the reagents. 6.2.1.6 Pros and cons Table 6.1 summarizes the main advantages and disadvantages of the FRAP assay. 6.2.1.7 Precursor techniques and related techniques The FRAP assay was first developed to measure the “antioxidant power” in plasma (Benzie & Strain, 1996). Although there have been various modifications, the main modification in the method described herein relates to reaction time. The FRAP assay is based on the hypothesis that the redox reaction proceeds quickly and is complete within 4 minutes (Prior et al., 2005), but this is not always the case. Therefore the reaction time has been increased in the assay described herein. Hence, in the original FRAP assay, Benzie and Strain (1996) recommended a 4 minute reaction time such that the total antioxidant capacity (TAC) could be measured without risk of the protein-associated changes in absorbance masking smaller, perhaps more important, changes occurring due to the antioxidant activity of the sample. Table 6.1: Pros and cons of the ferric-reducing antioxidant power (FRAP) assay. Pros Widely used, simple, easy, and inexpensive Assay reagents are stable and low toxicity, homogeneous assay formats, and low cost

Method is sensitive, robust, reproducible, and automatable Provides information about the antioxidant mechanism of peptides DTT, dithiothreitol; GSH, glutathione.

Cons The FRAP assay is carried out under acidic pH conditions (pH 3.6) which is nonphysiological Interference by detergents (TWEEN-20, TRITON X-100, NP-40), reducing compounds (DTT, 2-mercaptoethanol) and metal chelators (EDTA) which should not be present in the test sample No translation from in vitro to in vivo Not appropriate to assess proteins or antioxidant peptides (such as GSH) with a high content of thiol groups since they react very slowly

108 Chapter 6

6.2.2 Oxygen radical absorbance capacity (ORAC) assay The oxygen radical absorbance capacity (ORAC) assay measures the radical chain breaking ability of antioxidants by monitoring the inhibition of peroxyl radical
induced oxidation. The ORAC assay relies on free radical damage to a fluorescent probe, most commonly fluorescein (FL), caused by an oxidizing reagent resulting in a loss of fluorescent intensity over time (Ou, Hampsch-Woodill, & Prior, 2001). ORAC has emerged as a popular method to measure TAC due to its low cost and its amenability for adaption to high-throughput automation in a microplate format (Cao et al., 1999; Huang, Ou, Hampsch-Woodill, Flanagan, & Deemer, 2002). The ORAC assay is a robust analytical method to determine the antioxidant potential of a range of substances that may be found in food products (Rice-Evans & Miller, 1994). The assay has been widely used, for example, for assessment of the in vitro antioxidant activity of BAPs from milk (Contreras, Herna´ndez-Ledesma, ´ lvarez, & Recio, 2011; Le Maux, Nongonierma, Barre, & Fitzgerald, 2016; Amigo, Martı´n-A Lo´pez-Expo´sito, Quiro´s, Amigo, & Recio, 2007; O’Keeffe & FitzGerald, 2014; Power-Grant et al., 2016), soybean (Amigo-Benavent et al., 2014), egg (Carrillo et al., 2016), seaweed (Harnedy & FitzGerald, 2013), ham (Gallego, Mora, Reig, & Toldra´, 2018), brewer’s spent grain (Connolly et al., 2019), fish (Neves et al., 2017), and other protein sources. 6.2.2.1 Definition The ORAC assay is a simple, sensitive, and reliable method of quantitating the oxygen radical absorbance capacity (ORAC) of antioxidants. The ORAC assay is classified as a HAT-based method (Prior et al., 2005). It was originally developed for testing serum but now it is used widely to screen a range of different compounds (Cao, Alessio, & Cutler, 1993). In the protocol described herein, FL is used as the fluorescent probe, 2,20 -azo-bis (2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator and Trolox (a water-soluble vitamin E analog), as a positive control standard. This assay is based on the method developed by Ou et al. (2001). The uniqueness of this assay is that the TAC of a sample is estimated by taking the oxidation reaction to completion. The results are quantified by monitoring the fluorescence decay and calculating the net integrated area under the fluorescence decay curve. This approach accounts for lag time, the initial state, and the total extent of inhibition (Prior et al., 2005). Trolox is used to generate a standard curve (by plotting area under the curve (AUC) versus Trolox concentration) and to determine ORAC values for test samples. Thus ORAC results are expressed as TE/amount of sample as calculated from comparison to a Trolox calibration curve. 6.2.2.2 Materials, equipment, and reagents • •

Assay buffer: 75 mM phosphate buffered saline pH 7. 1.17 mM FL sodium salt stock solution. Dissolved to give a 0.78 μM FL working solution in assay buffer before use. Keep protected from light.

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Radical generator: 221 mM AAPH in assay buffer, dilute in prewarmed buffer immediately prior to use. Standard: 1 mM Trolox stock solution in 50 mL assay buffer. If there are problems with solubility, dissolved Trolox in 1 mL of methanol and make up to 50 mL with assay buffer. Prepare a Trolox calibration curve in a concentration range between 10 and 120 μM. Plate reader with fluorescence detection. Multichannel pipettes, tips, and black 96-well plates.

6.2.2.3 Protocols Add 50 μL of blank (buffer), Trolox standards, and samples to assigned wells. Add 50 μL of FL to each well with the multichannel pipette. Mix by repeated pipetting. Incubate in the plate reader for 15 minutes at 37 C. Determine the background FL (excitation: 485 nm, emission: 520 nm) every 60 seconds for 1 minute. 5. Add 25 μL of AAPH to each well. 6. Then immediately begin determination of the reaction fluorescence every 5 minutes for 120 minutes.

1. 2. 3. 4.

6.2.2.4 Analysis and statistics Calculate AUC for each sample from the relative fluorescence data. Calculate net AUC by subtracting AUCblank from the sample AUC using the formula net AUC 5 AUCSample 2 AUCblank. Plot the net AUC value for each standard as a function of the final concentration of Trolox. Interpolate the net AUC values for samples to calculate the equivalent Trolox concentration of the samples. The results are expressed as TE per mg, mL, or μmol of sample. 6.2.2.5 Safety considerations and standards A fluorescence decay curve (fluorescence intensity vs time) needs to be constructed. Final fluorescence should be less than 5% of the initial fluorescence reading to ensure that the reaction has gone to completion. All samples should be assayed as independent triplicates. A Trolox standard curve should be included on each microplate for antioxidant capacity determination and for conversion to TE. A blank, using buffer instead of sample, should be included to calculate net AUC. A control to assess fluorescence stability, where AAPH is substituted by an equal volume of buffer, should also be included. 6.2.2.6 Pros and cons Table 6.2 summarizes the main advantages and disadvantages of the ORAC assay.

110 Chapter 6 Table 6.2: Main pros and cons of the ORAC assay. Pros Low cost, high precision, rapid, and accurate High reproducibility and repeatability when the temperature is carefully controlled Allows for high-throughput analysis on automation

Cons A temperature-sensitive method Reducing compounds (DTT, 2-mercaptoethanol) and metal chelators (e.g., EDTA) may interfere with the assay No direct translation to in vivo antioxidant activity

DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid.

6.2.2.7 Precursor techniques and related techniques The ORAC assay is based largely on the work reported by Glazer (1990) as modified by Cao et al. (1993) and Cao and Prior (1998) and relies on assessing the effect of potential antioxidants by measuring fluorescence quenching. β-Phycoerythrin was initially employed as the fluorescent probe in early ORAC studies (Cao et al., 1993); however, this was unstable and produced high interference. It was therefore substituted with FL (Ou et al., 2001). Modification of the ORAC assay following the QUENCHER (quick, easy, new, cheap, and reproducible) approach for samples that present solubility problems has been described (Amigo-Benavent, del Castillo, & Fogliano, 2010). The ORAC assay appears to be the only antioxidant assay that brings the free radical action to completion and uses an AUC approach for quantitation. Therefore it combines both the extent of inhibition (%) and the duration of inhibition of the free radical action by antioxidants into a single readout.

6.2.3 Trolox-equivalent antioxidant capacity assay The Trolox-equivalent antioxidant capacity (TEAC) assay (Rice-Evans & Miller, 1994) measures the ability of antioxidants to scavenge a stable radical cation, that is, 2,20 -azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS•1). The TEAC assay is a colorimetric method that can be adapted to microplate format. It has been used to assess the TAC of serum or plasma to resolve the difficulty in measuring each antioxidant component separately and the interactions between antioxidants therein. The TEAC assay has also been employed to measure the TAC of pure substances, body fluids, and foods. The TEAC assay is classified as a SET reaction-based assay, the HAT mechanism may also apply depending on the structure of the antioxidant (Prior et al., 2005). The TEAC assay has been widely used, for example, to assess BAPs from dairy (Go´mez-Ruiz, Lo´pez-Expo´sito, Pihlanto, Ramos, & Recio, 2008; Herna´ndez-Ledesma, Quiro´s, Amigo, & Recio, 2007), soybean (Amigo-Benavent et al., 2014), seafood (Kleekayai et al., 2015), ham (Gallego et al., 2018), and brewer’s spent grain (Connolly et al., 2019) sources. 6.2.3.1 Definition The TEAC assay is based on the reduction in color (absorbance at 735 nm) of radical cations of ABTS•1 by antioxidants in test samples which converts the radical into its

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reduced colorless ABTS22 form (Huang, Boxin, & Prior, 2005). The degree of decolorization induced by a test compound is related to its antioxidant activity and it is calculated by using Trolox as a standard. The protocol described herein is a modification of the assay described by Re et al. (1999). 6.2.3.2 Materials, equipment, and reagents •

• • •

• • • • •

Assay buffer: 5 mM phosphate buffer saline pH 7.4. Weigh 0.5711 g Na2HPO4 (Mr 141.96 g/mol), 0.24 g KH2PO4 (Mr 136.09 g/mol), 8 g NaCl (Mr 58.44 g/mol), and 0.2 g KCl (Mr 74.55 g/mol) and dissolve in B900 mL distilled water. Adjust to pH 7.4 with 1 M HCl and make up to 1000 mL with distilled water. Oxidizing agent: 2.45 mM potassium persulfate (K2S2O8) in distilled water (50 mL). 7 mM ABTS solution (freshly prepared). Weigh 19.2 mg ABTS (Mr 548.68 g/mol) and make up to 5 mL with distilled water. Radical generator solution: ABTS•1 radical stock solution. Add oxidizing agent to ABTS solution at a ratio of 1:2 (v/v), mix and leave the mixture at room temperature in the dark for 16 hours to generate ABTS•1. ABTS•1 working solution. Dilute radical generator solution with assay buffer to obtain an absorbance at 734 nm of 0.7 6 0.2. Dissolve samples in assay buffer. Standard: Trolox calibration curve in the range 10
80 μM. Microplate reader. Pipettes, multichannel pipette, plastic vials, tips, and clear 96-well plates.

6.2.3.3 Protocol 1. Add 10 μL of sample at the appropriate dilution, standard, or buffer (control blank) to the assigned wells in a 96-well microplate. 2. Add 200 μL of ABTS•1 working solution to each well. 3. Mix and incubate in the dark at 30 C for 6 minutes (some test samples may need a longer time to reach the endpoint). 4. Determine the absorbance at 734 nm. 6.2.3.4 Analysis and statistics There are three ways to express the results of TEAC analyses: as a percentage of inhibitory activity, as a TEAC value or as the concentration of sample that gives a 50% reduction on ABTS•1 absorbance (EC50). The inhibitory activity of a test sample can be calculated using the following formula: Inhibitory activity ð%Þ 5

ðAC 2 ACB Þ 2 ðAS 2 ASB Þ 3 100 ðAC 2 ACB Þ

112 Chapter 6 where AC is the absorbance of control, ACB the absorbance of control blank, AS the absorbance of sample, and ASB the absorbance of sample blank. The TEAC is calculated by plotting the absorbance obtained as a function of the Trolox concentration and interpolating the net absorbance value obtained for samples (Re et al., 1999). EC50 estimation requires testing of different concentrations of the peptide/hydrolyzate of interest to generate data to graph the absorbance versus the log of the concentration of the sample, and the EC50 value corresponds to the concentration of antioxidant that causes a 50% decrease in the ABTS•1 absorbance (Kleekayai et al., 2015). 6.2.3.5 Safety considerations and standards The TEAC assay requires a reliable determination of the endpoint of the reaction for quantitative evaluation of antioxidant activity (Prior et al., 2005). It is recommended to test all test samples as independent triplicates. 6.2.3.6 Pros and cons Table 6.3 summarizes the main advantages and disadvantages of the TEAC assay. 6.2.3.7 Precursor and related techniques The TEAC assay was first reported by Miller and Rice-Evans (1997) using metmyoglobin and hydrogen peroxide to generate a ferrylmyoglobin radical, which was then reacted with ABTS to produce ABTS•1. Later on, Re et al. (1999) improved the protocol by using potassium persulfate as the oxidizing reagent. The TEAC assay has been automated and adapted for microplate and to flow injection formats (Milardovic, Kerekovi´c, Derrico, & Rumenjak, 2007). It may also be coupled with HPLC by including a postcolumn reaction with ABTS•1 to facilitate the search for individual antioxidants within complex mixtures. Modification of the TEAC assay following the QUENCHER approach for samples that present solubility problems has been described (Serpen, Capuano, Fogliano, & Go¨kmen, 2007). Table 6.3: Pros and cons of the Trolox-equivalent antioxidant capacity assay. Pros High reproducibility and repeatability Widely used, simple, and easy Sensitive, robust, and automatable Uses small amounts of sample and can be used over a wide pH range and with different solvents EC50 value estimation allows comparison between different samples ABTS, 2,20 -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid).

Cons ABTS 1 is a nonphysiological radical Radicals need to be generated before the assay (during an overnight reaction) It may take a long time to reach an endpoint for slow reactions Antioxidants with low redox potentials ( . 0.68 V) can reduce ABTS 1 to ABTS22 G

G

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6.2.4 Other antioxidant activity assays Estimation of a relative antioxidant capacity index (RACI) has been proposed as an integrated approach to evaluate overall food component antioxidant capacity. This approach combines the results from seven different in vitro assays, that is, the TEAC, FRAP, ORAC, hydroxyl radical absorbance capacity (HORAC), cupric ion
reducing antioxidant capacity (CUPRAC), total radical-trapping antioxidant parameter (TRAP) assays, and the phenol antioxidant index (PAOXI) (Sun & Tanumihardjo, 2007). PAOXi is calculated by dividing the total phenol concentration (determined by the Folin
Ciocalteu assay) by the half-maximal inhibitory concentration obtained from the inhibition assay associated with low-density lipoprotein (LDL) oxidation (Sun & Tanumihardjo, 2007). The HORAC assay determines the antioxidant activity against hydroxyl radicals (OH•). In this assay, OH• is generated by a Co(II)-mediated Fentonlike reaction. In similarity with the ORAC assay, FL is used as a probe and the area under the fluorescence decay curve is monitored in the presence and absence of the test antioxidant compound to allow determination of the antioxidant activity (Ou et al., 2002). The CUPRAC assay is a colorimetric ET-based assay. In similarity with the FRAP assay, it is based on a metallic redox reaction, the CUPRAC assay consists a redox reaction using a bis(neocuproine)copper(II) cation (Cu(II)-Nc), acting as the chromogenic oxidizing reagent, and a bis(neocuproine) copper(I) cation (Cu(I)-NC), acting as the chromophore. The reaction is developed at room temperature for 30 minutes and the absorbance at 450 nm is monitored allowing calculation of CUPRAC ¨ zyu¨rek, Gu¨c¸lu¨, & Apak, 2011). The TRAP assay determines antioxidant activity (O activity against peroxyl radicals generated as in the ORAC assay via thermal decomposition of AAPH using R-phycoerythrin as a fluorescence probe or a colorimetric (ABTS) probe. TRAP antioxidant activity is determined as the time to consume all the antioxidant, by extension of the lag time for the oxidized probe, its results are expressed as lag time or reaction time of the sample compared to corresponding times for Trolox (Prior et al., 2005). PAOXI is calculated by dividing the total phenol concentration (μmol/kg) from the Folin
Ciocalteu assay by the halfmaximal inhibitory concentration (EC50) in μM from the LDL oxidation inhibition assay. The LDL assay measures the antioxidant’s ability to inhibit autooxidation of LDL as caused by Cu21 or AAPH (Sun & Tanumihardjo, 2007). These methods are more frequently used in the study of the antioxidant activity of phenolic compounds. Apart from the antioxidant activity assays described herein, there are several other relevant assays such as the total antioxidant scavenging capacity, the photochemiluminescence assay, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay which have been reviewed by Prior et al. (2005). Moreover, the superoxide radical scavenging assay (Sun, Wang, Fang, Gao, & Tan, 2004), the DNA nicking assay for hydroxyl radical scavenging activity (Sun et al., 2004), the thiobarbituric acid reactive

114 Chapter 6 substances assay along with an antioxidant assessment electrophoresis assay (Wilson et al., 2002), ROS scavenging assays (Kim, Kang, & Yokozawa, 2008; Qian, Jung, Byun, ˇ ˇ & Kim, 2008; Stajner, Popovi´c, Canadanovi´ c-Brunet, & Boˇza, 2006), and a complex thiol assay (Thapa, Das, & Gunupuru, 2016) may also be used for assessment of the antioxidant properties of different test samples.

6.3 Enzyme inhibitory assays Enzymes play an important role in regulating key metabolic pathways in the human body, for example, angiotensin-I-converting enzyme (ACE) is a central component of the renin
angiotensin system (RAS), which controls blood pressure. Modification of the activity of enzymes is targeted during the development of various drug compounds for the management of different diseases. This includes the inhibition of ACE, for control of blood pressure, and dipeptidyl peptidase IV (DPP-IV) for glycemic management in type 2 diabetes (T2D). Therefore there has been an increasing interest in studying the enzyme inhibitory activity of food components, including proteins and their associated peptides, as naturally derived disease management agents in the form of functional food ingredients. Performance of in vitro enzyme inhibitory assays allows the screening and characterization of different food components as sources of compounds of potential benefit in the management of different diseases. The principle of these assays is based on measuring activity on incubating the enzyme with specific chromogenic, fluorogenic, or luminescent substrates and comparison of the catalytic activity in the presence and absence of a potential inhibitor. The enzyme inhibitory activities of BAPs derived from food proteins which have been most studied include inhibition of the following: ACE and renin for hypertension and cardiovascular disease; DPP-IV, α-amylase, and α-glucosidase for T2D; lipase activity for obesity; trypsin and chymotrypsin for anticancer activity and satiety; tyrosinase for UV protection and acetylcholinesterase (AChE) for neurodegenerative disorders. In this chapter, some of the inhibition assays employed for key metabolic enzymes are outlined and some pros and cons of the assays are provided.

6.3.1 Assay of angiotensin-I-converting enzyme inhibition Angiotensin-converting enzyme (ACE, EC 3.4.15.1), also known as peptidyl-dipeptide hydrolase, is a carboxypeptidase that plays a key role in a number of blood pressure controlling systems such as the kinin nitric oxide and renin
angiotensin pathways (Norris & FitzGerald, 2013; Fig. 6.1). Therefore the discovery of compounds that suppress ACE activity, thereby minimizing bradykinin (a potent vasodilator) breakdown and the formation of angiotensin II (a potent vasoconstrictor), is a strategy for the management and treatment of hypertension.

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Figure 6.1 The role of angiotensin-I-converting enzyme and renin in hypertension.

6.3.1.1 Definition A number of different methods are used to quantify ACE activity. However, the method developed by Carmel and Yaron (1978) is currently in routine use. This method is based on the intramolecularly quenched fluorescent tripeptide o-aminobenzoylglycyl-p-nitro-Lphenylalanyl-L-proline (Abz-Gly-Phe(NO2)-Pro). Hydrolysis of this substrate by ACE releases Abz-Gly which is quantified fluorimetrically. The positive control routinely used in this assay is Captopril (D-3-mercapto-2-methyl-propionyl-L-proline, C9H15NO3S), a synthetic drug compound prescribed for the control of blood pressure. Similar to many other synthetic ACE inhibitors, Captopril blocks the conversion of angiotensin I to angiotensin II along with blocking the breakdown of bradykinin (Odaka & Mizuochi, 2000). The ACE inhibitory assay described herein using Abz-Gly-Phe(NO2)-Pro is adapted from the methods of Carmel and Yaron (1978) and Norris, Casey, FitzGerald, Shields, and Mooney (2012). 6.3.1.2 Materials, equipment, and reagents • • • • •

• •

Assay buffer: 100 mM sodium borate (pH 8.3) containing 300 mM NaCl. Enzyme: Rabbit-lung ACE (alternatively bovine lung ACE can be used). Dissolve ACE in assay buffer to give an enzyme activity of 5 mU/mL. Substrate: Abz-Gly-Phe-(NO2)-Pro. Dissolve in assay buffer to give a concentration of 0.45 mM. Standard: Abz-Gly-OH. Dissolve in assay buffer to give concentrations ranging between 0 and 100 μM. Positive control inhibitor: Captopril. Dissolve in assay buffer to give a range of concentrations between 0 and 10,000 nM. The IC50 (concentration of inhibitor giving 50% inhibition of ACE) value for Captopril has been reported to be 17 nM (Ando et al., 1987). Microplate reader and water bath. Pipettes and multichannel pipette, tips, and black 96-well microplate.

116 Chapter 6 6.3.1.3 Protocol 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Background: Add 50 μL of assay buffer 1 200 μL of substrate. Negative control: Add 50 μL of assay buffer 1 200 μL of substrate. Positive control: Add 50 μL of Captopril solution 1 200 μL of substrate. Test sample: Add 50 μL of test sample 1 200 μL of substrate. Standard sample: Add 300 μL standard. Gently shake the microplate for a few seconds. Preincubate the microplate at 37 C for 10 minutes. Preincubate the enzyme solution at 37 C for 10 minutes. Add 50 μL of enzyme to the positive and negative controls, and the test sample wells, and add 50 μL assay buffer to the background well. Gently shake the microplate for a few seconds. Record the fluorescence using excitation and emission wavelengths of 360 and 400 nm, respectively at t0. Shake-incubate the plate at 37 C for 30 minutes. Record the fluorescence at t30.

6.3.1.4 Analysis and statistics One unit of ACE activity (U/mL) is defined as the amount of enzyme hydrolyzing 1 μmol of Abz-Gly-Phe-(NO2)-Pro per min at 37 C. A calibration curve generated with Abz-Gly within the concentration range 0
100 μM versus fluorescence units is plotted to calculate the activity units of ACE. The units of ACE activity can be calculated from the standard curve. The ACE inhibitory (%) activity of each sample in the different wells is related to the fluorescence generated during a 30 minutes incubation period (t30 2 t0) and it is calculated as follows: ACE inhibition ð%Þ 5

ðFN 2 FBÞ 2 ðFS 2 FBÞ 3 100 ðFN 2 FBÞ

where FN, FB, and FS represent the change in fluorescence during 30 minutes incubation for the negative control (enzyme 1 substrate without inhibitor which gives 100% activity), background, and the test samples/inhibitors, respectively. It is also possible to determine an IC50 value (concentration of inhibitor giving 50% inhibition of ACE) for a given inhibitor. This is achieved by assaying different concentrations of the inhibitor to obtain ACE inhibition (%) values at each concentration. Plotting the log of the concentration of inhibitor (x-axis) versus percentage ACE inhibition (y-axis) yields a sigmoidal curve (Vermeirssen, Van Camp, & Verstraete, 2002). The IC50 value corresponds to the x-value (concentration) where y 5 50%.

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6.3.1.5 Alternative methods/procedures A commercial ACE activity assay kit is available from Sigma-Aldrich. This kit has been developed on the basis of cleavage of a synthetic fluorogenic peptide. The measured fluorescence is directly proportional to the ACE activity present. The method developed by Cushman and Cheung (1971) has been used for many years to determine the ACE inhibitory potential of different compounds. It is based on the hydrolysis of the synthetic substrate hippuryl-L-histidine-leucine (HHL) by ACE. Spectrophotometric (λ 5 228 nm) measurement and fluorescence-based quantification of the hippuric acid released by ACE are employed to quantify ACE inhibitory activity. Another synthetic peptide that has been used as a substrate for ACE activity determination is N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG). This substrate is converted to FAP and GG by the action of ACE and the reaction can be monitored spectrophotometrically (Gorski & Campbell, 1991; Henda et al., 2013; Murray, Walsh, & FitzGerald, 2004). However, these methods have some limitations, such as the need to extract the HHL generated using an organic solvent in one instance and the need for continuous monitoring of the enzyme reaction in the case of the FAPGG assay.

6.3.2 Assay of renin inhibition Renin (EC 3.4.99.19) is an aspartyl protease which is released from the renal juxtaglomerular cells in response of some physiological changes such as sodium depletion, and blood volume and pressure reduction (Persson, 2003). Renin plays an important role in the RAS. Renin is a highly selective enzyme that catalyzes the rate-determining step in the conversion of angiotensinogen (the only known substrate of renin) to angiotensin I. This is considered as an advantage of renin over ACE inhibition. This is due to the fact that ACE is a nonselective enzyme that cleaves bradykinin in addition to angiotensin I and as a consequence certain side effects have been reported with ACE inhibitors (Yuan, Wu, Aluko, & Ye, 2006). Thus inhibition of renin provides an attractive alternative approach for the management of hypertension and associated cardiovascular diseases (Aluko, 2019) (Fig. 6.1). 6.3.2.1 Definition The renin-inhibitory assay described herein is based on the use of a synthetic peptide substrate containing a fluorophore 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS) at the N-terminal region and a nonfluorescent chromophore 4-(4dimethylaminophenylazo) benzoyl (DABCYL) at the C-terminal region. Cleavage of this substrate by renin leads to the release of Arg-Glu-EDANS-OH, which is highly fluorescent and thus can be fluorometrically quantified (Wang, Chung, Holzman, & Krafft, 1993). The

118 Chapter 6 renin-inhibitory assay described herein is adapted from the methods of Wang et al. (1993), Yuan et al. (2006), Bhullar, Ziaullah, and Rupasinghe (2014), Khalil, Motaal, Meselhy, and Abdel Khalek (2018), and Garcia-Vaquero, Mora, and Hayes (2019). 6.3.2.2 Materials, equipment, and reagents • • •

• • • •

Assay buffer: 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl. Substrate: 500 μM Arg-Glu-EDANS-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-LysDABCYL-Arg in dimethyl sulfoxide (DMSO). Inhibitor positive control: Dissolve Z-Arg-Arg-Pro-Phe-His-Sta-Ile-His-Lys(Boc)-OMe giving a concentration range between 0 and 10,000 nM. The IC50 value for Z-Arg-ArgPro-Phe-His-Sta-Ile-His-Lys(Boc)-OMe is ,1 μM (Fitzgerald et al., 2012). Sample solution: Dissolve the inhibitors in solvents such as DMSO, methanol, or ethanol based on the nature of the sample. Enzyme: Renin (human recombinant). Dissolve renin in assay buffer to give an enzyme activity of 5000 U/10 μL. Microplate reader and water bath. Pipettes, multichannel pipettes, and black 96-well plates.

6.3.2.3 Protocols 1. Background: Add 20 μL of substrate 1 150 μL of assay buffer 1 10 μL DMSO. 2. Negative control: Add 20 μL of substrate 1 150 μL of assay buffer 1 10 μL DMSO. 3. Positive control: Add 20 μL of substrate 1 150 μL of assay buffer 1 10 μL Z-Arg-ArgPro-Phe-His-Sta-Ile-His-Lys(Boc)-OMe solution. 4. Test sample: Add 20 μL of substrate 1 150 μL of assay buffer 1 10 μL test sample. 5. Gently shake the microplate for few seconds. 6. Preincubate the microplate at 37 C for 10 minutes. 7. Preincubate the enzyme solution at 37 C for 10 minutes. 8. Add 10 μL of renin solution to test samples and positive and negative controls, and add 10 μL assay buffer to background wells. 9. Determine the fluorescence using excitation and emission wavelengths of 340 and 490, respectively, at t0. 10. Shake-incubate the plate at 37 C for 45 minutes. 11. Determine the fluorescence at t45. 6.3.2.4 Analysis and statistics One unit of renin activity will produce one fluorescent unit from the hydrolysis of Arg-GluEDANS-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys-DABCYI-Arg per min at pH 8.0 and at 37 C. The renin-inhibitory (%) activity of each sample in the different wells is related to

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the fluorescence generated during a 45 minutes incubation period (t45 2 t0) and it is calculated as follows: Renin inhibition ð%Þ 5

ðFN 2 FBÞ 2 ðFS 2 FBÞ 3 100 ðFN 2 FBÞ

where FN, FB, and FS represent the change in fluorescence during 45 minutes incubation for the negative control (enzyme 1 substrate without inhibitor which gives 100% activity), background, and the test samples/inhibitors, respectively. It is also possible to determine an IC50 value (level of inhibitor giving 50% inhibition of renin) of a given inhibitor. This is achieved by assaying different concentrations of the inhibitor to obtain renin inhibition (%) values at each concentration. Plotting the log of the concentration of inhibitor (x-axis) versus percentage renin inhibition (y-axis) yields a sigmoidal curve. The IC50 value corresponds to the x-value (concentration) where y 5 50%. 6.3.2.5 Alternative methods/procedures As already mentioned, there is only one known substrate for renin and therefore alternative methods do not exist. A commercial renin-inhibitory assay kit is available from Cayman Chemicals (Ann Arbor, MI, United States) which has been widely used by many authors (Fu, Alashi, Young, Therkildsen, & Aluko, 2017; Yang et al., 2017). This kit has been developed on the basis of the same approach (i.e., quantification of the release of fluorenylmethyloxycarbonyl-Glu(EDANS)), as outlined earlier.

6.3.3 Assay of dipeptidyl peptidase IV inhibitory activity DPP-IV (EC 3.4.14.5) is a serine exopeptidase which can cleave Xaa-Pro, Xaa-Ala, and Xaa-HydroxyPro from the N-terminus of proteins/peptides. DPP-IV is ubiquitously distributed in the body, with the highest levels being observed in the proximal tubule of the kidney and on the luminal membrane of epithelial cells in the small intestine (Loijda, 1979). Inhibition of DPP-IV increases the half-life of the incretin hormones (glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide) and promotes insulinotropic activity (Kawanami, Matoba, Sango, & Utsunomiya, 2016). Synthetic DPP-IV inhibitors, known as gliptins, are in existence as validated drugs for the management of hyperglycemia. Given the side effects associated with synthetic drug compounds, interest in the discovery of food protein
derived peptide inhibitors of DPP-IV as natural glucoregulators has increased (Power, Nongonierma, Jakeman, & FitzGerald, 2014). 6.3.3.1 Definition This protocol describes the DPP-IV inhibitory activity assay for food protein
derived peptides. The assay measures the increase in fluorescence due to the liberation of 7-amino-

120 Chapter 6 4-methyl-coumarin (AMC) on cleavage of the peptide bond in Gly-Pro-AMC by DPP-IV. The protocol described herein was developed by Kato, Nagatsu, Kimura, and Sakakibara (1978) and modified by Harnedy and FitzGerald (2013). The protocol has been extensively used for the determination of the DPP-IV inhibitory activity of protein hydrolyzates/ peptides such as those from milk (Lacroix & Li-Chan, 2012; Nongonierma et al., 2019; Nongonierma & FitzGerald, 2013a, 2013b; Power-Grant et al., 2015), fish (Harnedy et al., 2018; Harnedy-Rothwell et al., 2020; Li-Chan, Hunag, Jao, Ho, & Hsu, 2012), macroalgal (Cermen˜o, Connolly et al., 2019; Harnedy & FitzGerald, 2013; Harnedy, O’Keeffe, & Fitzgerald, 2015; Pimentel, Alves, Harnedy, FitzGerald, & Oliveira, 2019), brewer’s spent grain (Cermen˜o, Stack et al., 2019; Connolly, Piggott, & FitzGerald, 2014), bean (de Souza Rocha, Hernandez, Chang, & de Mejı´a, 2014; Oseguera-Toledo, Gonzalez de Mejia, & Amaya-Llano, 2015), and insect (Nongonierma, Lamoureux, & FitzGerald, 2018) sources. 6.3.3.2 Materials, equipment, and reagents • • • •

• • •

Assay buffer: 20 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl and 1 mM EDTA. Standard: 20 mM AMC dissolved in DMSO. Substrate: 5 mM H-Gly-Pro-AMC stock solution. Prepare a calibration curve ranging in concentration from 1 to 6 μM in assay buffer. Inhibitor positive control: 1 mM N-(1-L-isoleucyl-L-prolyl)-L-isoleucine (Diprotin A) stock solution. The IC50 (concentration of inhibitor giving 50% inhibition of enzyme) value for Diprotin A has been reported to be B5 μM (Harnedy & FitzGerald, 2013). Enzyme: a working solution of 8 mU/mL DPP-IV (from human or porcine origin) freshly prepared in assay buffer immediately before use. Plate reader with fluorescence detection. Multichannel pipettes, tips, and black 96-well plates.

6.3.3.3 Protocols 1. Background: add 50 μL of assay buffer 1 50 μL of substrate. 2. Negative control: add 40 μL of assay buffer 1 50 μL of substrate. 3. Positive control: add 10 μL of 50 μM Diprotin A 1 50 μL of substrate 1 30 μL of assay buffer. 4. Test sample: add 10 μL of appropriate concentrations of sample 1 50 μL of substrate 1 30 μL of assay buffer. 5. Standard: add 100 μL of standard. 6. Mix and incubate at 37 C for 5 minutes in the plate reader. 7. Add 10 μL of DPP-IV to negative control, positive control, and sample designed wells to start the reaction, mix, and incubate at 37 C for 30 minutes. 8. Determine the fluorescence at time zero and at 30 minutes at excitation and emission wavelengths of 360 and 460 nm, respectively.

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6.3.3.4 Analysis and statistics One unit of DPP-IV activity is defined as the amount of enzyme that hydrolyzes 1 μmol of H-Gly-Pro-AMC per min at 37 C. Using the calibration curve for AMC, the concentration of AMC generated is calculated during a 30 minute period for the reaction in the absence (100% activity) and in the presence of test sample. There are two common ways to express the results: as a percentage of inhibition and as a half-maximal inhibitory concentration (IC50), that is, the concentration of sample needed to inhibit DPP-IV activity by 50%. When expressed as a percentage of inhibition, the results are calculated using the following equation: % inhibition 5 ((a 2 b)/a) 3 100; where a is the concentration of AMC in 100% enzyme activity and b is the concentration of AMC in the presence of test sample. When data are expressed as IC50, different concentrations of the sample are tested and their % of inhibition is calculated. Plotting % inhibition values versus the log of the sample concentration allows calculation of an IC50 value as described earlier for ACE inhibitory activity. 6.3.3.5 Precursor and related techniques The first assay described for measuring DPP-IV activity was based on the spectrophotometric quantification of p-nitroaniline (pNA) released from Gly-Pro-pNA (Nagatsu, Hino, Fuyamada, Hayakawa, & Sakakibara, 1976), and later on, Kato et al. (1978) developed a fluorogenic assay using 7-(Gly-Pro)-methylcoumarin. A real-time colorimetric assay has been described which is based on the aggregation of gold nanoparticles functionalized with peptide substrates such as Gly-Pro-Asp-Gly and Val-Pro-ethylene diamine-Asp-Cys (Aldewachi, Woodroofe, Turega, & Gardiner, 2017). This approach helps avoid interference when assaying DPP-IV activity in serum samples. 6.3.3.6 Alternative methods/procedures The assays to determine DPP-IV inhibitory activity employ colorimetric substrates such as glycine-proline-para-NA (Gly-Pro-pNA) and fluorescent derivatives such as gly-pro-7amino-4-methylcoumarin (Gly-Pro-AMC) and gly-pro-4-methoxy-β-naphthylamide (GlyPro-4-Me-β-NA). However, the latter is a carcinogenic agent that limits its use (Gard et al., 2017). Comparison of the characteristics of some of these assays has been published by Matheeussen et al. (2012).

6.3.4 Assay of α-amylase inhibitory activity α-Amylase (EC 3.2.1.1) or α-1,4-glucan-4-glucanohydrolase is an endoglucanase that hydrolyzes the endo α-(1,4)-glycosidic linkages in starch and related polysaccharides and therefore plays a key role in carbohydrate metabolism. α-Amylase is expressed in salivary and mammary glands, in the pancreas, and the jejunum (Groot et al., 1989). Mammalian

122 Chapter 6 amylases are composed of three structural domains: A, B, and C. Domain A is the largest and it contains the active site residues including two Asp and one Glu and a bound Cl2 ion. Domain B contains a bound Ca21 ion which is involved in stabilization of the protein’s conformation and domain C is located at the N-terminal region and is not involved in the catalytic mechanism (Butterworth, Warren, & Ellis, 2011). α-Amylase hydrolyzes both amylose and amylopectin in starch to produce maltose, maltotriose, and limit dextrins (Gropper, Smith, & Carr, 2018). Inhibition of α-amylase reduces the extent of digestion and thus the absorption of starch and carbohydrate breakdown products and thereby may contribute to a decrease in blood glucose level (Yan, Zhao, Yang, & Zhao, 2019). Therefore inhibition of this enzyme is a target for the management/treatment of T2D. 6.3.4.1 Definition This protocol describes the α-amylase activity assay used for the assessment of food protein
derived inhibitory peptides. α-Amylase inhibitory activity is determined by measuring the release of reducing sugars from starch in the presence and absence of inhibitor. Reducing sugars are quantified by reaction with 3,5-dinitrosalicylic acid (DNS) which oxidizes the aldehyde groups developing color which is measurable at 540 nm. The protocol described herein was originally developed by Bernfeld (1955) to quantify reducing sugars and was subsequently modified by Kwon, Vattem, and Shetty (2006). This protocol, with some modifications, has been used for the determination of α-amylase inhibitory activity of food protein
derived peptides such as those in egg (Yu, Yin, Zhao, Liu, & Chen, 2012), brewer’s spent grain (Connolly et al., 2014), beans (Oseguera-Toledo et al., 2015), cumin seed (Siow, Lim, & Gan, 2017), and red algae (Admassu, Gasmalla, Yang, & Zhao, 2018). 6.3.4.2 Materials, equipment, and reagents • • • •

•

•

Assay buffer: 20 mM sodium phosphate buffer (pH 6.9) containing 6 mM NaCl. Standard: 5 M maltose stock solution in distilled water. Prepare a calibration curve of maltose in the range 0.3
5.0 mM in assay buffer. Substrate: 1% (w/v) potato starch dissolved in assay buffer. Inhibitor positive control: acarbose used in the concentration range between 10 and 100 μg/mL. The IC50 value for acarbose has been reported to be B70 μg/mL (Chelladurai & Chinnachamy, 2018). Enzyme: porcine pancreatic α-amylase working solution (5 mU/mL) freshly prepared in assay buffer immediately before use. It is possible to substitute porcine with human α-amylase. Color reagent: DNS. Prepared by dissolving 1 g of DNS in 50 mL of distilled water and by slowly adding 30 g Na-K tartrate tetrahydrate, then add 20 mL of 2 N NaOH and make up to 100 mL with distilled water. Protect from carbon dioxide and store no longer than 2 weeks (Worthington, 2020).

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Spectrophotometer. Pipettes, tips, and tubes.

6.3.4.3 Protocols 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

Background: 700 μL of assay buffer. Negative control: 200 μL of assay buffer 1 500 μL of α-amylase solution. Positive control: 200 μL of inhibitor 1 500 μL of α-amylase solution. Test sample: 200 μL of an appropriate dilution of the peptide/hydrolyzate 1 500 μL of α-amylase solution. Standard sample: 200 μL of standard 1 1000 μL of assay buffer. Mix and incubate at 25 C for 5 minutes. Add 500 μL of a 1% (w/v) starch solution to start the reaction in background, negative control, positive control, and test sample assigned tubes and incubate all tubes at 25 C for exactly 10 minutes. Add 1 mL of DNS color reagent solution to stop the reaction. Incubate at 100 C for 5 minutes and cool to room temperature. Dilute the mixture by adding 10 mL of distilled water. Determine the absorbance at 540 nm.

6.3.4.4 Analysis and statistics One unit of α-amylase activity is defined as the amount of enzyme needed to release 1 μmol of reducing groups from soluble starch (calculated as maltose) per min at 25 C and pH 6.9 (Worthingong, 2020). Using the calibration curve for maltose, the concentration of reducing sugar released by α-amylase is calculated during a 10 minute period for reactions in the absence (100% activity) and in the presence of test sample. In similarity with the DPP-IV inhibitory assay, there are two common ways to express the results: as a percentage of inhibition and as an IC50 value (see earlier). 6.3.4.5 Precursor and related techniques The precursor of this assay was originally described by Sumner and Howell (1935) to determine saccharase activity with DNS reagent, and later it was reported by Bernfeld (1955) in Methods in Enzymology to determine α and β-amylase activity. 6.3.4.6 Alternative methods/procedures There are many methods to analyze amylase activity that can be used in turn to determine α-amylase inhibitory activity. These assays can be grouped into amyloclastic, saccharogenic, and chromogenic methods (Brena, Pazos, Franco-Fraguas, & Batista-Viera, 1996). Apart from the DNS assay, other spectrophotometric assays use amylopectin dyed with Reactone Red 26 (Babson & Babson, 1973) or Ostazin brilliant red H-2B (Biely, Mislovicova´, Markovic, & Kala´c, 1988) to determine α-amylase activity. Chromatographic

124 Chapter 6 methods are less popular in determining α-amylase activity, for a review see Brena et al. (1996).

6.3.5 Assay of α-glucosidase inhibitory activity α-Glucosidase (EC 3.2.1.20) is a carbohydrate hydrolase located in the brush border of the small intestine that catalyzes the hydrolysis of carbohydrates (oligosaccharides, disaccharides) to liberate glucose which is absorbed and enters the bloodstream (Matsui, Yoshimoto, Osajima, Oki, & Osajima, 1996). It is a key enzyme involved in the digestion and absorption of glucose in the small intestine. Inhibition of α-glucosidase slows down glucose release; therefore α-glucosidase inhibitors are used as therapeutic targets for the treatment/management of T2D (Ghosh & Collier, 2012). However, pharmacological α-glucosidase inhibitors for T2D treatment such as acarbose, miglitol, and voglibose can be associated with side effects including abdominal discomfort. Therefore there is a growing interest in the discovery of food-derived inhibitors of α-glucosidase as natural glucoregulators (Liu, Gao, Tang, & Nie, 2017). 6.3.5.1 Definition This assay describes a protocol to determine α-glucosidase inhibitory activity for BAPs and uses rat intestine as a source of α-glucosidase activity. The method colorimetrically determines α-glucosidase activity by quantifying the release of p-nitrophenol from p-nitrophenyl-α-Dglucopyranoside (pNPG) in the presence or absence of inhibitor at 405 nm. The protocol described herein was originally developed by Siegenthaler (1977) and subsequently modified by Jo et al. (2011). This assay, employing different sources of α-glucosidase, has been used for the determination of α-glucosidase inhibitory activity in food protein
derived peptides such as in egg (Yu et al., 2012), brewer’s spent grain (Connolly et al., 2014), beans (Castan˜eda-Pe´rez et al., 2019; Oseguera-Toledo et al., 2015), chia seed (Sosa Crespo, Laviada Molina, ChelGuerrero, Ortiz-Andrade, & Betancur-Ancona, 2018), millet grain (Kara´s et al., 2019), cumin (Siow et al., 2017), and microalgae (Hu, Fan, Qi, & Zhang, 2019). 6.3.5.2 Materials, equipment, and reagents • • •

•

Assay buffer: 0.1 M phosphate buffer (pH 6.9). Substrate: 5 mM pNPG solution in assay buffer. Positive control inhibitor: Acarbose. Prepare a calibration curve in the concentration range between 10 and 100 μg/mL in assay buffer. The IC50 value for acarbose has been reported to be B65 μg/mL using the assay with commercial porcine α-glucosidase (Chelladurai & Chinnachamy, 2018). Enzyme: α-glucosidase from rat intestine (see below for extraction), alternatively commercially available rat intestine α-glucosidase activity can be used. Dilute to 36 mU/mL with assay buffer.

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Microplate reader. Multichannel pipettes, tips, and clear 96-well plates.

6.3.5.3 Protocols Extraction of α-glucosidase activity from rat intestine: 1. Grind 1 g of rat-intestinal acetone powder with 4 mL of 0.1 M phosphate buffer (pH 6.9) using a mortar and pestle and then stir gently at 4 C overnight. 2. Centrifuge at 10,000g at 4 C for 15 minutes and the resulting supernatant consists of the α-glucosidase extract. 3. Dilute the α-glucosidase extract with assay buffer to a working solution containing 36 mU/mL. Determination of α-glucosidase inhibition: Background: Add 150 μL of assay buffer. Negative control: Add 50 μL of assay buffer 1 100 μL of α-glucosidase solution. Positive control: Add 50 μL of inhibitor 1 100 μL of α-glucosidase solution. Test sample: Add 50 μL of appropriately diluted test sample 1 100 μL of α-glucosidase solution. 5. Mix and incubate at 37 C for 10 minutes. 6. Add 50 μL of substrate to each well and mix. 7. Incubate at 37 C for 30 minutes in a microplate reader and monitor the absorbance at 405 nm every 2 minutes. 1. 2. 3. 4.

6.3.5.4 Analysis and statistics One unit of α-glucosidase activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 μmol of p-nitrophenyl per min at 37 C and pH 6.9 (Berthelot & Delmotte, 1999). Using the maltose calibration curve, the concentration of reducing sugars released can be calculated during a 10 minute incubation period for the reaction in the absence (100% activity) and in the presence of test sample. As in the case of the DPP-IV inhibitory assay, there are two common ways to express the results: as a percentage of inhibition and an IC50 value (see earlier). 6.3.5.5 Precursor and related techniques The colorimetric assay presented herein is the most common technique employed to determine α-glucosidase inhibitory activity and there are also commercially available kits (e.g., Sigma-Aldrich; FUJIFILM Wako Pure Chemical Corporation) based on this assay. Quantification of p-nitrophenol at 400 nm has also been used for screening of α-glucosidase inhibition by phenolic compounds using capillary electrophoresis (Hamdan & Afifi, 2010) along with HPLC-based approaches (Chen & Guo, 2017).

126 Chapter 6 6.3.5.6 Alternative methods/procedures Alternative methods for the assessment of α-glucosidase inhibitory activity have been reviewed (Zhang et al., 2020). These include the use of new colorimetric substrates, along with fluorescence and electrochemical detection strategies.

6.3.6 Assay of lipase inhibitory activity Obesity is developed when there is an imbalance between energy intake and energy expenditure, this in turn may lead to a variety of diseases including diabetes, atherosclerosis, hypertension, and functional depression in certain organs (Karri, Sharma, Hatware, & Patil, 2019). Current strategies for the prevention of obesity are associated with the regulation of food intake and a reduction in the absorption of dietary lipid. Inhibition of dietary triglyceride digestion is also a promising approach in the reduction of lipid absorption (Mukherjee, 2003). Lipase (EC 3.1.1.3) activity plays a key role in the efficient digestion of triglycerides (Fig. 6.2). Inhibition of lipase is therefore one of the most widely studied mechanisms for determining the potential efficacy of natural products as antiobesity agents. 6.3.6.1 Definition Two main methods are used for the measurement of lipase activity (assays A and B). The principle of method A is based on the hydrolysis of p-nitrophenyl esters by lipase and the release of the yellow chromogen, p-nitrophenol, which can be measured spectrophotometrically. It has been shown that this measurement is linearly related to the release of titratable free fatty acids (Blake, Koka, & Weimer, 1996). The principle of method B is based on the hydrolysis of 4-methylumbelliferyl oleate (4-MUO) by lipase and the release 4-MU which can be fluorometrically quantified (Jacks & Kircher, 1967). The synthetic drug, Orlistat, with a structural formula of [(S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]-dodecyl ester] and a chemical formula of C29H53NO5 is used as a positive control for both assays. Orlistat is a chemically synthesized derivative of lipstatin, derived from Streptomyces toxytricini, with the ability to irreversibly inhibit pancreatic and gastric lipase (Heck, Yanovski, & Calis, 2000).

Figure 6.2 The action of lipase in the hydrolysis of triglycerides.

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6.3.6.2 Assay A The lipase inhibitory assay described herein is adapted from the methods of Blake et al. (1996), Bendicho, Trigueros, Herna´ndez, and Martı´n (2001), Wang et al. (2010), Chedda, Kaikini, Bagle, and Seervi (2016), and Jakubczyk, Szymanowska, Karas´, Złotek, and Kowalczyk (2019). 6.3.6.2.1 Materials, equipment, and reagents

• • • •

• •

Assay buffer: 100 mM potassium phosphate buffer (pH 7.5). Enzyme: Porcine pancreatic lipase. Dissolve in assay buffer to give an activity of 50 mU/mL. Substrate: 4-Nitrophenyl acetate. Dissolve in DMSO to give a concentration of 5 mM. Inhibitor positive control: Orlistat. Dissolve in DMSO to give a final concentration of 20 μg/mL in the reaction mixture. The IC50 value for Orlistat with method A has been reported to be 25
35 μM (Conforti et al., 2012; Jaradat et al., 2017). Microplate reader and water bath. Pipettes, multichannel pipettes, tips, and 96-well plates.

6.3.6.2.2 Protocols

1. 2. 3. 4. 5. 6. 7. 8. 9.

Background: Add 2 μL of lipase 1 142 μL assay buffer 1 5 μL DMSO. Negative control: Add 2 μL of lipase 1 142 μL assay buffer 1 5 μL DMSO. Positive control: Add 2 μL of lipase 1 142 μL assay buffer 1 5 μL Orlistat. Test Sample: Add 2 μL of lipase 1 142 μL assay buffer 1 5 μL appropriately diluted test samples. Preincubate at 30 C for 3 minutes. Preincubate the substrate at 30 C for 3 minutes. Add 1 μL of the substrate to test sample, and positive and negative control wells, and 1 μL DMSO to the background wells. Shake-incubate at 30 C for 10 minutes. Determine the absorbance at 405 nm at t10.

6.3.6.2.3 Analysis and statistics

The lipase inhibitory (%) activity of each sample in the different wells is related to the absorbance after 10 minutes and it is calculated as follows: Lipase inhibition ð%Þ 5

ðAN 2 ABÞ 2 ðAS 2 ABÞ 3 100 ðAN 2 ABÞ

where AN, AB, and AS represent the absorbance after 10 minutes incubation for the negative control (enzyme 1 substrate without inhibitor which gives 100% activity), background, and the test samples/inhibitors, respectively.

128 Chapter 6 It is also possible to determine an IC50 value (level of inhibitor giving 50% inhibition of lipase) of any given inhibitor by assaying different concentrations of the inhibitor to obtain lipase inhibition (%) values at each concentration. Plotting the concentration of inhibitor (x-axis) versus percentage lipase inhibition (y-axis) yields a linear curve. The IC50 value corresponds to the x-value (concentration) where y 5 50%. 6.3.6.3 Assay B The lipase inhibitory assay described herein is adapted from the methods of Jacks and Kircher (1967), Shimura, Tsuzuki, Kobayashi, and Suzuki (1992), Kurihara, Asami, Shibata, Fukami, and Tanaka (2003), and Buchholz and Melzig (2016). 6.3.6.3.1 Materials, equipment, and reagents

• • • •

• • • •

Assay buffer: 13 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 1.3 mM CaCl2. Enzyme: Porcine pancreatic lipase. Dissolve in assay buffer to give an activity of 50 mU/mL. Substrate: 4-MUO. Dissolve in DMSO to reach a concentration of 0.5 mM. Positive control inhibitor: Orlistat. Dissolve in DMSO to reach a concentration of 20 μg/mL in the reaction mixture. The IC50 value for Orlistat with method B has been reported to be 32 μM (Sergent, Vanderstraeten, Winand, Beguin, & Schneider, 2012). Sample solution: Dissolve the test samples in DMSO. Stop-reaction reagent: 0.1 M sodium citrate (pH 4.2). Microplate reader. Pipettes, multichannel pipette, tips, and black 96-well microplates.

6.3.6.3.2 Protocols

Background: Add 50 μL of substrate 1 25 μL DMSO 1 100 μL assay buffer. Negative control: Add 50 μL of substrate 1 25 μL DMSO 1 100 μL assay buffer. Positive control: Add 50 μL of substrate 1 25 μL Orlistat 1 100 μL assay buffer. Test Sample: Add 50 μL of substrate 1 25 μL test samples 1 100 μL assay buffer. Gently shake the microplate for a few seconds. Add 25 μL of lipase solution to samples and positive and negative controls and add 25 μL assay buffer to the background wells. 7. Shake-incubate the plate at 25 C for 30 minutes. 8. Add 100 μL of 0.1 M sodium citrate. 9. Determine the fluorescence using excitation and emission wavelengths of 335 and 460, respectively, at t30.

1. 2. 3. 4. 5. 6.

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6.3.6.3.3 Analysis and statistics

The lipase inhibitory (%) activity of each sample in the different wells is related to the fluorescence generated after 30 minutes and it is calculated as follows: Lipase inhibition ð%Þ 5

ðFN 2 FBÞ 2 ðFS 2 FBÞ 3 100 ðFN 2 FBÞ

where FN, FB, and FS represent the fluorescence after 30 minutes incubation for the negative control (enzyme 1 substrate without inhibitor which gives 100% activity), background, and the test samples/inhibitors, respectively. It is also possible to determine an IC50 value (level of inhibitor giving 50% inhibition of lipase) of an inhibitor by assaying different concentrations of the inhibitor to obtain lipase inhibition (%) values at each concentration by plotting the log of the concentration of inhibitor (x-axis) versus percentage lipase inhibition (y-axis) which yields a sigmoidal curve. The IC50 value corresponds to the x-value (concentration) where y 5 50%.

6.3.7 Assay of tyrosinase inhibitory activity Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that has two activities associated with its catalytic action, that is, it catalyzes the ortho-hydroxylation of monophenols to catechols and their subsequent oxidation to ortho-quinones (Schurink, van Berkel, Wichers, & Boeriu, 2007). Tyrosinase activity is essential for melanogenesis and pigmentation, and it is synthesized by epithelial, mucosal, retinal, and ciliary body melanocytes (Merimsky, Shoenfeld, Baharav, Zigelman, & Fishman, 1996). Inhibition of tyrosinase activity is important in food (to avoid enzymatic browning), in cosmetics (to produce whitening agents), and in medical applications (to treat skin disorders and skin cancer). Kojic acid, arbutin, and hydroquinone are known tyrosinase inhibitors but these compounds may be toxic at certain doses (European Commission, 2012); therefore the discovery of alternative natural tyrosinase inhibitors is of interest. 6.3.7.1 Definition This protocol describes the tyrosinase inhibitory assay used for testing food protein
derived peptides. Tyrosinase inhibitory activity is spectrophotometrically determined (at 475 nm) by measuring the increase in dopachrome formation following the hydrolysis of L-Tyr. The protocol presented herein was described by Likhitwitayawuid and Sritularak (2001) and was modified for assessment of the inhibitory activity of peptides/ hydrolyzates by Harnedy, Soler-Vila, Edwards, and FitzGerald (2014). Tyrosinase inhibitory activity has been reported in several food protein
derived hydrolyzates (Addar, Bensouici, Si Ahmed Zennia, Boudjenah Haroun, & Mati, 2019; Harnedy et al., 2014; Nam, You, & Kim, 2008; Schurink et al., 2007; Zhuang et al., 2009).

130 Chapter 6 6.3.7.2 Materials, equipment, and reagents • • • • • •

Assay buffer: 50 mM potassium phosphate buffer (pH 6.8). Substrate: 7.5 mM L-Tyr in assay buffer. Inhibitor positive control: Kojic acid (0.1
0.5 mM). According to Saghaie, Pourfarzam, Fassihi, and Sartippour (2013), the IC50 for kojic acid is 0.28 mM. Enzyme: mushroom tyrosinase ( . 1000 U/mg solid) from Agaricus bisporus (T3824, Sigma-Aldrich). Dissolve in assay buffer to obtain 186 U/mL. Plate reader. Multichannel pipettes, tips, and clear 96-well microplates.

6.3.7.3 Protocols 1. Background: Add 160 μL of assay buffer 1 40 μL of L-Tyr solution. 2. Negative control: Add 120 μL of assay buffer 1 40 μL of L-Tyr solution. 3. Positive control: Add 40 μL of inhibitor 1 80 μL of assay buffer 1 40 μL of L-Tyr solution. 4. Test sample: Add 40 μL of the appropriately diluted protein hydrolyzate in assay buffer 1 80 μL of assay buffer 1 40 μL of L-Tyr solution. 5. Mix and incubate at 25 C for 5 minutes. 6. Add 40 μL of tyrosinase to start the reaction in negative and positive controls and test sample assigned wells. 7. Mix and monitor the absorbance at 475 nm over a 30 minute period using a plate reader. 6.3.7.4 Analysis and statistics One unit of tyrosinase activity represents a change of one absorbance unit per min at 475 nm at 25 C. Calculate the slope, by choosing two time points (t1 and t2) in the linear range, and their corresponding values for absorbance (A1 and A2). The slope is obtained by dividing the net absorbance (A2 2 A1) by the net time (t2 2 t1). The percentage inhibition is then calculated using the following equation:    % inhibition 5 Slope100% enzyme activity 2 SlopeSample =Slope100% enzyme activity 3 100 The IC50 value can be determined by plotting the % inhibition as a function of inhibitor concentration and calculating the concentration as previously described for the ACE inhibition assay (see earlier). 6.3.7.5 Precursor and related techniques Mason (1948) described a spectrophotometric method to determine tyrosinase activity using L-dihydroxyphenylalanine in test tube format. Later on, the method was modified and

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adapted for use in microplate format (Masuda, Yamashita, Takeda, & Yonemori, 2005). Other substrates have been described that can be used for the determination of tyrosinase inhibitory activity and these have been reviewed by Garcı´a-Molina et al. (2007). 6.3.7.6 Alternative methods/procedures Apart from the assay described herein, different chromatographic (Garcı´a & Furlan, 2015), electrophoretic (Jiang, Liang, Wang, Zhang, & Lv, 2013), and electrochemical (Ruzza et al., 2017) methods have been described to determine tyrosinase inhibition.

6.3.8 Assay of trypsin inhibitory activity Trypsin (EC 3.4.21.4) is a serine protease that hydrolyzes peptides on the carboxylic acid side of lysine and arginine (Yu & Ahmedna, 2012). Trypsin is synthetized as a proenzyme in the pancreas and is transported to the small intestine where it is activated and participates in the digestion of proteins and peptides (Baird & Craik, 2013). Protease inhibitors can regulate various cellular activities preventing or ameliorating unregulated proteolysis, which may be involved in the progression of cancer (De Mejia & Dia, 2010). It has also been reported that protease inhibitors could help to regulate appetite and energy balance involving the satiety hormones, thereby indicating their potential application in the management of obesity and metabolic syndrome
related conditions (de Lima, Piuvezam, Leal Lima Maciel, & Heloneida de Arau´jo Morais, 2019). 6.3.8.1 Definition This protocol describes a trypsin inhibitory assay to test food protein
derived hydrolyzates/ peptides. Trypsin activity is determined by measuring the increase in nitroanilide released during the hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA). The protocol described herein was developed by Kakade (1974) and modified by Domoney and Welham (1992). The Worthingong (2020) describes a similar assay using benzoyl-L-arginine ethyl ester as substrate. These assays have been used to determine trypsin inhibitory activity of low molecular mass proteins/peptides, such as the Bowman
Birk inhibitor, from soybean (Amigo-Benavent, Nitride, Bravo, Ferranti, & Del Castillo, 2013; Clemente, MacKenzie, Jeenes, & Domoney, 2004; Clemente, Moreno, Marı´n-Manzano, Jime´nez, & Domoney, 2010) and pea (Clemente, Carmen Marı´n-Manzano, Jime´nez, Carmen Arques, & Domoney, 2012), and also for food-derived peptides from fish (Manikkam, Mathai, Street, Donkor, & Vasiljevic, 2016), and pea (Awosika & Aluko, 2019) sources. 6.3.8.2 Materials, equipment, and reagents • •

Assay buffer: 50 mM Tris-HCl, pH 8.2. Substrate: BAPNA—dissolve in DMSO (40 mg/mL) and store aliquots at 220 C in the dark. Dilute to 0.4 mg/mL in assay buffer before use.

132 Chapter 6 •

Positive control inhibitor: Trypsin-chymotrypsin Bowman Birk inhibitor (BBI) from Glycine max (soybean) (ref T9777 from Sigma-Aldrich). The IC50 value for BBI has been reported to be 33 ng using N-acetyl-DL-phenylalanine-β-naphthylester (APNE) as substrate (Yakoby & Raskin, 2004) although no IC50 value was found for the protocol described herein; however, this value can be calculated using a range of concentrations as in the case for test samples. Enzyme: N-p-Tosyl-L-phenylalanine chloromethyl ketone
treated trypsin from bovine pancreas $ 10,000 U/mg protein (ref T1426 from Sigma-Aldrich). Dissolve to give 200 U/mL using 1 mM HCl. 30% (v/v) acetic acid. Spectrophotometer. Pipettes, tips, tubes, and cuvettes.

•

• • •

6.3.8.3 Protocols 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Background: Add 200 μL of assay buffer 1 200 μL of 1 mM HCl into a test tube. Negative control: Add 200 μL of assay buffer 1 200 μL of trypsin solution into a test tube. Positive control: Add 200 μL of inhibitor 1 200 μL of trypsin solution into a test tube. Test sample: Add 200 μL of appropriately diluted peptide/hydrolyzate sample 1 200 μL of trypsin solution into a test tube. Mix and preincubate at 37 C for 60 seconds. Add 500 μL of substrate to start the reaction and incubate at 37 C for 10 minutes. Add 100 μL of acetic acid to stop the reaction. Determine the absorbance at 410 nm. Reaction tubes without inhibitor/sample are considered to represent 100% enzyme activity. Trypsin inhibitor from soybean can be used as a positive control of enzyme inhibition.

6.3.8.4 Analysis and statistics One trypsin inhibitory unit is defined as that amount of sample which gives a reduction of 0.01 absorbance units at A410 nm, relative to trypsin control reactions, in an assay volume of 10 mL. Alternatively, as in the case of the DPP-IV inhibitory assay, the results can be expressed as a percentage of inhibition and as an IC50 value (see earlier). 6.3.8.5 Precursor and related techniques The most common approaches used to determine trypsin activity are based on colorimetric methods using substrates such as p-toluene-sulfonyl-L-arginine methyl ester (Hummel, 1959) but also it is possible to determine trypsin activity fluorometrically with substrates containing an aminomethylcoumarin group such as Z-Gly-Pro-Arg-AMC (Zimmerman, Ashe, Yurewicz, & Patel, 1977).

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6.3.8.6 Alternative methods/procedures Electrophoresis and zymogram analysis can be used to provide confirmation of the trypsin inhibitory activity of Bowman
Birk inhibitors and this approach has been employed with large molecular mass peptides or protein hydrolyzates (Clemente et al., 2010).

6.3.9 Assay of chymotrypsin inhibitory activity Chymotrypsin (EC 3.4.21.1) is a serine protease that hydrolyzes peptide bonds formed with the carboxyl groups of Tyr, Phe, Trp, and Leu (Gra´f, Szila´gyi, & Venekei, 2012). Chymotrypsin is synthesized in an inactive form by the pancreas and is transported in the pancreatic juice into the duodenum where it is activated. As was the case with trypsin, chymotrypsin has an important role in proteolysis and its inhibition can have potential as an anticarcinogen (De Mejia & Dia, 2010) and well as in satiety management. 6.3.9.1 Definition This protocol describes an assay to determine the chymotrypsin inhibitory activity in food proteins and food protein
derived peptides. This involves a continuous spectrophotometric rate determination (A256 nm)-based assay on the hydrolysis of Nbenzoyl-L-tyrosine ethyl ester (BTEE) by chymotrypsin which produces N-benzoyl-Ltyrosine and ethanol. The protocol described herein was developed by Hummel (1959) and modified by Rick (1974). This protocol has been extensively used, for example, in the determination of the chymotrypsin inhibitory activity of Bowman
Birk inhibitors from pea and soybean (Clemente et al., 2004; Amigo-Benavent et al., 2013) and pea protein hydrolyzates (Awosika & Aluko, 2019). 6.3.9.2 Materials, equipment, and reagents • • •

• • •

Assay buffer: 0.1 M Tris-HCl buffer pH 7.8 containing 0.1 M CaCl2. Substrate BTEE dissolve to 1 mM in 1:1 methanol:water. Positive control inhibitor: Chymotrypsin inhibitor from G. max (soybean) (ref T9777 from Sigma-Aldrich). The IC50 value for BBI has been reported to be 53 ng using APNE as a substrate (Yakoby & Raskin, 2004), although no IC50 value was found for the protocol described herein, this value can be calculated using a range of concentrations as in the case for the test sample. Enzyme: α-Chymotrypsin from bovine pancreas, type VII, $ 40 U/mg protein (Ref C3142 from Sigma-Aldrich). Dissolved to 0.8 U/mL in 1 mM HCl. Spectrophotometer. Pipettes, tips, tubes, and quartz cuvettes.

134 Chapter 6 6.3.9.3 Protocols 1. Background: Add 35 μL of 1 mM HCl 1 465 μL of assay buffer in a quartz cuvette. 2. Negative control: Add 35 μL of chymotrypsin solution 1 465 μL of assay buffer in a quartz cuvette. 3. Positive control: Add 35 μL of chymotrypsin solution 1 50 μL of inhibitor 1 415 μL of assay buffer in a quartz cuvette. 4. Test sample: Add 35 μL of chymotrypsin solution 1 50 μL of appropriate dilution of the hydrolyzates/peptides 1 415 μL in a quartz cuvette. 5. Mix and preincubate at 30 C for 2 minutes. 6. Add 500 μL of 1 mM BTEE solution to start the reaction. 7. Determine the change in absorbance at 256 nm over 5 minutes. 6.3.9.4 Analysis and statistics One unit of chymotrypsin activity is defined as the amount that hydrolyzes 1 μM of BTEE per min at pH 7.8 at 30 C. One chymotrypsin inhibitory unit (CIU) is defined as that amount of sample which gives a reduction at A256 nm of 0.01 relative to chymotrypsin reaction controlin a defined assay volume (10 mL). Specific chymotrypsin inhibitory activity is expressed as CIU per mg of hydrolyzate/peptide.
ΔA256nm =min test 2 ΔA256nm =min blank 3 ðTVÞ 3 ðdf Þ Enzyme ðU=mLÞ 5 ð0:964Þ 3 ðVtest Þ In the above equation, TV is the total volume (mL) of the reaction mixture in the cuvette; df is the dilution factor; 0.964 is the millimolar extinction coefficient of BTEE at 256 nm; and Vtest is the volume (mL) of enzyme solution used in the assay. Alternatively, results can be expressed as a percentage of inhibition and as an IC50 (see earlier). 6.3.9.5 Precursor and related techniques The most common protocols developed to determine chymotrypsin activity are colorimetric assays using pNA as leaving group, that is, N-succinyl-Ala-Ala-Pro-Phe-pNA (Schechter & Berger, 1967). There are also fluorogenic assays using Suc-Ala-Ala-Pro-Phe-SBzl (Harper, Ramirez, & Powers, 1981) and bioluminescence assays using 6-(N-acetyl-L-phenylalanyl)-aminoluciferin (Monsees, Miska, & Geiger, 1994). Commercial assay kits based on fluorescent substrates are available from Abcam (Cambridge, United Kingdom). 6.3.9.6 Alternative methods/procedures Methods based on enzyme immobilization and HPLC-UV (Chui & Wainer, 1992) and using electrophoresis (Yang, Sun, Fu, Jiang, & Tang, 2012) have also been described.

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6.3.10 Assay of acetylcholinesterase inhibitory activity AChE (EC 3.1.1.7) is a serine hydrolase that catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. AChE is distributed in nerve and muscle, in central and peripheral tissues, in motor and sensory fibers, and in cholinergic ˇ c, Krsti´c, Lazarevi´c-Paˇsti, Bondˇzi´c, & Vasi´c, 2013). The and noncholinergic fibers (Colovi´ biological role of AChE is the termination of impulse transmission at cholinergic synapses by rapid hydrolysis of acetylcholine (Dvir, Silman, Harel, Rosenberry, & Sussman, 2010). The inhibition of brain AChE is one of the major therapeutic targets in the treatment of Alzheimer’s disease (AD). Drug inhibitors such as Donepezil, Rivastigmine, and Galantamine are currently being used for AD treatment; however, these compounds are associated with different side effects. Therefore there is a growing interest in the discovery of food protein
derived peptide inhibitors of AChE (Prasasty, Radifar, & Istyastono, 2018). 6.3.10.1 Definition This protocol describes the AChE inhibitory assay for food protein
derived peptides. In this assay, AChE catalyzes the hydrolysis of acetylthiocholine iodide (ATCI) liberating thiocholine, which reacts with 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) to generate a colored anion that is detected spectrophotometrically (Ja¨rvinen et al., 2010). The protocol presented herein was developed by Ellman, Courtney, Andres, and Featherstone (1961) and modified by Moyo, Ndhlala, Finnie, and Van Staden (2010). AChE inhibitory activity has been reported in several food protein
derived hydrolyzates such as from fruit (Zare-Zardini, Tolueinia, Hashemi, Ebrahimi, & Fesahat, 2013), edible fungus (Tsai, Yen, & Yang, 2015), hemp seeds (Malomo & Aluko, 2016), and eggs (Yu et al., 2020). 6.3.10.2 Materials, equipment, and reagents • • • • • • •

Assay buffer: 50 mM Tris-HCl buffer, pH8. Substrate: 15 mM ATCI dissolved in distilled water. Color reagent: 3 mM of DTNB dissolved in 50 mM Tris-HCl buffer, pH 8, containing 0.1 M NaCl and 0.02 M MgCl2  6H2O. Positive control inhibitor: 160 μM Galantamine in assay buffer. The IC50 value for Galantamine has been reported to be B 0.37 μM (Moyo et al., 2010). Enzyme: a working solution of 0.2 U/mL AChE from Electrophorus electricus type VI-S (Ref C3389 Sigma-Aldrich) in assay buffer. Plate reader. Multichannel pipettes, tips, and clear 96-well plates.

6.3.10.3 Protocols 1. Background: Add 50 μL of assay buffer 1 25 μL of 15 mM ATCI solution 1 125 μL of 3 mM DTNB 1 50 μL of assay buffer containing 0.1% bovine serum albumin.

136 Chapter 6 2. Negative control: Add 25 μL of assay buffer 1 25 μL of 15 mM ATCI solution 1 125 μL of 3 mM DTNB 1 50 μL of assay buffer containing 0.1% bovine serum albumin. 3. Positive control: Add 25 μL of inhibitor 1 25 μL of 15 mM ATCI solution 1 125 μL of 3 mM DTNB 1 50 μL of assay buffer containing 0.1% bovine serum albumin. 4. Test sample: Add 25 μL of appropriately diluted test sample 1 125 μL of 3 mM DTNB 1 50 μL of assay buffer containing 0.1% bovine serum albumin. 5. Mix and monitor the absorbance at 405 nm every 45 seconds for 225 seconds at 25 C. 6. Add 25 μL of a solution of AChE (0.2 U/mL in assay buffer) to start the reaction in negative and positive controls and in test sample assigned wells. 7. Determine the absorbance at 405 nm every 45 seconds for 360 seconds at 25 C. 6.3.10.4 Analysis and statistics One unit of AChE activity is defined as the amount of enzyme which hydrolyzes 1 μmol of ATCI per min at pH 8 and at 25 C. The rate of enzyme reaction is calculated by dividing the change in absorbance by the time, that is, choosing two time points (t1 and t2) in the linear range, and their corresponding values for the absorbance (A1 and A2). The rate is obtained by dividing the net absorbance (A2 2 A1) by the net time (t2 2 t1). Any increase in absorbance due to the spontaneous hydrolysis of the substrate is corrected by subtracting the rate of reaction before adding the enzyme from the rate after adding the enzyme. The percentage inhibition can be calculated using the following equation:

% inhibition 5 Rate100% enzyme activity 2 RateSample =Rate100% enzyme activity 3 100 In similarity with DPP-IV inhibitory assay, it is possible to generate IC50 values to express the results (see earlier). 6.3.10.5 Precursor and related techniques The first assay for measuring AChE activity was reported by Ellman et al. (1961) based on the same principle described herein; later on, a 96-well plate method was developed (Di Giovanni et al., 2008) followed by miniaturization into 384-well plate format for high-throughput screening (Ja¨rvinen et al., 2010). Other assays used to determine AChE inhibitory activity are based on fluorescence using 2,4-dinitrobenzenesulfonyl fluorescein (Maeda et al., 2005) or on chemiluminescence-based reactions (Birman, 1985). 6.3.10.6 Alternative methods/procedures The most common protocols used to determine AChE activity are based on Ellman’s colorimetric assay as presented herein. Apart from these assays, there are also techniques that use thin layer chromatography (Di Giovanni et al., 2008) and HPLC analysis (Wang, Liang, Chen, & Zhou, 2018).

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6.3.11 Pros and cons Generally, the selection of the enzyme inhibitory assay will depend on the interest in specific activity/activities, the budget, amount of sample, and the available equipment. If there is no limitation of equipment, the assay sensitivity increases in the order: absorbance, fluorescence, and luminescence. Since the ultimate goal is to translate the in vitro determined activity of BAPs to in vivo relevant outcomes, it is generally recommended to use human-derived enzyme activities versus the corresponding activities obtained from other species. It has been shown, for instance, that there are differences in the activity of human versus porcine-derived DPP-IV (Lacroix & Li-Chan, 2015). Furthermore, to generate results relevant to in vivo, it is important to test the inhibitory potential of physiologically relevant concentrations of BAPs. The use of in silico analyses can be useful in providing information about the potential activity and in confirming the active regions of specific proteins/peptides. A number of reviews are available on this topic such as that of Tu, Cheng, Lu, and Du (2018), Nongonierma et al. (2019), and Moura, Halder, and Cordeiro (2019). Table 6.4 summarizes Table 6.4: Pros and cons of using in vitro enzyme inhibition assays for assessment of bioactive peptides activity and potency. Pros Sensitive assays that allow the study of the inhibitory activity of BAPs in a concentration range between nM and μM Comparison with other samples and other studies possible by determining IC50 values Use of standards and inhibitor controls act as quality control assessment process between and within different laboratories

Most of the assays are well validated and allow relatively rapid analysis

Cons Cost of the enzymes, particularly if using human origin or human recombinant forms. Cost and availability of specific substrates. Reproducibility and comparability of the results between different laboratories are highly dependent on the number of units of enzyme activity employed in the assay, for example, in the case of ACE inhibition (Murray & FitzGerald, 2007). In vitro results may not be directly translated to in vivo, this usually requires more studies.

In the case of enzymatic extracts: potential differences in the properties/activities of the extracted enzymes from different sources. Many of the enzyme inhibition assays use nonhuman sourced enzymes. This may lead to some differences in catalytic activity as, for example, demonstrated in the case of DPP-IV inhibition assays (Lacroix & LiChan, 2015). Heterogeneity of the test samples (protein extracts ACE, renin, DPP-IV, α-amylase, α-glucosidase, or its hydrolyzates) and the presence of other lipase, chymotrypsin, and acetylcholinesterase inhibitory assays are continuous assays that allow to compounds (salts, phenolic compounds) other than proteins with the ability to activate or deactivate monitoring of reactant concentrations in real time some enzymes may cause an under- or and therefore are useful for kinetic and mode of overestimation of the effect of the test sample. inhibition studies

Analysis of multiple samples at the same time in microplate format allows the potential for highthroughput screening The assays allow the use of low amounts of test sample

DPP-IV, Dipeptidyl peptidase IV.

138 Chapter 6 the advantages and disadvantages of using in vitro enzyme inhibition assays for assessment of BAP activity and potency.

6.3.12 Troubleshooting and optimization Some general hints during the utilization of enzyme inhibition assays are as follows: retain the enzymes under appropriate storage conditions, avoid multiple freeze
thaw cycles, many substrates are photolabile therefore store in the dark, ensure that all assay buffers, solutions, and equipment are preincubated at the specified assay temperature, use the appropriate type of microplate/cuvette for the detection method, use pipettes appropriate for the volume range being dispensed, ensure pipettes are calibrated regularly Table 6.5: Problems and solutions in enzyme inhibition assays. Problem Assay not working

Samples with erratic readings between replicates Samples with erratic readings

No values of absorbance/ fluorescence in sample wells

Interference from the samples Precipitate formation in test tubes Lower/higher readings in samples, controls, inhibitors, and standards Sample readings above/below the linear range Nonlinear standard curve

BAP, Bioactive peptide.

Solution Review protocol steps Verify correct microplate/cuvette type has been used, for example, black plates for fluorescence assays and clear plates for colorimetric assays Review reagent preparation steps and preincubation/incubation at the correct assay temperature and use fresh enzymes Review spectrophotometer/microplate reader settings Ensure that the enzyme has been aliquoted and stored correctly and that it has not been through numerous freeze
thaw cycles Improve pipetting technique and use reverse pipetting to avoid bubbles Use fresh samples or aliquot and freeze samples if needed for multiple uses Dissolved and homogenized the sample properly Reduce the concentration of the inhibitor and re-assay Ensure that the samples do not contain high salt concentrations or other compounds which could denature or alter the catalytic activity of the enzyme Ensure the samples are soluble, if not try to change the solvent and include a control for the solvent in the plate/tube Centrifuge test tubes before absorbance/fluorescence reading Always thaw and prepare fresh reaction mixture before use Verify correct incubation times and temperatures as per the protocol Dilute or concentrate the sample accordingly, taking into account the maximum possible physiological doses of the BAPs Review standard curve calculation and repeat assay Verify the volume range of the pipette is appropriate and that the pipettes are calibrated Ensure when pipetting to gently express the aliquot against the wall of the microplate well/tube/cell and avoid bubble formation

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and employ the appropriate storage and incubation time(s). If an assay produces unexpected results, Table 6.5 indicates different issues that may arise and suggests some potential solutions to these problems.

6.4 Summary A range of commonly used in vitro/biochemical assays to assess the antioxidant and enzyme inhibitory activity of food protein
derived hydrolyzates/peptides have been described in this chapter. Radical scavenging assays are based on HAT such as in the ORAC assay or ET such as in the TEAC, FRAP, and DPPH assays. Therefore the use of a combination of these antioxidant activity assays is recommended to obtain the maximum information on antioxidant activity of a protein hydrolyzate/BAP. Moreover, the use of a range of different assays is recommended to calculate a RACI. As earlier mentioned, estimation of RACI was proposed as an integrated approach to evaluate food component antioxidant capacity by using seven in vitro assays, that is, the TEAC, FRAP, ORAC, CUPRAC, HORAC, LDL oxidation, and TRAP assays (Sun & Tanumihardjo, 2007). In situations when it is not feasible to use more than three or less antioxidant activity assays due to instrumentation, time, and budget limitations, Sun and Tanumihardjo (2007) have demonstrated that the ORAC assay had the best correlation with RACI and this correlation increased when FRAP and ABTS analyses were included. The enzyme inhibitory assays presented herein allow the screening of food protein
derived peptides and hydrolyzates. Moreover, they can also be used to study the type of inhibition (competitive, noncompetitive, uncompetitive, etc.) if a Lineweaver
Burk type analysis is carried out. For example, in the DPP-IV assay, this can be achieved by measuring the initial rate of the reaction at different substrate concentrations (0.2
6 mM) in the presence and absence of inhibitors at the inhibitor’s IC50 concentration. Subsequently, Km and Vmax values can be deducted from the Lineaweaver and Burk double reciprocal plots and thereby an indication of the type of inhibition (Nongonierma & FitzGerald, 2013a, 2013b). It is also possible to study the combination/synergy or agonistic/antagonistic effects of different inhibitor peptides/drugs by carrying out experiments alone and in combination with BAPs and comparing their effect on enzyme inhibition. Nongonierma and FitzGerald (2015) used an isobole approach to study the interactive effects between drugs and peptides, for example, in the case of Sitagliptin, a drug inhibitor of DPP-IV, and DPP-IV inhibitory peptides. The application of many of the in vitro assays described herein has contributed significantly to the development of BAPs for use as functional foods with approved health claims. This is exemplified in the case of the hypotensive lactotripeptides, Val-Pro-Pro or Ile-Pro-Pro, found in commercial fermented milks (Korhonen, 2009). The number of patents and the market for peptide-based functional food ingredients (and indeed peptide-based drugs) continues to grow. The process from discovery to production has many different phases which go from studies

140 Chapter 6 associated with in vitro screening, pharmacodynamics, pharmacokinetics, toxicity, and safety assessments. In addition, economic factors such as productivity, market competition, intellectual property, and consumer acceptability and demand are also highly relevant. In the case of novel peptide-based drug candidates, these factors result in only 10% of candidate compounds ultimately reaching the market (Uhlig et al., 2014). Data in relation to the success rate for BAPs currently does not appear to be generally available. The antioxidant and enzyme inhibitory activity assays described in this chapter have the advantages of being sensitive, feasible, reproducible, validated, and adaptable for highthroughput screening. In the case of the antioxidant assays, one of the main disadvantages arises from interference from other compounds present in the sample. In addition, the radicals studied in these assays, with the exception of peroxyl radicals, are not considered as physiologically relevant radicals. In the case of the enzyme inhibition assays, the disadvantage is some cases is associated with the high cost and availability of the enzymes and their cognate substrates. In addition, the translation of in vitro determined activity to a beneficial effect in vivo activity may not occur in all cases. However, the casein-derived ACE inhibitory peptides, Val-Pro-Pro and Ile-Pro-Pro, have been tested in humans showing a reduction in blood pressure in mildly hypertensive subjects (Mizuno et al., 2005). PowerGrant et al. (2016) selected a milk protein matrix with antioxidant BAPs, as determined by ORAC analysis, to carry out an acute study in which the milk matrix was consumed by healthy women resulting in a 23% increase in plasma antioxidant capacity. In the case of milk protein hydrolyzates with potent in vitro DPP-IV inhibitory activity, where different casein hydrolyzates were screened using the DPP-IV inhibitory outlined herein (Nongonierma, Maux, Esteveny, & FitzGerald, 2017), it was demonstrated that the in vitro results were translated during subsequent cell, animal, and human studies (Drummond et al., 2018). Harnedy et al. (2018) demonstrated the translation of in vitro DPP-IV inhibitory analysis to in vivo following administration of a blue whiting protein hydrolyzate to mice which led to significant blood glucose
lowering effects. Therefore these studies and many others highlight the relevance of these in vitro assays for compound screening and the potential for their translation to in vivo. In conclusion, in vitro/biochemical antioxidant and enzyme inhibitory assays are very useful in the discovery and screening of BAPs and can provide valuable information about their mechanism of action and interactions with other compounds.

Acknowledgments Funding for this research was provided under the Marine Research Programme 2014
2020, through the Marine Institute of Ireland under grant PBA/MB/16/01 “A National Marine Biodiscovery Laboratory of Ireland (NMBLI)” for MAB; European Union Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Career-FIT Grant Agreement No. 713654 for MK and through the Food Institutional Research Measure, administered by the Department of Agriculture, Food, and the Marine, Ireland under grant issue 14/F/873 for GT.

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References Addar, L., Bensouici, C., Si Ahmed Zennia, S., Boudjenah Haroun, S., & Mati, A. (2019). Antioxidant, tyrosinase and urease inhibitory activities of camel αS-casein and its hydrolysate fractions. Small Ruminant Research, 173, 30
35. Available from https://doi.org/10.1016/j.smallrumres.2019.01.015. Admassu, H., Gasmalla, M. A. A., Yang, R., & Zhao, W. (2018). Identification of bioactive peptides with α-amylase inhibitory potential from enzymatic protein hydrolysates of red seaweed (Porphyra spp). Journal of Agricultural and Food Chemistry, 66(19), 4872
4882. Available from https://doi.org/10.1021/acs.jafc.8b00960. Ahmed, T. A. E., & Hammami, R. (2019). Recent insights into structure
function relationships of antimicrobial peptides. Journal of Food Biochemistry, 43(1), e12546. Available from https://doi.org/10.1111/jfbc.12546. Aldewachi, H. S., Woodroofe, N., Turega, S., & Gardiner, P. H. E. (2017). Optimization of gold nanoparticlebased real-time colorimetric assay of dipeptidyl peptidase IV activity. Talanta, 169, 13
19. Available from https://doi.org/10.1016/j.talanta.2017.03.039. Aluko, R. E. (2019). Food protein-derived renin-inhibitory peptides: In vitro and in vivo properties. Journal of Food Biochemistry, 43(1), 1
12. Available from https://doi.org/10.1111/jfbc.12648. Amigo-Benavent, M., Clemente, A., Caira, S., Stiuso, P., Ferranti, P., & Del Castillo, M. D. (2014). Use of phytochemomics to evaluate the bioavailability and bioactivity of antioxidant peptides of soybean β-conglycinin. Electrophoresis, 35(11), 1582
1589. Available from https://doi.org/10.1002/elps.201300527. Amigo-Benavent, M., del Castillo, M. D., & Fogliano, V. (2010). Are the major antioxidants derived from soy protein and fructo-oligosaccharides model systems colored aqueous soluble or insoluble compounds? European Food Research Technology, 231, 545
553. Available from https://doi.org/10.1007/s00217-010-1308-5. Amigo-Benavent, M., Nitride, C., Bravo, L., Ferranti, P., & Del Castillo, M. D. (2013). Stability and bioactivity of a Bowman-Birk inhibitor in orange juice during processing and storage. Food and Function, 4(7), 1051
1060. Available from https://doi.org/10.1039/c3fo30354c. Ando, T., Okada, S., Uchida, I., Hemmi, K., Nishikawa, M., Tsurumi, Y., & Okuhara, M. (1987). WF-10129, a novel angiotensin converting enzyme inhibitor produced by a fungus, doratomyces putredinis. The Journal of Antibiotics, 40(4), 468
475. Available from https://doi.org/10.7164/antibiotics.40.468. Awosika, T., & Aluko, R. E. (2019). Enzymatic pea protein hydrolysates are active trypsin and chymotrypsin inhibitors. Foods, 8(6), 200. Available from https://doi.org/10.3390/foods8060200. Babson, S. R., & Babson, A. L. (1973). An improved amylase assay using dyed amylopectin. Clinica Chimica Acta, 44(2), 193
197. Baird, T. T., & Craik, C. S. (2013). Trypsin. Handbook of proteolytic enzymes (pp. 2594
2600). Elsevier, 3 pp. doi:10.1016/B978-0-12-382219-2.00575-5. Bendicho, S., Trigueros, M. C., Herna´ndez, T., & Martı´n, O. (2001). Validation and comparison of analytical methods based on the release of p-nitrophenol to determine lipase activity in milk. Journal of Dairy Science, 84(7), 1590
1596. Available from https://doi.org/10.3168/jds.S0022-0302(01)74592-4. Benzie, I. F. F., & Choi, S.-W. (2014). Antioxidants in food: Content, measurement, significance, action, cautions, caveats, and research needs. Advances in Food and Nutrition Research, 71, 1
53. Available from https://doi.org/10.1016/B978-0-12-800270-4.00001-8. Benzie, I. F. F., Chung, W. Y., & Strain, J. J. (1999). ‘Antioxidant’ (reducing) efficiency of ascorbate in plasma is not affected by concentration. Journal of Nutritional Biochemistry, 10(3), 146
150. Available from https://doi.org/10.1016/S0955-2863(98)00084-9. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: The FRAP assay. Analytical Biochemistry, 239(1), 70
76. Available from https://doi. org/10.1006/abio.1996.0292. Benzie, I. F. F., & Strain, J. J. (1999). Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods in Enzymology, 299, 15
27. Bernfeld, P. (1955). Amylases, α and β, . Methods in enzymology (Vol. I, p. 149). New York: Academic Press. Berthelot, K., & Delmotte, F. M. (1999). Purification and characterization of an α-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280. Applied and Environmental Microbiology, 65(7), 2907
2911.

142 Chapter 6 Bhullar, K. S., Ziaullah, Z., & Rupasinghe, V. (2014). In vitro regulation of enzymes of the renin-angiotensinaldosterone system by isoquercitrin, phloridzin and their long chain fatty acid derivatives. Functional Foods in Health and Disease, 4(5), 208. Available from https://doi.org/10.31989/ffhd.v4i5.11. Biely, P., Mislovicova´, D., Markovic, O., & Kala´c, V. (1988). A new chromogenic substrate for assay and detection of alpha-amylase. Analytical Biochemistry, 178(1), 176
179. Birman, S. (1985). Determination of acetylcholinesterase activity by a new chemiluminescence assay with the natural substrate. Biochemical Journal, 225(3), 825
828. Available from https://doi.org/10.1042/bj2250825. Blake, M. R., Koka, R., & Weimer, B. C. (1996). A semiautomated reflectance colorimetric method for the determination of lipase activity in milk. Journal of Dairy Science, 79(7), 1164
1171. Available from https://doi.org/10.3168/jds.S0022-0302(96)76470-6. Brena, B. M., Pazos, C., Franco-Fraguas, L., & Batista-Viera, F. (1996). Chromatographic methods for amylases. Journal of Chromatography B: Biomedical Applications, 684(1
2), 217
237. Available from https://doi.org/10.1016/0378-4347(96)00103-X. Buchholz, T., & Melzig, M. F. (2016). Medicinal plants traditionally used for treatment of obesity and diabetes mellitus—Screening for pancreatic lipase and α-amylase inhibition. Phytotherapy Research, 30(2), 260
266. Available from https://doi.org/10.1002/ptr.5525. Butterworth, P. J., Warren, F. J., & Ellis, P. R. (2011). Human α-amylase and starch digestion: An interesting marriage. Starch/Staerke, 63(7), 395
405. Available from https://doi.org/10.1002/star.201000150. Cao, G., Alessio, H. M., & Cutler, R. G. (1993). Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biology and Medicine, 14(3), 303
311. Available from https://doi.org/10.1016/0891-5849 (93)90027-R. Cao, G., & Prior, R. L. (1998). Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clinical Chemistry, 44(6), 1309
1315. Cao, G., Shukitt-Hale, B., Bickford, P. C., Joseph, J. A., McEwen, J., & Prior, R. L. (1999). Hyperoxia-induced changes in antioxidant capacity and the effect of dietary antioxidants. Journal of Applied Physiology, 86(6), 1817
1822. Available from https://doi.org/10.1152/jappl.1999.86.6.1817. Carmel, A., & Yaron, A. (1978). An intramolecularly quenched fluorescent tripeptide as a fluorogenic substrate of angiotensin-I-converting enzyme and of bacterial dipeptidyl carboxypeptidase. European Journal of Biochemistry, 87(2), 265
273. Available from https://doi.org/10.1111/j.1432-1033.1978.tb12375.x. Carrillo, W., Go´mez-Ruiz, J. A., Miralles, B., Ramos, M., Barrio, D., & Recio, I. (2016). Identification of antioxidant peptides of hen egg-white lysozyme and evaluation of inhibition of lipid peroxidation and cytotoxicity in the Zebrafish model. European Food Research and Technology, 242(10), 1777
1785. Available from https://doi.org/10.1007/s00217-016-2677-1. Castan˜eda-Pe´rez, E., Jime´nez-Morales, K., Quintal-Novelo, C., Moo-Puc, R., Chel-Guerrero, L., & BetancurAncona, D. (2019). Enzymatic protein hydrolysates and ultrafiltered peptide fractions from Cowpea Vigna unguiculata L bean with in vitro antidiabetic potential. Journal of the Iranian Chemical Society, 16(8), 1773
1781. Available from https://doi.org/10.1007/s13738-019-01651-0. Cermen˜o, M., Connolly, A., O’Keeffe, M. B., Flynn, C., Alashi, A. M., Aluko, R. E., & FitzGerald, R. J. (2019). Identification of bioactive peptides from brewers’ spent grain and contribution of Leu/Ile to bioactive potency. Journal of Functional Foods, 60, 103455. Available from https://doi.org/10.1016/j.jff.2019.103455. Cermen˜o, M., Stack, J., Tobin, P. R., O’Keeffe, M. B., Harnedy, P. A., Stengel, D. B., & Fitzgerald, R. J. (2019). Peptide identification from a Porphyra dioica protein hydrolysate with antioxidant, angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities. Food and Function, 10(6), 3421
3429. Available from https://doi.org/10.1039/c9fo00680j. Chedda, U., Kaikini, A., Bagle, S., & Seervi, M. (2016). In vitro pancreatic lipase inhibition potential of commonly used Indian spices. IOSR Journal of Pharmacy, 6(10), 10
13. Chelladurai, G. R. M., & Chinnachamy, C. (2018). Alpha amylase and alpha glucosidase inhibitory effects of aqueous stem extract of Salacia oblonga and its GC-MS analysis. Brazilian Journal of Pharmaceutical Sciences, 54(1), e17151. Available from https://doi.org/10.1590/s2175-97902018000117151. Chen, G., & Guo, M. (2017). Rapid screening for α-glucosidase inhibitors from Gymnema sylvestre by affinity ultrafiltration-HPLC-MS. Frontiers in Pharmacology, 8, 228. Available from https://doi.org/10.3389/ fphar.2017.00228.

Methodologies for bioactivity assay: biochemical study

143

Chui, W.-K., & Wainer, I. W. (1992). Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: The immobilization of trypsin and α-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support. Analytical Biochemistry, 201(2), 237
245. Available from https://doi.org/10.1016/0003-2697(92)90334-4. Clemente, A., Carmen Marı´n-Manzano, M., Jime´nez, E., Carmen Arques, M., & Domoney, C. (2012). The antiproliferative effect of TI1B, a major Bowman-Birk isoinhibitor from pea (Pisum sativum L.), on HT29 colon cancer cells is mediated through protease inhibition. British Journal of Nutrition, 108(Suppl. 1), S135
S144. Available from https://doi.org/10.1017/S000711451200075X. Clemente, A., MacKenzie, D. A., Jeenes, D. J., & Domoney, C. (2004). The effect of variation within inhibitory domains on the activity of pea protease inhibitors from the Bowman-Birk class. Protein Expression and Purification, 36(1), 106
114. Available from https://doi.org/10.1016/j.pep.2004.03.015. Clemente, A., Moreno, F. J., Marı´n-Manzano, M. C., Jime´nez, E., & Domoney, C. (2010). The cytotoxic effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases. Molecular Nutrition and Food Research, 54(3), 396
405. Available from https://doi.org/10.1002/mnfr.200900122. ˇ Colovi´ c, M. B., Krsti´c, D. Z., Lazarevi´c-Paˇsti, T. D., Bondˇzi´c, A. M., & Vasi´c, V. M. (2013). Acetylcholinesterase inhibitors: Pharmacology and toxicology. Current Neuropharmacology, 11(3), 315
335. Available from https://doi.org/10.2174/1570159X11311030006. Conforti, F., Perri, V., Menichini, F., Marrelli, M., Uzunov, D., Statti, G. A., & Menichini, F. (2012). Wild Mediterranean dietary plants as inhibitors of pancreatic lipase. Phytotherapy Research, 26(4), 600
604. Available from https://doi.org/10.1002/ptr.3603. Connolly, A., Cermen˜o, M., Crowley, D., O’Callaghan, Y., O’Brien, N. M., & FitzGerald, R. J. (2019). Characterisation of the in vitro bioactive properties of alkaline and enzyme extracted brewers’ spent grain protein hydrolysates. Food Research International, 121, 524
532. Available from https://doi.org/10.1016/j. foodres.2018.12.008. Connolly, A., Piggott, C. O., & FitzGerald, R. J. (2014). In vitro α-glucosidase, angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory properties of brewers’ spent grain protein hydrolysates. Food Research International, 56, 100
107. Available from https://doi.org/10.1016/j.foodres.2013.12.021. ´ lvarez, P. J., & Recio, I. (2011). Production Contreras, M. D. M., Herna´ndez-Ledesma, B., Amigo, L., Martı´n-A of antioxidant hydrolyzates from a whey protein concentrate with thermolysin: Optimization by response surface methodology. LWT—Food Science and Technology, 44(1), 9
15. Available from https://doi.org/ 10.1016/j.lwt.2010.06.017. Cushman, D. W., & Cheung, H. S. (1971). Spectrophotometric assay and properties of the angiotensinconverting enzyme of rabbit lung. Biochemical Pharmacology, 20(7), 1637
1648. Available from https:// doi.org/10.1016/0006-2952(71)90292-9. Daliri, E. B.-M., Lee, B. H., & Oh, D. H. (2018). Current trends and perspectives of bioactive peptides. Critical Reviews in Food Science and Nutrition, 58(13), 2273
2284. Available from https://doi.org/10.1080/ 10408398.2017.1319795. de Lima, V. C. O., Piuvezam, G., Leal Lima Maciel, B., & Heloneida de Arau´jo Morais, A. (2019). Trypsin inhibitors: Promising candidate satietogenic proteins as complementary treatment for obesity and metabolic disorders? Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 405
419. Available from https:// doi.org/10.1080/14756366.2018.1542387. De Mejia, E. G., & Dia, V. P. (2010). The role of nutraceutical proteins and peptides in apoptosis, angiogenesis, and metastasis of cancer cells. Cancer and Metastasis Reviews, 29(3), 511
528. Available from https://doi. org/10.1007/s10555-010-9241-4. de Souza Rocha, T., Hernandez, L. M. R., Chang, Y. K., & de Mejı´a, E. G. (2014). Impact of germination and enzymatic hydrolysis of cowpea bean (Vigna unguiculata) on the generation of peptides capable of inhibiting dipeptidyl peptidase IV. Food Research International, 64, 799
809. Available from https://doi. org/10.1016/j.foodres.2014.08.016. Di Giovanni, S., Borloz, A., Urbain, A., Marston, A., Hostettmann, K., Carrupt, P.-A., & Reist, M. (2008). In vitro screening assays to identify natural or synthetic acetylcholinesterase inhibitors: Thin layer

144 Chapter 6 chromatography versus microplate methods. European Journal of Pharmaceutical Sciences, 33(2), 109
119. Available from https://doi.org/10.1016/j.ejps.2007.10.004. Domoney, C., & Welham, T. (1992). Trypsin inhibitors in Pisum: Variation in amount and pattern of accumulation in developing seed. Seed Science Research, 2(3), 147
154. Available from https://doi.org/ 10.1017/S0960258500001276. Drummond, E., Flynn, S., Whelan, H., Nongonierma, A. B., Holton, T. A., Robinson, A., . . . Brennan, L. (2018). Casein hydrolysate with glycemic control properties: Evidence from cells, animal models, and humans. Journal of Agricultural and Food Chemistry, 66(17), 4352
4363. Available from https://doi.org/ 10.1021/acs.jafc.7b05550. Dvir, H., Silman, I., Harel, M., Rosenberry, T. L., & Sussman, J. L. (2010). Acetylcholinesterase: From 3D structure to function. Chemico-Biological Interactions, 187(1-3), 10
22. Available from https://doi.org/ 10.1016/j.cbi.2010.01.042. Ellman, G. L., Courtney, K. D., Andres, V., Jr., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88
90. Available from https://doi.org/10.1016/0006-2952(61)90145-9, IN1
IN2, 91
95. European Commission. (2012). Scientific Committee on Consumer Safety (SCCS) opinion on kojic acid (22 p.). European Commission. https://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_098. pdf. (Accessed 17 April 2020). Fitzgerald, C., Mora-Soler, L., Gallagher, E., O’Connor, P., Prieto, J., Soler-Vila, A., & Hayes, M. (2012). Isolation and characterization of bioactive pro-peptides with in vitro renin inhibitory activities from the macroalga Palmaria palmata. Journal of Agricultural and Food Chemistry, 60(30), 7421
7427. Available from https://doi.org/10.1021/jf301361c. Franco, D., Munekata, P. E. S., Agrega´n, R., Bermu´dez, R., Lo´pez-Pedrouso, M., Pateiro, M., & Lorenzo, J. M. (2020). Application of pulsed electric fields for obtaining antioxidant extracts from fish residues. Antioxidants, 9(2), 90. Available from https://doi.org/10.3390/antiox9020090. Fu, Y., Alashi, A. M., Young, J. F., Therkildsen, M., & Aluko, R. E. (2017). Enzyme inhibition kinetics and molecular interactions of patatin peptides with angiotensin I-converting enzyme and renin. International Journal of Biological Macromolecules, 101, 207
213. Available from https://doi.org/10.1016/j.ijbiomac.2017.03.054. Gallego, M., Mora, L., Reig, M., & Toldra´, F. (2018). Stability of the potent antioxidant peptide SNAAC identified from Spanish dry-cured ham. Food Research International, 105, 873
879. Available from https://doi.org/10.1016/j.foodres.2017.12.006. Garcı´a, P., & Furlan, R. L. E. (2015). Multiresponse optimisation applied to the development of a TLC autography for the detection of tyrosinase inhibitors. Phytochemical Analysis, 26(4), 287
292. Available from https://doi.org/10.1002/pca.2562. Garcı´a-Molina, F., Mun˜oz, J. L., Varo´n, R., Rodrı´guez-Lo´pez, J. N., Garcı´a-Ca´novas, F., & Tudela, J. (2007). A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase. Journal of Agricultural and Food Chemistry, 55(24), 9739
9749. Available from https://doi. org/10.1021/jf0712301. Garcia-Vaquero, M., Mora, L., & Hayes, M. (2019). In vitro and in silico approaches to generating and identifying angiotensin-converting enzyme I inhibitory peptides from green macroalga ulva lactuca. Marine Drugs, 17(4), 1
19. Available from https://doi.org/10.3390/md17040204. Gard, P. R., Fidalgo, S., Lotter, I., Richardson, C., Farina, N., Rusted, J., & Tabet, N. (2017). Changes of reninangiotensin system-related aminopeptidases in early stage Alzheimer’s disease. Experimental Gerontology, 89, 1
7. Available from https://doi.org/10.1016/j.exger.2017.01.006. Glazer, A. N. (1990). Phycoerythrin fluorescence-based assay for reactive oxygen species. Methods in Enzymology, 186(C), 161
168. Available from https://doi.org/10.1016/0076-6879(90)86106-6. ´ ., Lo´pez-Expo´sito, I., Pihlanto, A., Ramos, M., & Recio, I. (2008). Antioxidant activity of Go´mez-Ruiz, J. A ovine casein hydrolysates: Identification of active peptides by HPLC-MS/MS. European Food Research and Technology, 227(4), 1061
1067. Available from https://doi.org/10.1007/s00217-008-0820-3.

Methodologies for bioactivity assay: biochemical study

145

Gorski, T. P., & Campbell, D. J. (1991). Angiotensin-converting enzyme determination in plasma during therapy with converting enzyme inhibitor: Two methods compared. Clinical Chemistry, 37(8), 1390
1393. Available from https://doi.org/10.1093/clinchem/37.8.1390. Ghosh, S., & Collier, A. (2012). Section 3. Management of diabetes. Churchill’s pocketbook of diabetes (2nd ed., pp. 83
125). Elsevier. Gra´f, L., Szila´gyi, L., & Venekei, I. (2012). Chapter 582. Chymotrypsin. In N. D. Rawlings, A. J. Barrett, J. F. Jr Woessner, & J. F. Woessner (Eds.), Handbook of proteolytic enzymes (pp. 2626
2633). Elsevier. Groot, P. C., Bleeker, M. J., Pronk, J. C., Arwert, F., et al. (1989). The human α-amylase multigene family consists of haplotypes with variable numbers of genes. Genomics, 5, 29
42. Available from https://doi.org/ 10.1016/0888-7543(89)90083-9. Gropper, S. S., Smith, J. L., & Carr, T. P. (2018). Chapter 3. Carbohydrates. Advanced nutrition and human metabolism (7th ed.). Cengage Learning, ISBN: 978-1-305-62785-7. Guo, L., Harnedy, P. A., Zhang, L., Li, B., Zhang, Z., Hou, H., . . . Fitzgerald, R. J. (2015). In vitro assessment of the multifunctional bioactive potential of Alaska pollock skin collagen following simulated gastrointestinal digestion. Journal of the Science of Food and Agriculture, 95(7), 1514
1520. Available from https://doi.org/10.1002/jsfa.6854. Halliwell, B., & Gutteridge, J. M. (2015). Free radicals in biology and medicine. Oxford University Press. Hamdan, I. I., & Afifi, F. U. (2010). Capillary electrophoresis as a screening tool for alpha amylase inhibitors in plant extracts. Saudi Pharmaceutical Journal, 18(2), 91
95. Available from https://doi.org/10.1016/j. jsps.2010.02.004. Harnedy, P. A., & FitzGerald, R. J. (2013). In vitro assessment of the cardioprotective, anti-diabetic and antioxidant potential of Palmaria palmata protein hydrolysates. Journal of Applied Phycology, 25(6), 1793
1803. Available from https://doi.org/10.1007/s10811-013-0017-4. Harnedy, P. A., O’Keeffe, M. B., & FitzGerald, R. J. (2015). Purification and identification of dipeptidyl peptidase (DPP) IV inhibitory peptides from the macroalga Palmaria palmata. Food Chemistry, 172, 400
406. Available from https://doi.org/10.1016/j.foodchem.2014.09.083. Harnedy, P. A., Parthsarathy, V., McLaughlin, C. M., O’Keeffe, M. B., Allsopp, P. J., McSorley, E. M., . . . FitzGerald, R. J. (2018). Blue whiting (Micromesistius poutassou) muscle protein hydrolysate with in vitro and in vivo antidiabetic properties. Journal of Functional Foods, 40, 137
145. Available from https://doi. org/10.1016/j.jff.2017.10.045. Harnedy, P. A., Soler-Vila, A., Edwards, M. D., & FitzGerald, R. J. (2014). The effect of time and origin of harvest on the in vitro biological activity of Palmaria palmata protein hydrolysates. Food Research International, 62, 746
752. Available from https://doi.org/10.1016/j.foodres.2014.04.035. Harnedy-Rothwell, P. A., McLaughlin, C. M., O’Keeffe, M. B., Le Gouic, A. V., Allsopp, P. J., McSorley, E. M., . . . FitzGerald, R. J. (2020). Identification and characterisation of peptides from a boarfish (Capros aper) protein hydrolysate displaying in vitro dipeptidyl peptidase-IV (DPP-IV) inhibitory and insulinotropic activity. Food Research International, 131, 108989. Available from https://doi.org/10.1016/j.foodres.2020.108989. Harper, J. W., Ramirez, G., & Powers, J. C. (1981). Reaction of peptide thiobenzyl esters with mammalian chymotrypsinlike enzymes: A sensitive assay method. Analytical Biochemistry, 118(2), 382
387. Available from https://doi.org/10.1016/0003-2697(81)90597-. Heck, A. M., Yanovski, J. A., & Calis, K. A. (2000). Orlistat, a new lipase inhibitor for the management of obesity. Pharmacotherapy, 20(3), 270
279. Available from https://doi.org/10.1592/phco.20.4.270.34882. Henda, Y. B., Labidi, A., Arnaudin, I., Bridiau, N., Delatouche, R., Maugard, T., & Bordenave-Juchereau, S. (2013). Measuring angiotensin-I converting enzyme inhibitory activity by micro plate assays: Comparison using marine cryptides and tentative threshold determinations with captopril and losartan. Journal of Agricultural and Food Chemistry, 61(45), 10685
10690. Available from https://doi.org/10.1021/jf403004e. Herna´ndez-Ledesma, B., Quiro´s, A., Amigo, L., & Recio, I. (2007). Identification of bioactive peptides after digestion of human milk and infant formula with pepsin and pancreatin. International Dairy Journal, 17(1), 42
49. Available from https://doi.org/10.1016/j.idairyj.2005.12.012.

146 Chapter 6 Hu, S., Fan, X., Qi, P., & Zhang, X. (2019). Identification of anti-diabetes peptides from Spirulina platensis. Journal of Functional Foods, 56, 333
341. Available from https://doi.org/10.1016/j.jff.2019.03.02. Huang, D., Boxin, O. U., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841
1856. Available from https://doi.org/10.1021/jf030723c. Hummel, B. C. W. (1959). A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin. Canadian Journal of Biochemistry and Physiology, 37, 1393
1399. Available from https://doi. org/10.1139/o59-157. Jacks, T. J., & Kircher, H. W. (1967). Fluorometric assay for the hydrolytic activity of lipase using fatty acyl esters of 4-methylumbelliferone. Analytical Biochemistry, 21(2), 279
285. Available from https://doi.org/ 10.1016/0003-2697(67)90190-X. Jakubczyk, A., Szymanowska, U., Kara´s, M., Złotek, U., & Kowalczyk, D. (2019). Potential anti-inflammatory and lipase inhibitory peptides generated by in vitro gastrointestinal hydrolysis of heat treated millet grains. CYTA—Journal of Food, 17(1), 324
333. Available from https://doi.org/10.1080/19476337.2019.1580317. Jaradat, N., Zaid, A., Hussein, F., Zaqzouq, M., Aljammal, H., & Ayesh, O. (2017). Anti-lipase potential of the organic and aqueous extracts of ten traditional edible and medicinal plants in palestine: A comparison study with Orlistat. Medicines, 4(4), 89. Available from https://doi.org/10.3390/medicines4040089. Ja¨rvinen, P. P., Fallarero, A., Gupta, S., Mohan, G. C., Hatakka, A. I., & Vuorela, P. M. (2010). Miniaturization and validation of the Ellman’s reaction based acetylcholinesterase inhibitory assay into 384-well plate format and screening of a chemical library. Combinatorial Chemistry and High Throughput Screening, 13 (3), 278
284. Available from https://doi.org/10.2174/138620710790980559. Jiang, T.-F., Liang, T.-T., Wang, Y.-H., Zhang, W.-H., & Lv, Z.-H. (2013). Immobilized capillary tyrosinase microreactor for inhibitor screening in natural extracts by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis, 84, 36
40. Available from https://doi.org/10.1016/j.jpba.2013.05.023. Jo, S.-H., Ha, K.-S., Moon, K.-S., Lee, O.-H., Jang, H.-D., & Kwon, Y.-I. (2011). In vitro and in vivo antihyperglycemic effects of Omija (Schizandra chinensis) fruit. International Journal of Molecular Sciences, 12(2), 1359
1370. Available from https://doi.org/10.3390/ijms12021359. Kakade, M. L. (1974). Biochemical basis for the differences in plant protein utilization. Journal of Agricultural and Food Chemistry, 22(4), 550
555. Available from https://doi.org/10.1021/jf60194a014. Karami, Z., & Akbari-adergani, B. (2019). Bioactive food derived peptides: A review on correlation between structure of bioactive peptides and their functional properties. Journal of Food Science and Technology, 56 (2), 535
547. Available from https://doi.org/10.1007/s13197-018-3549-4. Kara´s, M., Jakubczyk, A., Szymanowska, U., Je˛derka, K., Lewicki, S., & Złotek, U. (2019). Different temperature treatments of millet grains affect the biological activity of protein hydrolyzates and peptide fractions. Nutrients, 11(3), 550. Available from https://doi.org/10.3390/nu11030550. Karri, S., Sharma, S., Hatware, K., & Patil, K. (2019). Natural anti-obesity agents and their therapeutic role in management of obesity: A future trend perspective. Biomedicine and Pharmacotherapy, 110, 224
238. Available from https://doi.org/10.1016/j.biopha.2018.11.076. Kato, T., Nagatsu, T., Kimura, T., & Sakakibara, S. (1978). Fluorescence assay of X-prolyl dipeptidylaminopeptidase activity with a new fluorogenic substrate. Biochemical Medicine, 19(3), 351
359. Available from https://doi.org/10.1016/0006-2944(78)90035-2. Kawanami, D., Matoba, K., Sango, K., & Utsunomiya, K. (2016). Incretin-based therapies for diabetic complications: Basic mechanisms and clinical evidence. International Journal of Molecular Sciences, 17 (8), 1223. Available from https://doi.org/10.3390/ijms17081223. Kehinde, B. A., & Sharma, P. (2020). Recently isolated antidiabetic hydrolysates and peptides from multiple food sources: A review. Critical Reviews in Food Science and Nutrition, 60(2), 322
340. Available from https://doi.org/10.1080/10408398.2018.1528206. Khalil, N. A., Motaal, A. A., Meselhy, K., & Abdel Khalek, S. M. (2018). Renin and angiotensin converting enzyme inhibition of standardized bioactive fractions of hyphaene thebaica L. mart growing in Egypt. Pharmacognosy Journal, 10(4), 622
627. Available from https://doi.org/10.5530/pj.2018.4.102.

Methodologies for bioactivity assay: biochemical study

147

Kim, Y. J., Kang, K. S., & Yokozawa, T. (2008). The anti-melanogenic effect of pycnogenol by its anti-oxidative actions. Food and Chemical Toxicology, 46, 2466
2471. Available from https://doi.org/10.1016/j.fct.2008.04.002. Kleekayai, T., Harnedy, P. A., O’Keeffe, M. B., Poyarkov, A. A., Cunhaneves, A., Suntornsuk, W., & FitzGerald, R. J. (2015). Extraction of antioxidant and ACE inhibitory peptides from Thai traditional fermented shrimp pastes. Food Chemistry, 176, 441
447. Available from https://doi.org/10.1016/j.foodchem.2014.12.026. Kondrashina, A., Brodkorb, A., & Giblin, L. (2020). Dairy-derived peptides for satiety. Journal of Functional Foods, 66, 103801. Available from https://doi.org/10.1016/j.jff.2020.103801. Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications. Journal of Functional Foods, 1(2), 177
187. Available from https://doi.org/10.1016/j.jff.2009.01.007. Kumar, M. S. (2019). Peptides and peptidomimetics as potential antiobesity agents: Overview of current status. Frontiers in Nutrition, 6, 11. Available from https://doi.org/10.3389/fnut.2019.00011. Kurihara, H., Asami, S., Shibata, H., Fukami, H., & Tanaka, T. (2003). Hypolipemic effect of Cyclocarya paliurus (Batal) Iljinskaja in lipid-loaded mice. Biological and Pharmaceutical Bulletin, 26(3), 383
385. Available from https://doi.org/10.1248/bpb.26.383. Kwon, Y. I., Vattem, D. A., & Shetty, K. (2006). Evaluation of clonal herbs of Lamiaceae species for management of diabetes and hypertension. Asia Pacific Journal of Clinical Nutrition, 15(1), 107
118. Lacroix, I. M. E., & Li-Chan, E. C. Y. (2012). Dipeptidyl peptidase-IV inhibitory activity of dairy protein hydrolysates. International Dairy Journal, 25(2), 97
102. Available from https://doi.org/10.1016/j.idairyj.2012.01.003. Lacroix, I. M. E., & Li-Chan, E. C. Y. (2015). Comparison of the susceptibility of porcine and human dipeptidyl peptidase IV to inhibition by protein-derived peptides. Peptides, 69, 19
25. Available from https://doi.org/10.1016/j.peptides.2015.03.016. Le Maux, S., Nongonierma, A. B., Barre, C., & FitzGerald, R. J. (2016). Enzymatic generation of whey protein hydrolysates under pH-controlled and non pH-controlled conditions: Impact on physicochemical and bioactive properties. Food Chemistry, 199, 246
251. Available from https://doi.org/10.1016/j. foodchem.2015.12.021. Li-Chan, E. C. Y., Hunag, S.-L., Jao, C.-L., Ho, K.-P., & Hsu, K.-C. (2012). Peptides derived from Atlantic salmon skin gelatin as dipeptidyl-peptidase IV inhibitors. Journal of Agricultural and Food Chemistry, 60 (4), 973
978. Available from https://doi.org/10.1021/jf204720q. Likhitwitayawuid, K., & Sritularak, B. (2001). A new dimeric stilbene with tyrosinase inhibitory activity from Artocarpus gomezianus. Journal of Natural Products, 64(11), 1457
1459. Available from https://doi.org/ 10.1021/np0101806. Liu, D., Gao, H., Tang, W., & Nie, S. (2017). Plant non-starch polysaccharides that inhibit key enzymes linked to type 2 diabetes mellitus. Annals of the New York Academy of Sciences, 1401(1), 28
36. Available from https://doi.org/10.1111/nyas.13430. Loijda, Z. (1979). Studies on dipeptidyl(amino)peptidase IV. II. Blood vessels. Histochemistry, 59, 153
166. Lo´pez-Expo´sito, I., Quiro´s, A., Amigo, L., & Recio, I. (2007). Casein hydrolysates as a source of antimicrobial, antioxidant and antihypertensive peptides. Dairy Science and Technology, 87(4
5), 241
249. Available from https://doi.org/10.1051/lait:2007019. Lorenzo, J. M., Munekata, P. E. S., Go´mez, B., Barba, F. J., Mora, L., Pe´rez-Santaescola´stica, C., & Toldra´, F. (2018). Bioactive peptides as natural antioxidants in food products
A review. Trends in Food Science and Technology, 79, 136
147. Available from https://doi.org/10.1016/j.tifs.2018.07.003. Maeda, H., Matsuno, H., Ushida, M., Katayama, K., Saeki, K., & Itoh, N. (2005). 2,4-Dinitrobenzenesulfonyl fluoresceins as fluorescent alternatives to Ellman’s reagent in thiol-quantification enzyme assays. Angewandte Chemie
International Edition, 44(19), 2922
2925. Available from https://doi.org/10.1002/ anie.200500114. Malomo, S. A., & Aluko, R. E. (2016). In vitro acetylcholinesterase-inhibitory properties of enzymatic hemp seed protein hydrolysates. JAOCS, Journal of the American Oil Chemists’ Society, 93(3), 411
420. Available from https://doi.org/10.1007/s11746-015-2779-0.

148 Chapter 6 Manikkam, V., Mathai, M. L., Street, W. A., Donkor, O. N., & Vasiljevic, T. (2016). Biofunctional and physicochemical properties of fish scales collagen-derived protein powders. International Food Research Journal, 23(4), 1614
1622. Mason, H. S. (1948). The chemistry of melanin; mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. The Journal of Biological Chemistry, 172(1), 83
99. Masuda, T., Yamashita, D., Takeda, Y., & Yonemori, S. (2005). Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica. Bioscience, Biotechnology, and Biochemistry, 69, 197
201. Available from https://doi.org/10.1271/bbb.69.197. Matheeussen, V., Lambier, A.-M., Jungraithmayr, W., Gomez, N., Entee, K., Van der Veken, P., . . . De Meester, I. (2012). Method comparison of dipeptidyl peptidase IV activity assays and their application in biological samples containing reversible inhibitors. Clinica Chimica Acta, 413, 456
462. Available from https://doi.org/10.1016/j.cca.2011.10.031. Matsui, T., Yoshimoto, C., Osajima, K., Oki, T., & Osajima, Y. (1996). In vitro survey of α-glucosidase inhibitory food components. Bioscience, Biotechnology, and Biochemistry, 60(12), 2019
2022. Available from https://doi.org/10.1271/bbb.60.2019. Merimsky, O., Shoenfeld, Y., Baharav, E., Zigelman, R., & Fishman, P. (1996). Reactivity to tyrosinase: Expression in cancer (melanoma) and autoimmunity (vitiligo). Human Antibodies, 7(4), 151
156. Available from https://doi.org/10.3233/HAB-1996-7402. Milardovic, S., Kerekovi´c, I., Derrico, R., & Rumenjak, V. (2007). A novel method for flow injection analysis of total antioxidant capacity using enzymatically produced ABTS{radical dot} 1 and biamperometric detector containing interdigitated electrode. Talanta, 71(1), 213
220. Available from https://doi.org/ 10.1016/j.talanta.2006.03.042. Miller, N. J., & Rice-Evans, C. A. (1997). Cinnamates and hydroxybenzoates in the diet: Antioxidant activity assessed using the ABTS• 1 radical cation. British Food Journal, 99(2), 57
62. Available from https:// doi.org/10.1108/00070709710165197. Mizuno, S., Matsuura, K., Gotou, T., Nishimura, S., Kajimoto, O., Yabune, M., . . . Yamamoto, N. (2005). Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension. British Journal of Nutrition, 94(1), 84
91. Available from https://doi.org/10.1079/ bjn20051422. Monsees, T., Miska, W., & Geiger, R. (1994). Synthesis and characterization of a bioluminogenic substrate for α-chymotrypsin. Analytical Biochemistry, 221(2), 329
334. Available from https://doi.org/10.1006/ abio.1994.1421. Moura, A. S., Halder, A. K., & Cordeiro, M. N. D. S. (2019). From biomedicinal to in silico models and back to therapeutics: A review on the advancement of peptidic modeling. Future Medicinal Chemistry, 11(17), 2313
2331. Available from https://doi.org/10.4155/fmc-2018-0365. Moyo, M., Ndhlala, A. R., Finnie, J. F., & Van Staden, J. (2010). Phenolic composition, antioxidant and acetylcholinesterase inhibitory activities of Sclerocarya birrea and Harpephyllum caffrum (Anacardiaceae) extracts. Food Chemistry, 123(1), 69
76. Available from https://doi.org/10.1016/j.foodchem.2010. 03.130. Mukherjee, M. (2003). Human digestive and metabolic lipases
A brief review. Journal of Molecular Catalysis B: Enzymatic, 22(5
6), 369
376. Available from https://doi.org/10.1016/S1381-1177(03)00052-3. Murray, B., & FitzGerald, R. (2007). Angiotensin converting enzyme inhibitory peptides derived from food proteins: Biochemistry, bioactivity and production. Current Pharmaceutical Design, 13(8), 773
791. Available from https://doi.org/10.2174/138161207780363068. Murray, B. A., Walsh, D. J., & FitzGerald, R. J. (2004). Modification of the furanacryloyl-Lphenylalanylglycylglycine assay for determination of angiotensin-I-converting enzyme inhibitory activity. Journal of Biochemical and Biophysical Methods, 59(2), 127
137. Available from https://doi.org/10.1016/ j.jbbm.2003.12.009. Nagatsu, T., Hino, M., Fuyamada, H., Hayakawa, T., & Sakakibara, S. (1976). New chromogenic substrates for X-prolyl dipeptidyl-aminopeptidase. Analytical Biochemistry, 74, 466
476. Available from https://doi.org/ 10.1016/0003-2697(76)90227-X.

Methodologies for bioactivity assay: biochemical study

149

Nam, K. A., You, S. G., & Kim, S. M. (2008). Molecular and physical characteristics of squid (Todarodes pacificus) skin collagens and biological properties of their enzymatic hydrolysates. Journal of Food Science, 73(4), C249
C255. Available from https://doi.org/10.1111/j.1750-3841.2008.00722.x. Neves, A. C., Harnedy, P. A., O’Keeffe, M. B., Alashi, M. A., Aluko, R. E., & FitzGerald, R. J. (2017). Peptide identification in a salmon gelatin hydrolysate with antihypertensive, dipeptidyl peptidase IV inhibitory and antioxidant activities. Food Research International, 100, 112
120. Available from https://doi.org/10.1016/ j.foodres.2017.06.065. Nita, M., & Grzybowski, A. (2016). The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxidative Medicine and Cellular Longevity, 2016, 3164734. Available from https://doi. org/10.1155/2016/3164734. Nongonierma, A. B., Cadamuro, C., Le Gouic, A., Mudgil, P., Maqsood, S., & FitzGerald, R. J. (2019). Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of a camel whey protein enriched hydrolysate preparation. Food Chemistry, 279, 70
79. Available from https://doi.org/10.1016/j.foodchem.2018.11.142. Nongonierma, A. B., & FitzGerald, R. J. (2013a). Dipeptidyl peptidase IV inhibitory properties of a whey protein hydrolysate: Influence of fractionation, stability to simulated gastrointestinal digestion and food-drug interaction. International Dairy Journal, 32(1), 33
39. Available from https://doi.org/10.1016/j.idairyj.2013.03.005. Nongonierma, A. B., & FitzGerald, R. J. (2013b). Dipeptidyl peptidase IV inhibitory and antioxidative properties of milk protein-derived dipeptides and hydrolysates. Peptides, 39(1), 157
163. Available from https://doi.org/10.1016/j.peptides.2012.11.016. Nongonierma, A. B., & FitzGerald, R. J. (2015). Utilisation of the isobole methodology to study dietary peptide-drug and peptide-peptide interactive effects on dipeptidyl peptidase IV (DPP-IV) inhibition. Food and Function, 6(1), 313
320. Available from https://doi.org/10.1039/c4fo00883a. Nongonierma, A. B., Lamoureux, C., & FitzGerald, R. J. (2018). Generation of dipeptidyl peptidase IV (DPPIV) inhibitory peptides during the enzymatic hydrolysis of tropical banded cricket (Gryllodes sigillatus) proteins. Food and Function, 9(1), 407
416. Available from https://doi.org/10.1039/c7fo01568b. Nongonierma, A. B., Maux, S. L., Esteveny, C., & FitzGerald, R. J. (2017). Response surface methodology applied to the generation of casein hydrolysates with antioxidant and dipeptidyl peptidase IV inhibitory properties. Journal of the Science of Food and Agriculture, 2017(97), 1093
1101. Available from https:// doi.org/10.1002/jsfa.7834. Norris, R., Casey, F., FitzGerald, R. J., Shields, D., & Mooney, C. (2012). Predictive modelling of angiotensin converting enzyme inhibitory dipeptides. Food Chemistry, 133(4), 1349
1354. Available from https://doi. org/10.1016/j.foodchem.2012.02.023. Norris, R., & FitzGerald, R. J. (2013). Antihypertensive peptides from food proteins. Bioactive food peptides in health and disease (pp. 45
72). InTech. Available from http://doi.org/10.5772/51710. Odaka, C., & Mizuochi, T. (2000). Angiotensin-converting enzyme inhibitor captopril prevents activationinduced apoptosis by interfering with T cell activation signals. Clinical and Experimental Immunology, 121 (3), 515
522. Available from https://doi.org/10.1046/j.1365-2249.2000.01323.x. O’Keeffe, M. B., & FitzGerald, R. J. (2014). Antioxidant effects of enzymatic hydrolysates of whey protein concentrate on cultured human endothelial cells. International Dairy Journal, 36(2), 128
135. Available from https://doi.org/10.1016/j.idairyj.2014.01.013. Oseguera-Toledo, M. E., Gonzalez de Mejia, E., & Amaya-Llano, S. L. (2015). Hard-to-cook bean (Phaseolus vulgaris L.) proteins hydrolyzed by alcalase and bromelain produced bioactive peptide fractions that inhibit targets of type-2 diabetes and oxidative stress. Food Research International, 76, 839
851. Available from https://doi.org/10.1016/j.foodres.2015.07.046. Ou, B., Hampsch-Woodill, M., Flanagan, J., Deemer, E. K., Prior, R. L., & Huang, D. (2002). Novel fluorometric assay for hydroxyl radical prevention capacity using fluorescein as the probe. Journal of Agricultural and Food Chemistry, 50(10), 2772
2777. Available from https://doi.org/10.1021/jf011480w. Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry, 49(10), 4619
4626. Available from https://doi.org/10.1021/jf010586o.

150 Chapter 6 ¨ zyu¨rek, M., Gu¨c¸lu¨, K., & Apak, R. (2011). The main and modified CUPRAC methods of antioxidant O measurement. TrAC—Trends in Analytical Chemistry, 30(4), 652
664. Available from https://doi.org/ 10.1016/j.trac.2010.11.016. Peng, S. (2009). Methods and applications of antioxidant activity assays. Pharmaceutical Bioassays: Methods and Applications, 205
221. Available from https://doi.org/10.1002/9780470522202.ch12. Persson, P. B. (2003). Renin: Origin, secretion and synthesis. Journal of Physiology, 552(3), 667
671. Available from https://doi.org/10.1113/jphysiol.2003.049890. Pimentel, F. B., Alves, R. C., Harnedy, P. A., FitzGerald, R. J., & Oliveira, M. B. P. P. (2019). Macroalgalderived protein hydrolysates and bioactive peptides: Enzymatic release and potential health enhancing properties. Trends in Food Science and Technology, 93, 106
124. Available from https://doi.org/10.1016/j. tifs.2019.09.006. Power, O., Jakeman, P., & FitzGerald, R. J. (2013). Antioxidative peptides: Enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids, 44(3), 797
820. Available from https://doi.org/10.1007/s00726-012-1393-9. Power, O., Nongonierma, A. B., Jakeman, P., & FitzGerald, R. J. (2014). Food protein hydrolysates as a source of dipeptidyl peptidase IV inhibitory peptides for the management of type 2 diabetes. Proceedings of the Nutrition Society, 73(1), 34
46. Available from https://doi.org/10.1017/S0029665113003601. Power-Grant, O., Bruen, C., Brennan, L., Giblin, L., Jakeman, P., & FitzGerald, R. J. (2015). In vitro bioactive properties of intact and enzymatically hydrolysed whey protein: Targeting the enteroinsular axis. Food and Function, 6(3), 972
980. Available from https://doi.org/10.1039/c4fo00983e. Power-Grant, O., McCormack, W. G., Ramia De Cap, M., Amigo-Benavent, M., FitzGerald, R. J., & Jakeman, P. (2016). Evaluation of the antioxidant capacity of a milk protein matrix in vitro and in vivo in women aged 50-70 years. International Journal of Food Sciences and Nutrition, 67(3), 325
334. Available from https://doi.org/10.3109/09637486.2016.1153607. Prasasty, V., Radifar, M., & Istyastono, E. (2018). Natural peptides in drug discovery targeting acetylcholinesterase. Molecules (Basel, Switzerland), 23(9), 2344. Available from https://doi.org/10.3390/ molecules23092344. Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53(10), 4290
4302. Available from https://doi.org/10.1021/jf0502698. Qian, Z.-J., Jung, W.-K., Byun, H.-G., & Kim, S.-K. (2008). Protective effect of an antioxidative peptide purified from gastrointestinal digests of oyster, Crassostrea gigas against free radical induced DNA damage. Bioresource Technology, 99, 3365
3371. Available from https://doi.org/10.1016/j.biortech.2007.08.018. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26, 1231
1237. Available from https://doi.org/10.1016/S0891-5849(98)00315-3. Rice-Evans, C., & Miller, N. J. (1994). 241 Total antioxidant status in plasma and body fluids, . Methods in enzymology (234, pp. 279
293). Elsevier. Rick, W. (1974). In H. U. Bergmeyer (Ed.), Methods of enzymatic analysis (2nd ed., pp. 1006
1012). Academic Press. Ruzza, P., Serra, P. A., Fabbri, D., Dettori, M. A., Rocchitta, G., & Delogu, G. (2017). Hydroxylated biphenyls as tyrosinase inhibitor: A spectrophotometric and electrochemical study. European Journal of Medicinal Chemistry, 126, 1034
1038. Available from https://doi.org/10.1016/j.ejmech.2016.12.028. Saghaie, L., Pourfarzam, M., Fassihi, A., & Sartippour, B. (2013). Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid. Research in Pharmaceutical Sciences, 8(4), 233
242. Santos-Sa´nchez, N. F., Salas-Coronado, R., Villanueva-Can˜ongo, C., & Herna´ndez-Carlos, B. (2019). Antioxidant compounds and their antioxidant mechanism. Antioxidants. IntechOpen. Schechter, I., & Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications, 27(2), 157
162. Available from https://doi.org/10.1016/S0006291X(67)80055-X.

Methodologies for bioactivity assay: biochemical study

151

Schieber, M., & Chandel, N. S. (2014). ROS function in redox signaling and oxidative stress. Current Biology, 24(10), R453
R462. Available from https://doi.org/10.1016/j.cub.2014.03.034. Schurink, M., van Berkel, W. J. H., Wichers, H. J., & Boeriu, C. G. (2007). Novel peptides with tyrosinase inhibitory activity. Peptides, 28(3), 485
495. Available from https://doi.org/10.1016/j. peptides.2006.11.023. Sergent, T., Vanderstraeten, J., Winand, J., Beguin, P., & Schneider, Y. J. (2012). Phenolic compounds and plant extracts as potential natural anti-obesity substances. Food Chemistry, 135(1), 68
73. Available from https://doi.org/10.1016/j.foodchem.2012.04.074. Serpen, A., Capuano, E., Fogliano, V., & Go¨kmen, V. (2007). A new procedure to measure the antioxidant activity of insoluble food components. Journal of Agricultural and Food Chemistry, 55(19), 7676
7681. Available from https://doi.org/10.1021/jf071291z. Shimura, S., Tsuzuki, W., Kobayashi, S., & Suzuki, T. (1992). Inhibitory effect on lipase activity of extracts from medicinal herbs. Bioscience, Biotechnology, and Biochemistry, 56(9), 1478
1479. Available from https://doi.org/10.1271/bbb.56.1478. Siegenthaler, U. (1977). Simple and rapid method for determination of α glucosidase (saccharase) in honey [EINE EINFACHE UND RASCHE METHODE ZUR BESTIMMUNG DER α GLUCOSIDASE (SACCHARASE) IM HONIG]. Mitteilungen aus den Gebiete der Lebensmitteluntersuchung und Hygiene, 68(2), 251
258. Siow, H. L., Lim, T. S., & Gan, C. Y. (2017). Development of a workflow for screening and identification of aamylase inhibitory peptides from food source using an integrated bioinformatics-phage display approach: Case study
Cumin seed. Food Chemistry, 214, 67
76. Available from https://doi.org/10.1016/j. foodchem.2016.07.069. Sosa Crespo, I., Laviada Molina, H., Chel-Guerrero, L., Ortiz-Andrade, R., & Betancur-Ancona, D. (2018). Inhibitory effect of peptide fractions derivatives from chia (Salvia hispanica) hydrolysis against α-amylase and α-glucosidase enzymes [Efecto inhibitorio de fracciones peptı´dicas derivadas de la hidro´lisis de semillas de chı´a (Salvia hispanica) sobre las enzimas α-amilasa y α-glucosidasa]. Nutricion Hospitalaria, 35(4), 928
935. Available from https://doi.org/10.20960/nh.1713. Stack, J., Tobin, P. R., Gietl, A., Harnedy, P. A., Stengel, D. B., & FitzGerald, R. J. (2017). Seasonal variation in nitrogenous components and bioactivity of protein hydrolysates from Porphyra dioica. Journal of Applied Phycology, 29(5), 2439
2450. Available from https://doi.org/10.1007/s10811-017-1063-0. ˇ ˇ Stajner, D., Popovi´c, B., Canadanovi´ c-Brunet, J., & Boˇza, P. (2006). Free radical scavenging activity of three Equisetum species from Fruˇska gora mountain. Fitoterapia, 77, 601
604. Available from https://doi.org/ 10.1016/j.fitote.2006.06.006. Sumner, J. B., & Howell, S. F. (1935). A method for determination of saccharase activity. Journal of Biological Chemistry, 108(1), 51
54. Available from https://doi.org/10.1016/S0021-9258(18)75307-6. Sun, C., Wang, J.-W., Fang, L., Gao, X.-D., & Tan, R.-X. (2004). Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108. Life Sciences, 75, 1063
1073. Available from https://doi.org/10.1016/j.lfs.2004.02.015. Sun, T., & Tanumihardjo, S. A. (2007). An integrated approach to evaluate food antioxidant capacity. Journal of Food Science, 72(9), R159
R165. Available from https://doi.org/10.1111/j.17503841.2007.00552.x. Thapa, G., Das, D., & Gunupuru, L. R. (2016). Expression of Echmr gene from Eichhornia offers multiple stress tolerance to Cd sensitive Escherichia coli Δgsh mutants. Biochemical and Biophysical Research Communications, 478(1), 101
109. Available from https://doi.org/10.1016/j.bbrc.2016.07.086. Tsai, C.-H., Yen, Y.-H., & Yang, J. P.-W. (2015). Finding of polysaccharide-peptide complexes in Cordyceps militaris and evaluation of its acetylcholinesterase inhibition activity. Journal of Food and Drug Analysis, 23(1), 63
70. Available from https://doi.org/10.1016/j.jfda.2014.05.006. Tu, M., Cheng, S., Lu, W., & Du, M. (2018). Advancement and prospects of bioinformatics analysis for studying bioactive peptides from food-derived protein: Sequence, structure, and functions. TrAC—Trends in Analytical Chemistry, 105, 7
17. Available from https://doi.org/10.1016/j.trac.2018.04.005.

152 Chapter 6 Uhlig, T., Kyprianou, T., Martinelli, F. G., Oppici, C. A., Heiligers, D., Hills, D., . . . Verhaert, P. (2014). The emergence of peptides in the pharmaceutical business: From exploration to exploitation. EuPA Open Proteomics, 4, 58
69. Available from https://doi.org/10.1016/j.euprot.2014.05.003. Vermeirssen, V., Van Camp, J., & Verstraete, W. (2002). Optimisation and validation of an angiotensinconverting enzyme inhibition assay for the screening of bioactive peptides. Journal of Biochemical and Biophysical Methods, 51(1), 75
87. Available from https://doi.org/10.1016/S0165-022X(02)00006-4. Wang, G. T., Chung, C. C., Holzman, T. F., & Krafft, G. A. (1993). A continuous fluorescence assay of renin activity. Analytical Biochemistry, 210(2), 351
359. Available from https://doi.org/10.1006/ abio.1993.1207. Wang, X.-Y., Liang, Q., Chen, H.-G., & Zhou, X. (2018). Establishment of an HPLC method for testing acetylcholinesterase inhibitory activity and compared with traditional spectrophotometry. Chemical Papers, 72(9), 2255
2264. Available from https://doi.org/10.1007/s11696-018-0459-x. Wang, Y., Zhao, J., Xu, J. H., Fan, L. Q., Li, S. X., Zhao, L. L., & Mao, X. B. (2010). Significantly improved expression and biochemical properties of recombinant serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester. Applied Biochemistry and Biotechnology, 162(8), 2387
2399. Available from https://doi.org/10.1007/s12010-010-9011-3. Wilson, T., March, H., Banz, W. J., Hou, Y., Adler, S., Meyers, C. Y., . . . Maher, M. A. (2002). Antioxidant effects of phyto-and synthetic-estrogens on cupric ion-induced oxidation of human low-density lipoproteins in vitro. Life Sciences, 70, 2287
2297. Available from https://doi.org/10.1016/S0024-3205(02)01483-2. Wong, F.-C., Xiao, J., Wang, S., Ee, K.-Y., & Chai, T.-T. (2020). Advances on the antioxidant peptides from edible plant sources. Trends in Food Science and Technology, 99, 44
57. Available from https://doi.org/ 10.1016/j.tifs.2020.02.012. Worthington. (2020). Worthingong enzyme manual. http://www.worthington-biochem.com/AA/assay.html Yakoby, N., & Raskin, I. (2004). A simple method to determine trypsin and chymotrypsin inhibitory activity. Journal of Biochemical and Biophysical Methods, 59(3), 241
251. Available from https://doi.org/10.1016/ j.jbbm.2004.02.009. Yan, J., Zhao, J., Yang, R., & Zhao, W. (2019). Bioactive peptides with antidiabetic properties: A review. International Journal of Food Science and Technology, 54(6), 1909
1919. Available from https://doi.org/ 10.1111/ijfs.14090. Yang, D., Sun, S., Fu, C., Jiang, X., & Tang, D. (2012). Kinetic study of α-chymotrypsin by electrophoretically mediated microanalysis combined with partial filling technique. Chromatographia, 75(13
14), 755
760. Available from https://doi.org/10.1007/s10337-012-2250-4. Yang, Y. J., He, H. Y., Wang, F. Z., Ju, X. R., Yuan, J., Wang, L. F., . . . He, R. (2017). Transport of angiotensin converting enzyme and renin dual inhibitory peptides LY, RALP and TF across Caco-2 cell monolayers. Journal of Functional Foods, 35, 303
314. Available from https://doi.org/10.1016/j. jff.2017.05.053. Yu, J., & Ahmedna, M. (2012). Functions/applications of trypsin in food processing and food science research. Trypsin: Structure, biosynthesis and functions (pp. 75
96). ResearchGate. Yu, Z., Dong, W., Wu, S., Shen, J., Zhao, W., Ding, L., . . . Zheng, F. (2020). Identification of ovalbuminderived peptides as multi-target inhibitors of AChE, BChE, and BACE1. Journal of the Science of Food and Agriculture, 100(6), 2648
2655. Available from https://doi.org/10.1002/jsfa.10295. Yu, Z., Yin, Y., Zhao, W., Liu, J., & Chen, F. (2012). Anti-diabetic activity peptides from albumin against α-glucosidase and α-amylase. Food Chemistry, 135(3), 2078
2085. Available from https://doi.org/ 10.1016/j.foodchem.2012.06.088. Yuan, L., Wu, J., Aluko, R. E., & Ye, X. (2006). Kinetics of renin inhibition by sodium houttuyfonate analogs. Bioscience, Biotechnology, and Biochemistry, 70(9), 2275
2280. Available from https://doi.org/10.1271/ bbb.60213. Zare-Zardini, H., Tolueinia, B., Hashemi, A., Ebrahimi, L., & Fesahat, F. (2013). Antioxidant and cholinesterase inhibitory activity of a new peptide from Ziziphus jujuba fruits. American Journal of Alzheimer’s Disease and Other Dementias, 28(7), 702
709. Available from https://doi.org/10.1177/1533317513500839.

Methodologies for bioactivity assay: biochemical study

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Zhang, X., Li, G., Wu, D., Yu, Y., Hu, N., Wang, H., . . . Wu, Y. (2020). Emerging strategies for the activity assay and inhibitor screening of alpha-glucosidase. Food and Function, 11(1), 66
82. Available from https://doi.org/10.1039/c9fo01590f. Zhuang, Y., Sun, L., Zhao, X., Wang, J., Hou, H., & Li, B. (2009). Antioxidant and melanogenesis-inhibitory activities of collagen peptide from jellyfish (Rhopilema esculentum). Journal of the Science of Food and Agriculture, 89(10), 1722
1727. Available from https://doi.org/10.1002/jsfa.3645. Zimmerman, M., Ashe, B., Yurewicz, E. C., & Patel, G. (1977). Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates. Analytical Biochemistry, 78(1), 47
51. Available from https://doi.org/10.1016/0003-2697(77)90006-9.

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CHAPTER 7

Methodologies for bioactivity assay: cell study Nan Shang, Khushwant S. Bhullar and Jianping Wu 4
10 Agricultural/Forestry Centre, Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

7.1 Introduction Bioactive peptides are specific small fragments of protein that can provide positive impacts on body functions or conditions and thus have the potential to improve human health (Kang et al., 2020). They can be obtained from food proteins through digestive proteolysis in the gastrointestinal tract, enzymatic hydrolysis in vitro, food processing including microbial fermentation (Korhonen & Pihlanto, 2006), from endogenous proteins in living organisms during biological activities (Sasaki, Takahashi, Satoh, Yamasaki, & Minamino, 2010; Tinoco & Saghatelian, 2011), by recombinant DNA technology, chemical synthesis, or extraction from food stuffs (Ahn & Je, 2019). The past decade has seen an exponential increase in bioactive peptide research, and the bioactive peptide market value is expected to reach USD 48 billion by 2025 (Ghosh, 2016). Bioactive peptides play important roles in the pathophysiology of many diseases that are involved in inflammation, hemostasis, neurotransmission, immune response, cell proliferation, hormone responses, and oxidative stress (Bhandari et al., 2020). Food proteins are good sources of bioactive peptides. A great number of bioactive peptides has been identified from food proteins and processed foods (Sa´nchez & Va´zquez, 2017). In addition to providing essential amino acids, bioactive peptides can target various body systems to exert beneficial effects; thus bioactive peptides have vast potential for the prevention and mitigation of numerous chronic diseases (Cicero, Fogacci, & Colletti, 2017). Most studies use activity-guided empirical approach to identify peptides that often require multistep fractionation and then peptide characterization; over the past decade, there is a surge of interest to discover bioactive peptides using bioinformatics approach or an integrated/hybrid approach (Daliri, Lee, & Oh, 2018; Kang et al., 2020). What is essential in bioactive peptide discovery is to validate the bioactivity through in vitro and in vivo experiments. Although in vivo study is preferred, it is not practical and costly to apply in vivo study during the early stage of peptide discovery. Additionally, it is also important Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00013-3 © 2021 Elsevier Inc. All rights reserved.

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156 Chapter 7 to provide preliminary results to minimize the animal uses as far as possible since the right to use animal to benefit human purpose is also debated in our modern societies. Thus in vitro experiment is commonly applied first to validate and/or evaluate the bioactivity and functionality of the peptide of interest before developing animal study and/or clinical trials. Compared to pure chemical assay, cell study is considered more biological relevant (Bacharach, 1945; Jahandideh, Chakrabarti, Davidge, & Wu, 2016). Cells are the building blocks of life; every organ in the body is composed of different types of cells. In vitro cell culture is a laboratory method used for studying cell behavior in a controlled environment, free of systemic variations (Arango, Quintero-Ronderos, Castiblanco, & Montoya-Ortı´z, 2013; Thorpe, 2007). This technique was first developed in the early 20th century and now is a versatile tool to study basic cell biology and to explore disease pathogenesis (Segeritz & Vallier, 2017). Modern cell culture techniques can recapitulate many fundamental pathophysiological systems in the laboratory, and the effects of externally supplied agents (such as bioactive peptides) can be successfully tested in cultured cells. Therefore prior to animal study, a key step is to test potential bioactive peptides in cell culture systems. Cell culture is also widely used to understand the fundamental mechanisms of bioactive peptides. Results from cell culture study are used as the rationale for future animal study. In this chapter the basic knowledge of cell culture technique, including lab safety and biohazard management, aseptic technique, contamination control, basic techniques for cell culture, as well as basic cell culture protocols are introduced.

7.2 Cell culture basics 7.2.1 Basic equipment for cell culture This section lists the basic equipment and supplies required for any cell culture facility, and additional equipment that can improve work efficiency or permit a wider range of assay and analyses (Table 7.1). Note: 1. The requirements for any cell culture facility depend on the type of work conducted. 2. For more information on setting up and maintaining a cell culture facility, refer to Setting up a Cell Culture Laboratory, Cell Biology, a Laboratory Handbook (O’Connor & O’Driscoll, 2006).

7.2.2 Safety aspects of cell culture 7.2.2.1 Risk assessment The main aims of risk assessment are to prevent injury, to protect property, and to avoid harm to individuals and the environment. In addition to many common risks in a laboratory,

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Table 7.1: Recommended equipment/supply for the cell culture laboratory. Equipment

Purposes Basic equipment

Biosafety cabinet Incubator Centrifuge Water bath Fridge and freezer Liquid nitrogen Autoclave

To provide a clean working environment and to protect the operator from aerosols To provide a controlled environment for cell growth To collect cells for subculture To warm-up cell culture media To store cell media, samples, buffers needed for cell study To store cells To sterilize supplies such as pipettes and buffers Expanded equipment

Vacuum pump Inverted light microscope Cell counter Roller racks Cell scraper

To aspirate cell culture medium To assess cell morphology and to count cells To count cells, determine growth kinetics, and prepare suitable plating densities To scale-up monolayer cultures To detach adhesive cells from their tissue-culture-treated substrate Additional supplies

Plastic ware and consumables Syringes and needles Pipettes and pipettors Cell media and supplementary components Containers for waster Ethanol and bleach

To culture cells in different formats, such as flasks, Petri dishes, 96-well plates To aliquot different volumes To culture cells in desirable components To correctly dispose of waste To reduce the contaminations

a cell culture facility has a number of specific hazards associated with handling and manipulating human or animal cells and tissues, as well as toxic, corrosive, and mutagenic solvents and regents. The fundamental objective of any biosafety program is to avoid or eliminate exposure of potential harmful biological agents to laboratory workers and the outside environment. The regulations and recommendations for biosafety are contained in the document Biosafety in Microbiological and Biomedical Laboratories, prepared by the Centers for Disease Control (CDC) and the National Institutes of Health (NIH), published by the US Department of Health and Human Service. The document defines four ascending levels of containment, referred to as biosafety levels (BSL) 1 through 4, and described the microbiological practices, safety equipment, and facility safeguards for the corresponding level of risk associated with handing a particular agent. Most cell culture labs should be at least BSL-2, but the exact requirements depend upon the cell line used and the type of work conducted.

158 Chapter 7 Safety Data Sheet (SDS), previously referred to as Material Safety Data Sheet, is a form containing information regarding the properties of a particular substance. The SDS includes physical data such as melting point, boiling point, and flash point, information on the substance’s toxicity, reactivity, health effects, storage, and disposal, as well as recommendation protective equipment and procedures for handling spills. 7.2.2.2 Biohazards Viruses are one of the most likely biohazards presented by cell culture. Other potential biohazards include components of the cell culture medium, other adventitious agents, and cell products, some of which may be biologically active molecules with pharmacological, immunomodulating, or sensitizing properties. In addition, the generation and use of modified cells, for example, hybrids, transformed cells, and cells-containing recombinant DNA can be hazardous. Safety equipment in a cell culture laboratory includes primary barrier such as biosafety cabinet, enclosed container, and other engineering control designed to remove or minimize exposure to hazardous material, as well as personal protective equipment (PPE) that is often used in conjunction with the primary barrier. The biosafety cabinet is the most important workspace to provide containment of infectious splashes or aerosols generated by many microbiological procedures as well as to prevent contamination of your own cell culture. 7.2.2.3 Disinfection Methods designed for disinfection/decontamination of culture waste, work surfaces, and equipment are important means for minimizing the risk of harm. •

•

•

Hypochlorites, such as sodium hypochlorite, are disinfectant used for general purpose. They are active against virus, but should be made fresh daily and cannot be used on metal surfaces. Alcohol, such as ethanol and isopropanol, is effective against bacteria at an optimal concentration of 70% for ethanol or 60%B70% for isopropanol. Ethanol is effective against most viruses but not nonenveloped viruses, whereas isopropanol is not effective against viruses. Aldehydes, such as formaldehyde, are used to fumigate laboratories. The formaldehyde is heated in a device so it will vaporize, and all exposed surfaces are coated with the disinfectant. Generally, the use of aldehydes for disinfection and fumigation purpose can be hazardous.

7.2.2.4 Waste disposal Every employer has a “duty of care” to dispose all biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture

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waste should be decontaminated and disposed safely. • • • •

Tissue culture waste (culture medium) should be inactived for at least 2 h in a solution of hypochlorite (10,000 ppm) prior to disposal to drain with an excess of water Contaminated pipettes should be placed in hypochlorite solution (2500 ppm) overnight before disposal by autoclaving and incineration Solid waste such as flasks, centrifuge tubes, contaminated gloved, and tissues should be placed in side heavy-duty sacks for contaminated wastes and autoclaved or incinerated Waste from specially licensed laboratories requires specific treatment and tracking

Note: For more information on standard microbiological practices and for specific biosafety level guideline, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th Edition at www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.

7.2.3 Aseptic technique and contamination control In cell culture, contaminations represent the main problem. Contaminants are most commonly of biological nature and can include bacteria, fungi, viruses, and parasites. It is important to limit biological contaminants since they can alter the phenotype and genotype of the cultured cell line through competition for nutrients, synthesis of alkaline, acidic or toxic by-products, and the potential interference of viral components with the cell culture genome (Segeritz & Vallier, 2017). Aseptic technique, designed to provide a barrier between the microorganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probity to contaminations from these sources. The elements of aseptic technique are a good personal hygiene, sterile work area, sterile reagents and media, and sterile handling. 7.2.3.1 Personal hygiene The human skin harbors a naturally occurring and vigorous population of bacterial and fungal inhabitants that shed microscopically and ubiquitously. Thus it is important to prevent the introduction of human skin flora during aseptic culture manipulations (Cote´, 1998). Laboratory staff can contribute to a clean work surface by washing hands with soap before and after working with cell culture. Disposable gloves sprayed with 70% ethanol, and lab coats can further reduce the introduction of contaminants carried by hair, skin, or dust (Segeritz & Vallier, 2017). 7.2.3.2 Sterile work area—biosafety cabinet The simplest and most economical way to reduce contamination from airborne particles and aerosols is to use a biosafety cabinet. Class II and III biosafety cabinet is recommended to create sterile work surface for cell work. Most biosafety cabinets require a warm-up time after which the work surface should be decontaminated with an antifungal detergent, such as 5% Trigene, followed by 70% ethanol. Some biosafety cabinet may also have ultraviolet

160 Chapter 7 light for decontamination. All equipment and item that comes into the biosafety cabinet must be sterile by spraying or wiping with 70% ethanol. The number of items entering the biosafety cabinet should be kept at a minimum to avoid any obstruction of airflow and the introduction of contamination. Regular maintenance and routine service through biosafety cabinet engineers can ensure correct airflow and full filter capacity of this important piece of cell culture equipment. In addition, it is also critical to keep all other surfaces in contact with the cell culture vessels or media components clean, which includes the incubator, centrifuge, microscope, water bath, fridge, and freezer. 7.2.3.3 Sterile reagent and media In addition to the impact of laboratory staff and environment, another main source of contamination is the cell culture medium. Commercially sourced media and supplementary cell culture products are generally supplied in sterile condition. For selfprepared culture medium, filter-sterilizing is recommended by forcing the liquid through a 0.22 μM polyethersulfone low-binding filter system using a vacuum pump, whereas autoclaving is conventionally used to sterilize equipment. Adding antibiotics, such as penicillin/streptomycin and antibiotic/antimycotic, can further limit the risk of bacterial growth in media bottles after opening and in cell culture vessels. However, the use of antibiotics should be refrained in some conditions since it may facilitate the emergence of resistant bacteria strains and lead to interference with cell metabolism and experimental outcomes. Note: For an in-depth review of aseptic technique, refer to Culture of Animal Cells: A manual of Basic Technique and Specialized Application (Freshney, 2016).

7.2.4 Cell types and sourcing of cell lines 7.2.4.1 Primary cultures Primary cultures are derived directly from excised, normal animal tissue and cultures either as an explant culture or following dissociation into a single-cell suspension by enzyme digestion. Such cultures are initially heterogeneous but later become dominated by fibroblasts. The preparation of primary culture is labor intensive, and they can be maintained in vitro only for a limited period of time. During their relatively limited lifespan, primary cells usually retain many of the differentiated characteristics of the cell in vivo. Note: Primary cultures by definition have not been passaged. As soon as they are passaged, they become a cell line and are no longer primary. “Primary” cells sourced from most suppliers are in fact low-passage cell lines.

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7.2.4.2 Continuous cultures Continuous cultures are comprised of a single-cell type that can be serially propagated in culture either for a limited number of cell division or otherwise indefinitely. Continuous cell lines that can be propagated indefinitely generally have this ability because they have been transformed into tumor cells. Tumor cell lines are often derived from actual clinical tumors, but transformation may also be induced using viral oncogenes or by chemical treatment. Transformed cell lines present the advantage of almost limitless availability, but the disadvantage of having retained very little of the original in vivo characteristics. 7.2.4.3 Selecting the appropriate cell line Selecting an appropriate cell is considered as a starting point in the experiment. The choice of a cell line for cell culture depends heavily on the functional properties and specific readouts required of the cell model (Pan, Kumar, Bohl, Klingmueller, & Mann, 2009). The following criteria should be considered while selecting the appropriate cell lines. •

• •

•

•

•

Species: nonhuman and nonprimate cell lines usually have fewer biosafety restrictions, but ultimately your experiment will dictate whether to use species-specific culture or not. Functional characteristics: what is the purpose of your experiments? For example, liverand kidney-derived cell line may be more suitable for toxicity testing. Finite or continuous: while choosing from finite cell lines may give you more options to express the correct functions, continuous cell lines are often easier to clone and maintain. Normal or transformed: transformed cell lines usually have an increased growth rate and higher plating efficiency, are continuous, and require less serum in media, but they have undergone a permanent change in their phenotype through a genetic transformation. Growth conditions and characteristics: what are your requirements with respect to growth rate, saturation density, cloning efficiency, and the ability to grow in suspension? For example, to express a recombinant protein in high yield, you might want to choose a cell line with a fast growth rate and an ability to grow in suspension. Table 7.2 shows comparisons between adherent and suspension culture that can help the researchers with experimental design. Other criteria: if you are using a finite cell line, are there sufficient stocks available? Is the cell line well characterized, or do you have to perform the validation yourself? If you are using an abnormal cell line, do you have an equivalent normal cell line that you can use as a control? Is the cell line stable? If not, how easy it is to clone it and generate sufficient frozen stocks for your experiments?

162 Chapter 7 7.2.4.4 Sourcing cell lines You may establish your own culture from primary cells, or you may choose to buy established cell cultures from commercial or nonprofit suppliers, like cell banks. The information of several well-known cell banks is shown in Table 7.3. It is recommended to obtain cell lines from reputable suppliers, where certain quality control measures are in place that guarantee genomic stability and the absence of contaminants. You may also obtain cultures from other laboratories, but make sure that they are free from contaminants. Table 7.2: The comparisons between adherent and suspension culture. Adherent culture

Suspension culture

Appropriate for most cell type, including primary culture. Requires periodic passaging, but allows easy visual inspection under inverted microscope. Cells are dissociated enzymatically (i.e., trypsin) or mechanically (i.e., cell scraper). Growth is limited by surface area, which may limit product yields. Requires tissue-culture-treated vessel.

Used for cytology, harvesting products continuously, and many research applications.

Appropriate for cells adapted to suspension culture and a few other cell lines that are nonadhesive, such as hematopoietic. Easier to passage, but requires daily cell counts and viability determination to follow growth patterns; culture can be diluted to stimulate growth. Does not require enzymatic or mechanical dissociation. Growth is limited by concentration of cells in the medium, which allows easy scale-up. Can be maintained in culture vessels that are not tissueculture-treated, but requires agitation (i.e., shaking or stirring) for adequate gas exchange. Used for bulk protein production, batch harvesting, and many research applications.

Table 7.3: Cell culture banks. Cell bank name










American Type Culture Collection (ATCC) CellBank Australia Coriell Cell Repository Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) European Collection of Animal Cell Cultures (ECACC) Health Science Research Resources Bank (HSRRB), Japan Japanese Collection of Research Bioresources (JCRB) Interlab Cell Line Collection (ICLB) NIH Stem Cell Unit


RIKEN Gene Bank
UK Stem Cell Bank (UKSCB)
WiCell

Website (accessed by August 2020) http://www.atcc.org http://www.cellbankaustralia.com http://ccr.coriell.org http://www.dsmz.de http://www.phe-culturecollections.org.uk http://jhsf.or.jp/English/index_e.html http://cellbank.nihs.go.jp http://www.iclc.it http://stemcells.nih.gov/research/ nihresearch/scunit http://en.brc.riken.jp http://www.ukstemcellbank.org.uk http://www.wicell.org

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7.2.5 Cell culture conditions In general terms, cultured cells require a sterile environment and a supply of nutrient for growth. In addition, the culture environment should be stable in terms of temperature, pH, osmotic pressure, O2, and CO2 tension. 7.2.5.1 Culture media The culture medium is the most important component of the culture environment, because it provides the necessary nutrients, growth factor, and hormones for cell growth, as well as regulating the pH and osmotic pressure of the culture. The basic constituents of media include inorganic salts, carbohydrates, amino acids, vitamins, fatty acids and lipids, proteins and peptides, serum, and trace elements. •

•

•

•

Inorganic salts The inclusion of inorganic salts in media performs several functions. Primarily, they help to retain the osmotic balance of the cells and help regulate membrane potential by provision of sodium, potassium, and calcium ions. All of these are required in the cell matrix for cell attachment and as enzyme cofactors. Carbohydrates The main source of energy is derived from carbohydrates generally in the form of sugars. The major sugars used are glucose and galactose; however, some media contain maltose or fructose. The concentration of sugar varies form basal media containing 1
4.5 g/L in some more complex media. Media containing the higher concentration of sugars are able to support the growth of a wider range of cell types. Pyruvate is included in the formulation of some media, as an alternative energy source. Amino acids Amino acids are the building blocks of proteins. “Essential” amino acids must be added to culture media as cells are not able to synthesize themselves. The concentration of amino acids in the culture medium will determine the maximum cell density that can be achieved—once depleted the cells will no longer be able to proliferate. In relation to cell culture, glutamine, an essential amino acid, is particularly significant. Optimal cell performance usually requires supplementation of the media with glutamine prior to use. Some media formulations include L-alanyl glutamine, which is a more stable form of glutamine, and do not require supplementation. Adding supplements of nonessential amino acids to media both stimulates growth and prolongs the viability of the cells in culture. Serum Serum is a complex mix of albumins, growth factors, and growth inhibitors and is probably one of the most important components of cell culture medium. Fetal bovine serum (FBS) is the most commonly used serum in cell culture. Other types of serum are

164 Chapter 7

•

•

•

•

available including newborn calf serum and horse serum. The quality, type, and concentration of serum can all affect the growth of cells, and it is therefore important to screen batches of serum for their ability to support the growth of cells. Serum is also able to increase the buffering capacity of cultures that can be important for low growing cells or where the seeding density is low. It also helps to protect against mechanical damage which may occur in stirred cultures or whilst using a cell scraper. A further advantage of serum is the wide range of cell types with which it can be used despite the varying requirements of different culture in terms of growth factors. In addition, serum is able to bind and neutralize toxins. There is also a risk of contamination associated with the use of serum. In particular, serum is screened for the presence of bovine viral diarrhea virus and mycoplasma. Heat inactivation of serum (incubate at 56 C for 20
30 min) can help to reduce the risk of contamination; however, this process may also denature some proteins and destroy nutrients in the serum. Vitamins Serum is an important source of vitamins in cell culture. However, many media are also enriched with vitamins making them consistently more suitable for a wider range of cell lines. Vitamins are precursors for numerous cofactors. Many vitamins, especially B group vitamins, are necessary for cell growth and proliferation, and for some cell lines, the presence of B12 is essential. Protein and peptides These are particularly important in serum-free media. The most common protein and peptides include albumin, transferrin, fibronectin, and fetuin and are used to replace those normally present through addition of serum to the medium. Fatty acids and lipids Like protein and peptides these are important in serum-free media since they are normally present in serum. Trace elements

These include trace elements such as zinc, copper, selenium, and tricarboxylic acid intermediates. 7.2.5.2 Temperature, pH, CO2, and O2 levels The desired temperature for cell cultures depends on the body temperature of the species, and the microenvironment from which the cultured cell types were isolated. Usually most human and mammalian cell lines are incubated at 36 C
37 C, whereas cell lines originating from cold-blooded animals can be maintained at wider temperature ranges between 15 C and 26 C. The pH level for most human and mammalian cell lines cultured in the lab should be tightly controlled and kept at a physiological pH level of 7.2
7.4. In contrast, some

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fibroblast cell lines favor slightly more alkaline conditions between pH 7.4 and7.7, whereas transformed cell lines prefer more acidic environments between pH 7.0 and 7.4 (Schwartz, Both, & Lechene, 1989). As the cell propagate, their growth requires energy supplied in the medium, for example, in the form of glucose. After metabolism, its by-products such as pyruvic acid, lactic acid, and CO2 are formed. Since the pH level is dependent on the balance of CO2 and HCO32 (bicarbonate), the addition of bicarbonate-based buffers to cell culture media can equilibrate the CO2 concentrations. Other pH buffers can be of organic nature and include 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (10
25 mM) or 3-(N-morpholino)propanesulfonic acid (20 mM). Many cell culture media contain pH indicator, such as phenol red, which display a color range between acidic (yellow) and alkaline (pink) conditions. Furthermore, fluctuations in atmospheric CO2 concentrations can also alter the pH level. Cells should therefore be cultured in incubators that also allow for CO2 tensions to be adjusted to 5%
7%. 7.2.5.3 Subculturing Subculturing, also referred to as “passaging,” is a procedure that enable the further propagation of the cell lines when the available space in the cell culture vessel reaches B80% confluency. This process generates subcultures or subclones and requires enzymatic digestion or mechanical disruption of the adherent cell monolayer to detach cells from their tissue-culture-treated substrate. While the growth of adherent cells is limited or enabled by the available surface area, it is the concentration of cells in the medium that creates the rate-limiting step in suspension cultures. It is therefore essential to monitor the growth rates in suspension cultures over time.

7.3 Basic cell culture protocols This section provides the basic protocols required for the maintenance of cell cultures. Since some of these protocols may need to be amended to accommodate the specific requirements of various cell type, it is helpful to review the recommendations of the cell line supplier or the information on the American Type Culture Collection (ATCC) website. The following protocols are a general procedure for most mammalian cells. For detailed protocols, always refer to the cell-specific protocol.

7.3.1 Protocol 1. Subculturing adherent cultures 1. Remove and discard the spent cell culture medium from the culture vessel. [Note 1] 2. Wash cells with prewarmed wash solution, such as phosphate-buffered saline (PBS) free of Mg21 and Ca21 (approximately 2 mL per 10 cm2 culture surface area). [Note 2] 3. Remove and discard the wash PBS from the culture vessel.

166 Chapter 7 4. Add prewarmed dissociation reagent, such as digestive enzymes or chelating agent to cover the cell layer (approximately 0.5 mL per 10 cm2) or mechanically dissociated with cell scraper. [Note 3] 5. Incubate the culture vessel with dissociation reagent at 37 C for approximately 5 min. [Note 4 and 5] 6. Add the equivalent of 2 volumes of prewarmed complete growth medium and disperse the medium by pipetting over the cell layer surface several times. 7. Transfer the dissociated cells to a sterile Falcon tube and collect them by centrifuge at 200 3 g for 5
10 min. [Note 6] 8. Resuspened the cell pellet in prewarmed complete growth medium and seed into new cell culture vessels. [Note 7] 9. Culture the new vessels in incubator. Note: 1. In order to maintain cell culture in optimum condition, it is essential to keep cells in the log phase of growth as far as it is practicable. Therefore it is importance to subculture cells at the end of log phase or early of stationary phase. However, the frequency of subculture is dependent on a number of factors, including inoculation density, growth rate, plating efficiency, and saturation density. These factors will vary between cell lines. Usually, the adhesive cells should be subcultured when reach to 80%
90% confluence, and the suspension cells should be subcultured when the density reach to 105 to 106 cells/mL. 2. The wash step removes dead cells and any traces of serum, calcium, and magnesium that may inhibit the action of the dissociation reagent. 3. Detach adherent cells can be performed by enzymatic or mechanical means. Mechanical means include shake-off and scraping, and enzymatic dissociation agent include trypsin, and collagenase, dispase, and TrypLE dissociation enzyme. The selection of dissociation procedures depends on the cell type. 4. The time required to detach the anchored cells from their substrate, and cell
cell interactions can take 1
60 min and depends on the cell type and the digestive enzymes used. 5. The extent of dissociation can be monitored under a light microscope and once complete, tapping the culture vessel should dislodge remaining adherent cells. 6. The centrifuge speed and time vary based on the cell type. Cells can be quite fragile, and it is advisable to not centrifuge at higher speeds or to pipette them vigorously. 7. The total number of cells and percent viability can be determined with Protocol 3. 8. It is a good lab practice to record the number of passages that have taken place since the culture has been initiated. Some cell lines are not suitable for experimental work beyond a given passage number since chromosomal abnormalities tend to increase in mammalian lines with cell divisions over time.

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7.3.2 Protocol 2. Subculturing suspension cultures 1. When the cells are ready for passaging, take a small sample from the culture flask and determine the total number of the cells and percent viability as described in Protocol 3. [Note 1] 2. Calculate the volume of media needed to add to dilute the culture down to the recommended seeding density. [Note 2] 3. Aseptically add the appropriate volume of prewarmed growth medium into the culture flask. [Note 3] 4. Culture the new vessels in an incubator. Note: 1. Subculturing of suspension cultures can also be simply achieved by aseptically removing one-third of the cell suspension solution and replacing the volume with prewarmed complete medium. 2. You can split the culture into multiple flasks if needed. 3. To minimize the accumulation of cell debris and metabolic waste by-product, gently centrifuge the cell suspension at 100 3 g for 5
10 min and resuspend the cell pellet in fresh growth medium once every 3 weeks or as needed.

7.3.3 Protocol 3. Quantification of total cell number and cell viability 1. Suspend the cells in a suitable medium volume. [Note 1] 2. Mix the cell suspension with 0.4% Trypan blue (Bio-rad) in a 1:1 dilution in an Eppendorf tube. [Note 2] 3. Load 10 μL of the cell mixture in Trypan Blue onto a cell counter or a hemocytometer. 4. The automated cell counter can automatically provide information of total cell number and cell viability. If using a hemocytometer, the total cell number and cell viability can be counted under an inverted microscope. [Note 3] Note: 1. Since cells are commonly cultured in millions, the number of cells are first counted in a small volume and then extrapolated to the full cell volume. 2. Trypan Clue dye permeates only nonviable cells that can therefore be excluded from the subsequent quantification (Strober, 2015). 3. A healthy cell culture is characterized by 80%
95% cell viability.

7.3.4 Protocol 4. Freezing cells Since cell line is a valuable resource, and its replacement is expensive, and time consuming is vitally important that they should be preserved for long-term storage. Therefore as soon

168 Chapter 7 as a surplus of cells becomes available from subculturing, they should be frozen as a seed stock for future use. The working cells can be prepared and replenished from frozen seed stocks (Protocol 5). The most common method of cryopreserving cultured cell is storing them in liquid nitrogen in complete medium in the presence of a cryoprotective agent such as dimethylsulfoxide (DMSO). However, as with other cell culture procedures, it is recommended to follow the specific instruction provided by the supplier for the best result. The following protocol is a general procedure for cryopreserving cultured cells. 1. Prepare fresh freezing medium and store at 2 C
8 C until use. [Note 1] 2. Collect cells and resuspend in the growth medium depending on the cell type following the Protocol 1 or 2. 3. Determine the total number of cells and viability following the Protocol 3 and calculate the required volume of freezing medium according to the desired viable cell density. [Note 2] 4. Centrifuge the cell suspension at 200 3 g for 5
10 min and collect the cell pellet. [Note 3] 5. Resuspened the cell pellet in cold freezing medium at the recommended viable cell density. 6. Dispense aliquots of the cell suspension into cryogenic storage vials. [Note 4] 7. Freeze the cells in a controlled-rate freezing apparatus, decreasing the temperature approximately 1
2 C/min or place the cryovials in an isopropanol chamber and store at 280 C overnight. 8. Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen for long-term storage. Note: 1. The freezing medium depends on the cell type. Usually the freezing medium is the growth medium supplemented with 10% DMSO. While glycerol and DMSO are both suitable cryoprotective agents, DMSO is toxic to personnel and cultured cells and therefore cannot be added to cells without prior dilution. This toxicity also affects cells in freezing medium containing 10% DMSO when left for several hours at room temperature, highlighting the need to transfer cells to 280 C for storage within 30 min. In general, chemically protective gloves should be worn to safeguard personnel from the hazards of DMSO as its solutes can easily penetrate membranes, including the skin. 2. Freeze cultured cells at a high concentration and at as low a passage number as possible. Make sure that the cells are at least 90% viable before freezing. 3. Centrifugation speed and duration varies depending on the cell type. 4. Usually each cryovial should have no less than 1 3 106 cells.

7.3.5 Protocol 5. Thawing cryopreserved cells 1. Remove the cryovial from liquid nitrogen storage and immediately place it into a 37 C water bath.

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2. Quickly thaw the cells (,1 min) by gently swirling the vial in the water bath until twothird of the content are thawed. 3. Wipe the outside of the vial with 70% ethanol before transfer to a biosafety cabinet. 4. Transfer the thawed cells drop-wise into the centrifuge tube containing the desired amount of prewarmed complete growth medium appropriate for the cell line. [Note 1 and 2] 5. Centrifuge the cell suspension at approximately 200 3 g for 5
10 min. [Note 3] 6. Remove and discard the supernatant carefully by pipetting without disturbing the cell pellet. 7. Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and culture in incubator. [Note 4 and 5] Note: 1. The desired amount of medium used to transfer the thawed cells depends on the volume, density and viability of the cryopreserved cells. Usually, 9 mL of medium is used for 1 mL of cryopreserved cells. 2. The drop-wise fashion minimizes the osmotic stress imposed upon the cells when DMSO is diluted. 3. After aspirating the supernatant, the cell pellet can be further washed once in medium to remove residual cryopreservatives. 4. The appropriate flask size depends on the number of cells frozen in the cryovial. Usually, it is suggested to use a 25 flask. 5. Usually, cell attachment should occur within 24 h.

7.4 Study bone health-promoting peptide 7.4.1 Bone formation cells 7.4.1.1 Protocol 6. In vitro osteoblasts culturing MC3T3-E1 cell line (ATCC CRL-2593)

The osteoblastic cell line MC3T3-E1 was established from a C57BL/6 mouse calvaria and selected on the basis of high alkaline phosphatase activity in the resting state. Cells have the capacity to differentiate into osteoblast and osteocytes and have been demonstrated to form calcified bone tissue in vitro. This cell line is a good model for studying in vitro osteoblast differentiation, particularly extracellular matrix signaling. It shows behavior similar to primary calvarial osteoblasts. Materials, equipment, and reagents

1. Base medium: Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mM L-glutamine, and 1 mM sodium pyruvate, but without ascorbic acid.

170 Chapter 7 2. 3. 4. 5. 6. 7. 8.

Complete medium: Base medium 1 10% FBS 1 1% Pen-strep solution PBS, pH 7.2. Trypsin-EDTA solution T-25 and T-75 tissue culture flasks Tissue culture plates Biosafety cabinet Cell culture incubator

Method

This method is based on the recommended procedure of the American Type Culture Collection (MC3T3-E1 Subclone 4 ATCC ® CRL-2593t, 2020). 1. If staring from a frozen (liquid nitrogen) vial of MC3T3-E1 cells, quickly (,2 min) thaw the vial by gentle agitation in a 37 C water bath. 2. Remove the vial from water bathe as soon as the contents are thawed, and transfer the vial into biosafety cabinet after decontaminate by spraying with 70% ethanol. 3. Transfer drop-wise the contents to a centrifuge tube containing 9 mL complete culture medium and centrifuge at 125 3 g for 5 min. 4. Resuspend the cell pellet with the complete medium and dispense into a T-25 or T-75 culture flask. [Notes 1 and 2] 5. Incubate the culture flask at 37 C until confluent (typically 5
7 days in T-25 flask). During the culture, remove and discard culture medium and replace with fresh complete medium every 2
3 days. 6. After confluence, briefly rinse the cells once with prewarmed PBS and add 5 mL of trypsin-EDTA solution to disperse the cell layers for subculture (Usually within 5
15 min). [Note 3] 7. Add 10 mL prewarmed complete medium and disperse the medium by pipetting over the cell layer surface several times. 8. Transfer the dissociated cells to a sterile centrifuge tube and centrifuge at 200 3 g for 5 min to collect the pellet. 9. Resuspened the cell pellet in a designed volume of complete medium, measure the cell number, and seed into new cell culture vessels for the following experiment, such as mineralization and western blot. [Note 4] 10. Incubate the new vessels in incubator and treated with the bioactive peptide of interest according the design of the following experiments. Note: 1. The appropriate flask size depends on the number of cells frozen in the cryovial. Usually, it is suggested to use a 25 flask to revive the cells first and then subculture to a 75 flask to expand the number of cells for the following study.

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2. It is suggested to place the culture vessel containing the complete medium into the incubator for at least 15 min to allow the medium to reach its normal pH to avoid excessive alkalinity during the recovery of the cells. 3. Observe the cells under an inverted microscope to check the disperse. To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place the flask at 37 C to help with the dispersal. 4. The use of culture vessels and seeding concentrations vary in different experiments. In our group, we usually use six-well plates to collect cells for western blot and qPCR (5 3 105 to 1 3 106 cells/well), and 48-well plate for other experiments (1 3 104 to 5 3 104 cells/well). 7.4.1.2 Protocol 7. Mineralization assay—Alizarin Red S staining assay Alizarin Red S (ARS), an anthraquinone dye, has been widely used to evaluate calcium deposits in cell culture, especially in the evaluation of osteogenic activity in osteoblast cells. Materials, equipment, and reagents

1. Base medium: Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2 mM L-glutamine, and 1 mM sodium pyruvate, but without ascorbic acid. 2. Complete medium: Base medium 1 10% FBS 1 1% Pen-strep solution 3. Mineralization medium: complete medium 1 10 mM β-glycerophosphate 1 50 μg/mL ascorbic acid 4. PBS, pH 7.2. 5. 40 mM ARS in ddH2O 6. 10 nM ethylpyridinium chloride 7. Six-well tissue culture plate 8. Biosafety cabinet 9. Cell culture incubator 10. Microplate reader Method

This method is based on the published procedure of Kim et al. (2017) and Shang and Wu (2018). 1. Seed MC3T3-E1 cells in six-well plates at a density of 1 3 104 cells/well in complete medium for overnight. 2. Wash the cells with prewarmed PBS to remove the unattached dead cells. 3. Add 2 mL complete medium and the bioactive peptide of interest into each well, and culture until reach 70%
80% confluence (usually 3
4 days).

172 Chapter 7 4. To initiate the mineralization, remove the culture medium and replace with 2 mL mineralization medium and the bioactive peptide of interest. 5. Culture the cells with mineralization medium for designated time to produce calcium deposits. [Note 1] 6. After designated culture time, remove culture medium from each well and gently wash cells three times with PBS. 7. Fix the cells in 4% formaldehyde for 15 min at room temperature. 8. Remove fixative and wash the cells three times with ddH2O. [Note 2] 9. Remove ddH2O completely and add 1 mL of 40 mM ARS in each well. Incubate the plate at room temperature for 30 min with gentle shaking. 10. Remove the dye and wash the cells five times with ddH2O. 11. Inspect the cells using a phase microscope and take images. Calcium nodes are stained with ARS showing dark red color. 12. After capture the images, destain the ARS with 10 nM ethylpyridinium chloride for 30 min at room temperature with gentle shaking. [Note 3] 13. Transfer 200 μL destain solution into a 96-well plate and measure the absorbance at 405 nm using a microplate reader to quantify the calcium deposits. Note: 1. The mineralization assay can take up to 30 days. In our group, we usually measure the mineralization using ARS assay in 5, 15, and 30 days. 2. It is recommended to use ddH2O in all steps during the mineralization assay to avoid the effect of Ca21 in water. 3. The ethylpyridinium chloride may cause skin corrosion and/or irritation, and serious eye damage and/or irritation. Use proper PPE to protect from the hazard of the chemical.

7.4.2 Bone resorption cells 7.4.2.1 Protocol 8. In vitro macrophage RAW 264.7 cell culture RAW 264.7 cell line (ATCC TIB-71)

The RAW 264.7 cells are monocyte/macrophage-like cells, which was established from a tumor induced by the Abelson murine leukemia virus. It is widely used in the in vitro study of inflammation response. Meanwhile, it is also commonly used in the bone health study, especially the osteoclast-related study. Osteoclasts can be obtained by isolating primary bone marrow monocytes (BMMs) or by using macrophage cell lines. In both cases, the cells need to be differentiated into the mature osteoclast by macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Unlike with BMMs and other macrophage cell lines, RAW 264.7 can secrete M-CSF on its own thus become the most widely used cell line for osteoclast generation.

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Materials, equipment, and reagents

1. Base medium: Dulbecco’s modified Eagle medium (DMEM) (contained with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 1.0 mM sodium pyruvate) 2. Complete medium: Base medium 1 10% fetal bovine serum 1 1% of Anti-Anti 3. PBS, pH 7.2 4. Rubber cell scrapers 5. T-25 and T-75 tissue culture flasks 6. Tissue culture plates 7. Biosafety cabinet 8. Cell culture incubator Method This method is based on the recommended procedure of the ATCC (RAW 264.7 ATCC® TIB-71t, 2020). 1. If staring from a frozen (liquid nitrogen) vial of RAW cells, revive the cells as described above and culture the cells into a T-25 tissue flask. Increase the volume in the flask to 6 mL with additional culture medium and place into a tissue culture incubator (day 0). 2. Culture cells until confluent (typically 4B5 days). Remove the culture medium and replace with fresh complete medium every 2 days. 3. To subculture the confluent RAW 264.7 cells, remove the culture medium, add 6 mL of fresh complete medium to the flask, and scrape the cell layer into this fresh medium using a rubber scraper. 4. Measure the cell number and calculate the cell concentrations. Plate the RAW 264.7 cells at B1.5 3 105 cells/cm2 into tissue culture dishes of the desired size. [Notes 1 and 2]. 5. Incubate the new vessels in incubator and treat with RANKL to differentiate into osteoclast cells according to the following experiment. Note: 1. Twelve-well tissue culture plate is mostly used for osteoclasts staining, and 24-well tissue culture plate was mostly used for other experiments in our lab. 2. Typically, one confluent T-25 flask will provide a sufficient number of RAW 264.7 cells to seed two 100 mm dishes or 24-well dishes. Increase these volumes with additional medium as needed to yield 8 mL per 100 mm dish or 0.5 mL per well of a 24-well dish and then place the cells into a tissue culture incubator. 7.4.2.2 Protocol 9. The generation of osteoclast from macrophage RAW 264.7 Materials, equipment, and reagents

1. Base medium: DMEM (contained with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 1.0 mM sodium pyruvate)

174 Chapter 7 2. Complete medium: Base medium 1 10% fetal bovine serum 1 1% of Anti-Anti 3. PBS, pH 7.2 4. RANKL (R&D Systems or other vendors): reconstitute and store as a concentrated stock solution (typically 100 μg/mL in PBS) in aliquots (B10
50 μL) at 220 C or 280 C as recommended by the manufacture. Briefly thaw and dilute the RANKL stock into culture medium to a final concentration of 35B100 ng/mL (depending on experiment) immediately before use with RAW cells, and refreeze remaining RANKL (aim to thaw individual vials no more than three times to retain optimal bioactivity). 5. Tissue culture plates 6. Biosafety cabinet 7. Cell culture incubator Method

This method is based on the published procedure of Collin-Osdoby and Osdoby (2012) and Shang and Wu (2020). 1. Subculture confluent RAW cells into 12-well culture plate at the density of B1 3 104 cells/well. 2. Immediately add soluble recombinant RANKL to the plate at a final concentration of 50 ng/mL to initiate osteoclast development. [Note 1] 3. Option 1: To evaluate the effect of the bioactive peptide of interest on inhibiting osteoclast formation, add the bioactive peptide of interest at the same time with RANKL. [Note 2] 4. Option 2: Culture the cells without adding the bioactive peptide of interest. 5. At day 3, briefly examine the cells under a microscope to see if RAW 264.7 cells are beginning to fuse into multinucleated RAW-osteoclasts. 6. Refeed the developing RAW-osteoclasts cell culture with 0.5 mL (or 1 mL) of fresh medium containing 50 ng/mL RANKL. [Note 3] 7. Culture until many multinucleated RAW-osteoclasts have formed but have not completely covered the dishes (usually 5
7 days). 8. To evaluate the effect of the bioactive peptide of interest on regulating osteoclastic activity, add the bioactive peptide of interest at this moment for designated time. (Optional) 9. After treatment, the RAW-osteoclast populations can be used for cytochemical or immunological staining, harvested for biochemical or molecular studies, or analyzed for bone resorption according to the following experimental design. Note: 1. The final concentration of RANKL can vary from 10 to 200 ng/mL depending on the experimental design. In our group, we usually use 50 and 100 ng/mL RANKL.

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2. For different objectives, the bioactive peptide of interest can be added at different time. 3. There is no need to replace the medium with fresh medium. Always refeed the medium containing RANKL instead completely replace it. 7.4.2.3 Protocol 10. Tartrate resistant acid phosphatase staining Osteoclasts express very high level of the enzyme tartrate resistant acid phosphatase (TRAP) that can be easily visualized by staining. Therefore the TRAP staining is a commonly used method to exam and count the number of generated osteoclasts. Materials, equipment, and reagents

1. 4% formaldehyde 2. Naphtol-AS-BI-phosphate stock: 10 mg/mL Naphtol-AS-BI-phosphate in dimethylformamide. Stable B1 week at 4 C. 3. Veronal buffer: 1.17 g sodium acetate anhydrous, 2.94 g veronal (sodium barbiturate). Dissolve in 100 mL distilled water. 4. Acetate buffer 0.1 N, pH 5.2: a. Dissolve 0.82 g sodium acetate anhydrous in 100 mL distilled water. b. 0.6 mL Glacial acetic acid, make up to 100 mL with distilled water. c. Adjust the pH of solution (a) to pH 5.2 with solution (b). 5. 100 mM tartrate: dissolve 2.3 g of sodium tartrate in 100 mL of acetate buffer. 6. Pararosaniline: add 1 g Pararosaniline to 20 mL distilled water and add 5 mL concentrated hydrochloric acid, heat carefully for 15 min in a 95 C water bath while stirring, and filter once the solution has cooled down. [Note 1] Method

This method is based on the published procedure of Ghayor et al. (2011), Kim, Kim, and Leem (2008), and Nan and Wu (2020). 1. 2. 3. 4. 5. 6. 7. 8. 9.

Rinse the cell culture plate prepared in Protocol 9 with PBS for three times. Fix the cells with 4% formaldehyde for 5 minutes and then rinse three times with PBS. Prepare the staining solution: Solution 1: In a glass container, add 150 μL Naphtol-AS-BI-phosphate stock to 750 μL Veronal buffer (pH 10.1). Then, add 0.9 mL acetate buffer with 100 mM Tartrate. Solution 2: Mix 120 μL Pararosaniline and 120 μL 4% NaNO2. Mix solutions 1 and 2, filter through a 0.45-μm filter and use immediately. Incubate the cells for 30
60 min at 37 C with staining solution (250 μL/well of a 24-well plate). Remove the staining solution and rinse three times with distilled water. Osteoclasts and mononuclear osteoclast precursors should be visible as bright red stained cells and can be captured and counted under light. [Notes 2 and 3]

176 Chapter 7 Note: 1. Solution (2)
(5) are stable for months in a refrigerator protected from light. 2. The stained cells can be stored in 70% ethanol in 4 C for several days. 3. As an alternative to the protocol described here, a staining kit from Sigma (387-A, Leukocyte Acid Phosphatse staining kit) can be used to avoid preparing the staining solutions. This kit uses fast Garnet as the dye, and this leads to a very dark purple stain. The detailed protocol of this kit can be found in their manufacture’s instruction. 7.4.2.4 Protocol 11. Osteoclastic resorption assay The osteoblastic resorptive activity is a direct indicator of osteoclastic activity and its bone resorptive capacity. Therefore the test of osteoclastic resorption plays an important role to evaluate the effect of the bioactive peptide of interest on inhibiting bone resorption. The resorptive activity can be evaluated using a bone resorption assay kit from Cosmo Bio (SCR-BRA-24KIT) descried as follow. Materials, equipment, and reagents

1. Base medium: Phenol red-free DMEM (contained with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 1.0 mM sodium pyruvate) 2. Complete medium: Base medium 1 10% fetal bovine serum 1 1% of Anti-Anti. 3. Bone resorption assay kit (Cosmo Bio., SCR-BRA-24KIT) 4. Sodium hypochlorite Method

This method is based on the manufacture’s instruction of Bone Resorption Assay kit (Cosmo Bio, USA) and published procedure of Shang and Wu (2019) 1. Prepare osteoclast from RAW 264.7 using the Calcium phosphate (CaP)-coated 24-well plate provided in the kit as described in Protocol 9. 2. Culture for B5
6 days with the bioactive peptide of interest according to the experimental design. [Note 1] 3. After 5
6 days, transfer 100 μL culture medium from each well into a 96-well plate. [Note 2] 4. Add 50 μL Bone Resorption Assay Kit Buffer to each well and mix using a plate shaker. 5. Measure fluorescence intensity with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. 6. To measure the pit area, remove the cells in the well by treating the plate with 5% sodium hypochlorite for 5 min. 7. Rinse the plate three times with distilled water and air dry. 8. Use microscope to photograph the regions in each well and measure the pit areas with image analyzing software (e.g., ImageJ). [Note 3]

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Note: 1. During the culture, do not discard the culture medium as the plate was coated with fluorescence. Only refeed with fresh complete medium. 2. Use black plate for fluorescence measurement. 3. The osteoclastic resorptive activity can be evaluated by the fluorescence intensity and the pit areas.

7.5 Biochemical and molecular analysis of cell study In addition to determining the biological activity as described above, in vitro cell culture technique is also an important tool to explore the mechanisms of the bioactive peptide of interest. Signal transduction is the process by which extracellular information is received, relayed, and translated into a cellular response. Over the last decade, there has been a tremendous increase in understanding of the signal transduction pathways that regulate bone cell differentiation, survival, and function in the presence of systemic hormones, local regulatory factors, and drug treatments. In the following sections, we mainly introduce the application of western blotting and Quantitative reverse transcription polymerase chain reaction (RT-qPCR), two essential biochemical and molecular analyses that are commonly used in cell-based bioactive peptides investigation.

7.5.1 Protocol 12. Western blotting Western blotting is used to determine if a protein of interest is present within a specific cell type, to assess protein abundance and to assess phosphorylation status or other posttranslational modification such as prenylation. The technique uses denaturing gel electrophoresis to separate proteins present in cell extracts according to their molecular weight. Once separated, proteins are transferred to nitrocellulose or polyvinylidene fluoride (PVDF) membrane and incubated with a primary antibody directed against the proteins of interest. If the protein of interest is present within the lysate, the primary antibody will bind to the membrane-bound protein and this can be detected by incubating the membrane with a secondary antibody linked to an amplification system such as horseradish peroxidase (HRP) that uses HRP-conjugated secondary antibody that can be visualized using enhanced chemiluminescence imager or transferred on a film. 7.5.1.1 Materials, equipment, and reagents 1. Lysis buffer [Note 1] a. Trish-HCl lysis buffer: 20 mM Tris-HCl in distilled water (pH to 7.5). b. Tris-Triton X-100 lysis buffer: 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate in distilled water.

178 Chapter 7

2.

3. 4.

5. 6. 7. 8. 9. 10.

11. 12. 13.

c. NP-40 lysis buffer: 150 mM NaCl, 1% NP-40, and 50 mM Tris-HCl (pH 8.0) in distilled water. d. RIPA lysis buffer: 150 nM NaCl, 1% NP-40 (or Triton X-100 or IGEPAL CA-630), 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl (8.0) in distilled water. Separating gel: 40% acrylamide (30% acrylamide/bis-acrylamide solution, 19:1), 25% Tris (1.5 M, pH 8.8), 0.1% SDS (10%), 0.1% ammonium persulfate (10%), and 0.04 μL TEMED, 38.4% distilled water. [Note 2] Stacking gel: 12.5% acrylamide (30%), 25% Tris (1.5 M, pH 8.8), 1% SDS (10%), 0.5% ammonium persulfate (10%), and 0.1% TEMED, 60.9% distilled water. Sample-loading buffer (2 3 Laemmli buffer): 4% SDS, 10% β-mercaptoethanol (or 0.3 M DTT), 20% glycerol, 0.004% bromophenol blue, 0.15 M Tris-HCl in distilled water and adjust pH to 6.8. [Note 3] Electrophoresis running buffer: 3% Tris base, 1% SDS, 1.1% glycine in distilled water. Transfer buffer: 48 mM Tris base, 39 mM glycine, 0.04% (wt./vol.) SDS, 20% methanol in distilled water. 100% methanol. Pads or standard chromatography papers. Nitrocellulose or PVDF membrane. Tris-HCl buffer: dissolve 10% (vol./wt.) Tris base in 60 mL of distilled water, adjust pH by adding HCl (1 M), make up volume to 150 mL in distilled water, and store at 4 C. Tris-buffered saline (TBS): 2.4 g Tris base and 8.8 g NaCl in 900 mL distilled water, adjusting pH to 7.6 with 12 N HCl, and then add distilled water to final volume of 1 L. TBS with Tween-20 (TBST): 0.1% Tween-20 in TBS buffer. Blocking buffer: 5% (wt./vol.) dried nonfat milk in TBST. [Note 4]

7.5.1.2 Method This method is based on the published procedure of Idris (2012) and modified western blotting protocol of Wu’s lab (Chakrabarti, Liao, Davidge, & Wu, 2017; Liao, Chakrabarti, Davidge, & Wu, 2016; Shang, Bhullar, Hubbard, & Wu, 2019). 7.5.1.3 Preparation of cell lysate 1. Culture bone cells in tissue culture plates in the presence or absence of the bioactive peptide of interest for the desired period of time as described above. [Notes 5 and 6] 2. Remove the culture medium and rinse the cells three times with ice-cold PBS. 3. Add lysis buffer into each well and incubate for 10 min. [Note 7] 4. Collect the cell suspension and vigorous vortex for 10 s. Incubate the suspension on ice for 10 min. [Note 8]

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5. Centrifuge cell lysate at 14,000 3 g at 4 C for 10 min and transfer the supernatant to a clean Eppendorf. [Note 9] 6. Determine the protein concentration using a standard commercially available protein assay such as BCA Pierce protein assay. 7.5.1.4 Preparation of SDS polyacrylamide gel [Note 10] 1. To prepare SDS polyacrylamide gels in the laboratory, assemble a glass-plate gel assembly using in-house components or those supplied by commercially available kit. 2. Once the glass-plate assembly is in place, carefully transfer separating gel allowing about three cm from the top. Add a layer of 100% ethanol on the top of the separating gel to ensure an even surface. 3. Once the resolving gel is polymerized, rinse the separating gel surface completely with distilled water. Ensure that the gel surface is dry by wicking off all the moisture with filter paper. 4. Pipette the stacking gel solution smoothly between the glass plates to the top of the short plate, avoiding excess bubbles. 5. Quickly insert the comb between the short plate and spacer plate and between the spacers on the spacer plate. Be careful not to trap bubbles behind the comb. Be sure the appropriate comb is chosen. The flat side of the comb is its backside and should rest against the taller spacer plate. Ensure that the comb is seated properly by aligning the comb’s front ridge with the top of the short plate. 6. Allow the gel to polymerize completely, usually 5
15 min, depending on the amount of TEMED and 10% APS. 7.5.1.5 Electrophoresis The procedure separates the denatured proteins according to their molecular weight. 1. 2. 3. 4. 5.

Mix cell lysate with the appropriate volume of sample-loading buffer. Boil sample mixture at 95 C for 5 min to denature protein. Place precast gel into a vertical electrophoresis tank filled with electrophoresis running buffer. Load carefully into the designated well on the precast gel. [Note 11] Place gel-plates assembly into electrophoresis tank filled with electrophoresis running buffer and run at constant current of 120 V for 60
90 min. [Note 12]

7.5.1.6 Electrophoretic transfer from gel to membrane The procedure transfers proteins from the SDS polyacrylamide gel to a nitrocellulose or PVDF membrane, prior to incubation with the primary and secondary antibodies and detection step. 1. Remove gel from the glass-plate assembly and immerse gel into transfer buffer for 3
5 min. 2. Meanwhile, cut nitrocellulose or PVDF membrane to the size of gel, immerse in 100% methanol, and then allow it to equilibrate in transfer buffer for 5 min. [Note 13]

180 Chapter 7 3. Prepare a blotting sandwich by arranging successive layers of three pads presoaked in transfer buffer, nitrocellulose or PVDF membrane, SDS polyacrylamide gel, three presoaked pads. 4. Run transfer at a constant current 100 V for 1 h. [Note 14] 7.5.1.7 Protein detection 1. Incubate nitrocellulose or PVDF membrane at room temperature for 1 h in blocking solution containing 5% dried nonfat milk in TBST. 2. Rinse nitrocellulose or PVDF membrane in TBST buffer for at least 15 min while changing the buffer every 5 min. 3. Incubate membranes with the appropriate amount of primary polyclonal or monoclonal antibody at 4 C for 16
24 h with continuous gentle agitation. 4. Rinse nitrocellulose or PVDF membrane as described in step 2 and then incubate with the appropriate amount of the HRP-conjugated secondary antibody for 1 h at room temperature or overnight at 4 C with continuous gentle agitation. 5. Repeat step 2 and visualize protein of interest using a standard chemiluminescence imager (e.g., Odyssey Imaging System, Licor). The intensities of the bands can be quantified using software supplied with most commercially available imagers (e.g., ImageStudio). 6. To probe the membrane from a different protein, remove primary and secondary antibodies by incubating the membrane in stripping buffer at 56 C for 5
10 min depending on the strength of the signal obtained with previous antibody. Revisualize blot using a chemiluminescence imager to ensure that the antibody has been completely removed. 7. Proceed with detection from step 1 above using a different antibody at step 3. Membrane can be stripped and reprobed with a different primary and secondary antibody many times. [Notes 15 and 16] Note: 1. Lysis buffer vary form gentle, containing no detergents, to harsher denaturing solution containing SDS and other ionic detergents. Choosing a lysis buffer depends on the solubilization of the protein. Typically, mild nonionic detergents, such as NP-40 and Trish-HCl, are used for extraction of soluble cytoplasmic proteins. Harsher buffer, such as RIPA buffer and Tris-Triton X-100, are used for isolation of membrane-bound proteins and nuclear protein. In our lab, we also use a simple lysis buffer by adding 2%B5% DTT into 2 3 Laemmli buffer to roughly extract whole soluble proteins, which can be immediately used for loading, but cannot be used for determining the protein concentration and is low in purity. 2. This recipe is for making two 12% separating gels. Depending on the targeting biomarkers, the concentration of the gels can be modified to 8%
15%. Usually, for

Methodologies for bioactivity assay: cell study

3.

4. 5.

6.

7. 8. 9. 10. 11.

12.

13. 14. 15.

16.

181

proteins of molecular weight over 100 kDa use 8%, 50
100 kDa use 10%, 20
50 kDa use 12%, ,20 kDa use 15%. The loading buffer can also use 4 3 Laemmli buffer to increase the volume of samples. Recipe: 8% SDS, 10% β-mercaptoethanol (or 0.3 M DTT), 30% glycerol, 0.02% bromophenol blue, 0.25 M Tris-HCl in distilled water and adjust pH to 6.8. Five percentage BSA in TBST can also be used as blocking buffer. The selection of blocking buffer may vary from the target proteins, the cell lines, the antibodies, etc. The selection of tissue culture plate depends on the condition of the cells and the experimental design. In our lab, we usually use six-well plate to collect enough cells for western blotting. The treatment time of the bioactive peptide of interest also depends on the target protein. Usually, for functional proteins, the cells are treated with the bioactive peptide of interest for 12
24 h. For the test of protein phosphorylation, the cells are treated with the bioactive peptide of interest for 10
60 min. Use lysis buffer supplemented with proteases and phosphatases inhibitor cocktails for studies involving extraction of phosphorylated proteins. The cell scrapers can be used to collect cells. It is possible to store the sample frozen at 220 C for up to a year at this point. Alternatively precast SDS polyacrylamide gels can be used. These can be obtained in a wide range of sizes and thicknesses from a number of suppliers. The loading amount is depended on the protein concentration, the expression level of the target protein in the cells, and the sensitivity of the antibodies used to test the target protein. Therefore it is always recommended to develop a preliminary experiment to determine the best loading amount of each target protein. The current of the electrophoresis may different in each experiment from 80 to 200 V. In our lab, we usually use 80
150 V to avoid the denaturation of the target protein due to the increase of temperature in running buffer caused by high volume. To avoid the increase of temperature, it is also recommended to put the tank in the ice or run the gels in cold room. The nitrocellulose or PVDF membrane can also be immersed in transfer buffer instead of 100% methanol to reduce the risk of hazard. It is also recommended to put the tank in the ice or run the gels in cold room during transfer. Although membrane can be stripped and reprobed with a different primary and secondary antibody, it is not recommended to do it too many times as it may affect the quality and sensitivity of the target protein. For an in-depth review of western blot technique and related knowledge, refer to Laboratory Techniques in Biochemistry and Molecular Biology (Work & Work, 1969) and Biochemistry Laboratory: Modern theory and techniques, second Edition (Boyer, 2006).

182 Chapter 7

7.5.2 Protocol 13. Quantitative reverse transcription polymerase chain reaction The polymerase chain reaction (PCR) is a technique that can amplify small amounts of target DNA in an exponential manner by using sequence-specific oligonucleotide primers, whereas the RT-qPCR is used when the starting material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). Then, the cDNA is used as the template for the qPCR reaction. During the reaction, the amount of the DNA amplification can be calculated or measured by monitoring the reaction in real-time, which can eventually provide the information to reflect the gene expression level. In this section, we provide the methodology for performing RT-qPCR, linked to a total RNA extraction step and a reverse transcription step, thereby allowing the researchers to analyze levels of gene expression in bioactive peptide treated cells. 7.5.2.1 Materials, equipment, and reagents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

TRIzol reagent Chloroform Isopropanol 75% ethanol RNase-free water DEPC-treated water Reverse transcription kit containing reaction buffer, dNTPs mix, DTT, and reverse transcriptase [Note 1] SYBR Green Water bath or heat block NanoDrop Spectrophotometer qPCR machine

7.5.2.2 Method This method is based on the published procedure of Hughes (2012), Majumder et al. (2015), and Shang and Wu (2019) 7.5.2.3 RNA extraction by TRIzol reagent [Note 2] 1. Culture bone cells in tissue culture plates in the presence or absence of the bioactive peptide of interest for the desired period of time as described above. 2. Remove the culture medium and rinse the cells three times with ice-cold PBS. 3. Collect the cell pellet in 1.5 mL Eppendorf using cell scrapers or follow the Protocol 1 step 1
7. 4. Add 0.3
0.4 mL of TRIzol Reagent per 1 3 105 to 107 cells and homogenize by pipetting the content up and down several times.

Methodologies for bioactivity assay: cell study

183

5. Incubate at room temperature for 5 min to complete dissociation of the nucleoprotein complex. 6. Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for lysis, vortex, and then incubate at room temperature for 2
3 min. 7. Centrifuge the sample for 15 min at 12,000 3 g at 4 C. 8. Transfer the colorless upper aqueous phase into a new tube. 9. Add 0.5 mL isopropanol to the aqueous phase per 1 mL TRIzol Reagent used for lysis, vortex and then incubate at room temperature for 10 min. 10. Centrifuge for 10 min at 12,000 3 g at 4 C. 11. Discard the supernatant and resuspend the pellet in 1 mL of 75% ethanol per 1 mL TRIzol Reagent used for lysis. 12. Vortex the sample briefly, and centrifuge for 5 min at 7500 3 g at 4 C. 13. Discard the supernatant and vacuum or air dry the RNA pellet for 5
10 min. 14. Resuspend the pellet in 20
50 μL of RNase-free water and pipet up and down. 15. Incubate in a water bath or heat block set at 55
60 C for 10
15 min. 16. Dilute the extracted RNA in RNase-free water, and measure concentration and absorbance at 260 nm and 280 nm using the NanoDrop Spectrophotometer. [Note 3] 7.5.2.4 Reverse transcription 1. Aliquot 2 μg of RNA into duplicate RNase-free tubes and adjust the total volume to 11 μL with DEPC-treated water. Hold on ice. 2. Add 1 μL of 2 μg/mL random hexamer primers to each sample, flick tube to mix and pulse on a centrifuge to collect the reaction mix at the bottom of the tube. 3. Heat at 70 C for 10 min on a thermocycler and then immediately cool on ice for 2
3 min. 4. Add 4 μL of 5 3 Reaction Buffer, 2 μL of 0.1 M DTT, and 1 μL of 10 mM dNTPs mix to each tube, and then add 200 U of SuperScriptII M-MLV reverse transcriptase to reverse transcrib samples or an equivalent volume of DEPC-treated water to no-RT controls. 5. Mix tubes by flicking and collect the reaction mix at the bottom of the tube by pulsing briefly on a centrifuge. 6. Incubate the reaction mix at room temperature for 10 min. 7. Heat the tube to 42 C for 50 min in a thermal cycler then at 95 C for 5 min before cooling on ice for 2
3 min. 8. Adjust reaction volume to 100 μL with DEPC-treated water and store at 220 C for several months or 4 C for a week. 7.5.2.5 Design primers for SYBR Green qPCR assay Quantitative PCR assay using SYBR green is more sensitive and cheaper than those employ hydrolysis probes. The primers can be designed use the National Center for

184 Chapter 7 Biotechnology Information’s (NCBI) Primer-BLAST tool and the Primer3 primer design software. 7.5.2.6 Perform quantitative reverse transcription polymerase chain reaction using SYBR Green assay 1. Normalize the primer concentration and mix the gene-specific forward and reverse primer pair. [Note 4] 2. Prepare the RT-PCR reaction mixture for either 50 μL/well or 25 μL/well as follows [Note 5] SYBR Green Mix (2 3) cDNA Primer pair mix H2O Total volume

25 μL 0.5 μL 2 μL 22.5 μL 50 μL

12.5 μL 0.2 μL 1 μL 11.3 μL 25 μL

3. Run on a qPCR machine for 40 cycles, using the thermal cycling conditions as follows [Note 6] Cycle

Step

Temperature

Hold time

13 40 3

Enzyme activation Denaturation Annealing Extension Denaturation Strand annealing Melting curve

95 95 Avg. primer Tm minus 2
5 72 95 65 65
97

5 min 10 s 15 s (Amplicon length [bp]/25) s 10 s 1 min —

13

4. Perform melting curve analysis to validate amplification is specific and not due to primer
dimer or mispriming events. Plot the first derivative (dF/dT) of the melting curve data generated against temperature. Specific primers will generate a single, strong peak, whereas nonspecific primers will generate multiple peaks. Primer dimerization will cause “hump” evident at lower temperatures. 7.5.2.7 Analysis of quantitative reverse transcription polymerase chain reaction data: comparative CT methods [Note 7] Relative quantification uses an internal control (reference gene) and/or a control group (untreated group) to quantify the mRNA of interest relative to these references, which is sufficient to draw conclusion in most of bioactive peptide applications. A few methods have been developed to perform the relative quantification depending on the difference between assumptions and models. The most common method, comparative CT method, is introduced here. [Note 8]

Methodologies for bioactivity assay: cell study

185

Comparative CT method Assumption

Relative expression

1. This method assumes that the cDNA templates of the genes of interest as well as the control/reference gene have similar and near perfect amplification efficiency. 2. The expression difference between two genes/can be calculated by subtracting one (gene of interest) from another (reference gene). 3. The reference gene does not change with the treatment. Step 1. Normalize the CT value the gene of interest/target gene to the reference gene (e.g., GAPDH or tubulin)

ΔCT 5 CTðtarget geneÞ 2 CTðreference geneÞ Step 2. Exponentially transformed the ΔCT to the ΔCT expression

ΔCT expression 5 2ð2ΔCT Þ Step 3. Normalize the ΔCT expression in treatment group to untreated control

ΔΔCT expression 5

ΔCT expressionðtreatmentÞ ΔCT expressionðcontrolÞ

Example showing ΔΔCT calculation
To determine the gene expression of runx2, a bone formation biomarker, after treated with a peptide of interest

Peptide

Control

A 1 2 3 4 5 6

CT

CT

(runx2)

(gapdh)

B 22.2 22.5 22.6 27.6 27.3 27.6

C 20.6 20.8 20.9 20.8 20.2 20.9

ΔCT CT (runx2) 2 CT (gapdh) D 1.6 1.7 1.7 6.8 7.1 6.7

ΔCT expression 22ΔCT E 0.330 0.308 0.308 0.009 0.007 0.010

(E1 1 E2 1 E3)/3 and (E4 1 E5 1 E6)/3 F 0.315

ΔΔCT expression F1/F4 and F4/F4 G 36.5

0.009

1

Mean ΔCT expression

Note: 1. The name and content of reverse transcription kit from different company may different. Check the manufacture’s instruction before use. 2. TRIzol reagent is the most widely used chemical for RNA extraction. This section briefly introduced the RNA extraction method using TRIzol reagent. More detailed information of TRIzol reagent can be found in the manufacture’s instruction. In addition to using TRIzol reagent, several companies also provide RNA extraction kit. 3. The RNA concentration can be calculated using the formula A260 3 dilution 3 40 5 μg RNA/mL. The purity of extracted RNA can be calculated by the ratio of A260/A280. A ratio of B2 is considered pure. 4. Usually the final concentration of each primer in the mixture is 5 pmol/μL.

186 Chapter 7 5. The total volume of the reaction mixture vary depending on the experimental design and the plan of the following studies. 6. The thermal cycling condition may vary depending on the supplier. Check the manufacture’s instruction before use. 7. Currently, most of qRT-PCR machines are supplied with programmed software that can be easily used for data analysis. In addition, the qPCR data can be analyzed automatically using free online REST spreadsheet (available at www.genequantification.de/download.html) or free download PCR/qPCR package at CRAN (available at https://cran.r-project.org/web/packages/qpcR/index.html). 8. CT (cycle threshold), the number of cycles required for the fluorescent signal to cross the threshold, is also known as Cq (quantification cycle). 9. The formal derivation of the double delta CT model is described in Kenneth and Schmittgen (2001). 10. For an in-depth review of PCR/RT-qPCR technique and related knowledge, refer to Laboratory Techniques in Biochemistry and Molecular Biology (Work & Work, 1969) and Molecular Biology Techniques: A Classroom Laboratory Manual, fourth Edition (Carson et al., 2020).

7.6 Summary Over the years, there is a surge interest in identifying novel bioactive peptides from food proteins for uses as functional foods and nutraceuticals. Cell culture is a widely used tool to determine the toxicity and bioactivity of bioactive peptides, in particularly at early stage of study. Although cell culture cannot completely replace in vivo animal study, it can mimic many fundamental pathophysiological conditions in a highly controlled condition. In comparison to animal study, cell culture is robust, reproducible, and cost-effective; cell culture also significantly reduces the uses of animals in scientific researches and avoids the ethics constraints of using living animals. Admittedly, there are some limitations using in vitro cell culture technique; for example, the culture techniques need to be carried out under strict aseptic conditions, and there is a high possibility of contamination if operated inappropriately. Another important limitation is that cell culture technique cannot reflect the biological efficacy, pharmacokinetic profile and immunogenicity of the target bioactive peptides. Recent development of three-dimensional (3D) cell culture is believed to provide a more physiologically relevant condition than the conventional cell culture; therefore its application in bioactive peptide study is expected to increase.

References Ahn, C. B., & Je, J. Y. (2019). Bone health-promoting bioactive peptides. Journal of Food Biochemistry, 43, e12529, January 1, Blackwell Publishing Ltd. Available from https://doi.org/10.1111/jfbc.12529. Arango, M.T., Quintero-Ronderos, P., Castiblanco, J., & Montoya-Ortı´z, G. (2013). Cell culture and cell analysis. Autoimmunity: From bench to beside. Retrieved from , https://www.ncbi.nlm.nih.gov/books/NBK459464/ . .

Methodologies for bioactivity assay: cell study

187

Bacharach, A. L. (1945). Biological assay and chemical analysis. The Analyst, 70(836), 394. Available from https://doi.org/10.1039/an9457000394. Bhandari, D., Rafiq, S., Gat, Y., Gat, P., Waghmare, R., & Kumar, V. (2020). A review on bioactive peptides: Physiological functions, bioavailability and safety. International Journal of Peptide Research and Therapeutics, 26, 139
150, March 1, Springer. Available from https://doi.org/10.1007/s10989019-09823-5. Boyer, R. (2006). Biochemistry laboratory: modern theory and techniques (2nd ed.). Pearson Education, Inc. Carson, S., Miller, H. B., Witherow, D. S., & Srougi, M. C. (2020). Molecular biology techniques: a classroom laboratory manual (4th ed). Academic Press. Chakrabarti, S., Liao, W., Davidge, S. T., & Wu, J. (2017). Milk-derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) differentially modulate angiotensin II effects on vascular smooth muscle cells. Journal of Functional Foods, 30(January), 151
158. Available from https://doi.org/10.1016/j. jff.2016.12.022. Cicero, A. F. G., Fogacci, F., & Colletti, A. (2017). Potential role of bioactive peptides in prevention and treatment of chronic diseases: A narrative review. British Journal of Pharmacology, 174, 1378
1394, John Wiley and Sons Inc. Available from https://doi.org/10.1111/bph.13608. Collin-Osdoby, P., & Osdoby, P. (2012). RANKL-mediated osteoclast formation from murine RAW 264.7 cells. Methods in Molecular Biology, 816, 187
202. Cote´, R. J. (1998). Aseptic technique for cell culture. Current Protocols in Cell Biology, 00(1), 1.3.1
1.3.10. Available from https://doi.org/10.1002/0471143030.cb0103s00. Daliri, E. B.-M., Lee, B. H., & Oh, D. H. (2018). Current trends and perspectives of bioactive peptides. Critical Reviews in Food Science and Nutrition, 58(13), 2273
2284. Available from https://doi.org/10.1080/ 10408398.2017.1319795. Freshney, R.I. (2016). Culture of animal cells: A manual of basic technique and specialized applications. Retrieved August 19, 2020, from , https://www.wiley.com/en-ca/Culture 1 of 1 Animal 1 Cells: 1 A 1 Manual 1 of 1 Basic 1 Technique 1 and 1 Specialized 1 Applications, 1 7th 1 Edition-p9781118873656 . . Ghayor, C., Correro, R. M., Lange, K., Karfeld-Sulzer, L. S., Gra¨tz, K. W., & Weber, F. E. (2011). Inhibition of osteoclast differentiation and bone resorption by N-methylpyrrolidone. Journal of Biological Chemistry, 286(27), 24458
24466. Available from https://doi.org/10.1074/jbc.M111.223297. Ghosh, S. (2016). Peptide therapeutics market: Forecast and analysis 2015-2025. Oligos & Peptides, 34(2), 5
7. Retrieved from , https://www.teknoscienze.com/tks_article/peptide-therapeutics-market-forecast-andanalysis-2015-2025/ . . Hughes, A. (2012). Analysis of gene expression in bone by quantitative RT/PCR. Methods in Molecular Biology, 816, 261
275. Idris, A. I. (2012). Analysis of signaling pathways by western blotting and immunoprecipitation. Methods in Molecular Biology, 816, 223
232. Jahandideh, F., Chakrabarti, S., Davidge, S. T., & Wu, J. (2016). Antioxidant peptides identified from ovotransferrin by the ORAC method did not show anti-inflammatory and antioxidant activities in endothelial cells. Journal of Agricultural and Food Chemistry, 64(1), 113
119. Available from https://doi. org/10.1021/acs.jafc.5b04230. Kang, N. J., Jin, H. S., Lee, S. E., Kim, H. J., Koh, H., & Lee, D. W. (2020). New approaches towards the discovery and evaluation of bioactive peptides from natural resources. Critical Reviews in Environmental Science and Technology, 50(1), 72
103. Available from https://doi.org/10.1080/10643389.2019.1619376. Kenneth, L. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the double delta CT method. Methods (San Diego, Calif.), 25(4). Available from http://doi.org/10.1006/meth.2001.1262. Kim, H. K., Kim, M.-G., & Leem, K. H. (2008). Inhibitory effects of egg yolk soluble protein on bone resorption. Bioscience, Biotechnology, and Biochemistry, 72(7), 1929
1931. Available from https://doi.org/ 10.1271/bbb.70491.

188 Chapter 7 Kim, H. M., Kim, D. H., Han, H. J., Park, C. M., Ganipisetti, S. R., Arasu, M. V., & Soung, N. K. (2017). Ginsenoside re promotes osteoblast differentiation in mouse osteoblast precursor MC3T3-E1 cells and a zebrafish model. Molecules (Basel, Switzerland), 22(1), 1
9. Available from https://doi.org/10.3390/ molecules22010042. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16(9), 945
960. Available from https://doi.org/10.1016/j.idairyj.2005.10.012. Liao, W., Chakrabarti, S., Davidge, S. T., & Wu, J. (2016). Modulatory effects of egg white ovotransferrinderived tripeptide IRW (Ile-Arg-Trp) on vascular smooth muscle cells against Angiotensin II stimulation. Journal of Agricultural and Food Chemistry, 64(39), 7342
7347. Available from https://doi.org/10.1021/ acs.jafc.6b03513. Majumder, K., Liang, G., Chen, Y., Guan, L., Davidge, S. T., & Wu, J. (2015). Egg ovotransferrin-derived ACE inhibitory peptide IRW increases ACE2 but decreases proinflammatory genes expression in mesenteric artery of spontaneously hypertensive rats. Molecular Nutrition & Food Research, 59(9), 1735
1744. Available from https://doi.org/10.1002/mnfr.201500050. MC3T3-E1 Subclone 4 ATCC ® CRL-2593t. (2020). Mus musculus bone/calv. Retrieved October 22, 2020, from , https://www.atcc.org/products/all/CRL-2593.aspx . . O’Connor, R., & O’Driscoll, L. (2006). Setting up a cell culture laboratory, . Cell biology, four-volume set (Vol. 1, pp. 5
11). Elsevier Inc. Available from https://doi.org/10.1016/B978-012164730-8/ 50002-2. Pan, C., Kumar, C., Bohl, S., Klingmueller, U., & Mann, M. (2009). Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Molecular and Cellular Proteomics, 8(3), 443
450. Available from https://doi.org/10.1074/mcp.M800258-MCP200. RAW 264.7 ATCC® TIB-71t. (2020). Retrieved October 22, 2020, from , https://www.atcc.org/products/all/ TIB-71.aspx . . Sa´nchez, A., & Va´zquez, A. (2017). Bioactive peptides: A review, . Food Quality and Safety (1, pp. 29
46). March 1, Oxford University Press. Available from https://doi.org/10.1093/fqsafe/fyx006. Sasaki, K., Takahashi, N., Satoh, M., Yamasaki, M., & Minamino, N. (2010). A peptidomics strategy for discovering endogenous bioactive peptides. Journal of Proteome Research, 9(10), 5047
5052. Available from https://doi.org/10.1021/pr1003455. Schwartz, M. A., Both, G., & Lechene, C. (1989). Effect of cell spreading on cytoplasmic pH in normal and transformed fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 86(12), 4525
4529. Available from https://doi.org/10.1073/pnas.86.12.4525. Segeritz, C. P., & Vallier, L. (2017). Cell culture: growing cells as model systems in vitro. Basic Science Methods for Clinical Researchers (pp. 151
172). Elsevier Inc. Available from https://doi.org/10.1016/ B978-0-12-803077-6.00009-6. Shang, N., Bhullar, K. S., Hubbard, B. P., & Wu, J. (2019). Tripeptide IRW initiates differentiation in osteoblasts via the RUNX2 pathway. Biochimica et Biophysica Acta (BBA)—General Subjects, 1863(6), 1138
1146. Available from https://doi.org/10.1016/j.bbagen.2019.04.007. Shang, N., & Wu, J. (2018). Egg white ovotransferrin shows osteogenic activity in osteoblast cells. Journal of Agricultural and Food Chemistry, 66(11), 2775
2782. Available from https://doi.org/10.1021/acs. jafc.8b00069. Shang, N., & Wu, J. (2019). Egg white ovotransferrin attenuates RANKL-induced osteoclastogenesis and bone resorption. Nutrients, 11(9), 2254. Available from https://doi.org/10.3390/nu11092254. Shang, N., & Wu, J. (2020). Egg-derived tripeptide IRW attenuates LPS-induced osteoclastogenesis in RAW 264.7 Macrophages via inhibition of inflammatory responses and NF-κB/MAPK activation, Journal of Agricultural and Food Chemistry 68(22), 6132
6141. Strober, W. (2015). Trypan blue exclusion test of cell viability. Current Protocols in Immunology, 111(1), A3. B.1
A3.B.3. Available from https://doi.org/10.1002/0471142735.ima03bs111.

Methodologies for bioactivity assay: cell study

189

Thorpe, T. A. (2007). History of plant tissue culture. Molecular Biotechnology, 37, 169
180. Available from https://doi.org/10.1007/s12033-007-0031-3. Tinoco, A. D., & Saghatelian, A. (2011). Investigating endogenous peptides and peptidases using peptidomics. Biochemistry, 50(35), 7447
7461. Available from https://doi.org/10.1021/bi200417k. Work, T. S., & Work, E. (1969). Laboratory techniques in biochemistry and molecular biology (Vol. 1). Amsterdam & London: North Holland Publishing Co.

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CHAPTER 8

Methodologies for bioactivity assay: animal study Feiran Xu1,2 and Elvira Gonzalez de Mejia1,3 1

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Champaign, IL, United States, 2School of Food Science and Technology, Jiangnan University, Wuxi, P.R. China, 3Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Champaign, IL, United States

Abbreviations ACE ACE2 DPP-IV FDA HHS RAAS SHRs SPF STZ USDA

angiotensin-converting enzyme angiotensin-converting enzyme 2 dipeptidyl peptidase-IV US Food and Drug Administration US Department of Health and Human Services renin
angiotensin
aldosterone system spontaneously hypertensive rats specific pathogen free streptozotocin US Department of Agriculture.

8.1 Introduction There are numerous studies directed toward the identification of dietary or nondietary natural peptides for the prevention and treatment of various human diseases. Two to twenty amino acid residues are the pivotal components of the vast majority of bioactive peptides, and they are hidden in the primary structure of proteins, requiring proteolysis for their release (Martinez-Villaluenga, Pen˜as, & Frias, 2017; Wang, Ding, Du, Yu, & Liu, 2019). Moreover, the human small intestine can absorb oligopeptides containing 2
10 amino acid residues (Sangsawad et al., 2018; Shen & Matsui, 2017; Xu et al., 2017). Cell experiments in vitro, such as the human colon adenocarcinoma cell lines (Caco-2) monolayer cell model, cannot fully simulate the absorption of oligopeptides in the small intestine (Vij, Reddi, Kapila, & Kapila, 2016; Xu et al., 2017). The absorption rate of food peptides in cell experiments is often higher than that of the everted rat sacs (Lundquist & Artursson, 2016; Wang et al., 2019), and the metabolites also vary significantly due to the metabolism in the Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00022-4 © 2021 Elsevier Inc. All rights reserved.

191

192 Chapter 8 animal experiment (Sanchez-Rivera et al., 2016). Animal studies have been crucial in the advancement of new options for the therapy or prevention of cancer, diabetes, and hypertensive diseases. Indeed, the functional investigation of bioactive peptides can no longer be limited to in vitro assays, including antioxidant activity (Aluko, 2015), angiotensin-converting enzyme (ACE) inhibitory activity (Wu, Liao, & Udenigwe, 2017), and dipeptidyl peptidase-IV (DPP-IV) inhibitory activity (Nongonierma & FitzGerald, 2019). Without physiological conditions, it is not possible to reflect the actual biological activity of food peptides because their absorption and metabolism must be considered (Xu et al., 2018). Different animal models (Fig. 8.1) have been used to validate and discover the biological activity of peptides, for instance, spontaneously hypertensive rats (SHRs) (Miralles, Amigo, & Recio, 2018), streptozotocin (STZ)-induced diabetic mice (Marthandam Asokan, Wang, Su, & Lin, 2019), specific knockout mice (Edelsbrunner, Herzog, & Holzer, 2009), and dietary intervention with mice (Gao, Song, Du, & Mao, 2019). Animal studies can increase the accuracy in revealing the molecular mechanism of bioactive peptides regarding a specific disease. Herein, mice and rats account for more than 85% of the yearly use of animals for scientific research (Kapila, Kapila, & Vij, 2017). However, so far, methods of animal models for assessing active food peptides are not well classified. For instance, which administration route is more effective? The dosage selection of active peptides in animal experiments is especially challenging (Aguirre et al., 2016).

Figure 8.1 Flow chart of animal experiments in the study of food peptides.

Methodologies for bioactivity assay: animal study

193

Failure to establish an adequate animal model often affects the experimental results and even the presentation of the molecular mechanism of the active peptides. Also, the processing of extensive data and multiple methods of data analysis are increasingly valued (Asgharpour et al., 2016; Sztuka & Jasinska-Stroschein, 2017). This chapter presents detailed methodologies about animal models for the evaluation of bioactivity of food peptides. Several publications were obtained from the Web of Science, Elsevier databases, the website of the US Department of Agriculture, among others, especially for the prevention of hypertension, metabolic disease, and metabolic dysfunction. The objectives of this chapter were to focus on the description of methods to administer peptides from food sources in animal models. In addition the classification of assays and the optimal selection for animal models to evaluate hypertension and its related cardiovascular diseases and metabolic dysfunction were investigated. Finally, the multivariate data analysis methods targeted at extensive sample data were enumerated.

8.2 Administration of food peptides and animal safety 8.2.1 Safety and toxicological evaluation of peptides The potential toxicity of the bioactive peptides is in itself often neglected due to the continuous research on the efficacy and mechanisms of absorption. Li, Xu, Cui, Huang, and Sun (2017) indicated that among the arginine-rich peptides, R11 (RRRRRRRRRRR), a peptide composed purely of arginine residues, had the lowest LD50 value (16.5 mg/kg) and manifested the highest toxicity. In contrast, the arginine-free transdermal peptide with the sequence ACSSSPSKHCG showed lower toxicity in a mice model. The coliform bacteria count was decreased, and the short-chain fatty acid levels were increased by antimicrobial peptide Microcin J25 with a medium (9.1 mg/kg) or a low dosage (4.6 mg/kg) in mice (Yu et al., 2018). Unexpectedly, a high dosage of antimicrobial peptide Microcin J25 in mice (18.2 mg/kg) increased intestinal permeability and imbalance of intestinal bacteria. In addition, some safety issues with glucagon-like peptide-1 (GLP-1) receptor agonists were found associated with an increased risk of cholelithiasis (Monami et al., 2017). Therefore before considering functional active peptides for therapeutic purpose and food addition, it is first necessary to evaluate the safety and toxicity of peptides. By contrast to synthetic drugs, active peptides derived from natural sources are less toxic and safer. The safety and toxicity of food peptides are the prerequisites for their extensive clinical use and addition to foods as functional ingredients. It is essential to study the potential biological mechanisms by which food peptides exert their active actions, as well as their safety (Bhandari et al., 2020; Chalamaiah, Yu, & Wu, 2018). The concentration of food peptides taken orally or injected should be strictly controlled; otherwise, the peptides

194 Chapter 8 that did not get metabolized on time might cause acute toxicity to the liver (Hanh et al., 2017; Sanchez-Rivera et al., 2016). For instance, the allergenic effect may be the main problem for the peptides derived from milk. There are two peptides β-casomorphin-5 and β-casomorphin-7 derived from casein with a strong allergic reaction (Dalziel et al., 2014; Fiedorowicz et al., 2014). However, β-casomorphin-7 can alter the μ-opioid receptor and DPP-IV gene expression (Fiedorowicz et al., 2014). Also, there was no adverse reaction in a murine model when the highest dose of lactotripeptide Ile-Pro-Pro (IPP) was 4% in the diet equivalent to an IPP intake of 40 mg/kg BW/day (Beltra´n-Barrientos et al., 2017). Peptides derived from plant-based soybean and peanut may exhibit immunoglobulin E-mediated allergenic effect, such as VTVRGGLRILSPDRK (Di Stasio et al., 2017), and a hydrolysate from soybean protein using sequential pepsin and chymotrypsin (Sung, Ahn, Lim, & Oh, 2014). Interestingly, soybean hydrolysates, with the same treatment, were also found to possess DPP-IV and ACE-inhibitory activities (Singh, Vij, & Hati, 2014). Herein, the dosage is particularly significant for food peptides that have bioactive effects without causing adverse consequences.

8.2.2 Meal feeding information Determining meal feeding is an essential precondition for the study of toxicity and the biological activity of food peptides in animal experiments. Physiologic and metabolic adaptations that occur in the ad libitum-fed rodent versus the meal fed may have consequences in shorter life span, alteration of circadian rhythms, higher body temperature, slower growth of the stomach, and small intestines (Mattson, Longo, & Harvie, 2017). The US Food and Drug Administration (FDA) provides the regulations and guidelines for animal feed manufacturers to follow when making animal feed decisions. These rules assure that the feedstuff used in research is safe and high quality (https://www.fda.gov/animalveterinary/animal-health-literacy/animal-feed-regulations). Also, hypertriglyceridemia, hypercholesterolemia, metabolic disease, and dietary-induced obesity in ad libitum-fed rats may be less acceptable compared to that in rats fed a restricted diet (Godar et al., 2015; Guo, 2014). Thus animal experiments on food peptides have established exclusive feeding diets and feeding times (Mykkanen et al., 2014; Reidelberger, Haver, Chelikani, & Buescher, 2008). Table 8.1 presents the recommended good practices and maximal volume for mouse and rat studies (Diehl et al., 2001). Table 8.2 shows the doses of food peptides that have been tested on different animal models. It includes both oligopeptides and protein hydrolysates. Protein hydrolysates were often initially evaluated using animal models, and then the active components (unique peptide sequence) were purified and identified (Salem et al., 2018; Shi, Hou, Guo, & He, 2019; Zarei et al., 2015). To comply with government regulations [(Public Health Service and US

Methodologies for bioactivity assay: animal study

195

Table 8.1: Good practice and maximal volume recommended for mice and rat studies. Oral (mL/kg) Species OD MD Mouse Rat

10 10

50 50

Subcutaneous (mL/kg)

Intraperitoneal (mL/kg)

Intramuscular (mL/kg)

OD

OD

OD

10 5

MD 40 10

20 10

MD 80 20

a

0.05 0.1a

MD a

0.1 0.2a

IV rapid bolus (mL/kg)

IV slow infusion (mL/kg)

OD

MD

OD

MD

5 5

N/A N/A

N/A N/A

25 20

MD, maximal dose; OD, optimum dose. a Values in milliliters per site. Source: Referred from Diehl, K.H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., . . . Vorstenbosch, C.V.D. (2001). A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology: An International Journal, 21(1), 15
23.

Department of Agriculture (USDA)] and Animal Care and Use Committees (ACUC), relevant regulations on the use of drugs, anesthetics, are particularly important. Hohlbaum et al. (2017) reported that repeated isoflurane anesthesia is better than single isoflurane anesthesia. In the case of an operation, the intensity and duration of pain must be minimized through the use of appropriate anesthetics, analgesics, and sedatives based on acceptable standards in veterinary medicine. The requirement for the alleviation of pain applies at not only the time the procedure is being conducted but also following the procedure until the pain is reduced (Carrizzo et al., 2019).

8.2.3 Distribution of gender and age There have been reports showing differences in symptoms and complications between males and females and even some differences in pathogenesis. For example, results from trials with hormone-regulated food peptides demonstrated interferences from hormones associated with menstrual cycles in female mice (Klein & Flanagan, 2016; Org et al., 2016). As male mice are more likely to develop diabetes than females, and the fluctuation of female hormones, male mice were used in the study of antidiabetic food peptides Shi et al. (2019). In terms of cardiovascular disease, existing hypertension did much more damage to pregnant female mice than nonpregnant or male mice. This effect may be related to the decrease of plasma renin activity and urine aldosterone during pregnancy (Malha, Sison, Helseth, Sealey, and August, 2018). In addition, there is a difference between females and males in the blood pressure sensitivity and inflammation (Mair et al., 2014). The lower number of females with hypertension is mainly due to the cardioprotective effects of estrogen. Consequently, the ratio of female to male mice plays an essential role in acquiring accurate results. Furthermore, age is also a significant factor to consider in animal experiments for bioactive peptides. Hanh et al. (2017) reported a higher absorption of dipeptide GS and tripeptide

Table 8.2: Food peptides that have been tested in vivo.

Peptide sequence

Molecular weight (Da)

Corn

Hydrolysates

N/A

Rice

IFRF

581.72

Food source

Thermolysin-digested rice bran LRA YY Hydrolysates

N/A 358.427 344.352 N/A

Wheat

Hydrolysates

N/A

Black bean

Hydrolysates

N/A

Chickpea

Hydrolysates

Rapeseed

VFVRN Hydrolysates

,1 kDa and 1
3 kDa 633.79 N/A

GHS LY RALP

299.27 294.34 455.54

Biological activity Development of T1D with antiinflammatory ability Hypotensive Anorexigenic activities Antihypertensive effect

Dosage range (mg/kg BW)

Optimum dosage (mg/kg BW)

Animal model used to evaluate

Pharmacological intervention

C57BL/6J mice

Intraperitoneal injection of STZ 45 mg/kg

50, 500

500

Sun, Zhang, Mu, Zhang, and Chen (2019)

Male SHRs/Izm (12
15 weeks old) ddY mice (4 or 7 weeks old) Male SHRs/Izm (12 weeks old)

N/A

5, 15

15

Kaushik and Vaswani (2018)

N/A

50, 500

500

Shobako et al. (2018)

N/A N/A N/A

0.25 1.0 100, 500, 1000, 2000 500

0.25 1.0 2000

Ishikawa et al. (2015)

500

Sun et al. (2019)

100, 150, 200

150, 200

50, 100, 200

N/A

Mojica, Gonzalez de Mejia, GranadosSilvestre, and Menjivar (2017) Shi et al. (2019)

Stimulating GLP-1 secretion and reducing GLP-1 degradation Development of T1D with antiinflammatory ability Management of blood glucose

Male SD rats (6 weeks old, 150
170 g)

Antiobesity

Wistar rats (8 weeks)

ACE inhibition

N/A Male SD rats (adult, 185
314 g)

Regulation of renin
angiotensin

Male SHRs and WisterKyoto rats (10 weeks)

References

N/A

NOD mice 3 weeks

N/A

Male Wistar rats (4 weeks old, 250 g)

Intraperitoneal injection of STZ 30 mg/kg and 15 mg/kg N/A

N/A

N/A 600

600

N/A

30

30

Makinen, Streng, Larsen, Laine, and Pihlanto (2016) He et al. (2019)

(Continued)

Table 8.2: (Continued)

Peptide sequence

Molecular weight (Da)

Biological activity

Vglycin (37 residues)

3786.40

Antitype 2 diabetes

Peptides Maillard reaction products

N/A

Potato

DIKTNKPVIF

1174.38

Arthrospira platensis Red Seaweed Palmaria palmata

Hydrolysates

N/A

IRLIIVLMPLIMA

1495.80

Alleviating cognitive impairment and systemic inflammation Ameliorating type 1 diabetes mellitusassociated damages Antitype 1 diabetes mellitus Hypotensive effect

Hydrolysates

N/A

GIVAGDVTPI

941.07

Bovine α-lactalbumin

Hydrolysates

N/A

Casein

Hydrolysates

N/A

Whey protein

LIL IW Hydrolysates

357.48 317.37 N/A

Yoghurt

Hydrolysates

# 10 kDa

AVFQHNCQE QVGPLIGRYCG Hydrolysates

1075.20 1162.40 N/A

Food source Soybean

Spirulina platensis

Chicken foot

Vascular NO misregulation

Dosage range (mg/kg BW)

Optimum dosage (mg/kg BW)

References

Animal model used to evaluate

Pharmacological intervention

Male Wistar rats (220
250 g) Male nude mice (2
3 weeks, 15
25 g) Male ICR mice (16 months old, 48 6 2 g; 6 weeks old, 24 6 1 g)

Intraperitoneal injection of STZ 30 mg/kg

0.04

0.08

Jiang et al. (2014)

D-galactose (500 mg/kg)induced

200, 400, 800

800

Zhao et al. (2019)

Male ICR mice

Intraperitoneal injection of STZ 60 mg/kg Injection of alloxan 70 mg/kg N/A

25, 50

50

Marthandam Asokan et al. (2019)

50, 100, 200 50

200

2, 5, 50, 100, 200 2.5, 5, 10 100, 200, 400

200

Ou, Ren, Wang, and Yang (2016) Fitzgerald, Aluko, Hossain, Rai, and Hayes (2014) Carrizzo et al. (2019)

200

200

Wang et al. (2020)

N/A 19 770

N/A 19 770

Martin et al. (2015)

Male ICR mice (18
22 g) Adult SHRs (20 weeks, 360
400 g)

50

Male eNOS knockout mice (8 weeks, 24 6 1.0 g)

N/A

Ameliorate adipose insulin resistance and inflammation Alleviating liver damage in diabetes DPP-IV inhibition Antihypertensive and cardioprotective effects Antiobesity

Male C57BL/6J mice 5 weeks old

N/A

Male SD rats

Intraperitoneal injection of STZ 25 mg/kg N/A

Male C57BL/6 (6 weeks)

N/A

N/A

0.2

Shi et al. (2019)

ACE inhibition

Male SHRs (17
20 weeks, 300
400 g)

N/A N/A N/A

10 10 10

10 10 10

Bravo, Mas-Capdevila, Margalef, Arola-Arnal, and Muguerza (2019)

Male SHRs over 14 weeks

10 400

Gao, Song, Du, and Mao (2018)

(Continued)

Table 8.2: (Continued)

Peptide sequence

Molecular weight (Da)

Cuttlefish

VELYP

620.10

Egg ovotransferrin

LKP IQW

356.47 445.52

Egg ovalbumin

RVPSL

570.68

QIGLF

577.41

IRW

455.59

Regulation of renin
angiotensin

Hydrolysates

N/A

DPP-IV inhibition and GLP-1 stimulation

IPGDPGPPGPPGP LPGERGRPGAPGP GPKGDRGLPGPPGRDGM Hydrolysates

919.53 1026.58 1358.76 N/A

MGP

303.38

Food source

Egg white

Fish skin gelatin

Porcine skin gelatin

Biological activity Antihypertensive effect Antihypertensive effect Regulation of renin
angiotensin Antihypertensive effects

ACE-inhibitory and hypotensive activity

Animal model used to evaluate

Pharmacological intervention

Dosage range (mg/kg BW)

Male SHRs (17
20 weeks) Male SHRs (12
14 weeks, 270.0 6 10.5 g)) Male SHRs (9
10 weeks, 220
270 g)) Male SHRs and normotensive Wistar rats (9
10 weeks, 220
270 g) Adult male SHRs (13
15 weeks, 290.0 6 10 g)) Male SD rats (7 weeks, 230
250 g)

N/A

10

10

Balti et al. (2015)

N/A N/A

15 15

15 15

Majumder et al. (2015)

N/A

2, 10, 50

50

N/A

2, 10, 50

50

Yu, Yin, Zhao, Chen, and Liu (2014) Yu et al. (2017)

MasR antagonist A779 (48 μg/kg)

15

15

Liao, Fan, Liu, and Wu (2019)

Intraperitoneal injection of STZ 65 mg/kg N/A N/A N/A N/A

750

750

Zarei et al. (2015)

N/A

N/A

20, 50

50

N/A

N/A

N/A N/A N/A Male SHRs (6 weeks) N/A

Optimum dosage (mg/kg BW)

References

O’Keeffe, Norris, Alashi, Aluko, and FitzGerald (2017)

ACE, angiotensin-converting enzyme; DPP-IV, dipeptidyl peptidase-IV; GLP-1, glucagon-like peptide-1; NO, nitric oxide; NOD, nonobese diabetic; SHR, spontaneously hypertensive rats; STZ, streptozotocin; T1D, type 1 diabetes.

Methodologies for bioactivity assay: animal study

199

GSS in aged SHRs compared with young SHRs. Results indicated that the effect of aging might be restrictive to small peptides, but not oligopeptides, which suggest age-related changes in Peptide transporter 1 (PepT1) expression (Hanh et al., 2017). Aging is the leading risk factor for multiple chronic diseases and decline in physical function. Alzheimer’s disease is an age-dependent neurodegenerative disease, which exacerbates hormones associated with type 2 diabetes in mice (Mehla, Chauhan, & Chauhan, 2014). Therefore mice use in the study of diabetes mellitus is younger than 10 weeks. Notably, physiological changes related to age also take place in a specific cell, secretory profiles, insulin resistance, and inflammatory status of tissues, which lead to metabolic dysfunction (Stout, Justice, Nicklas, & Kirkland, 2017). Age in animal models and the design of models will significantly affect the experimental results. It is necessary to indicate whether the experimental rats or mice are aged in the evaluation of food peptides.

8.2.4 Development of oral and injectable peptides derived from food Peptides from food have been reported to have antihypertensive, antidiabetes, and antiobesity potential (Manikkam, Vasiljevic, Donkor, & Mathai, 2016; Mojica et al., 2017). Food peptide products fed orally include various staple food protein-derived hydrolysates (Girgih, Nwachukwu, Onuh, Malomo, & Aluko, 2016; Iba et al., 2016), commercial oligopeptides (Miralles et al., 2018), and medical oral peptides used in the treatment of diseases (Fosgerau & Hoffmann, 2015; Zhang, 2011). In recent years, the development of injectable agents is another interesting aspect related to food peptides. Injectable peptide hydrogel is one of the types of flexible material with a three-dimensional network structure, which can retain a large amount of water inside, maintaining structural integrity in vivo (Jin et al., 2018; Wu et al., 2017; Zhao et al., 2019). It can be easily applied to the target site by a syringe and then quickly gelled in situ, which will also display low cytotoxicity than synthetic drugs (Xing et al., 2017). Magnesium ions were used as enhancers to prepare an injectable hydrogel from hydrolyzed lysozyme peptide fragments (Yang, Li, Yao, Yu, & Ma, 2019). Stability and antidiabetic properties of IAVPTGVA derived from soybean and LTFPGSAED derived from lupin can be increased through encapsulation and gelation with the RADA16 peptide (Pugliese, Bollati, Gelain, Arnoldi, & Lammi, 2019). Food peptides have been developed as therapeutic agents, and related animal experiments have also been carried out.

8.3 Animal models to evaluate hypertension In consideration of the side effects produced by prolonged use of antihypertensive drugs, food protein-derived antihypertensive peptides have been tested in vivo as a safer alternative (Ganguly, Sharma, & Majumder, 2019; Wu et al., 2017) Several sources of antihypertensive food peptides have been tested, such as milk, seafood, legumes, cereals, and from a diversity of

200 Chapter 8 plant material. Table 8.2 shows the antihypertensive activity of food protein-derived bioactive peptides. Such peptides could potentially be used as therapeutic agents or functional food ingredients to improve changes of structure and function in the cardiovascular system.

8.3.1 Classical animal models to evaluate hypertension The renin
angiotensin
aldosterone system (RAAS) is well-known to play an important role in salt/fluid homeostasis and the regulation of blood pressure (Martı´nez-Sa´nchez, Gabaldo´nHerna´ndez, & Montoro-Garcı´a, 2020). Animal models have demonstrated that food peptides may exert their blocking RAAS activity through potential mechanisms such as ACE-inhibitory effects (Bravo et al., 2019), angiotensin-converting enzyme 2 (ACE2) agonist (Liu et al., 2018; Majumder et al., 2015), and renin inhibition (Udenigwe & Mohan, 2014). Moreover, the arginine
nitric oxide pathway is another hypotensive mechanism, including nitric oxidemediated vasodilation (Mas-Capdevila et al., 2019), angiotensin receptor blocking (FernandezMusoles et al., 2014), calcium channel blocking (Kumrungsee et al., 2014), and enhancement of vascular antioxidative action (Wassmann, Wassmann, & Nickenig, 2004) Fig. 8.2 illustrates the different molecular mechanisms of antihypertensive peptides. The selection of animal models is greatly determined by the mechanism of hypertension to be studied. Herein, SHRs are the most commonly used murine model in the research of hypertension and cardiovascular disease. SHRs strain was produced by Okamoto and Aoki (1963) through selective inbreeding of Wistar-Kyoto hypertensive rats. Food peptides controlled elevated blood pressure during hypertension, for better cardiovascular health, using SHRs models (Miralles et al., 2018). The blood pressure response of rats was better than that of rabbits, and the blood volume was more than that of mice (Milani-Nejad & Janssen, 2014). Wu’s group (Liao, Fan, Davidge, & Wu, 2019) listed detailed experimental procedures with little modification. Male SHRs (290.0 6 10 g) from 9 to 14 weeks old were used and kept under controlled conditions (23 C, 12/12 h light/dark cycle). Antihypertensive tripeptide IRW was the peptide tested. Animals were fed specific pathogen-free (SPF) pellets with free access to saline. After 7
10 days of acclimatization, their systolic blood pressure was measured by the tail-cuff method using a programmed electro-sphygmomanometer, and then the rats were randomly assigned into four groups: untreated (n 5 6), IRW (15 mg/kg body weight, n 5 6), MasR antagonist A779 (48 μg/kg of body weight/h, n 5 6), and IRW 1 A779 (n 5 6). Peptide samples were dissolved in saline and administered orally once a day by intubation for 4 weeks; the control rats were given the same volume of saline. Basal blood pressure (day-7) was recorded, and then an osmotic pump (ALZET, Cupertino, USA) loaded with A779 was implanted subcutaneously into the animal. A779 was infused for 7 days prior to the start of IRW treatment. Blood pressure was measured by a sphygmomanometer every week only before the administration of the peptide treatments. After repeated oral administration trials, the rats were sacrificed by bleeding from the

Methodologies for bioactivity assay: animal study

201

Figure 8.2 Hypotensive mechanisms of certain food peptides. The sequence of amino acid residues of the peptide is abbreviated.

abdominal aorta after fasting for at least 16 h. Vital organs were collected. The collection of blood was used to prepare serum by centrifugation with 800 g for 15 min at 4 C. The specific method is shown in Fig. 8.3A.

Figure 8.3 Three examples for the stages of the animal models use in the study of food peptides. (A) SHRs use in the research of antihypertensive peptides. (B) STZ-induced diabetic C57BL/6J mice. STZ was injected into the C57BL/6J mice (n 5 at least 6/group) on the first day, and the mice with obesity and diabetes phenotype were selected after about 4
5 weeks. Then, food peptides were added, notably, high-fat and high-sugar feed can be used cooperatively. (C) SD rats with HFD. Experimental design: 5-week-old male diet-induced obese SD mice (n 5 at least 6/group) were (Continued)

Methodologies for bioactivity assay: animal study

203

Although rats have many advantages in the aspects of measuring blood pressure and collecting blood, it is challenging to construct the model of deletion or low expression of a specific genotype. The knockout technique has been successful for mice, not in other rodents (Huang, 2009; Rabelo, Nunes-Souza, & Bader, 2015). The generation of transgenic and knockout mice was widely used to study the role of RASS components in cardiovascular control. For example, the ACE2 knockout mouse model was established to study the activator of ACE2 in fighting hypertension. Embryonic stem cells (ESCs) technology led to the development of the mouse for generating new animal modeling of human disease (Saha & Jaenisch, 2009). The gene-targeting construct is introduced by transfection or electroporation into ESCs. Those cells that have been correctly targeted by homologous recombination are selected and injected into blastocysts. However, there is a need for a knockout mouse model applied to ACE2 upregulating food peptides. The targeted genetic alteration of the expression of single-hormone system components is the most straightforward method to analyze their functions in cardiovascular and metabolic homeostasis and disorders. Multiperspective approaches to gain a better understanding of the pathophysiology of food peptides are required to come up with more appropriate models.

8.3.2 Newfangled animal models to evaluate hypertension and cardiovascular disease

L

As explained in Section 9.3.1, RAAS is a complex system of hormone regulation and response in the human body. Thus the discovery and development of ACE-inhibitory peptides derived from food are only part of the RAAS system. Subsequently, the development of newfangled animal models must be combined with the study of various antihypertensive randomized into groups. After prescriptive weeks of baseline measures, mice were randomized into one of these treatment groups. After an additional week of measurements to determine the effect of the treatment, all mice were started on a high-fat diet (HFD). After about 3 weeks of the HFD, the mice began to develop obesity and hyperglycemia phenotype. Source: (A) The above protocol was adapted from Majumder, K., Chakrabarti, S., Morton, J.S., Panahi, S., Kaufman, S., Davidge, S.T., & Wu, J. (2015). Egg-derived ACE-inhibitory peptides IQW and LKP reduce blood pressure in spontaneously hypertensive rats. Journal of Functional Foods, 13, 50
60; Liao, W., Fan, H., Davidge, S.T., & Wu, J. (2019). Egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) reduces blood pressure in spontaneously hypertensive rats via the ACE2/Ang (1
7)/mas receptor axis. Molecular Nutrition and Food Research, 63(9), e1900063; and Khedr, S., Deussen, A., Kopaliani, I., Zatschler, B., & Martin, M. (2018). Effects of tryptophan-containing peptides on angiotensin-converting enzyme activity and vessel tone ex vivo and in vivo. European Journal of Nutrition, 57(3), 907
915. (B) The above protocol was adapted from Yuan, Q., Zhan, B., Chang, R., Du, M., & Mao, X. (2020). Antidiabetic effect of casein glycomacropeptide hydrolysates on high-fat diet and STZ-induced diabetic mice via regulating insulin signaling in skeletal muscle and modulating gut microbiota. Nutrients, 12(1), 220. (C) The above protocol was adapted from Wang, C., Zheng, L., Su, G., Zeng, X.-A., Sun, B., & Zhao, M. (2020). Evaluation and exploration of potentially bioactive peptides in casein hydrolysates against liver oxidative damage in STZ/HFD-induced diabetic rats. Journal of Agricultural and Food Chemistry, 68(8), 2393
2405.

204 Chapter 8 pathways. It has been reported that RAAS also has a significant role in inflammation (Li et al., 2015; Pacurari, Kafoury, Tchounwou, & Ndebele, 2014), antiobesity (Lee et al., 2017), and relief of hepatic steatosis (Cao et al., 2016). For instance, the Ang-(1
7)/ACE2/Mas axis may regulate lipid metabolism genes through the ATP/P2 receptor/CaM signaling pathway, partially reducing liver lipid accumulation (Cao et al., 2016). Milk casein-derived tripeptide valine
proline
proline (VPP) has an antiinflammatory effect on the adipose tissue of highfat diet (HFD)-fed mice (Aihara, Osaka, & Yoshida, 2014) In an in vivo antiobesity study, male C57BL/6 mice were fed with HFD containing silk peptide and a silkworm pupa peptide for 8 weeks. Then the parameters of blood and tissue related to obesity were analyzed (Lee et al., 2017). In addition, ACE2 mutant and ACE2 wild-type mice were used to investigate how amino acid malnutrition can cause intestinal inflammation and diarrhea (Hashimoto et al., 2012). ACE2 mutant mice model can be used in the study of food peptides, which have great potential for activating ACE2. Based on SHRs, several upgraded and research-specific rat models were developed. For example, high-sodium diet-fed SHRs have a higher content of urinary protein excretion and damage of glomerular (Mohammed-Ali, Carlisle, Nademi, & Dickhout, 2017). SHRs have been crossed with other strains to establish newfangled murine models, such as the SHRs heart failure rat, to model congestive heart failure secondary to essential hypertension. In addition, the stroke-prone SHRs is a unique model for studying severe hypertension and hemorrhagic stroke (Otani et al., 2009). Notably, marketed antihypertensive peptides were not only applied in clinical trials but also used in chronic hindlimb ischemia surgery and myocardial ischemiareperfusion injury in rats (Miralles et al., 2018). Intake of VPP and IPP might be beneficial for preventing atherosclerosis caused by hypercholesterolemia (Nakamura et al., 2013). The animal model used in this study was 5-week-old male B6.129P2-Apoetm1Unc/J mice instead of SHRs. Therefore SHRs are not the only models for studying antihypertensive peptides, especially those related to cardiovascular disease or specific surgical treatments. In summary, the study of an antihypertensive peptide can use SHRs, SD rats, and Wistar rats. However, if hypertension-related diseases such as inflammation, stroke, cardiovascular, and cerebrovascular conditions are involved, specific gene knockout mice or restricted diet intervention mice may be selected for the modeling studies. Recommended animal models in the study of hypertension and cardiovascular disease are presented in Table 8.3. HFD, high-fat diet; NOD, nonobese diabetic; SHR, spontaneously hypertensive rats; STZ, streptozotocin; T1D, type 1 diabetes.

8.4 Animal models to evaluate metabolic dysfunction A metabolic dysfunction occurs when the metabolism process fails and causes the body to have either too much or too little of the essential nutrients needed to stay healthy. Bioactive

Methodologies for bioactivity assay: animal study

205

Table 8.3: Pros and cons, problems and solutions of the method recommended to evaluate (A) hypertension and (B) metabolic dysfunction. Pros

Cons

Problems

Solutions

a. How to set up normal control group? b. The choices of age and weight c. The use of specific pathway inhibitors

a. Wistar rats are often used as the normal control group b. Male, 250
400 g c. MasR antagonist A779 (48 μg/kg) used in the regulation of renin
angiotensin by hypotensive peptides 10 weeks, 250
400 g

(A) Method recommended to evaluate hypertension SHRs

Almost all blood pressure value controled by hypotensive peptides can be evaluated using SHRs; the large size is conducive to the measurement of blood pressure and blood drawing

Unable to set up a control group; the regulation of key proteins in specific signaling pathways cannot be investigated; lack of normal control group

Wistar rats

Used as a normal control for SHRs

C57BL/6J mice fed with HFD

To evaluate the efficacy of antihypertensive peptides from the perspective of diet and nutrition; hypertensionrelated obesity and cardiovascular disease can be evaluated by this model The regulation of key proteins in specific signaling pathways can be investigated

Generally not used in the evaluation of antihypertensive peptides alone The decrease in blood pressure was not as significant as that of the SHRs; smaller bodies is adverse to the measurement of blood pressure

Specific knockout mice

Only the hypotensive peptides with specific molecular mechanisms have actions on this model, do not have broad spectrum

The choices of age and weight

a. The choices of age and weight b. HFD formula and modeling time period

a. Ethical requirements for transgene b. Selection of knockout methods

a. More than 8 weeks, N/A b. Different models have different formulations, the principle being high in fat and sugar a. See details in the Section 9.6.1 b. The main methods of knockout include ZFN and TALEN

(B) Method recommended to evaluate metabolic dysfunction SD rats

STZinduced diabetic C57BL/6J mice ICR mice

NOD mice

The large size is conducive to the measurement of blood glucose, weight and blood drawing Drug intervention is beneficial to the construction of type I and type II diabetes models.

No specific capacity in the research of metabolic dysfunction

The choices of age and weight

Smaller bodies is adverse to the measurement of blood glucose, weight and blood drawing

a. The choices of age and weight b. Dosage and duration of use

An ideal model for immunodrug screening and pathological replication

Some differences occur incidental in genetic characteristics between groups; Smaller bodies is adverse to the measurement of blood glucose, weight and blood drawing

a. The choices of age and weight b. Removal of disadvantaged individuals

A polygenic model for T1D that is charactrerized by leukocytic infiltration of the pancreatic islets.

Smaller bodies is adverse to the measurement of blood glucose, weight and blood drawing

The choices of age and weight

Male, 150
350 g

a. More than 6 weeks, N/A b. Intraperitoneal injection of STZ 15
65 mg/kg a. Male or female, 15
50 g b. The male mice are combative, the dominant group kept their beards, while the disadvantaged lost their hair More than 3 weeks

(Continued)

206 Chapter 8 Table 8.3: (Continued) Pros

Cons

C57BL/6J mice fed with HFD

To evaluate the efficacy of antihypertensive peptides from the perspective of diet and nutrition

Smaller bodies is adverse to the measurement of blood glucose, weight and blood drawing

a. The choices of age and weight b. HFD formula and modeling time period

Problems

Specific knockout mice

The regulation of key proteins in specific signaling pathways can be investigated

Only the antimetabolic dysfunction peptides with specific molecular mechanisms have actions on this model, do not have broad spectrum

a. Ethical requirements for transgene b. Selection of knockout methods

Solutions a. More than 8 weeks, N/A b. Different models have different formulations, the principle being high in fat and sugar. a. See details in the Section 9.6.1 b. The main methods of knockout include ZFN and TALEN

HFD, high-fat diet; NOD, nonobese diabetic; SHR, spontaneously hypertensive rats; STZ, streptozotocin; T1D, type 1 diabetes.

peptides, as products of hydrolysis of diverse food proteins, exert various biological roles against metabolic dysfunction, including two crucial activities of antidiabetes and antiobesity (Manikkam et al., 2016; Oseguera-Toledo, de Mejia, & Amaya-Llano, 2015). Moreover, the role of peptides on specific receptors is highly selective, and their pharmacokinetic and pharmacodynamic properties can be adjusted through structural modifications Bruno, Miller, & Lim, 2013). This section reviews typical animal models to evaluate the effects of peptides from food sources on the prevention of obesity and diabetes. Table 8.2 lists peptides derived from food sources on the prevention of obesity and diabetes. Fig. 8.4 presents the main molecular mechanisms of food peptides against obesity and diabetes. The main mechanisms are achieved by regulating gastrointestinal hormones and inhibiting cellular inflammation.

8.4.1 Animal models to evaluate metabolic dysfunction Several studies have demonstrated that food peptides offer a spectrum of potential therapeutic opportunities to improve blood glucose control in diabetes. Sun et al. (2019) suggested that the effect of cereal peptides on the development of type 1 diabetes (T1D) is associated with their antiinflammatory ability. Hydrolysates or peptides derived from food proteins that inhibit DPP-IV may also have potential in the management of type 2 diabetes (T2D) (Nongonierma & FitzGerald, 2016). Hydrolysates from casein, rice, and chicken promote GLP-1 release in T2D model of cells and animals (Casanova-Marti et al., 2019; Ishikawa et al., 2015; O’Halloran et al., 2018). Dietary peptides (γ-[Glu](n51,2)-Phe/-Met/-Val) and soybean β51
63 peptide triggered the release of CCK and GLP-1 to alleviate T2D by activating the calcium-sensing receptor (CaSR) (Nakajima, Hira, Eto, Asano, & Hara, 2010; Yang et al., 2019). Animal models can be selected based on targeting specific mechanisms of food peptides in the prevention of diabetes. Male and female Wistar rats or Sprague-Dawley rats are used to study food peptides or hydrolysates in the release of CCK and GLP-1 to

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Figure 8.4 Antimetabolic dysfunction (diabetes mellitus, and obesity) mechanisms of certain food peptides. The sequence of amino acid residues of the peptide is abbreviated.

alleviate T2D (Kato, Nakanishi, Tani, & Tsuda, 2017; Li, Liu, He, & Wu, 2018). Male C57BL/6J mice age 5-weeks-old were used to elucidate the prevention of hepatic insulin resistance and management of T2D. Besides the control group, mice were fed an HFD (20% of calories from protein, 60% of calories from fat, and 20% of calories from carbohydrates) to establish a mice model of T2D (Song, Gao, Du, & Mao, 2018). Mice models of T2D include diet intervention combined with STZ-induced have been developed. For instance, the HFD-fed and low-dose STZ-induced diabetic rats were used as animal models in the study of the DPP-IV inhibitory effects of sodium caseinate hydrolysate (Hsieh et al., 2016). T1D is characterized by the infiltration of immune cells into the pancreas, and consequent inflammation selectively leads to dysfunction and finally death of β-cells (Majumder, Mine, & Wu, 2016). It has been reported that C57BL/6J mice except for the controls were injected with STZ to establish an inflammatory model based on TD1 (Sun et al., 2019). In addition, the nonobese diabetic (NOD) mice model has also been used in T1D which, suggests that a mixture of corn and wheat peptide could prevent diabetes with pancreatitis in NOD mice (Sun et al., 2019). The NOD mouse is a polygenic model for T1D that is characterized by leukocytic infiltration of the pancreatic islets. After about 40 years since the original inbred strain was developed in Japan, substrains of NOD mice have been established all over the world (Simecek et al., 2015). Appropriate animal models should be used to evaluate food peptides in the prevention and treatment of diabetes (T1D and T2D).

208 Chapter 8 Obesity is characterized by an expansion of adipose tissue resulting from adipocyte hyperplasia and hypertrophy, which is an imbalance between energy intake and expenditure (Su, Huang, & Zhu, 2016). Synthetic drugs frequently used for antiobesity purposes have the disadvantage of showing multiple adverse side effects. For this reason, the search for natural peptides derived from food sources is of high interest. For example, treating HFD-induced obese rats (6 weeks-old male Sprague-Dawley) with kefir peptides reduced the fatty acid synthase protein significantly. It blocked lipogenesis in the liver by increasing the p-acetyl-CoA carboxylase protein (Tung et al., 2018). Chickpea peptide (VFVRN) had high hypolipidemic effects (Shi et al., 2019). Obesity is a metabolic dysfunction with low-grade systemic chronic inflammation (Ouchi, Parker, Lugus, & Walsh, 2011). Fat tissue can induce abnormal levels of pro-inflammatory cytokines, reactive oxygen species, and inflammatory mediators, with a potent risk for insulin resistance, T2D, and cardiovascular disease (Ellulu, Patimah, Khaza’ai, Rahmat, & Abed, 2017). Food peptides, due to the structural similarity with endogenous peptides, can interact with receptors and play significant roles modulating metabolic processes that can modify immune regulators Luna-Vital, Mojica, Gonza´lez de Mejı´a, Mendoza, & LoarcaPin˜a, 2015). Gomes et al. (2020) reported that bean protein hydrolysate showed hypocholesterolemic activity preventing inflammation and dysfunction of vascular endothelium in male BALB/c mice fed an atherogenic diet. In addition, dietary dipeptide γ-glutamylvaline-activated CaSR preventing the accumulation of adipocytokines and adipokines secreted by dysfunctional adipocytes Xing, Zhang, Majumder, Zhang, & Mine, 2019). The mechanism of food peptides controlling dietary intake and blood glucose through CaSR (Colombini et al., 2013) were in vitro rather than in vivo.

8.4.2 Knockout mice models to evaluate metabolic dysfunction Knockout mice can facilitate a better understanding of the specific mechanisms of antidiabetes and antiobesity. The adiponectin gene is located at 3q27, which is also a susceptible site for metabolic dysfunction and coronary heart disease (Joshaghani et al., 2020). It has been reported that the adiponectin-knockout mice model was used in the study of alleviating insulin resistance and hepatoprotective effect Ferreira, de Sousa Filho, Marinovic, Rodrigues, & Otton, 2020). Therapeutic effects of GLP-1 agonists exendin-4 and liraglutide were reported using knockout mice models Tashiro et al., 2014). However, the application of this model in animal experiments with antidiabetic food peptides is needed. Involving mechanisms of antiobesity, specific animal model knockout of p85α in brown adipose tissue (Gomez-Hernandez et al., 2020), nescient basic helix-loop-helix 2 (Nhlh2) knockout (N2KO) mice (Kim, Gilliard, Good, & Park, 2012), and CD14 knockout mice (Roncon-Albuquerque et al., 2008) were used. Flow charts of the models for STZ-induced diabetic C57BL/6J mice and SD rats with HFD are shown in Fig. 8.3B and C.

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More research is needed for further development of food peptides in clinical applications and commercialization. There is a need for robust pharmacokinetic data and standardized procedures. Specific knockout mice models need to be applied to confirm the effects of food peptides in preventing metabolic dysfunction and being used as treatments. The pros, cons, problems, and solutions of animal models in the study of metabolic dysfunction are presented in Table 8.3.

8.5 Analysis and statistics 8.5.1 Sample size: power analysis Determining sample size for an animal study has always been a significant problem that must be solved. Power analysis combines statistics, subject domain knowledge, and specific requirements to derive the optimal sample size for studying food peptides in animal models. For continuous data in an animal study, power analysis can also be used to assess sample sizes through ANOVA and DOE designs (Bate & Clark, 2014). Power curve graph is often used to determine how the power varies by the difference. Moreover, there are hypothesis tests for other types of data, such as proportions tests (binomial data) and incidence rates (Poisson data) (Fleiss, Levin, & Paik, 2013). These tests have their corresponding capabilities and sample analysis.

8.5.2 Handling of normal and nonnormal distributed data Advanced consistency and completeness of data reporting provide a large number of opportunities for improving mathematical models. Principal component analysis and orthogonal partial least square analysis are powerful tools for processing large data sets and enable the discrimination of different samples (Zhang et al., 2013). The better visualization of group clustering patterns such as heat map representation of the one-way hierarchical clustering analysis of data in vivo is essential in the study of biological activity of food peptides (Huang et al., 2019; Siddik et al., 2020; Wang et al., 2020).

8.5.3 Multivariate analysis of animal studies Because of the bioactivity of peptides, some of them may be used as food ingredients. There is a large number of peptide sequences from hydrolysates, and each sequence has a different biological activity. Analysis of these data can provide a better basis for animal experiments Iwaniak, Darewicz, Mogut, & Minkiewicz, 2019). In silico tools help in predicting proteases and hydrolysis conditions that can be used in the production of bioactive peptides. For instance, peptides from casein hydrolysates were effective on oxidative liver damage in STZ/ HFD-induced diabetic rats based on the analysis in silico and in vivo (Wang et al., 2020).

210 Chapter 8 There are techniques recommended to look for the distribution of peptides from hydrolysates, such as UpSet Venn diagram (http://www.omicshare.com/tools). The variation analysis of the peak areas in common peptides can be shown by the volcano plot (http://www.omicshare. com/tools); and the potential bioactivities of the peptides by using the Peptide Ranker. The CPPpred (distilldeep.ucd.ie/CPPpred/) can predict the intestinal permeability. Lacroix and Li-Chan (2012) assessed DPP-IV inhibition of peptides produced from 34 proteins in nine food commodities using an in silico approach. In silico, combined with in vivo mimicking the action of intestinal enzymes, was used predicting the release of bioactive peptides from meat (Sayd et al., 2018). Therefore according to the progress in the development of information technologies, scientists combine in silico with in vivo studies when analyzing food peptides in the prevention of cardiovascular and metabolic diseases. A meta-analysis of animal experiments has been considered an effective way to guide animal experiments to clinical research. It can effectively reduce the risks of translation from animal to clinical studies. Further, it can highlight standard methodological practices and provide a basis for assessing the areas in which improvement is warranted (Tang, Zhao, Zhong, Zheng, & Feng, 2020). The meta-analysis can obtain the source of heterogeneity in experimental animals and determine the size of heterogeneity by using I2-test and H-test (Higgins, Thompson, Deeks, & Altman, 2003; Jafarnejad, Mirzaei, Clark, Taghizadeh, & Ebrahimzadeh, 2020). If significant heterogeneity is detected among studies by the above methods (P , 0.05 or I2 . 50%), the source of heterogeneity can be explored through subgroup analysis, meta-regression analysis, and other heterogeneity treatments (Annane et al., 2004). In addition, the data collection that allows further access and sharing can be selected by meta-analysis (Barili, Parolari, Kappetein, & Freemantle, 2018). Compared with single-experimental animal research, the results of a meta-analysis based on high-quality animal research are more reliable. It has been reported that meta-analysis was used in correlating structure of bioactive peptides in foods of animal origin with regard to effect and stability (Maestri, Pavlicevic, Montorsi, & Marmiroli, 2019), and effect of peptides derived from food proteins on blood pressure (Hugo Pripp, 2008). These results can effectively evaluate and optimize the best innovative animal model and avoid waste of resources.

8.6 Safety considerations and standards during the development of animal models 8.6.1 Bioethics considerations Ethical principles are crucial in the use of animals for the evaluation of food components, such as peptides. Fundamental principles and ethical issues are presented in this section. First and foremost, animal experiments need to be followed with the approval of the Institutional Animal Care and Use Committee (IACUC). As with other compounds and

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drugs, animal studies on the function of food peptides need to be committed to the highest ethical standards (Doke & Dhawale, 2015). Whether studying antihypertensive peptides or antimetabolic dysfunction peptides, the ethical statement of animals is mandatory in the conduction of scientific experiments. In brief, all animals receive humane care according to the criteria outlined in the national guidelines “Guide for the Care and Use of Laboratory Animals” used following the international law on the protection and use of animals. This regulatory document provides guidelines for the environment, housing, and management of laboratory animals used or produced for research, including various animal models for bioactivity assays of food peptides. In recent years, the use of transgenic mice has increased significantly in the study of clinical evaluation of drugs. Animal welfare and evolutionary constraints need to be fully considered (Bovenkerk, 2020). When animal testing is done to support applications for medical products regulated by FDA, manufacturers or sponsors are required to follow FDA’s regulation, Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58). Animals have been saved from various diseases through the testing of vaccinations; without this testing, millions of animals would have died (Kaushik & Vaswani, 2018). For example, ACE-inhibitory peptides from food sources have extensive hypotensive effects on SHRs (Balti et al., 2015; Liao et al., 2019). The principle of the “3 Rs” (Replace, Reduce, and Refine) for research must be followed (MacArthur Clark, 2018).

8.6.2 Clinical evaluation of sick animals Some studies require SPF animals (Al-Asmakh & Zadjali, 2015). Sick or dead animals need to be processed and analyzed. Then, the reason for death from the drug dose, environment, feed, and other factors need to be investigated (Dunbar, Higa, Jones, Kaminski, & Panicker, 2012). It should be noted that no matter what the scientific application of animals, there are restrictions on the methods of their disposal once they have been euthanized or dead (Kaushik & Vaswani, 2018). The USDA, https://www.usda.gov/, and the US Department of Health and Human Services (HHS), https://www.hhs.gov/, offer specific guidelines in this regard.

8.7 Summary In the search for functional activities of food peptides, many kinds of animal models have been applied, including classical and innovative models. The animal model approach used needs to be tailored to the specific bioactivity of the food peptides. This chapter described the rat and mouse models used in the prevention of hypertension and metabolic dysfunction. Furthermore, since the animal models cannot replace human clinical tests, commercial food peptides require a lengthy evaluation cycle. Also, there are considerable

212 Chapter 8 commercial challenges to provide a consistent quality product with guaranteed sterility and an adequate shelf-life.

References Aguirre, T. A., Teijeiro-Osorio, D., Rosa, M., Coulter, I. S., Alonso, M. J., & Brayden, D. J. (2016). Current status of selected oral peptide technologies in advanced preclinical development and in clinical trials. Advanced Drug Delivery Reviews, 106, 223
241. Aihara, K., Osaka, M., & Yoshida, M. (2014). Oral administration of the milk casein-derived tripeptide Val-Pro-Pro attenuates high-fat diet-induced adipose tissue inflammation in mice. British Journal of Nutrition, 112(4), 513
519. Al-Asmakh, M., & Zadjali, F. (2015). Use of germ-free animal models in microbiota-related research. Journal of Microbiology Biotechnology, 25(10), 1583
1588. Aluko, R. E. (2015). Chapter 5-Amino acids, peptides, and proteins as antioxidants for food preservation. In F. Shahidi (Ed.), Handbook of antioxidants for food preservation (pp. 105
140). Woodhead Publishing. Annane, D., Bellissant, E., Bollaert, P. E., Briegel, J., Keh, D., & Kupfer, Y. (2004). Corticosteroids for severe sepsis and septic shock: a systematic review and meta-analysis. BMJ (Clinical Research ed.), 329(7464), 480. Asgharpour, A., Cazanave, S. C., Pacana, T., Seneshaw, M., Vincent, R., Banini, B. A., . . . Sanyal, A. J. (2016). A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. Journal of Hepatology, 65(3), 579
588. Balti, R., Bougatef, A., Sila, A., Guillochon, D., Dhulster, P., & Nedjar-Arroume, N. (2015). Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) muscle protein hydrolysates and antihypertensive effect of the potent active peptide in spontaneously hypertensive rats. Food Chemistry, 170, 519
525. Barili, F., Parolari, A., Kappetein, P. A., & Freemantle, N. (2018). Statistical Primer: heterogeneity, random- or fixed-effects model analyses? Interactive Cardiovascular and Thoracic Surgery, 27(3), 317
321. Bate, S. T., & Clark, R. A. (2014). The design and statistical analysis of animal experiments. Cambridge University Press. Beltra´n-Barrientos, L. M., Garcı´a, H. S., Torres-Llanez, M. J., Gonza´lez-Co´rdova, A. F., Herna´ndez-Mendoza, A., & Vallejo-Cordoba, B. (2017). Safety of milk-derived bioactive peptides. International Journal of Dairy Technology, 70(1), 16
22. Bhandari, D., Rafiq, S., Gat, Y., Gat, P., Waghmare, R., & Kumar, V. (2020). A review on bioactive peptides: Physiological functions, bioavailability and safety. International Journal of Peptide Research and Therapeutics, 26(1), 139
150. Bovenkerk, B. (2020). Ethical perspectives on modifying animals: Beyond welfare arguments. Animal Frontiers, 10(1), 45
50. Bravo, F. I., Mas-Capdevila, A., Margalef, M., Arola-Arnal, A., & Muguerza, B. (2019). Novel antihypertensive peptides derived from chicken foot proteins. Molecular Nutrition and Food Research, 63(12), e1801176. Bruno, B. J., Miller, G. D., & Lim, C. S. (2013). Basics and recent advances in peptide and protein drug delivery. Therapeutic Delivery, 4(11), 1443
1467. Cao, X., Yang, F., Shi, T., Yuan, M., Xin, Z., Xie, R., . . . Yang, J.-K. (2016). Angiotensin-converting enzyme 2/angiotensin-(1
7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis. Scientific Reports, 6, 21592. Carrizzo, A., Conte, G. M., Sommella, E., Damato, A., Ambrosio, M., Sala, M., . . . Vecchione, C. (2019). Novel potent decameric peptide of Spirulina platensis reduces blood pressure levels through a PI3K/AKT/ eNOS-dependent mechanism. Hypertension, 73(2), 449
457. Casanova-Marti, A., Bravo, F. I., Serrano, J., Ardevol, A., Pinent, M., & Muguerza, B. (2019). Antihyperglycemic effect of a chicken feet hydrolysate via the incretin system: DPP-IV-inhibitory activity and GLP-1 release stimulation. Food and Function, 10(7), 4062
4070.

Methodologies for bioactivity assay: animal study

213

Chalamaiah, M., Yu, W., & Wu, J. (2018). Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins: A review. Food Chemistry, 245, 205
222. Colombini, A., Perego, S., Ardoino, I., Marasco, E., Lombardi, G., Fiorilli, A., . . . Ferraretto, A. (2013). Evaluation of a possible direct effect by casein phosphopeptides on paracellular and vitamin D controlled transcellular calcium transport mechanisms in intestinal human HT-29 and Caco-2 cell lines. Food and Function, 4(8), 1195
1203. Dalziel, J. E., Spencer, N. J., Dunstan, K. E., Lynch, A. T., Haggarty, N. W., Gopal, P. K., & Roy, N. C. (2014). An in vitro rat model of colonic motility to determine the effect of beta-casomorphin-5 on propagating contractions. Food and Function, 5(11), 2768
2774. Diehl, K. H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., . . . Vorstenbosch, C. V. D. (2001). A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology: An International Journal, 21(1), 15
23. Di Stasio, L., Picariello, G., Mongiello, M., Nocerino, R., Berni Canani, R., Bavaro, S., . . . Mamone, G. (2017). Peanut digestome: Identification of digestion resistant IgE binding peptides. Food and Chemical Toxicology, 107(Part A), 88
98. Doke, S. K., & Dhawale, S. C. (2015). Alternatives to animal testing: A review. Saudi Pharmaceutical Journal, 23(3), 223
229. Dunbar, G., Higa, J., Jones, T., Kaminski, B., & Panicker, S. (2012). Guidelines for ethical conduct in the care and use of nonhuman animals in research. American Psychological Association Committee on Animal Rights Research and Ethics, 1
9, 2010-2011. Edelsbrunner, M. E., Herzog, H., & Holzer, P. (2009). Evidence from knockout mice that peptide YY and neuropeptide Y enforce murine locomotion, exploration and ingestive behaviour in a circadian cycle- and gender-dependent manner. Behavioural Brain Research, 203(1), 97
107. Ellulu, M. S., Patimah, I., Khaza’ai, H., Rahmat, A., & Abed, Y. (2017). Obesity and inflammation: The linking mechanism and the complications. Archives of Medical Science: AMS, 13(4), 851
863. Fernandez-Musoles, R., Castello-Ruiz, M., Arce, C., Manzanares, P., Ivorra, M. D., & Salom, J. B. (2014). Antihypertensive mechanism of lactoferrin-derived peptides: Angiotensin receptor blocking effect. Journal of Agricultural and Food Chemistry, 62(1), 173
181. Ferreira, L. T., de Sousa Filho, C. P. B., Marinovic, M. P., Rodrigues, A. C., & Otton, R. (2020). Green tea polyphenols positively impact hepatic metabolism of adiponectin-knockout lean mice. Journal of Functional Foods, 64, 103679. Fiedorowicz, E., Kaczmarski, M., Cieslinska, A., Sienkiewicz-Szlapka, E., Jarmolowska, B., Chwala, B., & Kostyra, E. (2014). β-Casomorphin-7 alters mu-opioid receptor and dipeptidyl peptidase IV genes expression in children with atopic dermatitis. Peptides, 62, 144
149. Fitzgerald, C., Aluko, R. E., Hossain, M., Rai, D. K., & Hayes, M. (2014). Potential of a renin inhibitory peptide from the red seaweed Palmaria palmata as a functional food ingredient following confirmation and characterization of a hypotensive effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 62(33), 8352
8356. Fleiss, J. L., Levin, B., & Paik, M. C. (2013). Statistical methods for rates and proportions. John Wiley and Sons. Fosgerau, K., & Hoffmann, T. (2015). Peptide therapeutics: Current status and future directions. Drug Discovery Today, 20(1), 122
128. Ganguly, A., Sharma, K., & Majumder, K. (2019). Food-derived bioactive peptides and their role in ameliorating hypertension and associated cardiovascular diseases. Advances in Food and Nutrition Research, 89, 165
207. Gao, J., Song, J., Du, M., & Mao, X. (2018). Bovine alpha-lactalbumin ydrolysates (alpha-LAH) ameliorate adipose insulin resistance and inflammation in high-fat diet-fed C57BL/6J mice. Nutrients, 10(2), 242. Gao, J., Song, J., Du, M., & Mao, X. (2019). Bovine α-lactalbumin hydrolysates (α-LAH) attenuate high-fat diet induced nonalcoholic fatty liver disease by modulating hepatic lipid metabolism in C57BL/6J mice. Journal of Functional Foods, 54, 254
262.

214 Chapter 8 Girgih, A. T., Nwachukwu, I. D., Onuh, J. O., Malomo, S. A., & Aluko, R. E. (2016). Antihypertensive properties of a pea protein hydrolysate during short-and long-term oral administration to spontaneously hypertensive rats. Journal of Food Science, 81(5), H1281
H1287. Godar, R. J., Ma, X., Liu, H., Murphy, J. T., Weinheimer, C. J., Kovacs, A., . . . Diwan, A. (2015). Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury. Autophagy, 11(9), 1537
1560. Gomes, M. J. C., Lima, S. L. S., Alves, N. E. G., Assis, A., Moreira, M. E. C., Toledo, R. C. L., . . . Martino, H. S. D. (2020). Common bean protein hydrolysate modulates lipid metabolism and prevents endothelial dysfunction in BALB/c mice fed an atherogenic diet. Nutrition, Metabolism and Cardiovascular Diseases, 30(1), 141
150. Gomez-Hernandez, A., Lopez-Pastor, A. R., Rubio-Longas, C., Majewski, P., Beneit, N., Viana-Huete, V., . . . Benito, M. (2020). Specific knockout of p85alpha in brown adipose tissue induces resistance to high-fat diet-induced obesity and its metabolic complications in male mice. Molecular Metabolism, 31, 1
13. Guo, S. (2014). Insulin signaling, resistance, and the metabolic syndrome: insights from mouse models into disease mechanisms. The Journal of Endocrinology, 220(2), T1
T23. Hanh, V. T., Shen, W., Tanaka, M., Siltari, A., Korpela, R., & Matsui, T. (2017). Effect of aging on the absorption of small peptides in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 65(29), 5935
5943. Hashimoto, T., Perlot, T., Rehman, A., Trichereau, J., Ishiguro, H., Paolino, M., . . . Penninger, J. M. (2012). ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation. Nature, 487(7408), 477
481. He, R., Yang, Y. J., Wang, Z., Xing, C. R., Yuan, J., Wang, L. F., . . . Ju, X. R. (2019). Rapeseed proteinderived peptides, LY, RALP, and GHS, modulates key enzymes and intermediate products of reninangiotensin system pathway in spontaneously hypertensive rat. NPJ Science of Food, 3(1), 1
6. Higgins, J. P., Thompson, S. G., Deeks, J. J., & Altman, D. G. (2003). Measuring inconsistency in metaanalyses. BMJ (Clinical Research ed.), 327(7414), 557
560. Hohlbaum, K., Bert, B., Dietze, S., Palme, R., Fink, H., & Thone-Reineke, C. (2017). Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice-Assessing the degree of distress. PLoS One, 12(6), e0179588. Hsieh, C.-H., Wang, T.-Y., Hung, C.-C., Jao, C.-L., Hsieh, Y.-L., Wu, S.-X., & Hsu, K.-C. (2016). In silico, in vitro and in vivo analyses of dipeptidyl peptidase IV inhibitory activity and the antidiabetic effect of sodium caseinate hydrolysate. Food and Function, 7(2), 1122
1128. Huang, P. L. (2009). eNOS, metabolic syndrome and cardiovascular disease. Trends in Endocrinology and Metabolism, 20(6), 295
302. Huang, Z., Peng, H., Sun, Y., Zhu, X., Zhang, H., Jiang, L., . . . Xiong, H. (2019). Beneficial effects of novel hydrolysates produced by limited enzymatic broken rice on the gut microbiota and intestinal morphology in weaned piglets. Journal of Functional Foods, 62, 103560. Hugo Pripp, A. (2008). Effect of peptides derived from food proteins on blood pressure: A meta-analysis of randomized controlled trials. Food and Nutrition Research, 52(1), 1641. Iba, Y., Yokoi, K., Eitoku, I., Goto, M., Koizumi, S., Sugihara, F., . . . Yoshimoto, T. (2016). Oral administration of collagen hydrolysates improves glucose tolerance in normal mice through GLP-1-dependent and GLP-1-independent mechanisms. Journal of Medicinal Food, 19(9), 836
843. Ishikawa, Y., Hira, T., Inoue, D., Harada, Y., Hashimoto, H., Fujii, M., . . . Hara, H. (2015). Rice protein hydrolysates stimulate GLP-1 secretion, reduce GLP-1 degradation, and lower the glycemic response in rats. Food and Function, 6(8), 2525
2534. Iwaniak, A., Darewicz, M., Mogut, D., & Minkiewicz, P. (2019). Elucidation of the role of in silico methodologies in approaches to studying bioactive peptides derived from foods. Journal of Functional Foods, 61, 103486. Jafarnejad, S., Mirzaei, H., Clark, C. C. T., Taghizadeh, M., & Ebrahimzadeh, A. (2020). The hypotensive effect of salt substitutes in stage 2 hypertension: a systematic review and meta-analysis. BMC Cardiovascular Disorders, 20(1), 98.

Methodologies for bioactivity assay: animal study

215

Jiang, H., Feng, J., Du, Z., Zhen, H., Lin, M., Jia, S., . . . Chen, Z. (2014). Oral administration of soybean peptide Vglycin normalizes fasting glucose and restores impaired pancreatic function in Type 2 diabetic Wistar rats. The Journal of Nutritional Biochemistry, 25(9), 954
963. Jin, H., Wan, C., Zou, Z., Zhao, G., Zhang, L., Geng, Y., . . . Yang, K. (2018). Tumor ablation and therapeutic immunity induction by an injectable peptide hydrogel. ACS Nano, 12(4), 3295
3310. Joshaghani, H. R., Kokhaei, P., Barati, M., Pakdel, A., Mohammadzadeh, G., Bandegi, N., & Bandegi, A. R. (2020). Association of adiponectin gene polymorphisms and their haplotypes with type 2 diabetes and related metabolic traits in an Iranian population. International Journal of Diabetes in Developing Countries, 40, 1
7. Kapila, R., Kapila, S., & Vij, R. (2017). Chapter 23-Efficacy of milk-derived bioactive peptides on health by cellular and animal models. In R. R. Watson, R. J. Collier, & V. R. Preedy (Eds.), Nutrients in dairy and their implications on health and disease (pp. 303
311). Academic Press. Kato, M., Nakanishi, T., Tani, T., & Tsuda, T. (2017). Low-molecular fraction of wheat protein hydrolysate stimulates glucagon-like peptide-1 secretion in an enteroendocrine L cell line and improves glucose tolerance in rats. Nutrition Research, 37, 37
45. Kaushik, K., & Vaswani, R. (2018). Research on animals and current UGC guidelines on animal dissection and experimentation: A critical analysis. Bioethics Update, 4(2), 119
139. Khedr, S., Deussen, A., Kopaliani, I., Zatschler, B., & Martin, M. (2018). Effects of tryptophan-containing peptides on angiotensin-converting enzyme activity and vessel tone ex vivo and in vivo. European Journal of Nutrition, 57(3), 907
915. Kim, J. H., Gilliard, D., Good, D. J., & Park, Y. (2012). Preventive effects of conjugated linoleic acid on obesity by improved physical activity in nescient basic helix-loop-helix 2 knockout mice during growth period. Food and Function, 3(12), 1280
1285. Klein, S. L., & Flanagan, K. L. (2016). Sex differences in immune responses. Nature Reviews. Immunology, 16 (10), 626
638. Kumrungsee, T., Wang, Z. Q., Matsumura, S., Saiki, T., Tanaka, M., & Matsui, T. (2014). Identification of peptides from soybean protein, glycinin, possessing suppression of intracellular Ca2 1 concentration in vascular smooth muscle cells. Food Chemistry, 152, 218
224. Lacroix, I. M. E., & Li-Chan, E. C. Y. (2012). Evaluation of the potential of dietary proteins as precursors of dipeptidyl peptidase (DPP)-IV inhibitors by an in silico approach. Journal of Functional Foods, 4(2), 403
422. Lee, E. J., Hur, J., Ham, S. A., Jo, Y., Lee, S., Choi, M. J., & Seo, H. G. (2017). Fish collagen peptide inhibits the adipogenic differentiation of preadipocytes and ameliorates obesity in high fat diet-fed mice. International Journal of Biological Macromolecules, 104, 281
286. Li, Q., Xu, M., Cui, Y., Huang, C., & Sun, M. (2017). Arginine-rich membrane-permeable peptides are seriously toxic. Pharmacology Research and Perspectives, 5(5), e00334. Li, S., Liu, L., He, G., & Wu, J. (2018). Molecular targets and mechanisms of bioactive peptides against metabolic syndromes. Food and Function, 9(1), 42
52. Li, Y., Cao, Y., Zeng, Z., Liang, M., Xue, Y., Xi, C., . . . Jiang, W. (2015). Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis prevents lipopolysaccharide-induced apoptosis of pulmonary microvascular endothelial cells by inhibiting JNK/NF-kappaB pathways. Scientific Reports, 5, 8209. Liao, W., Fan, H., Davidge, S. T., & Wu, J. (2019). Egg white-derived antihypertensive peptide IRW (Ile-Arg-Trp) reduces blood pressure in spontaneously hypertensive rats via the ACE2/Ang (1-7)/mas receptor axis. Molecular Nutrition and Food Research, 63(9), e1900063. Liao, W., Fan, H., Liu, P., & Wu, J. (2019). Identification of angiotensin converting enzyme 2 (ACE2) upregulating peptides from pea protein hydrolysate. Journal of Functional Foods, 60, 103395. Liu, P., Wysocki, J., Souma, T., Ye, M., Ramirez, V., Zhou, B., . . . Jin, J. (2018). Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation. Kidney International, 94(1), 114
125. Luna-Vital, D. A., Mojica, L., Gonza´lez de Mejı´a, E., Mendoza, S., & Loarca-Pin˜a, G. (2015). Biological potential of protein hydrolysates and peptides from common bean (Phaseolus vulgaris L.): A review. Food Research International, 76, 39
50.

216 Chapter 8 Lundquist, P., & Artursson, P. (2016). Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues. Advanced Drug Delivery Reviews, 106, 256
276. MacArthur Clark, J. (2018). The 3Rs in research: A contemporary approach to replacement, reduction and refinement. British Journal of Nutrition, 120(S1), S1
S7. Maestri, E., Pavlicevic, M., Montorsi, M., & Marmiroli, N. (2019). Meta-analysis for correlating structure of bioactive peptides in foods of animal origin with regard to effect and stability. Comprehensive Reviews in Food Science and Food Safety, 18(1), 3
30. Mair, K. M., Wright, A. F., Duggan, N., Rowlands, D. J., Hussey, M. J., Roberts, S., . . . MacLean, M. R. (2014). Sex-dependent influence of endogenous estrogen in pulmonary hypertension. American Journal of Respiratory and Critical Care Medicine, 190(4), 456
467. Majumder, K., Chakrabarti, S., Morton, J. S., Panahi, S., Kaufman, S., Davidge, S. T., & Wu, J. (2015). Egg-derived ACE-inhibitory peptides IQW and LKP reduce blood pressure in spontaneously hypertensive rats. Journal of Functional Foods, 13, 50
60. Majumder, K., Mine, Y., & Wu, J. (2016). The potential of food protein-derived anti-inflammatory peptides against various chronic inflammatory diseases. Journal of the Science of Food and Agriculture, 96(7), 2303
2311. Makinen, S., Streng, T., Larsen, L. B., Laine, A., & Pihlanto, A. (2016). Angiotensin I-converting enzyme inhibitory and antihypertensive properties of potato and rapeseed protein-derived peptides. Journal of Functional Foods, 25, 160
173. Malha, L., Sison, C. P., Helseth, G., Sealey, J. E., & August, P. (2018). Renin-angiotensin-aldosterone profiles in pregnant women with chronic hypertension. Hypertension, 72(2), 417
424. Manikkam, V., Vasiljevic, T., Donkor, O. N., & Mathai, M. L. (2016). A review of potential marine-derived hypotensive and anti-obesity peptides. Critical Reviews in Food Science and Nutrition, 56(1), 92
112. Marthandam Asokan, S., Wang, T., Su, W. T., & Lin, W. T. (2019). Antidiabetic effects of a short peptide of potato protein hydrolysate in STZ-induced diabetic mice. Nutrients, 11(4), 779. Martin, M., Kopaliani, I., Jannasch, A., Mund, C., Todorov, V., Henle, T., & Deussen, A. (2015). Antihypertensive and cardioprotective effects of the dipeptide isoleucine-tryptophan and whey protein hydrolysate. Acta Physiologica, 215(4), 167
176. Martı´nez-Sa´nchez, S. M., Gabaldo´n-Herna´ndez, J. A., & Montoro-Garcı´a, S. (2020). Unravelling the molecular mechanisms associated with the role of food-derived bioactive peptides in promoting cardiovascular health. Journal of Functional Foods, 64, 103645. Martinez-Villaluenga, C., Pen˜as, E., & Frias, J. (2017). Chapter 2-Bioactive peptides in fermented foods: Production and evidence for health effects. In J. Frias, C. Martinez-Villaluenga, & E. Pen˜as (Eds.), Fermented foods in health and disease prevention (pp. 23
47). Academic Press. Mas-Capdevila, A., Iglesias-Carres, L., Arola-Arnal, A., Aragones, G., Aleixandre, A., Bravo, F. I., & Muguerza, B. (2019). Evidence that nitric oxide is involved in the blood pressure lowering effect of the peptide AVFQHNCQE in spontaneously hypertensive rats. Nutrients, 11(2), 225. Mattson, M. P., Longo, V. D., & Harvie, M. (2017). Impact of intermittent fasting on health and disease processes. Ageing Research Reviews, 39, 46
58. Mehla, J., Chauhan, B. C., & Chauhan, N. B. (2014). Experimental induction of type 2 diabetes in aging-accelerated mice triggered Alzheimer-like pathology and memory deficits. Journal of Alzheimer’s Disease, 39(1), 145
162. Milani-Nejad, N., & Janssen, P. M. (2014). Small and large animal models in cardiac contraction research: advantages and disadvantages. Pharmacology & Therapeutics, 141(3), 235
249. Miralles, B., Amigo, L., & Recio, I. (2018). Critical review and perspectives on food-derived antihypertensive peptides. Journal of Agricultural and Food Chemistry, 66(36), 9384
9390. Mohammed-Ali, Z., Carlisle, R. E., Nademi, S., & Dickhout, J. G. (2017). Chapter 16-Animal models of kidney disease. In P. M. Conn (Ed.), Animal models for the study of human disease (2nd Ed., pp. 379
417). Academic Press.

Methodologies for bioactivity assay: animal study

217

´ ., & Menjivar, M. (2017). Evaluation of the Mojica, L., Gonzalez de Mejia, E., Granados-Silvestre, M. A hypoglycemic potential of a black bean hydrolyzed protein isolate and its pure peptides using in silico, in vitro and in vivo approaches. Journal of Functional Foods, 31, 274
286. Monami, M., Nreu, B., Scatena, A., Cresci, B., Andreozzi, F., Sesti, G., & Mannucci, E. (2017). Safety issues with glucagon-like peptide-1 receptor agonists (pancreatitis, pancreatic cancer and cholelithiasis): Data from randomized controlled trials. Diabetes, Obesity and Metabolism, 19(9), 1233
1241. Mykkanen, O. T., Huotari, A., Herzig, K. H., Dunlop, T. W., Mykkanen, H., & Kirjavainen, P. V. (2014). Wild blueberries (Vaccinium myrtillus) alleviate inflammation and hypertension associated with developing obesity in mice fed with a high-fat diet. PLoS One, 9(12), e114790. Nakajima, S., Hira, T., Eto, Y., Asano, K., & Hara, H. (2010). Soybean β51
63 peptide stimulates cholecystokinin secretion via a calcium-sensing receptor in enteroendocrine STC-1 cells. Regulatory Peptides, 159(1-3), 148
155. Nakamura, T., Hirota, T., Mizushima, K., Ohki, K., Naito, Y., Yamamoto, N., & Yoshikawa, T. (2013). Milk-derived peptides, Val-Pro-Pro and Ile-Pro-Pro, attenuate atherosclerosis development in apolipoprotein e-deficient mice: A preliminary study. Journal of Medicinal Food, 16(5), 396
403. Nongonierma, A. B., & FitzGerald, R. J. (2016). Prospects for the management of type 2 diabetes using food protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Current Opinion in Food Science, 8, 19
24. Nongonierma, A. B., & FitzGerald, R. J. (2019). Features of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from dietary proteins. Journal of Food Biochemistry, 43(1), e12451. O’Halloran, F., Bruen, C., McGrath, B., Schellekens, H., Murray, B., Cryan, J. F., . . . Giblin, L. (2018). A casein hydrolysate increases GLP-1 secretion and reduces food intake. Food Chemistry, 252, 303
310. Okamoto, K., & Aoki, K. (1963). Development of a strain of spontaneously hypertensive rats. Japanese Circulation Journal, 27(3), 282
293. O’Keeffe, M. B., Norris, R., Alashi, M. A., Aluko, R. E., & FitzGerald, R. J. (2017). Peptide identification in a porcine gelatin prolyl endoproteinase hydrolysate with angiotensin converting enzyme (ACE) inhibitory and hypotensive activity. Journal of Functional Foods, 34, 77
88. Org, E., Mehrabian, M., Parks, B. W., Shipkova, P., Liu, X., Drake, T. A., & Lusis, A. J. (2016). Sex differences and hormonal effects on gut microbiota composition in mice. Gut Microbes, 7(4), 313
322. Oseguera-Toledo, M. E., de Mejia, E. G., & Amaya-Llano, S. L. (2015). Hard-to-cook bean (Phaseolus vulgaris L.) proteins hydrolyzed by alcalase and bromelain produced bioactive peptide fractions that inhibit targets of type-2 diabetes and oxidative stress. Food Research International, 76, 839
851. Otani, L., Ninomiya, T., Murakami, M., Osajima, K., Kato, H., & Murakami, T. (2009). Sardine peptide with angiotensin I-converting enzyme inhibitory activity improves glucose tolerance in stroke-prone spontaneously hypertensive rats. Bioscience, Biotechnology, and Biochemistry, 73(10), 2203
2209. Ou, Y., Ren, Z., Wang, J., & Yang, X. (2016). Phycocyanin ameliorates alloxan-induced diabetes mellitus in mice: Involved in insulin signaling pathway and GK expression. Chemico-Biological Interactions, 247, 49
54. Ouchi, N., Parker, J. L., Lugus, J. J., & Walsh, K. (2011). Adipokines in inflammation and metabolic disease. Nature Reviews. Immunology, 11(2), 85
97. Pacurari, M., Kafoury, R., Tchounwou, P. B., & Ndebele, K. (2014). The renin-angiotensin-aldosterone system in vascular inflammation and remodeling. International Journal of Inflammation, 2014, 689360. Pugliese, R., Bollati, C., Gelain, F., Arnoldi, A., & Lammi, C. (2019). A supramolecular approach to develop new soybean and lupin peptide nanogels with enhanced dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Journal of Agricultural and Food Chemistry, 67(13), 3615
3623. Rabelo, L. A., Nunes-Souza, V., & Bader, M. (2015). Chapter 22-Animal models with a genetic alteration of the ACE2/Ang-(1-7)/Mas axis. In T. Unger, U. M. Steckelings, & R. A. S. dos Santos (Eds.), The protective arm of the renin angiotensin system (RAS): Functional aspects and therapeutic implications (pp. 161
168). Academic Press.

218 Chapter 8 Reidelberger, R. D., Haver, A. C., Chelikani, P. K., & Buescher, J. L. (2008). Effects of different intermittent peptide YY (3-36) dosing strategies on food intake, body weight, and adiposity in diet-induced obese rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 295(2), R449
R458. Roncon-Albuquerque, R., Jr., Moreira-Rodrigues, M., Faria, B., Ferreira, A. P., Cerqueira, C., Lourenco, A. P., . . . Leite-Moreira, A. F. (2008). Attenuation of the cardiovascular and metabolic complications of obesity in CD14 knockout mice. Life Sciences, 83(13-14), 502
510. Saha, K., & Jaenisch, R. (2009). Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell, 5(6), 584
595. Salem, R. B. S. B., Ktari, N., Bkhairia, I., Nasri, R., Mora, L., Kallel, R., . . . Nasri, M. (2018). In vitro and in vivo anti-diabetic and anti-hyperlipidemic effects of protein hydrolysates from Octopus vulgaris in alloxanic rats. Food Research International, 106, 952
963. Sanchez-Rivera, L., Santos, P. F., Miralles, B., Carro´n, R., Jose´ Montero, M., & Recio, I. (2016). Peptide fragments from β-casein f (134
138), HLPLP, generated by the action of rat blood plasma peptidases show potent antihypertensive activity. Food Research International, 88, 348
353. Sangsawad, P., Choowongkomon, K., Kitts, D. D., Chen, X.-M., Li-Chan, E. C. Y., & Yongsawatdigul, J. (2018). Transepithelial transport and structural changes of chicken angiotensin I-converting enzyme (ACE) inhibitory peptides through Caco-2 cell monolayers. Journal of Functional Foods, 45, 401
408. Sayd, T., Dufour, C., Chambon, C., Buffie`re, C., Remond, D., & Sante´-Lhoutellier, V. (2018). Combined in vivo and in silico approaches for predicting the release of bioactive peptides from meat digestion. Food Chemistry, 249, 111
118. Shen, W., & Matsui, T. (2017). Current knowledge of intestinal absorption of bioactive peptides. Food and Function, 8(12), 4306
4314. Shi, W., Hou, T., Guo, D., & He, H. (2019). Evaluation of hypolipidemic peptide (Val-Phe-Val-Arg-Asn) virtual screened from chickpea peptides by pharmacophore model in high-fat diet-induced obese rat. Journal of Functional Foods, 54, 136
145. Shobako, N., Ogawa, Y., Ishikado, A., Harada, K., Kobayashi, E., Suido, H., . . . Ohinata, K. (2018). A novel antihypertensive peptide identified in thermolysin-digested rice bran. Molecular Nutrition and Food Research, 62(4), 1700732. Siddik, M. A. B., Chaklader, M. R., Foysal, M. J., Howieson, J., Fotedar, R., & Gupta, S. K. (2020). Influence of fish protein hydrolysate produced from industrial residues on antioxidant activity, cytokine expression and gut microbial communities in juvenile barramundi Lates calcarifer. Fish & Shellfish Immunology, 97, 465
473. Simecek, P., Churchill, G. A., Yang, H., Rowe, L. B., Herberg, L., Serreze, D. V., & Leiter, E. H. (2015). Genetic analysis of aubstrain divergence in non-obese diabetic (NOD) mice. G3: Genes, Genomes, Genetics, 5(5), 771
775. Singh, B. P., Vij, S., & Hati, S. (2014). Functional significance of bioactive peptides derived from soybean. Peptides, 54, 171
179. Song, J.-j, Gao, J., Du, M., & Mao, X.-y (2018). Casein glycomacropeptide hydrolysates ameliorate hepatic insulin resistance of C57BL/6J mice challenged with high-fat diet. Journal of Functional Foods, 45, 190
198. Stout, M. B., Justice, J. N., Nicklas, B. J., & Kirkland, J. L. (2017). Physiological aging: Links among adipose tissue dysfunction, diabetes, and frailty. Physiology, 32(1), 9
19. Su, M., Huang, W., & Zhu, B. (2016). Acetylshikonin from Zicao prevents obesity in rats on a high-fat diet by inhibiting lipid accumulation and inducing lipolysis. PLoS One, 11(1), e0146884. Sun, S., Zhang, G., Mu, H., Zhang, H., & Chen, Y. Q. (2019). The mixture of corn and wheat peptide prevent diabetes in NOD mice. Journal of Functional Foods, 56, 163
170. Sun, S., Zhang, H., Shan, K., Sun, T., Lin, M., Jia, L., & Chen, Y. Q. (2019). Effect of different cereal peptides on the development of type 1 diabetes is associated with their anti-inflammatory ability: In vitro and in vivo studies. Molecular Nutrition and Food Research, 63(11), e1800987. Sung, D., Ahn, K. M., Lim, S. Y., & Oh, S. (2014). Allergenicity of an enzymatic hydrolysate of soybean 2S protein. Journal of the Science of Food and Agriculture, 94(12), 2482
2487.

Methodologies for bioactivity assay: animal study

219

Sztuka, K., & Jasinska-Stroschein, M. (2017). Animal models of pulmonary arterial hypertension: A systematic review and meta-analysis of data from 6126 animals. Pharmacological Research, 125, 201
214. Tang, J. W. Y., Zhao, M., Zhong, H., Zheng, J., & Feng, F. (2020). Legume and soy intake and risk of type 2 diabetes: a systematic review and meta-analysis of prospective cohort studies. The American Journal of Clinical Nutrition, 111(3), 677
688. Tashiro, Y., Sato, K., Watanabe, T., Nohtomi, K., Terasaki, M., Nagashima, M., & Hirano, T. (2014). A glucagon-like peptide-1 analog liraglutide suppresses macrophage foam cell formation and atherosclerosis. Peptides, 54, 19
26. Tung, Y. T., Chen, H. L., Wu, H. S., Ho, M. H., Chong, K. Y., & Chen, C. M. (2018). Kefir peptides prevent hyperlipidemia and obesity in high-fat-diet-induced obese rats via lipid metabolism modulation. Molecular Nutrition and Food Research, 62(3), 1700505. Udenigwe, C. C., & Mohan, A. (2014). Mechanisms of food protein-derived antihypertensive peptides other than ACE inhibition. Journal of Functional Foods, 8, 45
52. Vij, R., Reddi, S., Kapila, S., & Kapila, R. (2016). Transepithelial transport of milk derived bioactive peptide VLPVPQK. Food Chemistry, 190, 681
688. Wang, C., Zheng, L., Su, G., Zeng, X.-A., Sun, B., & Zhao, M. (2020). Evaluation and exploration of potentially bioactive peptides in casein hydrolysates against liver oxidative damage in STZ/HFD-induced diabetic rats. Journal of Agricultural and Food Chemistry, 68(8), 2393
2405. Wang, L., Ding, L., Du, Z., Yu, Z., & Liu, J. (2019). Hydrolysis and transport of egg white-derived peptides in Caco-2 cell monolayers and everted rat sacs. Journal of Agricultural and Food Chemistry, 67(17), 4839
4848. Wassmann, S., Wassmann, K., & Nickenig, G. (2004). Modulation of oxidant and antioxidant enzyme expression and function in vascular cells. Hypertension, 44(4), 381
386. Wu, J., Liao, W., & Udenigwe, C. C. (2017). Revisiting the mechanisms of ACE inhibitory peptides from food proteins. Trends in Food Science and Technology, 69, 214
219. Xing, L., Zhang, H., Majumder, K., Zhang, W., & Mine, Y. (2019). Gamma-glutamylvaline prevents low-grade chronic inflammation via activation of a calcium-sensing receptor pathway in 3T3-L1mouse adipocytes. Journal of Agricultural and Food Chemistry, 67(30), 8361
8369. Xing, R., Li, S., Zhang, N., Shen, G., Mohwald, H., & Yan, X. (2017). Self-assembled injectable peptide yydrogels capable of triggering antitumor immune response. Biomacromolecules, 18(11), 3514
3523. Xu, F., Wang, L., Ju, X., Zhang, J., Yin, S., Shi, J., . . . Yuan, Q. (2017). Transepithelial transport of YWDHNNPQIR and its metabolic fate with cytoprotection against oxidative stress in human intestinal Caco-2 cells. Journal of Agricultural and Food Chemistry, 65(10), 2056
2065. Xu, F., Zhang, J., Wang, Z., Yao, Y., Atungulu, G. G., Ju, X., & Wang, L. (2018). Absorption and metabolism of peptide WDHHAPQLR derived from rapeseed protein and inhibition of HUVEC apoptosis under oxidative stress. Journal of Agricultural and Food Chemistry, 66(20), 5178
5189. Yang, L., Li, H., Yao, L., Yu, Y., & Ma, G. (2019). Amyloid-based injectable yydrogel derived from hydrolyzed hen egg white lysozyme. ACS Omega, 4(5), 8071
8080. Yu, H., Shang, L., Zeng, X., Li, N., Liu, H., Cai, S., . . . Qiao, S. (2018). Risks related to high-dosage recombinant antimicrobial peptide Microcin J25 in mice model: Intestinal microbiota, intestinal barrier function, and immune regulation. Journal of Agricultural and Food Chemistry, 66(43), 11301
11310. Yu, Z., Yin, Y., Zhao, W., Chen, F., & Liu, J. (2014). Antihypertensive effect of angiotensin-converting enzyme inhibitory peptide RVPSL on spontaneously hypertensive rats by regulating gene expression of the reninangiotensin system. Journal of Agricultural and Food Chemistry, 62(4), 912
917. Yu, Z., Zhao, W., Ding, L., Wang, Y., Chen, F., & Liu, J. (2017). Short- and long-term antihypertensive effect of egg protein-derived peptide QIGLF. Journal of the Science of Food and Agriculture, 97(2), 551
555. Yuan, Q., Zhan, B., Chang, R., Du, M., & Mao, X. (2020). Antidiabetic effect of casein glycomacropeptide hydrolysates on high-fat diet and STZ-induced diabetic mice via regulating insulin signaling in skeletal muscle and modulating gut microbiota. Nutrients, 12(1), 220. Zarei, M., Forghani, B., Ebrahimpour, A., Abdul-Hamid, A., Anwar, F., & Saari, N. (2015). In vitro and in vivo antihypertensive activity of palm kernel cake protein hydrolysates: Sequencing and characterization of potent bioactive peptides. Industrial Crops and Products, 76, 112
120.

220 Chapter 8 Zhang, A. H., Sun, H., Han, Y., Yan, G. L., Yuan, Y., Song, G. C., . . . Wang, X. J. (2013). Ultraperformance liquid chromatography-mass spectrometry based comprehensive metabolomics combined with pattern recognition and network analysis methods for characterization of metabolites and metabolic pathways from biological data sets. Analytical Chemistry, 85(15), 7606
7612. Zhang, L. (2011). Voluntary oral administration of drugs in mice. Protocol Exchange, 10. Zhao, Q., Xu, H., Hong, S., Song, N., Xie, J., Yan, Z., . . . Jiang, X. (2019). Rapeseed protein-derived antioxidant peptide RAP ameliorates nonalcoholic steatohepatitis and related metabolic disorders in mice. Molecular Pharmaceutics, 16(1), 371
381.

CHAPTER 9

Methodologies for bioavailability assessment of food-derived peptide Kenji Sato Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

9.1 Introduction It has been demonstrated that oral administration of peptides in food protein hydrolyzates has beneficial effects on human health beyond their conventional nutritional value as an amino acid source. Yoshikawa and his coworkers first developed this concept in the 1990s (Yokoyama, Chiba, & Yoshikawa, 1992) and it has been confirmed not only in animal experiments but also in placebo-controlled human trials as shown in different chapters in this book. However, peptides are degraded by proteases, including endoproteinases and exopeptidases. Endoproteinases such as pepsin, trypsin, and chymotrypsin are secreted into the gastrointestinal tract; in addition, exopeptidases are present in pancreatic juice, the apical surface, and cytosol of enterocytes, as well as in blood (Bai, 1994; Hedemann, Gabert, & Larsen, 1998; Mutoh et al., 2005). The majority of oligopeptides, including dipeptides, in enzymatic hydrolyzates are degraded by in vitro digestion with exopeptidases (carboxypeptidase A and leucine aminopeptidase) (Chen et al., 2019). To exert beneficial activity beyond the amino acid source, at least, some peptides in the enzymatic hydrolyzate must survive exopeptidase digestion and reach the target organ. However, it is difficult to detect food-derived peptides in the body via a single high-performance liquid chromatography (HPLC) using a nonspecific detector, such as an ultraviolet spectrophotometer, due to the presence of so many endogenous and food-derived compounds in the body as shown in Fig. 9.1A. To detect food-derived peptides in human and animal blood and tissue extracts, two-dimensional HPLC using the column-switching technique has been employed (Matsui, Tanaka, Kawasaki, & Osajima, 1999; Matsui, Tamaya, Osajima, Matsumoto, & Kawasaki, 2002; Matsui et al., 2004). Recently, LC
electron spray ionization tandem mass spectrometry (LC
ESI
MS/MS) in multireaction monitoring (MRM) mode has been frequently used to detect food-derived peptides in the body after ingestion of food protein hydrolyzates (Foltz et al., 2007; Ichikawa et al., 2010; Nakashima et al., 2011; Taga, Kusubata, Ogawa-Goto, & Hattori,

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00018-2 © 2021 Elsevier Inc. All rights reserved.

221

222 Chapter 9

Figure 9.1 Chromatograms by direct injection of deproteinized fraction of human blood plasma (A) and collagen peptide fraction (B) to reversed-phase HPLC. Peptides in collagen hydrolyzate (C, upper) and peptides in human blood 1 h after ingestion of collagen hydrolyzate (C, lower) were fractionated by size exclusion chromatography. Elution of peptides was monitored by absorbance at 214 nm (A and B) or amino acid analysis after HCl hydrolysis (C). Contents of total amino acids and Hyp were expressed by bar and solid line, respectively. Collagen peptide fraction as indicated with bar (C) was collected and injected to reversed-phase HPLC (B). HPLC, High-performance liquid chromatography; Hyp, hydroxyproline. Source: This figure was adapted with permission from Iwai, K., Hasegawa, T., Taguchi, Y., Morimatsu, F., Sato, K., Nakamura, Y., . . . Ohtsuki, K. (2005). Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates. Journal of Agricultural and Food Chemistry, 53, 6531
6536.

2014; Taga, Iwasaki, Shigemura, & Mizuno, 2019). LC
MS/MS in MRM mode provides highly specific detection of target peptides in complex matrices. In some studies, peptides have been derivatized with 2,4,6-trinitrobenzensulfonate (Hanh et al., 2017) or 6-amino quinolyl-N-hydroxysuccinimidyl carbamate (AccQ) (Asai et al., 2019) before LC
MS/MS analysis, to improve resolution and sensitivity. To adjust the interference of other compounds with the ionization of target peptides, stable isotope-labeled inner standards have been used for LC
MS/MS (Hanh et al., 2017; Nakashima et al., 2011; Taga et al., 2014, 2019). For the detection of relatively large peptides, such as lunasin (5.1 kDa), in human blood, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Methodologies for bioavailability assessment of food-derived peptide 223 (MALDI-TOF MS) has been used (Dia, Torres, De Lumen, Erdman, & De Mejia, 2009). Recently, food-derived peptides in the slice of tissues have been directly detected via mass imaging techniques that utilize MALDI-TOF MS (Hong, Tanaka, Yoshii, Mine, & Matsui, 2013). These techniques require structural information about the target peptides and/or standard peptides for analysis; in other words, the target peptide must be isolated and identified before detection and quantification in the body. For the last two decades, target peptides have been identified from food protein hydrolyzates and fermented foods using in vitro activity-guided fractionation. Lactotripeptides such as Val-Pro-Pro and Ile-Pro-Pro are good examples (Nakamura, Yamamoto, Sakai, Okubo et al., 1995); these peptides were identified via in vitro activity-guided fractionation using an angiotensin-converting enzyme inhibitory assay and have been assumed to be responsible for the attenuation of mild hypertension caused by oral administration of fermented cow milk. However, the levels of these peptides in human blood were very low (approximately 1 nM) (Foltz et al., 2007), and far less than the dose used in in vitro assays to identify them (Nakamura et al., 1995). Table 9.1 (Group A) Table 9.1: Maximum blood levels of food-derived peptides after ingestion of food protein hydrolyzate and a fermented food. Target peptide

Maximum content

Object

Test sample

Dose

Reference

Val-Tyr

Human

Sardine meat hydrolyzate

Human

Yogurt

1 nM 2 nM 1 nM

Matsui et al. (2002)

Ile-Prp-Pro

Beverage containing 6 and 12 mg of Val-Tyr 250 mL containing 20.4 mg of Ile-ProPro

Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human

Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate Collagen hydrolyzate

25 g 25 g 25 g 25 g 25 g 25 g 25 g 25 g 25 g 25 g 25 g 25 g 13 g 13 g 13 g 13 g 13 g 13 g 13 g 13 g

50 μM 12 μM 8 μM 7 μM 6 μM 6 μM 6 μM 5 μM 4 μM 3 μM 1 μM 1 μM 27 μM 3 μM 3 μM 3 μM 2 μM 2 μM 2 μM 1 μM

Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Taga et al. (2014) Asai et al. (2019) Asai et al. (2019) Asai et al. (2019) Asai et al. (2019) Asai et al. (2019) Asai et al. (2019) Asai et al. (2019) Asai et al. (2019)

Group A

Foltz et al. (2007)

Group B Pro-Hyp Ala-Hyp Hyp-Gly Leu-Hyp Phe-Hyp Glu-Hyp Ser-Hyp Ser-Hyp-Gly Ala-Hyp-Gly Glu-Hyp-Gly Gly-Pro-Hyp Pro-Hyp-Gly Pro-Hyp Hyp-Gly Ala-Hyp-Gly Ala-Hyp Pro-Hyp-Gly Leu-Hyp Phe-Hyp Ser-Hyp-Gly

(Continued)

224 Chapter 9 Table 9.1: (Continued) Maximum content

Reference

13 g 10 g/60 kg body weight

1 μM 18 μM

Asai et al. (2019) Shigemura et al. (2012)

9g 9g 9g 9g 9g 9g 9g 9g 9g 167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight

100 nM 8 nM 30 nM 30 nM 110 nM 10 nM 40 nM 60 nM 30 nM 25 nM

Ejima Ejima Ejima Ejima Ejima Ejima Ejima Ejima Ejima Ejima

10 nM

Ejima et al. (2019)

23 nM

Ejima et al. (2019)

167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight 167 mg/kg body weight

8 nM

Ejima et al. (2019)

3 nM

Ejima et al. (2019)

28 nM

Ejima et al. (2019)

14 nM

Ejima et al. (2019)

28 nM

Ejima et al. (2019)

34 nM

Ejima et al. (2019)

25 nM

Ejima et al. (2019)

38 nM

Ejima et al. (2019)

Target peptide

Object

Test sample

Dose

Gly-Pro-Hyp Pro-Gly

Human Human

Collagen hydrolyzate Elastin hydrolyzate

Human Human Human Human Human Human Human Human Human Rat

Corn gluten hydrolyzate Corn gluten hydrolyzate Corn gluten hydrolyzate Corn gluten hydrolyzate Wheat gluten hydrolyzate Wheat gluten hydrolyzate Wheat gluten hydrolyzate Wheat gluten hydrolyzate Wheat gluten hydrolyzate Porcine liver hydrolyzate

α-D-Asp-Val

Rat

Porcine liver hydrolyzate

β-D-Asp-Val

Rat

Porcine liver hydrolyzate

β-L-Asp-Phe

Rat

Porcine liver hydrolyzate

α-D-Asp-Phe

Rat

Porcine liver hydrolyzate

β-D-Asp-Phe

Rat

Porcine liver hydrolyzate

β-L-ASP-Ile

Rat

Porcine liver hydrolyzate

α-D-Asp-Ile/Leu

Rat

Porcine liver hydrolyzate

β-D-Asp-Ile

Rat

Porcine liver hydrolyzate

β-L-Asp-Leu

Rat

Porcine liver hydrolyzate

β-D-Asp-Leu

Rat

Porcine liver hydrolyzate

Group C pyroGlu-Pro pyroGlu-Leu-Pro pyroGlu-Gln-Pro Pro-Ala pyroGlu-Pro pyroGlu-Leu-Pro pyroGlu-Gln-Pro Pro-Gly Pro-Gln β-L-Asp-Val

et al. et al. et al. et al. et al. et al. et al. et al. et al. et al.

(2018) (2018) (2018) (2018) (2018) (2018) (2018) (2018) (2018) (2019)

summarizes the maximum levels of in vitro bioactive peptides in plasma after ingestion of fermented foods and food protein hydrolyzate. In all cases, the levels of in vitro bioactive peptides in plasma were very low (approximately 1
2 nM). On the other hand, exopeptidase-resistant peptides such as Gly-Sar (glycyl-sarcosine), Gly-Sar-Sar, Gly-Sar-Sar-Sar, and Gly-Sar-Sar-Sar-Sar showed relatively high levels in blood plasma (approximately 1
10 μM) after oral administration of synthetic peptides at a dose of 10 mg/kg body weight (Hanh et al., 2017); however, these sarcosyl peptides are not present in food protein hydrolyzates, and thus it has been assumed that blood contents of food-derived peptides in blood are too small to exert biological activity (Foltz et al., 2007). On the other hand, the presence of unexpectedly higher levels of food-derived

Methodologies for bioavailability assessment of food-derived peptide 225 collagen peptides (up to 100 μM) in human blood plasma has been reported (Iwai et al., 2005; Ohara, Matsumoto, Ito, Iwai, & Sato, 2007; Shigemura, Kubomura, Sato, & Sato, 2014). These facts indicate two important points: first, peptides in foods are generally degraded during in vivo digestion; second, nonnegligible amounts of food-derived peptides are present in the body, despite the very low levels of food-derived peptides in blood, as reported previously. Therefore identification of food-derived peptides in the body rather than that of peptides in foods is crucial to understand mechanisms of bioactivities of peptides in food. However, there is limited information about food-derived peptides in the body, except for food-derived collagen peptides. In this chapter, recent advances in methodologies for identifying food-derived peptides in the body are introduced. Finally, perspective metabolism of food-derived peptides in the digestive tract is discussed.

9.2 Structure of peptides in foods Naturally occurring peptides such as glutathione and imidazole peptides (carnosine and anserine) are present in foods. However, peptides for food ingredients are usually produced by partial hydrolysis of food proteins by using acid/alkaline treatments, high-temperature treatment, enzymatic digestion, and fermentation. In the fermentation process, enzymes derived from microorganisms are used for the production of peptides. Enzymatic hydrolysis is most often used for the preparation of food-grade peptides. Food-grade protease is prepared from animals, plants, fungi, and bacteria; fungal and bacterial proteases are preferentially used for this purpose because of high stability at 50 C
60 C, as the growth of contaminated microorganisms can generally be suppressed at this temperature. Bacterial proteases have a strong endoproteinase activity, which cleaves proteins into peptides with molecular weights less than 10 kDa. Fungal proteases have endoproteinase and exopeptidase activity, which releases amino acids or dipeptides from peptides (Sumantha, Larroche, & Pandey, 2006). Thus the fungal protease digests contain higher free amino acid levels and smaller peptides than bacterial protease digests. To inactivate proteases, heat treatment at temperature higher than 80 C is frequently used. During heat treatment, peptides with glutaminyl residues at the amino terminus can be changed into pyroglutamyl peptides, which can resist aminopeptidase digestion except for pyroglutamate aminopeptidases (Sato et al., 1998; Suzuki, Motoi, & Sato, 1999). In addition to pyroglutamyl peptides, the generation of some modified peptides during protease digestion has been reported. Ejima, Yamada, and Sato (2019) demonstrated that aspartyl dipeptides with D-aspartyl residues and/or β-peptide bonds are present in a porcine liver protein hydrolyzate. Racemization and isomerization of aspartyl residues in proteins, via reaction of the β-carboxyl group of the aspartyl residue to the peptide bond, have been reported (Fujii, Takata, Fujii, Aki, & Sakaue, 2018; Geiger & Clarke, 1987). Such a reaction may occur during the preparation of the enzymatic hydrolyzate. Taga, Kusubata, Ogawa-Goto, and Hattori (2016) reported that cyclic prolyl dipeptides, referred to as diketopiperazines, are generated during the digestion of gelatin with ginger rhizome protease. It has been

226 Chapter 9 mentioned in the literature that bioactive peptides are encrypted in the structure of the parent proteins (Daliri, Oh, & Lee, 2017; Mada, Ugwu, & Abarshi, 2020; Sa´nchez & Va´zquez, 2017); however, some peptides are not just fragments of the parent protein but have chemical and enzymatic modifications.

9.3 Presence of food-derived peptides with modified amino acid residues in blood As mentioned in Section 9.1, the bioavailability of in vitro active peptides, which can be identified using in vitro activity-guided fraction in food protein hydrolyzates, is very low (Table 9.1 Group A). Thus the following concept has been prevalent among some researchers. Most peptides in foods are degraded into amino acids during the digestion process. Some oligopeptides can be absorbed into enterocytes through peptide transporter 1 (PepT1) but are rapidly degraded into amino acids in enterocytes and blood (Foltz et al., 2007; Freeman, 2015; Vermeirssen, Van Camp, & Verstraete, 2004). If this concept is correct, then peptides in food can supply only amino acids, which is inconsistent with the vast evidence for the beneficial activities of oral administration of food protein hydrolyzates. The above concept of digestion and absorption of peptides in foods has been changed due to the detection of food-derived collagen peptides in human blood (Iwai et al., 2005; Ohara et al., 2007). Collagen consists of posttranslationally modified amino acids such as hydroxyproline (Hyp), hydroxylysine (Hyl), and other minor amino acids. Among them, Hyp accounts for approximately 10% of total amino acids in the molar ratio of collagen (Van der Rest & Garrone, 1991). It is specifically distributed in collagen, with rare exceptions. Thus Hyp containing peptide can be considered to be derived from collagen. Peptide-form Hyp can be estimated by subtracting free Hyp from total Hyp in the HCl hydrolyzate of the deproteinized fraction of plasma. After ingestion of collagen hydrolyzate, 30%
50% of Hyp was present in peptide form in human blood plasma (Iwai et al., 2005; Ohara et al., 2007). Peptide-form Hyp, namely, collagen peptide, increases in human blood plasma in a dose-dependent manner and reaches approximately 100 μM, which is much higher than the previously reported levels of food-derived peptides in blood. In some cases, significantly higher levels of collagen peptides compared to initial levels were observed 7 hours after the ingestion (Ohara et al., 2007). These observations clearly demonstrate that considerable amounts of food-derived collagen peptides are absorbed into the blood circulation and remain there for a few hours. As mentioned earlier, pyroglutamyl peptides are generated in the enzymatic hydrolyzate of food proteins after heat treatment (Sato et al., 1998; Suzuki et al., 1999). In some cases, this accounts for more than 10% of the total peptide (Suzuki et al., 1999). The pyroglutamyl residue can be specifically released by pyroglutamate aminopeptidase digestion; thus the pyroglutamyl peptide level can be estimated by determining the amounts of pyroglutamic

Methodologies for bioavailability assessment of food-derived peptide 227 acid released by pyroglutamate aminopeptidase digestion. Using this technique, Higaki-Sato et al. (2006) demonstrated that some pyroglutamyl peptides are absorbed into rat portal blood after ingestion of a wheat gluten hydrolyzate. To detect the presence of food-derived peptides after ingestion of soy protein hydrolyzate and other protein hydrolyzates, peptide levels in the deproteinized fraction of plasma were determined in a preliminary experiment conducted by us. However, it was difficult to distinguish the food-derived peptides and endogenous peptides in deproteinized fraction of blood due to the absence of marker amino acid for food-derived peptides such as Hyp and pyroGlu.

9.4 Direct identification of food-derived peptides in the body Food-derived collagen peptides in human blood have been resolved and identified by HPLC and LC
MS/MS. Early studies using reversed-phase HPLC with a nonspecific detector, such as an ultraviolet spectrophotometer, reported presence of casein-derived peptides in human blood at relatively high levels after ingestion of milk and yogurt (Chabance et al., 1998). However, these casein-derived peptides have not been detected by LC
MS/MS. As shown in Fig. 9.1A, direct injection of the deproteinized fraction of human plasma into reversed-phase HPLC generated too many peaks to separate food-derived peptides from other blood components (Aito-Inoue et al., 2006); thus the oligo-peptide fraction was prepared via size exclusion chromatography (SEC) before reversed-phase HPLC analysis (Fig. 9.1C). As shown in Fig. 9.1B, the resolution of peptides in the oligo-peptide fraction was still unsatisfactory due to the weak retention of hydrophilic peptides in the reversed-phase column, while the peak number decreased after prefractionation via SEC (Iwai et al., 2005). To improve the resolution of the hydrophilic peptides, precolumn derivatization techniques have been used. Peptides in the SEC fractions have been derivatized with phenyl isothiocyanate (Aito-Inoue et al., 2006; Shigemura et al., 2011). The resultant phenyl thiocarbamyl (PTC)-peptides have been resolved by reversed-phase HPLC. As shown in Fig. 9.2, excellent separation of PTC-peptides (Pro-Hyp and Hyp-Gly) and PTC-amino acids was obtained (Shigemura et al., 2011). Peptide sequencers based on Edman degradation can sequence the PTC-peptide, as the PTC-peptide is an intermediate product of Edman degradation. Collagen dipeptides, tripeptides, and elastin dipeptides have been identified in human blood after ingestion of collagen and elastin hydrolyzate using this technique (Shigemura et al., 2011, 2012; Shigemura, Suzuki, Kurokawa, Sato, & Sato, 2018), while these dipeptides and tripeptides are not contained in the collagen hydrolyzate as shown in Fig. 9.1C. Thus large peptides in collagen hydrolyzate are degraded into dipeptides and tripeptides during the digestion process and can then be absorbed into the blood circulation system.

228 Chapter 9

Std.

SEC Fr.35 Fr.36 Fr. 37 Fr. 38

Before After Before After Before After Before After

a

b c

Figure 9.2 Elution patterns of PTC-derivatives from reversed-phase HPLC. Deproteinized fraction of human plasma before and 1 h after ingestion of collagen hydrolyzate were fractionated by SEC. Aliquots of SEC fractions were derivatized with phenyl isothiocyanate and resultant PTC-derivatives were resolved by reversed-phase HPLC with detection by absorption at 254 nm. Std. represents amino acid mixture. Peak a, b, and c were identified as Pro-Hyp, Hyp-Gly, and free Hyp. HPLC, Highperformance liquid chromatography; Hyp, hydroxyproline; PTC, phenyl thiocarbamyl; SEC, size exclusion chromatography. Source: This figure was adapted with permission from Shigemura, Y., Akaba, S., Kawashima, E., Park E.-Y. Nakamura, Y., & Sato, K. (2011). Identification of a novel food-derived collagen peptide, hydroxyprolyl-glycine, in human peripheral blood by pre-column derivatisation with phenyl isothiocyanate. Food Chemistry, 129, 1019
1024.

To improve the specificity and detection limit, mass spectrometers have been used to detect peptide derivatives. However, PTC-peptides showed weak ionization in acidic solvents used for reversed-phase HPLC, such as 0.1% formic acid containing acetonitrile (from our preliminary experiment). Alternatively, other derivatization reagents, such AccQ, have been used for derivatization of peptides for LC
MS and LC
MS/MS analyses. The AccQ-peptide can be detected by ESI
MS. In addition, collision of an AccQ-peptide with argon gas generates an AccQ b1 ion with a mass-to-charge ratio (m/z) of 171. AccQ derivatives can be detected as precursor ions, which generate product ions having an m/z of 171 by precursor ion scan mode using quadrupole MS/MS. Thus compounds with primary and secondary amines can be specifically detected using this method. As shown in Fig. 9.3 (upper), some AccQ derivatives were detected in human blood after ingestion of collagen hydrolyzate by the precursor ion scans at narrow scan range. The peptide structure can be obtained through product ion scan of the AccQ-peptide. The peak with an m/z of 399.1 was identified as AccQ-Pro-Hyp on the basis of product ions as shown in Fig. 9.3 (lower). Other two peaks with m/z of 406.1 and 390.1 did not generate product ions from peptides.

Methodologies for bioavailability assessment of food-derived peptide 229

Figure 9.3 LC
MS/MS analysis of 6-amino quinolyl-N-hydroxysuccinimidyl carbamate (AccQ) derivatives. Deproteinized fraction of human blood was derivatized with AccQ. The derivatives were resolved by reversed-phase HPLC and detected by MS/MS in precursor ion scan mode targeting b1 ion of AccQ (m/z of 171.1) at scan rage between m/z of 380 and 410. The precursor ion (m/z of 399.1) was subjected to product ion scan using different collision energies. Immonium ion of Pro (P*), y1 ion of Hyp and y2 ion of Pro-Hyp were observed. On the basis of these data, the peak with m/z 399.1 can be identified as AccQ-Pro-Hyp. Peaks with m/z of 406.1 and 390.1 did not generate product ions for peptide. HPLC, High-performance liquid chromatography; LC, liquid chromatography; MS/MS, tandem mass spectrometric. Source: Nonpublished data.

Food-derived peptides in human blood higher than 1 μM levels can be detected using this technique. However, food-derived peptides after ingestion of noncollagen protein hydrolyzate have generally been less than 1 μM level. It has been difficult to directly detect and identify the food-derived peptides in the body at such levels by the above methods. In such cases, the food-derived peptides in the body have been identified by the approach as described in the following session. Food-derived peptides directly identified in human blood have been quantified using LC
MS/MS in MRM mode. The maximum levels of the food-derived peptides in human blood are approximately 1
50 μM as summarized in Table 9.1 Group B; they are extensively higher than the maximum levels of the in vitro active peptide in blood (Group A).

9.5 Detection of exopeptidase-resistant peptides in blood Ingestion of noncollagen protein hydrolyzates, such as plant, milk, fish, meat, and egg protein hydrolyzates, has been demonstrated to exert beneficial activities. Thus some specific peptides may reach target organs in the body. However, there is limited information about food-derived peptides in the body after ingestion of noncollagen

230 Chapter 9 protein hydrolyzates. Some peptides in food have been suggested to target cells in the digestive tract to exert biological activities such as regulation of absorption of nutrients (Shimizu, 2004). Even in such cases, peptides may suffer from protease digestion. Thus peptides reaching target organs may be resistant to protease digestion. It has been demonstrated that some peptides in food protein hydrolyzates resist pepsin and trypsin digestion (Fan, Wang, Liao, Jiang, & Wu, 2019; Gu & Wu, 2013). These peptides are thought to be stable in the gastrointestinal tract; however, Chen et al. showed that the majority of peptides in corn gluten hydrolyzate resisted pepsin and trypsin digestion (Fig. 9.4 upper) but were degraded by exopeptidases (carboxylpeptidase A and leucine aminopeptidase), and only prolyl and pyroglutamyl-dipeptides and tripeptides remained after exopeptidase digestion (Fig. 9.4 lower) (Chen et al., 2019). In addition to these peptides, Ejima et al. (2019) found α-D-aspratyl dipeptides and β-D/L-asparatyl isopeptides in porcine liver protein hydrolyzate (Fig. 9.5). LC
MS/MS in MRM mode revealed that some of these peptides significantly increased in human and animal blood after ingestion of protein hydrolyzates (Ejima, Nakamura, Suzuki, & Sato, 2018; Ejima et al., 2019), as summarized in Table 9.1 Group C. Surprisingly, all peptides have prolyl, pyroglutamyl, and

150 100 50

pEQ LP+IP pEQP pEP pEL pELP pEPQ VQ LQ pELLP AL LL VL ILLP pEQQ pEQQQ AV LFP IFP VLP NQL LLP IIGGA VANP

0

Exopeptidase digestion 4.0 3.0

Non digest Digested for 8 h

2.0 1.0 0.0

pEQ LP pE… pEP IP pEL pELP pE… VQ LQ pE… AL LL VL ILLP pE… pE… AV LPF IFP VLP NQL LLP II… V…

Content (%) in hydrolysate

% of non digest

Endoproteinase digestion

Non digest Pepsin digest Trypsin digest

Figure 9.4 Susceptibility of peptides in a corn gluten hydrolyzate to pepsin and trypsin (upper) and exopeptidases; mixture of leucine aminopeptidase and carboxypeptidase A (lower). One letter abbreviation for amino acid residue is used. pE represents pyroglutamyl residue. Source: This figure was adapted with permission from Chen, L., Ejima, A., Gu, R.Z., Lu, J., Cai, M., & Sato, K. (2019). Presence of exopeptidase resistant and susceptible peptides in a bacterial protease digest of corn gluten. Journal of Agricultural and Food Chemistry, 67, 11948
11954.

Methodologies for bioavailability assessment of food-derived peptide 231

Figure 9.5 Presence of α-D-asparatyl and β-L/D-asparatyl peptides in porcine liver hydrolyzate. Isoforms of Asp-Val, Asp-Ile, Asp-Leu, and Asp-Phe in standard (std.) and porcine liver hydrolyzate (digest) were resolved by LC
MS/MS in multireaction monitoring mode. LC, Liquid chromatography; MS/MS, tandem mass spectrometric. Source: This figure was adapted with permission from Ejima, A., Yamada, K., & Sato, K. (2019). Presence of isomerized aspartic dipeptides in a porcine liver protein hydrolysate and their bioavailability upon ingestion. Bioactive Compounds in Health and Disease, 2, 155
169.

aspartyl residues at the amino terminus. The maximum levels of these peptides (3
110 nM) were smaller than those of the directly identified collagen and elastin peptides in blood (Table 9.1 Group B) but higher than those of the in vitro active peptides (Table 9.1 Group A). Thus exopeptidase-resistant peptides are good candidates for food-derived peptides in the body. On the other hand, dipeptides with prolyl residue at the carboxy terminal did not increase after ingestion of corn and wheat gluten hydrolyzates, while these peptides also resisted in vitro exopeptidase digestion (Ejima et al., 2018). In human and animal blood, nonnegligible amounts of the dipeptides with prolyl residues at the carboxy terminal (such as Gly-Pro) were present, indicating that these peptides also resist in vivo exopeptidase digestion (Ejima et al., 2018). The reason why these peptides do not increase in the blood after ingestion but are present in blood remains to be elucidated. One possible explanation is that these peptides might be modified at the amino terminal or carboxy terminal during the absorption process.

9.6 Peptides pass through Caco-2 monolayer Caco-2 is a human colon carcinoma cell line. Caco-2 cells in a confluent monolayer differentiate into enterocyte-like cells. Caco-2 monolayers have been used as a model of intestinal epithelial monolayers. The permeability of peptides through Caco-2 monolayers has been examined extensively over the last two decades (Xu, Hong, Wu, & Yan, 2019; Xu, Yan, Zhang, & Wu, 2019). It has been considered that peptides pass through Caco-2 monolayers via three possible pathways: PepT1-mediated permeation, paracellular transportation, and transcytosis. Dipeptides and tripeptides are believed to pass through PepT1 expressed on the apical side. However, peptide transporters on the basal side of the monolayer have not been identified. Tetra peptides and lager peptides have been assumed to

232 Chapter 9 pass via the paracellular route and by transcytosis. In fact, larger peptides such as lunasin (Dia et al., 2009), Gly-Sar-Sar-Sar, and Gly-Sar-Sar-Sar-Sar (Hanh et al., 2017) were detected in blood after oral administration, while these peptides could not pass through PepT1. Therefore peptides larger than tripeptides might pass through the intestinal epithelial monolayer via the paracellular route or by transcytosis. Many peptides larger than tripeptides have been demonstrated to pass through Caco-2 monolayers and are supposed to be absorbed into the blood circulation and exert biological activities, such as antioxidant, antihypertensive, antiinflammatory, antiatherosclerotic, and dipeptidyl peptidase-4 inhibitory activities (Xu, Hong et al., 2019; Xu, Yan et al., 2019). Peptides that can pass through Caco-2 monolayers are candidates for peptides that can be absorbed into the body. However, there is very limited evidence for the presence of food-derived peptides larger than tripeptides in the body. For example, collagen-derived octapeptide, Gly-Ala-Hyp-GlyLeu-Hyp-Gly-Pro, has been demonstrated to pass through Caco-2 cell monolayers (Shimizu et al., 2010), but the presence of food-derived collagen peptides larger than tripeptides has not been detected in human blood after the ingestion of collagen hydrolyzate, as shown in Fig. 9.1C. These facts suggest that collagen octapeptide might be able to pass through the intestinal epithelial monolayer via the paracellular route but might be degraded before or after passing through the intestinal epithelial layer by exopeptidases. Therefore the presence of these peptides in the body should be confirmed using LC
MS/MS in MRM mode.

9.7 Biological activity of food-derived peptides in body Some food-derived peptides found in the body as summarized in Table 9.1 (Groups B and C) have been demonstrated to exert significant biological activities. Among collagen peptides, Pro-Hyp (Asai et al., 2020; Shigemura et al., 2009) and Hyp-Gly (Shigemura et al., 2011) can trigger the growth of fibroblasts cultured on collagen gel. Recently, ProHyp has been demonstrated to be specifically incorporated into fibroblasts with mesenchymal stem cell marker (low-affinity nerve growth factor receptor or p75 neurotrophin receptor) and initiate its growth (Asai et al., 2020). The fibroblasts expressing p75 neurotrophin infiltrate the wound area and form granulation tissue. Thus Pro-Hyp has been associated with the enhancement of wound healing of pressure ulcers by ingestion of collagen hydrolyzate. Furthermore, Pro-Hyp increases the production of hyaluronic acid (Ohara et al., 2010) and glycosaminoglycans (Nakatani, Mano, Sampei, Shimizu, & Wada, 2009) by fibroblasts and chondrocytes, respectively. As mentioned earlier, Pro-Hyp is generated by the limited degradation of larger peptides in the collagen hydrolyzate during the digestion and absorption process and absorbed into the blood. Thus identification of food-derived peptides in the body, rather than peptides in foods, is important to understand the mechanism of the ingestion of food protein hydrolyzates. It is worth noting that ProHyp and Hyp-Gly exert significant activities in in vitro assays at similar concentrations in

Methodologies for bioavailability assessment of food-derived peptide 233 blood after the ingestion of collagen hydrolyzate, which suggests that these peptides account for the biological response associated with the ingestion of collagen hydrolyzate. Pro-Gly was also identified in human blood after the ingestion of elastin (Shigemura et al., 2012) and corn gluten hydrolyzates (Ejima et al., 2018). It has been found that Pro-Gly increases elastin production by cultured fibroblasts (Shigemura et al., 2012) and also protects artery endothelial cells of spontaneously hypertensive rats (Takemori, Yamamoto, Ito, & Kometani, 2015), which have been associated with the improvement of skin elasticity and blood flow due to ingestion of elastin hydrolyzate. In addition to the peptides as listed in Table 9.1, pyroglutamyl-leucine (pyroGlu-Leu) increased in the rat blood (Sato et al., 2013) and mouse intestine (Wada et al., 2013) after oral administration of synthetic pyroGlu-Leu. PyroGlu-Leu was demonstrated to resist the activities of endoproteinases and exopeptidases (Chen et al., 2019; Ejima et al., 2018). It has been found that pyroGlu-Leu attenuates D-galactosamine-induced hepatitis in rat (Sato et al., 2013). In addition, it attenuates colitis and high-fat diet-induced disturbance of colon bacteria flora, referred to as dysbiosis, in mice (Wada et al., 2013) and rats (Shirako et al., 2019), respectively, by increasing the host antimicrobial peptide upon oral administration at 0.1
1.0 mg/kg body weight; moreover, it can be administered via consumption of wheat and corn gluten hydrolyzates, which contain pyroGlu-Leu at approximately 5
7 mg/g (Chen et al., 2019). The biological activities of other pyroglutamyl and prolyl peptides have not been fully examined.

9.8 Conclusion and future prospects Ingestion of peptides in food protein hydrolyzates exerts beneficial biological activities. Food-grade heat-stable bacterial proteases with strong endoproteinase activity and low exopeptidase activity (Sumantha et al., 2006) are frequently used for the preparation of food protein hydrolyzates. Numerous peptides have been identified in food protein hydrolyzates via in vitro activity-guided fractionation; however, a large proportion of the peptides in food protein hydrolyzates is degraded by in vitro digestion with exopeptidases present in the gastrointestinal tract and tissues. On the other hand, some peptides with specific structures can resist in vitro digestion from endoproteinases and exopeptidases and are absorbed into the body (Chen et al., 2019; Ejima et al., 2018). Recent advances in LC
MS and precolumn derivatization techniques have allowed the direct detection of food-derived peptides in blood and tissue extracts. Food-derived collagen and elastin peptides, such as Pro-Hyp and Pro-Gly, have been demonstrated to be present at unexpectedly high levels (approximately 1
50 μM) in human blood after ingestion of 2
10 g of the hydrolyzates. Recently, it has been demonstrated that part of the food-derived collagen peptides are metabolized into diketopiperazines in the body (Shigemura, Iwasaki et al., 2018; Taga et al., 2019). In addition, thin-layer chromatographic analysis has revealed that 14C-labeled

234 Chapter 9 Pro-Hyp is metabolized into peptides with unknown structure in rats (Kawaguchi, Nanbu, & Kurokawa, 2012). These facts indicate that food-derived peptides in the body can be metabolized to other modified peptides, in addition to constituting amino acids. Even using modern LC
MS/MS, it is still difficult to directly detect and identify foodderived peptides less than 1 μM levels in such complex matrices. In such cases, it can be considered that peptides that resist in vitro exopeptidase digestion have the potential to reach targets in the body. It is much easier to identify peptides in the exopeptidase digest of food protein hydrolyzate than peptides in the body. Peptides with a known structure can be detected and quantified with high sensitivity at low nM levels by LC
MS/MS in MRM mode even in the complex matrices. Using this approach, the presence of some foodderived peptides at maximum levels of 3
110 nM in the body has been demonstrated. Peptides increased in the body are dipeptides and tripeptides with prolyl and pyroglutamyl residues at the amino terminal (Ejima et al., 2018) as well as D- and β-asparatyl dipeptides (Ejima et al., 2019). On the other hand, peptides with other structures, such as dipeptides with prolyl residue at carboxy terminal, do not increase in the body after ingestion of food protein hydrolyzates, while these peptides also resist exopeptidase digestion. In addition, oral administration of a single peptide with a different structure from the abovementioned peptides, such as Val-Tyr, also exerts biological response; decrease of blood pressure of spontaneously hypertensive rat (Matsui et al., 2004; Nakamura, Yamamoto, Sakai, & Takano, 1995), while their bioavailability is very low (Foltz et al., 2007; Matsui et al., 2004). These facts suggest that these peptides might be metabolized into compounds other than amino acids. Diketopiperazines, which are observed in human urine after the ingestion of collagen hydrolyzate, could be formed, as could other metabolites such as N-formyl, N-acetyl, and N-propionyl peptides, as well as peptides with methylation. However, there is no evidence for metabolites of food-derived peptides in the body. It is possible that such metabolites might be responsible for the biological responses induced by the ingestion of food protein hydrolyzates; however, it may be difficult to directly detect these metabolites in the body. LC
MS/MS in MRM mode based on the predicted structure of the metabolites is one method with the potential to identify such metabolites.

References Aito-Inoue, M., Ohtsuki, K., Nakamura, Y., Park, E. Y., Iwai, K., Morimatsu, F., & Sato, K. (2006). Improvement in isolation and identification of food-derived peptides in human plasma based on precolumn derivatization of peptides with phenyl isothiocyanate. Journal of Agricultural and Food Chemistry, 54, 5261
5266. Asai, T., Takahashi, A., Ito, K., Uetake, T., Matsumura, Y., Ikeda, K., . . . Sato, K. (2019). Amount of collagen in the meat contained in Japanese daily dishes and the collagen peptide content in human blood after ingestion of cooked fish meat. Journal of Agricultural and Food Chemistry, 67, 2831
2838. Asai, T. T., Yoshikawa, K., Sawada, K., Fukamizu, K., Koyama, Y., Shigemura, Y., . . . Sato, K. (2020). Mouse skin fibroblasts with mesenchymal stem cell marker p75 neurotrophin receptor proliferate in response to

Methodologies for bioavailability assessment of food-derived peptide 235 prolyl-hydroxyproline. Journal of Functional Foods, 66. Available from: https://doi.org/10.1016/j. jff.2020.103792. Bai, J. P. F. (1994). Distribution of brush-border membrane peptidases along the rat intestine. Pharmaceutical Research, 11, 897
900. Chabance, B., Marteau, P., Rambaud, J. C., Migliore-Samour, D., Boynard, M., Perrotin, P., . . . Fiat, A. M. (1998). Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie, 80, 155
165. Chen, L., Ejima, A., Gu, R. Z., Lu, J., Cai, M., & Sato, K. (2019). Presence of exopeptidase resistant and susceptible peptides in a bacterial protease digest of corn gluten. Journal of Agricultural and Food Chemistry, 67, 11948
11954. Daliri, E. B.-M., Oh, D. H., & Lee, B. H. (2017). Bioactive peptides. Foods, 6, 32. Available from: https://doi. org/10.3390/foods6050032. Dia, V. P., Torres, S., De Lumen, B. O., Erdman, J. W., Jr., & De Mejia, E. G. (2009). Presence of lunasin in plasma of men after soy protein consumption. Journal of Agricultural and Food Chemistry, 57, 1260
1266. Ejima, A., Nakamura, M., Suzuki, A. Y., & Sato, K. (2018). Identification of food-derived peptides in human blood after ingestion of corn and wheat gluten hydrolysates. Journal of Food Bioactives, 2, 104
111. Ejima, A., Yamada, K., & Sato, K. (2019). Presence of isomerized aspartic dipeptides in a porcine liver protein hydrolysate and their bioavailability upon ingestion. Bioactive Compounds in Health and Disease, 2, 155
169. Fan, H., Wang, J., Liao, W., Jiang, X., & Wu, J. (2019). Identification and characterization of gastrointestinalresistant angiotensin-converting enzyme inhibitory peptides from egg white proteins. Journal of Agricultural and Food Chemistry, 67, 7147
7156. Foltz, M., Meynen, E. E., Bianco, V., van Platerink, C., Koning, T. M. M. G., & Kloek, J. (2007). Angiotensin converting enzyme inhibitory peptides from a lactotripeptide enriched milk beverage are absorbed intact into the circulation. Journal of Nutrition, 137, 953
958. Freeman, H. J. (2015). Clinical relevance of intestinal peptide uptake. World Journal of Gastrointestinal Pharmacology and Therapeutics, 6, 22
27. Fujii, N., Takata, T., Fujii, N., Aki, K., & Sakaue, H. (2018). D-Amino acids in protein: The mirror of life as a molecular index of aging. Biochimica et Biophysica Acta—Proteins and Proteomics, 1866, 840
847. Geiger, T., & Clarke, S. (1987). Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. The Journal of Biological Chemistry, 262, 785
974. Gu, Y., & Wu, J. (2013). LC-MS/MS coupled with QSAR modeling in characterising of angiotensin I-converting enzyme inhibitory peptides from soybean proteins. Food Chemistry, 141, 2682
2690. Hanh, V. T., Shen, W., Tanaka, M., Siltari, A., Korpela, R., & Matsui, T. (2017). Effect of aging on the absorption of small peptides in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 65, 5935
5943. Hedemann, M. S., Gabert, V. M., & Larsen, T. (1998). Pancreatic secretion of zinc and carboxypeptidase A and B in growing pigs. Reproduction Nutrition Development, 38, 391
400. Higaki-Sato, N., Sato, K., Inoue, N., Nawa, Y., Kido, Y., Nakabou, Y., . . . Ohtsuki, K. (2006). Occurrence of the free and peptide forms of pyroglutamic acid in plasma from the portal blood of rats that had ingested a wheat gluten hydrolysate containing pyroglutamyl peptides. Journal of Agricultural and Food Chemistry, 54, 6984
6988. Hong, S.-M., Tanaka, M., Yoshii, S., Mine, Y., & Matsui, T. (2013). Enhanced visualization of small peptides absorbed in rat small intestine by phytic-acid-aided matrix-assisted laser desorption/ionization-imaging mass spectrometry. Analytical Chemistry, 85, 10033
10039. Ichikawa, S., Morifuji, M., Ohara, H., Matsumoto, H., Takeuchi, Y., & Sato, K. (2010). Hydroxyprolinecontaining dipeptides and tripeptides quantified at high concentration in human blood after oral administration of gelatin hydrolysate. International Journal of Food Sciences and Nutrition, 61, 52
60.

236 Chapter 9 Iwai, K., Hasegawa, T., Taguchi, Y., Morimatsu, F., Sato, K., Nakamura, Y., . . . Ohtsuki, K. (2005). Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates. Journal of Agricultural and Food Chemistry, 53, 6531
6536. Kawaguchi, T., Nanbu, P. N., & Kurokawa, M. (2012). Distribution of prolylhydroxyproline and its metabolites after oral administration in rats. Biological & Pharmaceutical Bulletin, 35, 422
427. Mada, S. B., Ugwu, C. P., & Abarshi, M. (2020). Health promoting effects of food-derived bioactive peptides: A review. International Journal of Peptide Research and Therapeutics, 26, 831
848. Matsui, T., Imamura, M., Oka, H., Osajima, K., Kimoto, K., Kawasaki, T., & Matsumoto, K. (2004). Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration of spontaneously hypertensive rats. Journal of Peptide Science, 10, 535
545. Matsui, T., Tamaya, K., Osajima, K., Matsumoto, K., & Kawasaki, T. (2002). Absorption of Val-Tyr with in vitro angiotensin I-converting enzyme inhibitory activity into the circulating blood system of mild hypertensive subjects. Biological & Pharmaceutical Bulletin, 25, 1228
1230. Matsui, T., Tanaka, K., Kawasaki, T., & Osajima, Y. (1999). Determination of angiotensin metabolites in human plasma by fluorimetric high-performance liquid chromatography using a heart-cut column-switching technique. Journal of Chromatography B, 729, 89
95. Mutoh, H., Satoh, K., Kita, H., Sakamoto, H., Hayakawa, H., Yamamoto, H., . . . Sugano, K. (2005). Cdx2 specifies the differentiation of morphological as well as functional absorptive enterocytes of the small intestine. The International Journal of Developmental Biology, 49, 867
871. Nakamura, S., Yamamoto, N., Sakai, K., & Takano, T. (1995). Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. Journal of Dairy Science, 78, 1253
1257. Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., & Takano, T. (1995). Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. Journal of Dairy Science, 78, 777
783. Nakashima, E. M. N., Kudo, A., Iwaihara, Y., Tanaka, M., Matsumoto, K., & Matsui, T. (2011). Application of 13 C stable isotope labeling liquid chromatography
multiple reaction monitoring
tandem mass spectrometry method for determining intact absorption of bioactive dipeptides in rats. Analytical Biochemistry, 414, 109
116. Nakatani, S., Mano, H., Sampei, C., Shimizu, J., & Wada, M. (2009). Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo. Osteoarthritis and Cartilage, 17, 1620
1627. Ohara, H., Ichikawa, S., Matsumoto, H., Akiyama, M., Fujimoto, N., Kobayashi, T., & Tajima, S. (2010). Collagen-derived dipeptide, proline-hydroxyproline, stimulates cell proliferation and hyaluronic acid synthesis in cultured human dermal fibroblasts. The Journal of Dermatology, 37, 330
338. Ohara, H., Matsumoto, H., Ito, K., Iwai, K., & Sato, K. (2007). Comparison of quantity and structures of hydroxyproline-containing peptides in human blood after oral ingestion of gelatin hydrolysates from different sources. Journal of Agricultural and Food Chemistry, 55, 1532
1535. Sa´nchez, A., & Va´zquez, A. (2017). Bioactive peptides: A review. Food Quality and Safety, 1, 29
46. Sato, K., Egashira, Y., Ono, S., Mochizuki, S., Shimmura, Y., Suzuki, Y., . . . Sanada, H. (2013). Identification of a hepatoprotective peptide in wheat gluten hydrolysate against D-galactosamine-induced acute hepatitis in rats. Journal of Agricultural and Food Chemistry, 61, 6304
6310. Sato, K., Nishimura, R., Suzuki, Y., Motoi, H., Nakamura, Y., Ohtsuki, K., & Kawabata, M. (1998). Occurrence of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale. Journal of Agricultural and Food Chemistry, 46, 3403
3405. Shigemura, Y., Akaba, S., Kawashima, E., Park, E.-Y., Nakamura, Y., & Sato, K. (2011). Identification of a novel food-derived collagen peptide, hydroxyprolyl-glycine, in human peripheral blood by pre-column derivatisation with phenyl isothiocyanate. Food Chemistry, 129, 1019
1024. Shigemura, Y., Iwai, K., Morimatsu, F., Iwamoto, T., Mori, T., Oda, C., . . . Sato, K. (2009). Effect of prolylhydroxyproline (Pro-Hyp), a food-derived collagen peptide in human blood, on growth of fibroblasts from mouse skin. Journal of Agricultural and Food Chemistry, 57, 444
449.

Methodologies for bioavailability assessment of food-derived peptide 237 Shigemura, Y., Iwasaki, Y., Tateno, M., Suzuki, A., Kurokawa, M., Sato, Y., & Sato, K. (2018). A pilot study for the detection of cyclic prolyl-hydroxyproline (Pro-Hyp) in human blood after ingestion of collagen hydrolysate. Nutrient, 10, 1356. Available from: https://doi.org/10.3390/nu10101356. Shigemura, Y., Kubomura, D., Sato, Y., & Sato, K. (2014). Dose-dependent changes in the levels of free and peptide forms of hydroxyproline in human plasma after collagen hydrolysate ingestion. Food Chemistry, 159, 328
332. Shigemura, Y., Nakaba, M., Shiratsuchi, E., Suyama, M., Yamada, M., Kiyono, T., . . . Sato, K. (2012). Identification of food-derived elastin peptide, prolyl-glycine (Pro-Gly), in human blood after ingestion of elastin hydrolysate. Journal of Agricultural and Food Chemistry, 60, 5128
5133. Shigemura, Y., Suzuki, A., Kurokawa, M., Sato, Y., & Sato, K. (2018). Changes in composition and content of food-derived peptide in human blood after daily ingestion of collagen hydrolysate for 4 weeks. Journal of the Science of Food and Agriculture, 98, 1944
1950. Shimizu, K., Sato, M., Zhang, Y., Kouguchi, T., Takahata, Y., Morimatsu, F., & Shimizu, M. (2010). The bioavailable octapeptide Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro stimulates nitric oxide synthesis in vascular endothelial cells. Journal of Agricultural and Food Chemistry, 58, 6960
6965. Shimizu, M. (2004). Food-derived peptides and intestinal functions. Biofactors (Oxford, England), 21, 43
47. Shirako, S., Kojima, Y., Tomari, N., Nakamura, Y., Matsumura, Y., Ikeda, K., . . . Sato, K. (2019). Pyroglutamyl leucine, a peptide in fermented foods, attenuates dysbiosis by increasing host antimicrobial peptide. NPJ Science of Food, 3, 18. Sumantha, A., Larroche, C., & Pandey, A. (2006). Microbiology and industrial biotechnology of food-grade proteases. Food Technology and Biotechnology, 44, 211
220. Suzuki, Y., Motoi, H., & Sato, K. (1999). Quantitative analysis of pyroglutamic acid in peptides. Journal of Agricultural and Food Chemistry, 47, 3248
3251. Taga, Y., Iwasaki, Y., Shigemura, Y., & Mizuno, K. (2019). Improved in vivo tracking of orally administered collagen hydrolysate using stable isotope labeling and LC-MS techniques. Journal of Agricultural and Food Chemistry, 67, 4671
4678. Taga, Y., Kusubata, M., Ogawa-Goto, K., & Hattori, S. (2014). Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen. Journal of Agricultural and Food Chemistry, 62, 12096
12102. Taga, Y., Kusubata, M., Ogawa-Goto, K., & Hattori, S. (2016). Efficient absorption of X-hydroxyproline (Hyp)Gly after oral administration of a novel gelatin hydrolysate prepared using ginger protease. Journal of Agricultural and Food Chemistry, 64, 2962
2970. Takemori, K., Yamamoto, E., Ito, H., & Kometani, T. (2015). Prophylactic effects of elastin peptide derived from the bulbus arteriosus of fish on vascular dysfunction in spontaneously hypertensive rats. Life Sciences, 120, 48
53. Van der Rest, M., & Garrone, R. (1991). Collagen family of proteins. The FASEB Journal, 5, 2814
2823. Vermeirssen, V., Van Camp, J., & Verstraete, W. (2004). Bioavailability of angiotensin I converting enzyme inhibitory peptides. British Journal of Nutrition, 92, 357
366. Wada, S., Sato, K., Ohta, R., Wada, E., Bou, Y., Fujiwara, M., . . . Yoshikawa, T. (2013). Ingestion of low dose pyroglutamyl leucine improves dextran sulfate sodium-induced colitis and intestinal microbiota in mice. Journal of Agricultural and Food Chemistry, 61, 8807
8813. Xu, Q., Hong, H., Wu, J., & Yan, X. (2019). Bioavailability of bioactive peptides derived from food proteins across the intestinal epithelial membrane: A review. Trends in Food Science and Technology, 86, 399
411. Xu, Q., Yan, X., Zhang, Y., & Wu, J. (2019). Current understanding of transport and bioavailability of bioactive peptides derived from dairy proteins: A review. International Journal of Food Science & Technology, 54, 1930
1941. Yokoyama, K., Chiba, H., & Yoshikawa, M. (1992). Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Bioscience, Biotechnology, and Biochemistry, 56, 1541
1545.

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CHAPTER 10

Methodologies for studying the structure
function relationship of food-derived peptides with biological activities Advaita Ganguly1, Kumakshi Sharma2 and Kaustav Majumder3 1

Comprehensive Tissue Centre, UAH Transplant Services, Alberta Health Services, Edmonton, AB, Canada, 2Health, Safety and Environment Branch, National Research Council Canada, Edmonton, AB, Canada, 3Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, United States

10.1 Introduction Peptides are obtained by cleavage from precursor proteins (Mooney, Haslam, Holton, Pollastri, & Shields, 2013), and this distribution of various functions make peptides suitable therapeutic agents, such as antimicrobials (Fjell, Hiss, Hancock, & Schneider, 2012; Hancock & Sahl, 2006) and analgesics (Diochot et al., 2012). Peptides can be hormones or immune-modulating entities, associating with cytokines, receptors, and signaling proteins (Mo¨ller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008). Recent studies report their potential as inhibitors of protein
protein interactions (Boonen, Creemers, & Schoofs, 2009). Peptides also exhibit functions including quorum sensing in the regulation of gut bacteria (Swift, Vaughan, & de Vos, 2000) and inhibition or activation of enzymes such as angiotensin-converting enzyme inhibitors, membrane-bound protein channels, transporters, and receptors by toxin or venom peptides (Lewis & Garcia, 2003). Peptides can also disrupt cell membranes to eliminate microbes by antimicrobial peptides. Plants and bovine milk-derived bioactive peptides, as like other food sourced peptides, have immense health benefits (Clare & Swaisgood, 2000). Norris and group showed the role of bioactive dipeptides in angiotensin-converting enzyme inhibition (Norris, Casey, FitzGerald, Shields, & Mooney, 2012). Milk-derived peptides play a role in immunogenicity and nutrition (Newburg & Walker, 2007). Many bioactive peptides exhibit health-enhancing characteristics such as antithrombotic, antimicrobial, cytomodulatory, and blood pressure management (Hartmann & Meisel, 2007). Peptides also serve as bioactive

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00008-X © 2021 Elsevier Inc. All rights reserved.

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240 Chapter 10 additives in functional foods. More studies are investigating bioactive peptides in different food sources (Clare & Swaisgood, 2000). Many nutraceuticals products have been developed using various bioactive peptides (Korhonen & Pihlanto, 2006). However, the methods and analytical tools developed so far are limited, and thus further enhancement is required to improve the identification and characterization of novel bioactive peptides from food sources (Khaldi, 2012).

10.2 Bioactivity prediction of peptides The bioactivity of peptides involves the regulations of diverse body functions, such as lowering blood pressure (angiotensin-converting enzyme inhibitors), blood glucose level management (dipeptidyl peptidase inhibitors), and cholesterol modulation (Carrasco-Castilla, ´ lvarez, Jime´nez-Martı´nez, Fidel Gutie´rrez-Lo´pez, & Da´vila-Ortiz, 2012; Herna´ndez-A Iwaniak, Darewicz, & Minkiewicz, 2018). Bioactive peptides also have applications as antioxidative, antibacterial, immunomodulating, and antithrombotic agents (Li et al., 2019). The peptides also have important roles in flavor enhancement in addition to their bioactive potential (Temussi, 2012). The propensity of food hydrolysates comprising of bioactive peptides to develop bitterness constrains their food industry application (Fu, Chen, Bak, & Lametsch, 2018; Wu & Aluko, 2007). But due to the bioactive potential, certain peptides are used as food-based therapeutics for metabolic diseases (Tidona et al., 2009). Food-based angiotensin-converting enzyme inhibitors used in prehypertension regimen (Iwaniak, Minkiewicz, & Darewicz, 2014) are mild and safer than the synthetic inhibitory peptide therapeutics (Kumar, Kumar, Sharma, & Baruwa, 2010). Therapeutic peptides used as dietary do not exhibit undesirable side effects often associated with chemical drugs, and this can significantly reduce treatment costs (Ma¨kinen, Johannson, Vegarud, Pihlava, & Pihlanto, 2012). Peptides sourced from food-based proteins are characterized by in silico, in vitro tools, and methodologies as well as traditional ex vivo/in vivo approaches (Darewicz, Borawska, Vegarud, Minkiewicz, & Iwaniak, 2014; Udenigwe, 2014). In silico methods include computer technologies for the peptide database management (Agyei, Bambarandage, & Udenigwe, 2019; Kalmykova et al., 2018) and application of programs for studying proteins as peptide sources (Ke˛ska & Stadnik, 2017). The prediction of action mechanisms of bioactive peptides also involves bioinformatics approach (Nongonierma, Mooney, Shields, & FitzGerald, 2014). Multivariate techniques are employed for structure
function prediction of peptides (Pripp, Isaksson, Stepaniak, & Sørhaug, 2004; Wu, Aluko, & Nakai, 2006) and also used for analyzing chemical nature of peptides (chemoinformatics) (Iwaniak, Minkiewicz, Darewicz, Protasiewicz, & Mogut, 2015). The classical methods in evaluating bioactive peptides includes the identification of peptides in novel food sources and optimizing peptide production (Udenigwe, 2014). Classical in vitro and in vivo methods involve validations in animal models such as spontaneously hypertensive rats in particular (Koyama, Hattori, Amano, Watanabe, & Nakamura, 2014). Rapid development in the field of bioinformatics

Structure–function relationship of food-derived peptides 241 enabled researchers to combine in silico and traditional methods to study bioactive peptides (Agyei et al., 2019; Han et al., 2018), resulting in hybrid or integrated methods.

10.3 Mapping methods to predict structure
function of bioactive peptides Several studies have reported that food proteins contain different amounts of exogenous peptides (de Castro & Sato, 2015; Udenigwe & Aluko, 2012). The peptides constitute between 3 and 20 amino acids and are encrypted in protein sequences. During gastric digestion, these peptides are cleaved from proteins by digestive enzymes and absorbed in the intestine and finally released in circulation. Absorption mechanisms vary based on the number of amino acid residues and charge of the peptide. Low-molecular-weight dipeptides and tripeptides are absorbed in the enterocyte through the intestinal endothelium by the cotransporter PepT1 (Miner-Williams, Stevens, & Moughan, 2014). Studies show that di and tripeptides are unaffected by cytosolic hydrolysis and pass the basolateral membrane (Daniel, 2004). Other peptides transport through tight junctions and/or enter via vesiculation (Bougle & Bouhallab, 2017; Mo¨ller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008; Wang & de Mejia, 2005). The bioactive peptides in circulation influences the biological activity of several enzymes critical in the regulation of blood pressure, stimulating or suppressing the immune system, modulating the activity of the nervous system, exhibiting antiinflammatory effect, and facilitating reduction in cholesterolaemia (Cicero, Fogacci, & Colletti, 2016). Reports also suggest that biological effects of bioactive peptides are mostly dependent on its amino acid composition (Li, Li, He, & Qian, 2011; Pownall, Udenigwe, & Aluko, 2011) and their spatial arrangements (He, Aluko, & Ju, 2014; Pak, Koo, Kwon, & Yun, 2012). The peptides usually exhibit one biological function but, in some cases, can have more than one bioactivity (Meisel, 2004). Due to their health-enhancing and therapeutic potential, research and studies on bioactive peptide as functional foods or nutraceuticals have significantly increased (Korhonen & Pihlanto, 2006; Shahidi & Zhong, 2008; Udenigwe & Aluko, 2012). Functional food comprising bioactive compounds, as part of diets, helps in the prevention and management of disease (Martirosyan & Singh, 2015). Databases and bioinformatics tools have been developed to predict and study bioactive peptides in food proteins like PepBank (Shtatland, Guettler, Kossodo, Pivovarov, & Weissleder, 2007), Antimicrobial Peptide Database (APD) (Wang, Li, & Wang, 2016), and BIOPEP (Minkiewicz, Dziuba, Iwaniak, Dziuba, & Darewicz, 2008). Databases possess important information pertaining to incidence of bioactive peptides in proteins. Primary structure information of characterized peptides is used to find sequence homology to correctly predict occurrence of peptides in different food proteins. Sequence analyses are performed using BLAST and PSI-BLAST programs (Altschul et al., 1997). Primary structure information is also used to optimize machine

242 Chapter 10 learning algorithms to improve accuracy in predicting bioactive peptides (Mooney et al., 2013) as well as simulation of peptide release by action of digestive enzymes based on in silico simulation (Dave, Montoya, Rutherfurd, & Moughan, 2014). Sequence homology analyses of bioactive peptides and potential target sequences serve as an important step in identifying more potential peptides. Bioinformatics programs consider differential patterns of amino acid substitution (i.e., conservation or coevolution) to identify and predict regions in proteins. Analyzing peptides from novel substrates by classical approaches are extremely time intensive and financially overwhelming in comparison to modern in silico methods. In recent times, in silico methods like PeptideRanker, Pepdrew, and Pepcalc have been developed and are more user friendly. Some of these approaches are briefly highlighted in the next sections.

10.4 In silico methods predicting bioactivity in food-derived peptides A large number of human diseases like Alzheimer’s, Parkinson’s disease, and type II diabetes results from protein and peptide aggregation (Dobson, 2006). Evaluating aggregation is critical for functional proteins and peptides. Dobson and group have earlier highlighted the biophysical and biochemical methods used to analyze protein aggregation (Dobson, 2006). Bioinformatics and software tools have been developed such as AGGRESCAN (http://bioinf.uab.es/aggrescan/) and PASTA 2.0 server (http://protein.bio. unipd.it/pasta2/) to predict protein and peptide aggregation (Caflisch, 2006). Human calcitonin is a polypeptide hormone secreted by Thyroid C cells, which has therapeutic potential for osteoporosis, Paget’s disease, hypercalcemia, and musculoskeletal aches (Duchowicz, Talevi, Brunoblanch, & Castro, 2008). But calcitonin aggregation can be a limitation; hence, Fowler and coworkers developed aggregation-resistant bioactive peptides and human calcitonin-like variants, which overcame aggregation without impacting physiological function, adopting a semi empirical and quantitative approach using in silico tools (Fowler et al., 2005). A suitable method for changing aggregation factors of bioactive peptides is predicting small mutations, resulting in aggregation rate alteration with in silico tools, which can be extended to high-throughput screening. Aqueous solubility for peptides is a critical factor enabling absorption, distribution, and elimination of peptides in the body (Balakin, Savchuk, & Tetko, 2006). Studies also show that inadequate solubility of compounds conceals toxicity and adverse effects (Balakin et al., 2006). In silico methods have been developed for solubility prediction, as classical methods were not compatible with high-throughput screening (Balakin et al., 2006; Duchowicz et al., 2008; Hewitt et al., 2009). Fusion of target polypeptides to a solubilizing protein fusion partner, glycosylation with hydrophilic carbohydrates, addition of short solubility enhancement peptide tags, and site-specific modification circumvents increases peptide solubility (Xiao, Burn, & Tolbert, 2008). Betaine facilitates increase in solubility of peptides by site-specific modification (Xiao et al., 2008).

Structure–function relationship of food-derived peptides 243 Solubility of peptides also depends on physicochemical characteristics and amino acid content. Hydrophilic residues such as aspartic acid, glutamic acid, and serine positively enable peptide solubility compared to other hydrophilic residues like asparagine, glutamine, threonine, lysine, and arginine (Trevino, Scholtz, & Pace, 2007, 2008). Another tool is the PeptideRanker, which is an online server that helps in predicting the probability of biological activity of a particular peptide. The web-based program generates a peptide score of 0
1 representing potential peptide bioactivity and ranks them as per their structure
function pattern. Primary structures of bioactive peptides can be generated by the PepDraw software, and the online Pepcalc program can calculate theoretical molecular weight, isoelectric point, peptide charge, and solubility as well as extinction coefficient of bioactive peptides (Pooja, Rani, & Prakash, 2017).

10.5 Methods to analyze the physicochemical feature of bioactive peptide The degree of bioactivity displayed by a peptide is dependent on the secondary structure and its constituent physicochemical characteristics. Some software such as UniProt and Po^le Bioinformatique Lyonnais can analyze peptides up to 20 amino acids in length, and GOR V and PreSSAPro programs can process even smaller peptide sequences. The importance of physicochemical properties to determine hydrophobicity, electrostatic potential, and secondary structure properties is invaluable. AA index is a repository compiled with amino acid information. The AA index in combination with chemometric and quantitative structure
activity relation (QSAR) methods would facilitate assessment of bioactive peptide features as well as in elucidating homology trends and prediction simulation in docking experiments. These methods have been employed to determine toxicity of peptides (Carrasco-Castilla et al., 2012). The enzymes used during hydrolysis, parameters of food processing and peptide lengths, etc. are all considered important for discerning peptide bioactivity which impacts their absorption potential across the enterocytes and potential bioavailability (Udenigwe & Aluko, 2012). In silico and several in vitro methods and tools have been developed focused on generating and elucidating novel bioactive peptides, especially in the food matrix (Kussmann, Van, & Bladeren, 2011). In vitro approaches comprise identification of a food protein, specific enzymatic hydrolysis coupled with fermentation, and sometimes gastrointestinal digestion. This is followed with in vitro screening of the peptides for potential bioactivity and a fractionation step. The peptide structures are then studied and analyzed, followed by design of synthetic structural analogs or peptide mimetic to validate peptide biological activity both in vitro and in vivo (Saavedra, Hebert, Minahk, & Ferranti, 2013). Current OMICS and computational methods harness cell biology concepts, immunological insights, and other components of biochemistry along with the use of combinatorial library with mass spectrometry to select different peptide patterns and their

244 Chapter 10 respective bioactivity functions. Usually the method involves an initial sample purification step, followed by 2D-PAGE- or RP-HPLC-based separation. There is enzymatic digestion and identification of peptides by mass spectrometry or tandem mass spectrometry. Naturally occurring human milk peptides have been characterized by this approach (Picariello, Ferranti, Mamone, Roepstorff, & Addeo, 2008), resulting in the identification of bioactive peptides. Gomez-Ruiz’s group analyzed angiotensin-converting enzyme inhibitory sequences in milk (Gomez-Ruiz, Ramos, & Recio, 2007) and were also evaluated during cheese ripening (Hernandez-Ledesma, Amigo, Ramos, & Recio, 2004) by multiple reaction monitoring detection protocols. MALDI-TOF-MS off late has been an important analytical technique in screening of peptides and evaluation of their bioactive functions. Screening an assortment of complex food-derived peptides can be a limitation of this method. Peptide combinations may have hydrophobic or hydrophilic regions, which may impair analysis by MALDI-TOF-MS due to lack of desorbing because of matrix suppression. The adoption of several matrices can circumvent this drawback by evaluating a higher pool of peptides (Hernandez-Ledesma et al., 2004). Sinapinic acid is known to be apt for evaluation of intact proteins and large peptide fragments, whereas α-cyano-4-hydroxycinnamic acid helps in better screening of intermediate peptides. Studies have demonstrated a threshold setting of minimum two matrices (or higher) facilitates a twofold increase in protein and peptide coverage. The combinatorial employment of α-cyano-4-hydroxycinnamic acid and sinapinic acid matrix enabled enhanced hydrophilic proteins coverage. 2,5-Dihydroxybenzoicacid in combination with the sinapinic acid matrix led to similar results with hydrophobic proteins (Gonnet, Lemaitre, Waksman, & Tortajada, 2003). The development of better and accurate tools to screen bioactive low-molecular-weight peptides should be focused. The routine method for these small peptides is MALDI-TOF-MS analysis; but, matrix suppression or interference inhibits identification of low-molecular-weight entities. Recently, nanostructure-assisted laser desorption/ionization analysis is used to evaluate low-molecular-weight peptides of small peptides bypassing extensive sample pretreatment. This technique is matrix-independent and enables better signal intensity for small peptide components in comparison to MALDI with matrix requirement. This fairly novel technology has been applied in study of bovine milk and colostrum-based peptides (Ku¨tt, Malbe, & Stagsted, 2001).

10.6 Quantitative structure
activity relationship methods to assess food-derived peptide functions QSAR relates to the structural attributes of molecules to their biological or chemical functions (Nongonierma & Fitzgerald, 2016) and has been extensively studied in food chemistry, involving bioactive peptides, sensory peptides, etc. Such approaches can be advantageous for prediction and synthesis of active peptides simultaneously, providing an enhanced understanding of the inherent physicochemical mechanisms. Multivariate

Structure–function relationship of food-derived peptides 245 regression models such as partial least square and artificial neural networks (ANNs) have greatly increased application of QSAR modeling. The structure and activity relations of biologically active peptides have been studied on antimicrobial, ACE-inhibitory, antioxidant, and renin and dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. QSAR-based approaches generally comprise creating a bioactive peptide library, consisting of peptide sequences, defining peptides with scalar descriptors of amino acids, building the QSAR model and followed by confirmatory and validation studies with synthetic peptides in vivo (Nongonierma & Fitzgerald, 2016). Recently, studies estimated the activities of related compounds and predicted structures of highly functional peptides using QSAR models. Food-derived angiotensin-I converting enzyme (ACE) inhibitory peptides functionally restrict the ACE, which is known to reduce blood pressure and ameliorate hypertension (Ganguly, Sharma, & Majumder, 2019). Jing and coworkers generated a QSAR model with accurate functional prediction capability using ACE-inhibitory peptides. Milk-derived ACE-inhibitory tripeptides were selected and validated based on QSAR predictions (Nongonierma & Fitzgerald, 2016). In another study, Qi and group studied four potent tripeptides, GEF, VEF, VRF, and VKF, based on predictive QSAR models, and experimental evaluation validated the predicted values (Qi, Lin, Rong, & Wu, 2017). Generation of bitter peptides during the enzymatic process to produce functional bioactive peptide hydrolysates and during the aging process of fermented food products is considered a problem in the food industry (Kim & Li-Chan, 2006). Consumers instinctively detest bitterness and shun such food products (Maehashi & Huang, 2009). QSAR models-based methods were employed to study the peptide characteristics that constitute bitterness in the peptides. Hydrophobic amino acids at the C-terminus and basic amino acids at the N-terminus were found to be associated with the bitterness of peptides (Kim & Li-Chan, 2006). Another study also involved a QSAR model to analyze the relation between ACE-inhibitory activity and bitterness of peptides. Significant association between enhanced ACE inhibition and bitterness was noticed in dipeptides. The relation was mainly attributed to the role and importance of hydrophobicity pertaining to both ACE-inhibitory function and peptide bitterness. Wang and coworkers showed that intermediate length peptides have increased bitterness compared to other peptides (Wang & Mejia, 2010). Restricted structural variations in dipeptides result in difficulty to have attributes that hinder the effect of C-terminal hydrophobicity, necessary for ACE inhibition, and on peptide bitterness. QSAR modeling plays an important role in the development of functional protein foods by predicting functionally active peptides by detailed explanation and association of structure
activity relationships. However, QSAR approaches also have limitations in predicting rare types of bioactive peptides owing to problems in generating a model and their physical characteristics. Studies have focused on peptides with ACE inhibitory and antibacterial functions, along with some bitter peptides based on some regularity and coherence in their amino acid constitution.

246 Chapter 10

10.7 Artificial neural networking and quantitative structure
activity relationship integrative approach to assess bioactive of peptides Machine learning tools, like ANNs, were developed based on biological neural networks without the physicochemical characteristics (Baldi & Brunak, 1998, 2001; Haykin, 1999). ANN models comprise processing units also known as artificial neurons and a number of weighted connections or artificial synapses between the neurons (Wu, Whitson, Mclarty, Ermongkonchai, & Chang, 1992). The ANN development initially requires a learning module wherein the network automatically adjusts the strength of its synapses according to a set of measured data (Wu et al., 1992). The self-adjustment process is synonymous with the biological events and manifestations in the brain during the learning module. Neural networks have been extensively used in protein research, molecular biology (Diederichs, Freigang, Umhau, Zeth, & Breed, 1998; Dombi & Lawrence, 1994; Ferran, Pflugfelaer, & Ferrara, 1994; Jafarnia-Dabanloo, McLernon, Zhang, Ayatollahi, & Johari-Majd, 2007; Kenward, Wachtmeister, Ghirlanda, & Enquist, 2004; Leake & Anninos, 1976; Lohmann, Schneider, Behrens, & Wrede, 1994; Wang, Yang, & Chou, 2006), and also in cancer research and other infectious diseases (Zhou, Jiang, Yang, & Chen, 2002). The benefits of ANNs and similar machine learning methods and tools are easily accessible for research needs and generate reliable and verifiable results. One of the major limitations though is statistical characterization and clarity in physicochemical implications (King, 1996). Huang and coworkers developed a novel and improved version of ANN factoring in physics and chemistry-based inputs combined with advanced mathematical approaches of ANN. This Physio-Chem-based ANN integrates QSAR methods as well, and every layer and components have distinct physical and chemical implications. The improved version also generates important physical and chemical insights, resulting in increased accuracy in predicting the bioactivities of proteins and peptides (Huang et al., 2009). The primary equation of the novel version builds on the linear free energy equation of QSAR, and the binding free energy inputs also follow QSAR model. The neurons constituting the hidden layer serve as a primary structural and functional unit in peptide sequences. The free energy terms in the improved neural network model consist of physical implications. The Physio-Chem incorporated version makes better prediction compared to classical QSAR and also gives accurate structural information and physical insights than traditional neural network approaches. The novel Physio-Chem-based method can give a more accurate prediction of shorter peptides and can be significant for protein design and prediction when appropriate functional residues constitute the hidden layer. This novel combination of QSAR and ANN approaches can be beneficial in protein and peptide-based drug designing as well when the bioactivity input is IC50 values of proteins and peptides (Du, Huang, Wei, Wang, & Chou, 2007; Du, Wei, Pang, Chou, & Huang, 2007, Ganguly,

Structure–function relationship of food-derived peptides 247 Sharma, & Majumder, 2020). Improved data analysis tools can facilitate further development of QSAR modeling peptide, and food-derived peptide studies should incorporate knowledge gained from QSAR methods employed in other realms of life science research.

10.8 Limitations of classical bioinformatics and computational biology approach for peptide analysis Secretory proteins and peptides are generally developed as precursors, like pre-pro-proteins. These precursor proteins are subject to posttranslational proteolysis wherein the N-terminal preregion, which is the signal peptide, is cleaved by a signal peptidase (Dalbey, Von, & Heijne, 1992; Paetzel, Karla, Strynadka, & Dalbey, 2002), whereas different proteases release the active peptides from the pro-proteins (Seidah & Chretien, 1999). They often undergo proteolysis to mature. A large number of secretory peptides are generated as inactive precursors that need posttranslational actions to be biologically active peptides. But different methods employed to predict natural peptides are skewed by reduced accuracy of proteolytic site predictors as well as difficulty in pairing these sites. In silico analysis rapidly facilitates scalability of precursor proteins research and the specific release of bioactive peptides, simultaneously being cost effective. Although, in silico digestion assists in enzyme selection during protein hydrolysis, an accurate depiction of the peptides and their bioactive functionalities is still based on experimental procedures. Some therapeutic peptide hormones, whose proteolytic processing regulates their activities, include insulin, somatostatin, parathyroid hormone, and GLP-1. Identifying mature peptides involves in vitro and computational tools targeting and predicting novel proteolytic sites. Biochemical separation techniques have been the classical method for generating smaller peptides like GPCR ligands. This method is complex, as peptides exhibit limited expression, and scarcely found in complex biological matrices with other secreted proteins along with a wide range of physiological characteristics. Several trials with biochemical separation and purification along with mass spectrometry protocols have been studied (Kalkum, Lyon, & Chait, 2003; Ohyama, Ogawa, & Matsubayashi, 2008). Natural peptide prediction has become relatively simpler and accurate owing to rapid developments in computational biology tools. This facilitates the comprehensive analysis of genome and proteome data. Certain computational methods follow the classical furin and dibasic proteolytic sites, which can be identified using simple regular expressions (Shi, Ko, Abott, & Ko, 2012; Shichiri et al., 2003). Predicting tools analyzing complex pro-hormone convertase cleavage sites show considerably reduced performance (Duckert, Brunak, & Blom, 2004; Hummon et al., 2003; Kliger et al., 2008), resulting in erroneously predicted peptides. Hidden Markov model and machine learning-based

248 Chapter 10 computational peptide identification are restricted to cases where considerable sequence homology to prior prevalent peptides exists (Mirabeau et al., 2007; Shemesh et al., 2008; Sonmez et al., 2009). Studies suggest that sequence and structural differences complicate the computational identification of peptides by Markov model methods. Toporik and coworkers studied that the amino acid sequence of functional peptides is more conserved (among orthologous proteins) in comparison with the rest of the precursor sequence. This theory is validated in corticoliberin, tachykinin-3, and insulin. Adopting a secretome-wide approach, they showed that the relative conservation of peptide sequences enables significant accuracy in prediction of peptides. Machine learning tools, incorporating additional parameters, ensure increased prediction performance resulting in peptides as potentially novel hormones (Toporik, Borukhov, Apatoff, Gerber, & Kliger, 2014). QSAR-based methods to analyze proteins and peptides with longer sequences can also be cumbersome with the increase in number of terms. Amino acid distribution can sometimes be more useful than determining the location of the residue in the sequence (Carrasco-Castilla et al., 2012).

10.9 Conclusion and future directions Classical and in silico approaches like mass spectrometry, chemometrics, and bioinformatics and their use can be expanded. Until now, there has been little concern about safety of bioactive peptides since they are normally produced by digestive enzymes and food-grade enzymes, and processes are utilized for industrial production of peptides (Dave et al., 2014). Although bioactive peptides are generally safe, the use of processing techniques that would negatively affect peptide quality and safety should be avoided. In this sense, mass spectrometry and chemometrics have been successfully applied to identify modifications induced by food processing, through the application of mass spectrometry efficient to monitor procedures. The predictive nature of in silico programs and tools and the high-throughput analytical potential of peptidomics based technologies can develop suitable tools for the identification and evaluation of food-based functional peptides and peptides in other biological matrices. Advancements in bioinformatics facilitated efficient prediction and discovery of food-derived peptides with biological activities. Food-based peptide identification methods currently do not incorporate posttranslational modification, such as oxidation, methylation, deamination, and acetylation, of amino acid residues of peptides. Future in silico algorithms with emphasis on biological events such as posttranslational modifications would result in improved prediction of functional peptides. The hybrid methodology is considered complementary in terms of combination of in silico and in vitro approaches, but some drawbacks need to be resolved such as peptide stability and safety, bioavailability, toxicity, and solubility. Prevalent methods need further improvement to resolve these issues in the field of bioactive peptide research.

Structure–function relationship of food-derived peptides 249

References Agyei, D., Bambarandage, E., & Udenigwe, C. C. (2019). The role of bioinformatics in the discovery of bioactive peptides. In F. Shahidi, L. Melton, & P. Valeris (Eds.), Encyclopedia of food chemistry (pp. 337
344). Amsterdam; Kidlington, Oxford; Cambridge MA: Elsevier. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Research, 25, 3389
3402. Balakin, K. K. V., Savchuk, N. P., & Tetko, I. V. (2006). In silico approaches to prediction of aqueous and DMSO solubility of drug-like compounds: Trends, problems and solutions. Current Medicinal Chemistry, 13, 223
241. Baldi, P., & Brunak, S. (1998). Bioinformatics: The machine learning approach. Cambridge, MA: MIT Press. Baldi, P., & Brunak, S. (2001). Bioinformatics: The machine learning approach (2nd ed.). Cambridge, MA: MIT Press. Boonen, K., Creemers, J. W., & Schoofs, L. (2009). Bioactive peptides, networks and systems biology. Bioessays: News and Reviews in Molecular, Cellular and Developmental Biology, 31, 300
314. ˆ . D., & Bouhallab, S. (2017). Dietary bioactive peptides: Human studies. Critical Reviews in Food Bougle, A Science and Nutrition, 57, 335
343. Caflisch, A. (2006). Computational models for the prediction of polypeptide aggregation propensity. Current Opinion in Chemical Biology, 10, 437
444. ´ lvarez, A. J., Jime´nez-Martı´nez, C., Fidel Gutie´rrez-Lo´pez, G., & Da´vila-Ortiz, Carrasco-Castilla, J., Herna´ndez-A G. (2012). Use of proteomics and peptidomics methods in food bioactive peptide science and engineering. Food Engineering Reviews, 4, 224
243. Cicero, A. F. G., Fogacci, F., & Colletti, A. (2016). Potential role of bioactive peptides in prevention and treatment of chronic diseases: A narrative review. British Journal of Pharmacology, 174, 1378
1394. Clare, D., & Swaisgood, H. (2000). Bioactive milk peptides: A prospectus. Journal of Dairy Science, 83, 1187
1195. Dalbey, R. E., & Von Heijne, G. (1992). Signal peptidases in prokaryotes and eukaryotes—A new protease family. Trends in Biochemical Sciences, 17, 474
478. Daniel, H. (2004). Molecular and integrative physiology of intestinal peptide transport. Annual Review of Physiology, 66, 361
384. Darewicz, M., Borawska, J., Vegarud, G., Minkiewicz, P., & Iwaniak, A. (2014). Angiotensin I-converting enzyme (ACE) inhibitory activity and ACE inhibitory peptides of Salmon (Salmo salar) protein hydrolysates obtained by human and porcine gastrointestinal enzymes. International Journal of Molecular Sciences, 15, 14077
14101. Dave, L. A., Montoya, C. A., Rutherfurd, S. M., & Moughan, P. J. (2014). Gastrointestinal endogenous proteins as a source of bioactive peptides—An in silico study. PLoS One, 9, e98922. de Castro, R. J. S., & Sato, H. H. (2015). Biologically active peptides: Processes for their generation, purification and identification and applications as natural additives in the food and pharmaceutical industries. Food Research International, 74, 185
198. Diederichs, K., Freigang, J., Umhau, S., Zeth, K., & Breed, J. (1998). Prediction by a neural network of outer membrane {beta}-strand protein topology. Protein Science: A Publication of the Protein Society, 7, 2413
2420. Diochot, S., Baron, A., Salinas, M., Douguet, D., Scarzello, S., Anne- Dabert-Gay, S., . . . Lingueglia, E. (2012). Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature, 490, 552
555. Dobson, C. M. (2006). Protein aggregation and its consequences for human disease. Protein and Peptide Letters, 13, 219
227. Dombi, G. W., & Lawrence, J. (1994). Analysis of protein transmembrane helical regions by a neural network. Protein Science: A Publication of the Protein Society, 3, 557
566.

250 Chapter 10 Du, Q. S., Huang, R. B., Wei, Y. T., Wang, C. H., & Chou, K. C. (2007). Peptide reagent design based on physical and chemical properties of amino acid residues. Journal of Computational Chemistry, 28, 2043
2050. Du, Q. S., Wei, Y. T., Pang, Z. W., Chou, K. C., & Huang, R. B. (2007). Predicting the affinity of epitope
peptides with class I MHC molecule HLA-A 0201: An application of amino acid-based peptide prediction. Protein Engineering, Design & Selection: PEDS, 20, 417
423. Duchowicz, P. R., Talevi, A., Brunoblanch, L. E., & Castro, E. A. (2008). New QSPR study for the prediction of aqueous solubility of drug-like compounds. Bioorganic & Medicinal Chemistry, 16, 7944
7955. Duckert, P., Brunak, S., & Blom, N. (2004). Prediction of proprotein convertase cleavage sites. Protein Engineering, Design & Selection: PEDS, 17, 107
112. Ferran, E. A., Pflugfelaer, B., & Ferrara, P. (1994). Self-organized neural maps of human protein sequences. Protein Science: A Publication of the Protein Society, 3, 507
521. Fjell, C., Hiss, J. A., Hancock, R. E. W., & Schneider, G. (2012). Designing antimicrobial peptides: Form follows function. Nature Reviews. Drug Discovery, 11, 37
51. Fowler, S. B., Poon, S., Muff, R., Chiti, F., Dobson, C. M., & Zurdo, J. (2005). Rational design of aggregation-resistant bioactive peptides: Reengineering human calcitonin. Proceedings of the National Academy of Sciences of the United States of America, 102, 10105
10110. Fu, Y., Chen, J., Bak, K. H., & Lametsch, R. (2018). Valoristaion of protein hydrolysates from animal by-products: Perspectives on bitter taste and debittering methods: A review. International Journal of Food Science & Technology, 54, 978
986. Ganguly, A., Sharma, K., & Majumder, K. (2019). Food-derived bioactive peptides and their role in ameliorating hypertension and associated cardiovascular diseases. Advances in Food and Nutrition Research, 89, 165
207. Ganguly, A., Sharma, K., & Majumder, K. (2020). Peptides as biopolymers—Past, present, and future. In K. Pal, I. Banerjee, P. Sarkar, D. Kim, W.-P. Deng, N. K. Dubey, & K. Majumder (Eds.), Biopolymer-based formulations (pp. 87
104). Elsevier. Gomez-Ruiz, J. A., Ramos, M., & Recio, I. (2007). Identification of novel angiotensinconverting enzyme-inhibitory peptides from ovine milk proteins by CE-MS and chromatographic techniques. Electrophoresis, 28, 4202
4211. Gonnet, F., Lemaitre, G., Waksman, G., & Tortajada, J. (2003). MALDI/MS peptide mass fingerprinting for proteome analysis: Identification of hydrophobic proteins attached to eucaryote keratinocyte cytoplasmic membrane using different matrices in concert. Proteome Science, 1, 1
7. Han, J., Tang, S., Li, Y., Bao, W., Wan, H., Lu, C., . . . Su, X. (2018). In silico analysis and in vivo tests of the tuna dark muscle hydrolysate anti-oxidation effect. RSC Advances, 8, 14109
14119. Hancock, R., & Sahl, H. (2006). Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature Biotechnology, 24, 1551
1557. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: From research to food applications. Current Opinion in Biotechnology, 18, 163
169. Haykin, S. (1999). Neural Networks. New York: Macmillan College Publishing Company Inc. He, R., Aluko, R. E., & Ju, X.-R. (2014). Evaluating molecular mechanism of hypotensive peptides interactions with renin and angiotensin converting enzyme. PLoS One, 9, e91051. Hernandez-Ledesma, B., Amigo, L., Ramos, M., & Recio, I. (2004). Application of high-performance liquid chromatography-tandem mass spectrometry to the identification of biologically active peptides produced by milk fermentation and simulated gastrointestinal digestion. Journal of Chromatography A, 1049, 107
114. Hewitt, M., Cronin, M. T., Enoch, S. J., Madden, J. C., Roberts, D. W., & Dearden, J. C. (2009). In silico prediction of aqueous solubility: The solubility challenge. Journal of Chemical Information and Modeling, 49, 2572
2587. Huang, R. B., Du, Q.-S., Wei, Y.-T., Pang, Z.-W., Wei, H., & Chou, K.-C. (2009). Physics and chemistry-driven artificial neural network for predicting bioactivity of peptides and proteins and their design. Journal of Theoretical Biology, 256, 428
435.

Structure–function relationship of food-derived peptides 251 Hummon, A. B., Hummon, N. P., Corbin, R. W., Li, L., Vilim, F. S., Weiss, K. R., & Sweedler, J. V. (2003). From precursor to final peptides: A statistical sequence-based approach to predicting prohormone processing. Journal of Proteome Research, 2, 650
656. Iwaniak, A., Darewicz, M., & Minkiewicz, P. (2018). Peptides derived from foods as supportive diet components in the prevention of the metabolic syndrome. Comprehensive Reviews in Food Science and Food Safety, 17, 63
81. Iwaniak, A., Minkiewicz, P., & Darewicz, M. (2014). Food-originating ACE inhibitors, including antihypertensive peptides, as preventive food components in blood pressure reduction. Comprehensive Reviews in Food Science and Food Safety, 13, 114
134. Iwaniak, A., Minkiewicz, P., Darewicz, M., Protasiewicz, M., & Mogut, D. (2015). Chemometrics and cheminformatics in the analysis of biologically active peptides from food sources. Journal of Functional Foods, 16, 334
351. Jafarnia-Dabanloo, N., McLernon, D. C., Zhang, H., Ayatollahi, A., & Johari-Majd, V. (2007). A modified Zeeman model for producing HRV signals and its application to ECG signal generation. Journal of Theoretical Biology, 244, 180
189. Kalkum, M., Lyon, G. J., & Chait, B. T. (2003). Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 100, 2795. Kalmykova, S. D., Arapidi, G. P., Urban, A. S., Osetrova, M. S., Gordeeva, V. D., Ivanov, V. T., & Govorun, V. M. (2018). In silico analysis of peptide potential biological functions. Russian Journal of Bioorganic Chemistry, 44, 367
385. Kenward, B., Wachtmeister, C. A., Ghirlanda, S., & Enquist, M. (2004). Spots and stripes: The evolution of repetition in visual signal form. Journal of Theoretical Biology, 230, 407
419. Ke˛ska, P., & Stadnik, J. (2017). Taste-active peptides and amino acids of pork meat as components of of dry-cured meat products: An in silico study. Journal of Sensory Studies, 32(6), e12301. Khaldi, N. (2012). Bioinformatics approaches for identifying new therapeutic bioactive peptides in food. Functional Foods in Health and Disease, 2, 325
338. Kim, H. O., & Li-Chan, E. C. (2006). Quantitative structure-activity relationship study of bitter peptides. Journal of Agricultural and Food Chemistry, 54, 10102
10111. King, R. D. (1996). In M. J. E. Sternberg (Ed.), Protein structure prediction: A practical approach (pp. 79
97). Oxford: IRL Press. Kliger, Y., Gofer, E., Wool, A., Toporik, A., Apatoff, A., & Olshansky, M. (2008). Predicting proteolytic sites in extracellular proteins: Only halfway there. Bioinformatics (Oxford, England), 24, 1049
1055. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16, 945
960. Koyama, M., Hattori, S., Amano, Y., Watanabe, M., & Nakamura, K. (2014). Blood pressure-lowering peptides from neo-fermented buckwheat sprouts: A new approach to estimating ACE-inhibitory activity. PLoS One, 9(9), e105802. Kumar, R., Kumar, A., Sharma, R., & Baruwa, A. (2010). Pharmacological review on natural ACE inhibitors. Der Pharmacia Lettre, 2, 273
293. Kussmann, M., & Van Bladeren, P. J. (2011). The extended nutrigenomics—Understanding the interplay between the genomes of food, gut microbes, and human host. Frontiers in Genetics, 2, 1
13. Ku¨tt, M. L., Malbe, M., & Stagsted, J. (2001). Nanostructure-assisted laser desorption/ionization (NALDI) for analysis of peptides in milk and colostrums. Agronomy Research, 9, 415
420. Leake, B., & Anninos, P. A. (1976). Effect of connectivity on the activity of neural net models. Journal of Theoretical Biology, 58, 337
363. Lewis, R., & Garcia, M. L. (2003). Therapeutic potential of venom peptides. Nature Reviews. Drug Discovery, 2, 790
802.

252 Chapter 10 Li, S., Tingting, B., Zheng, J., Liu, L., He, G., & Wu, J. (2019). Preparation, bioavailability, and mechanism of emerging activities of Ile-Pro-Pro and Val-Pro-Pro. Comprehensive Reviews in Food Science and Food Safety, 18, 1097
1110. Li, Y.-W., Li, B., He, J., & Qian, P. (2011). Quantitative structure 6 activity relationship study of antioxidative peptide by using different sets of amino acids descriptors. Journal of Molecular Structure, 998, 53
61. Lohmann, R., Schneider, G., Behrens, D., & Wrede, P. A. (1994). Neural network model for the prediction of membrane-spanning amino acid sequences. Protein Science: A Publication of the Protein Society, 3, 1597
1601. Maehashi, K., & Huang, L. (2009). Bitter peptides and bitter taste receptors. Cellular and Molecular Life Sciences: CMLS, 66, 1661
1671. Ma¨kinen, S., Johannson, T., Vegarud, E. G., Pihlava, J. M., & Pihlanto, A. (2012). Angiotensin I-converting enzyme inhibitory and antioxidant properties of rapeseed hydrolysates. Journal of Functional Foods, 4, 575
583. Martirosyan, D. M., & Singh, J. (2015). A new definition of functional food by FFC: What makes a new definition unique? Journal of Functional Foods in Health and Disease, 6, 209
223. Meisel, H. (2004). Multifunctional peptides encrypted in milk proteins. Biofactors (Oxford, England), 21, 55
61. Miner-Williams, W. M., Stevens, B. R., & Moughan, P. J. (2014). Are intact peptides absorbed from the healthy gut in the adult human? Nutrition Research Reviews, 27, 308
329. Minkiewicz, P., Dziuba, J., Iwaniak, A., Dziuba, M., & Darewicz, M. (2008). BIOPEP database and other programs for processing bioactive peptide sequences. Journal of AOAC International, 91, 965
980. Mirabeau, O., Perlas, E., Severini, C., Audero, E., Gascuel, O., Possenti, R., . . . Gross, C. (2007). Identification of novel peptide hormones in the human proteome by hidden Markov model screening. Genome Research, 17, 320
327. Mo¨ller, N., Scholz-Ahrens, K. E., Roos, N., & Schrezenmeir, J. (2008). Bioactive peptides and proteins from foods: Indication for health effects. European Journal of Nutrition, 47, 171
182. Mooney, C., Haslam, N. J., Holton, T. A., Pollastri, G., & Shields, D. C. (2013). Peptide Locator: Prediction of bioactive peptides in protein sequences. Bioinformatics (Oxford, England), 29, 1120
1126. Newburg, D., & Walker, W. (2007). Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatric Research, 61, 2
8. Nongonierma, A., & Fitzgerald, D. (2016). Learnings from quantitative structure activity relationship (QSAR) studies with respect to food protein-derived bioactive peptides: A review. RSC Advances, 6, 75400
75413. Nongonierma, A. B., Mooney, C., Shields, D. C., & FitzGerald, R. J. (2014). In silico approaches to predict the potential of milk-protein derived milk peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors. Peptides, 57, 43
51. Norris, R., Casey, F., FitzGerald, R. J., Shields, D. C., & Mooney, C. (2012). Predictive modelling of angiotensin converting enzyme inhibitory dipeptides. Food Chemistry, 133, 1349
1354. Ohyama, K., Ogawa, M., & Matsubayashi, Y. (2008). Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis. The Plant Journal for Cell and Molecular Biology, 55, 152
160. Paetzel, M., Karla, A., Strynadka, N. C. J., & Dalbey, R. E. (2002). Signal peptidases. Chemical Reviews, 102, 4549
4580. Pak, V. V., Koo, M., Kwon, D. Y., & Yun, L. (2012). Design of a highly potent inhibitory peptide acting as a competitive inhibitor of HMG-CoA reductase. Amino Acids, 43, 2015
2025. Picariello, G., Ferranti, P., Mamone, G., Roepstorff, P., & Addeo, F. (2008). Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry. Proteomics, 8, 3833
3847. Pooja, K., Rani, S., & Prakash, B. (2017). In silico approaches towards the exploration of rice bran proteinsderived angiotensin-I-converting enzyme inhibitory peptides. International Journal of Food Properties, 20 (2), 2178
2191.

Structure–function relationship of food-derived peptides 253 Pownall, T. L., Udenigwe, C. C., & Aluko, R. E. (2011). Effects of cationic property on the in vitro antioxidant activities of pea protein hydrolysate fractions. Food Research International, 44, 1069
1074. Pripp, A. H., Isaksson, T., Stepaniak, L., & Sørhaug, T. (2004). Quantitative structure-activity relationship modelling of ACE inhibitory peptides derived from milk proteins. European Food Research and Technology, 219, 579
583. Qi, C., Lin, G., Rong, Z., & Wu, W. (2017). Studies on the bioactivities of ACE-inhibitory peptides with phenylalanine C-terminus using 3D-QSAR, molecular docking and in vitro evaluation. Molecular Informatics, 36, 1600157. Saavedra, L., Hebert, E. M., Minahk, C., & Ferranti, P. (2013). An overview of “omic” analytical methods applied in bioactive peptide studies. Food Research International, 54, 925
934. Seidah, N. G., & Chretien, M. (1999). Proprotein and prohormone convertases: A family of subtilases generating diverse bioactive polypeptides. Brain Research, 848, 45
62. Shahidi, F., & Zhong, Y. (2008). Bioactive peptides. Journal of AOAC International, 91, 914
931. Shemesh, R., Toporik, A., Levine, Z., Hecht, I., Rotman, G., Wool, A., . . . Cohen, Y. (2008). Discovery and validation of novel peptide agonists for G-protein-coupled receptors. The Journal of Biological Chemistry, 283, 34643
34649. Shi, L., Ko, M. L., Abott, L. C., & Ko, G. Y.-P. (2012). Identification of Peptide lv, a novel putative neuropeptide that regulates the expression of L-type voltage-gated calcium channels in photoreceptors. PLoS One, 7, e43091. Shichiri, M., Ishimaru, S., Ota, T., Nishikawa, T., Isogai, T., & Hirata, Y. (2003). Salusins: Newly identified bioactive peptides with hemodynamic and mitogenic activities. Nature Medicine, 9, 1166
1172. Shtatland, T., Guettler, D., Kossodo, M., Pivovarov, M., & Weissleder, R. (2007). PepBank—A database of peptides based on sequence text mining and public peptide data sources. BMC Bioinformatics, 8(280), 1
10. Sonmez, K., Zaveri, N. T., Kerman, I. A., Burke, S., Neal, C. R., Xie, X., . . . Toll, L. (2009). Evolutionary sequence modeling for discovery of peptide hormones. PLoS Computational Biology, 5, e1000258. Swift, S., Vaughan, E. E., & de Vos, W. M. (2000). Quorum sensing within the gut ecosystem. Microbial Ecology in Health and Disease, 12, 81
92. Temussi, P. A. (2012). The good taste of peptides. Journal of Peptide Science: An Official Publication of the European Peptide Society, 18, 73
82. Tidona, F., Criscione, A., Guastella, A. M., Zuccaro, A., Bordonaro, S., & Marletta, D. (2009). Bioactive peptides in dairy products. Italian Journal of Animal Science, 8, 315
3340. Toporik, A., Borukhov, I., Apatoff, A., Gerber, D., & Kliger, Y. (2014). Computational identification of natural peptides based on analysis of molecular evolution. Bioinformatics (Oxford, England), 30(15), 2137
2141. Trevino, S. R., Scholtz, J. M., & Pace, C. N. (2007). Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa. Journal of Molecular Biology, 366, 449
460. Trevino, S. R., Scholtz, J. M., & Pace, C. N. (2008). Measuring and increasing protein solubility. Journal of Pharmaceutical Sciences, 97, 4155
4166. Udenigwe, C. C. (2014). Bioinformatic approaches, prospects and challenges of food bioactive peptide research. Trends in Food Science and Technology, 36, 137
143. Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides: Production, processing, and potential health benefits. Journal of Food Science, 77, R11
R24. Wang, G., Li, X., & Wang, Z. (2016). APD3: The antimicrobial peptide database as a tool for research and education. Nucleic Acids Research, 44, D1087
D1093. Wang, S. Q., Yang, J., & Chou, K. C. (2006). Using stacked generalization to predict membrane protein types based on pseudo-amino acid composition. Journal of Theoretical Biology, 242, 941
946. Wang, W., & de Mejia, E. G. (2005). A new Frontier in soy bioactive peptides that may prevent age-related chronic diseases. Comprehensive Reviews in Food Science and Food Safety, 4, 63
78.

254 Chapter 10 Wang, W., & Mejia, E. G. D. (2010). A new frontier in soy bioactive peptides that may prevent age-related chronic diseases. Comprehensive Reviews in Food Science and Food Safety, 4, 63
78. Wu, C., Whitson, G., Mclarty, J., Ermongkonchai, A., & Chang, T. C. (1992). Protein classification artificial neural system. Protein Science: A Publication of the Protein Society, 1, 667
677. Wu, J., & Aluko, R. E. (2007). Quantitative structure-activity relationship study of bitter di- and tri-peptides including relationship with angiotensin I-converting enzyme inhibitory activity. Journal of Peptide Science: An Official Publication of the European Peptide Society, 13, 63
69. Wu, J., Aluko, R. E., & Nakai, S. (2006). Structural requiremenets of angiotensin I-converting enzyme inhibitory peptides: Quantitative structure-activity relationship study of di- and tripeptides. Journal of Agricultural and Food Chemistry, 54, 732
738. Xiao, J., Burn, A., & Tolbert, T. J. (2008). Increasing solubility of proteins and peptides by site-specific modification with betaine. Bioconj Chem, 19, 1113
1118. Zhou, Z. H., Jiang, Y., Yang, Y. B., & Chen, S. F. (2002). Lung cancer cell identification based on artificial neural network ensembles. Artificial Intelligence in Medicine, 24, 25
36.

CHAPTER 11

Methodologies for investigating the vasorelaxation action of peptides Mitsuru Tanaka1 and Toshiro Matsui2,3 1

Division of Integrated Research for Five-sense Devices, Research and Development Center for FiveSense Devices, Kyushu University, Fukuoka, Japan, 2Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka, Japan, 3Division of Taste Sensor/Odor Sensor, Research and Development Center for Five-Sense Devices, Kyushu University, Fukuoka, Japan

11.1 Introduction Antihypertensive peptides have attracted attention for their effective prevention of lifestylerelated diseases (Aluko, 2015). Thus far, various peptides from natural proteins have been reported to exhibit blood pressure lowering effects in spontaneously hypertensive rats and mildly hypertensive humans. Since blood pressure is defined as the pressure of circulating blood on the wall of blood vessels (i.e., arteries) (Burton & Stinson, 1960), enhancing flexibility or reducing vascular stiffness must therefore lead to a lowering of blood pressure. Thus the vascular relaxation effect has been investigated as a mechanism of antihypertensive peptides. As reviewed previously (Matsui, Wang, & Tanaka, 2011), di-/tripeptides such as Val-Tyr (Tanaka et al., 2006), Trp-His (Tanaka et al., 2008), Met-Tyr (Erdmann et al., 2006) from sardine muscle, Ile-Pro-Pro from casein (Miguel et al., 2007), and Ile-Arg-Trp from egg protein (Majumder et al., 2013) have been shown to exhibit vasorelaxation effects. Although ex vivo and/or in vitro investigations, including vascular tension measurements and/or intracellular Ca21 concentration ([Ca21]i), are important for providing insights into the underlying vasoprotective mechanism(s) of bioactive peptides, attention must also be given to their bioavailability in the gut intestinal tract in their intact form; intact intestinal absorption of peptides is determined by protease resistance and peptide length (Foltz, van der Pijl, & Duchateau, 2010; Matsui, 2018; Shen & Matsui, 2017). Peptide-induced vasorelaxation is achieved in either an endothelium-dependent or -independent manner (Fig. 11.1). Endothelium-dependent vasorelaxation is mainly caused by the activation of cyclooxygenase/cyclic adenosine monophosphate, nitric oxide/cyclic guanosine monophosphate (cGMP), and heme oxygenase-1/carbon monoxide (CO)/cGMP pathways. Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00011-X © 2021 Elsevier Inc. All rights reserved.

255

256 Chapter 11

Figure 11.1: Signaling cascades for contraction/relaxation in vessels. AC, Adenylyl cyclase; CaM, calmodulin; CaMK II, Ca21/CaM-dependent protein kinase II; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CO, carbon monoxide; COX, cyclooxygenase; DAG, 1,2-diacylglycerol; ER/SR, endoplasmic/sarcoplasmic reticulum; HO-1, heme oxygenase-1; IP3, inositol-1,4,5-triphosphate; IP3-R, IP3 receptor; MLC, myosin light chain; MLCK, myosin light chain kinase; MLC-P, phosphorylated myosin light chain; NO, nitric oxide; PGI2, prostaglandin I2; PKA, protein kinase A; PKG, protein kinase G; PLC, phospholipase C; SERCA pump, sarco/endoplasmic reticulum Ca21-ATPase; sGC, soluble guanylyl cyclase.

Endothelium-independent vasorelaxation can be achieved by the suppression of receptormediated signaling pathways such as angiotensin II (Ang II) type I receptor (AT1R), α-adrenergic receptor, and endothelin type I receptor. Hyperpolarization of vascular membranes by cGMP-induced K1 channel activation also induces vasorelaxation through the suppression of extracellular Ca21 influx via voltage-dependent L-type Ca21 channel. Although such various vasorelaxation-related signaling cascades regulate vessel function, the trigger for this is [Ca21]i in vascular smooth muscle cells (VSMCs). Trp-His, which is one of the representative endothelium-independent vasorelaxation peptides, can reduce elevated [Ca21]i in Ang IIstimulated VSMCs, probably by its two physiological actions in cells, the first being Ca21 channel blocking [by binding to the voltage-dependent L-type Ca21 channel (Wang et al., 2010)] and the second being inhibition of Ca21-calmodulin (CaM) complex formation

Methodologies for investigating the vasorelaxation action of peptides 257 [resulting in suppressed phosphorylation of the Ca21 channel via reduced CaMK II activity (Kobayashi et al., 2012; Kumrungsee et al., 2014)]. Therefore, in addition to vasorelaxation response, a series of investigations regarding intracellular Ca21-related signaling is required to understand the physiological roles of antihypertensive peptides in vessels. Hence, in this chapter, in the context of studying peptides with vasorelaxation potential, protocols for cellular [Ca21]i measurement and a Ca21
CaM complex formation assay are introduced.

11.2 Principles 11.2.1 Measurement of vascular tension Measurement of vascular tension is based on monitoring the physical isometric tension of the vessel. This monitoring is carried out in a microtissue organ bath, mounted on a vessel ring, and coupled with a force transducer system. The thoracic aorta (i.e., mesenteric artery) from rats is commonly used for this purpose. Successive measurements of change in tension (in g or mN) by target peptides in the presence of contractive agents (e.g., phenylephrine, KCl, and Ang II) or relaxant agents (e.g., acetylcholine) are recorded. Relaxation response is sequentially monitored amid cumulative addition of agents or peptides; a dose
response curve obtained from the cumulative addition experiments can indicate the vasorelaxation power of the peptides, given as the effective concentration that produces 50% vasorelaxation of the maximal contractive response (EC50 value) (Fig. 11.2).

Figure 11.2: A typical tension curve in ex vivo vasocontraction experiments. Vasorelaxation of peptides is evaluated by the percentage of relaxation tension (ΔT) against that of phenylephrine contraction (T0). ACh, Acetylcholine; PE, phenylephrine.

258 Chapter 11

11.2.2 Measurement of [Ca21]i Measurement of [Ca21]i is based on monitoring of the fluorescent intensity of a Ca21specific probe, such as a fluorescent Ca21 chelator (e.g., Fura-2 or Fluo-4). After incorporating the prodrug-type Ca21 probe (e.g., Fura-2/AM or Fluo-4/AM) into cells, an active probe formed by cellular hydrolysis can catch a free Ca21 through chelating action to form a Ca21-probe complex. As intracellular Ca21 is a trigger for vasocontraction in VSMCs, increasing [Ca21]i relates to vascular contractive response in VSMCs and vice versa. The source of elevated [Ca21]i is from either an influx of extracellular Ca21 into the intracellular cytosol through Ca21 channels (mainly L-type), or Ca21 release from intracellular Ca21 stores (i.e., the endoplasmic/sarcoplasmic reticulum). Thus [Ca21]i measurement is a useful tool to understand the mechanism(s) of peptides’ vasorelaxation properties. Generally, to evaluate the vasorelaxation mechanism of peptides in terms of [Ca21]i, agonistic stimulation by Ang II [via the Ang II type I receptor (AT1R)], phenylephrine (via the α1-adrenergic receptor), and KCl (via membrane depolarization) is used for the activation of contractive signaling cascades in VSMCs.

11.2.3 Assay for Ca21
CaM complex formation CaM is a regulatory protein involved in a variety of cellular Ca21-dependent signaling pathways, including vasocontraction. Upon binding up to four Ca21 in its four EF-hands (Fig. 11.3), converting it to an active form as a Ca21
CaM complex, CaM undergoes a conformational change, enabling it to activate a number of intracellular kinases, such as CaMK II (Schaub & Heizmann, 2008). Some peptides, termed calcium-like peptides [CALPs; e.g., VKFGVGFKVMVF (Villain et al., 2000)], that contain basic amino acid residues inhibit Ca21
CaM complex formation, resulting in the subsequent inhibition of Ca21
CaMdependent kinases. A fluorescence-based assay for indirect evaluation of Ca21
CaM complex formation has been proposed (Kumrungsee et al., 2014), since no direct assay for the detection of this complex has been reported. The principle of this assay lies in the reduction of free Ca21 through its consumption in solution during Ca21
CaM complex formation (Fig. 11.3). Free Ca21-chelating fluorescence probes (Fura-2 and Fluo-4) can be used for the indirect evaluation of Ca21
CaM complex inhibition of peptides. Stains-all (Caday & Steiner, 1985) is used as a positive inhibitor to validate the Ca21-fluorescence probe-aided method.

11.3 Materials, equipments, and reagents The following materials, equipments, and reagents are shown as representative examples for the corresponding experiments. Readers are free to choose equivalent available alternatives of similar quality.

Methodologies for investigating the vasorelaxation action of peptides 259

Figure 11.3: A fluorometric evaluation of Ca21
CaM complex formation. CaM, Calmodulin; F, fluorescence intensity.

11.3.1 Measurement of vascular tension 11.3.1.1 Materials The thoracic aorta (or mesenteric artery) is taken from rats or pigs. 11.3.1.2 Equipment 1. Microtissue organ bath to monitor vascular tension force: A chamber of microtissue organ bath with a pair of stainless wires coupled with a force transducer (Model MTOB-1Z, Labo Support, Osaka, Japan) etc. Data acquisition system (Bridge8 Modules Low Noise Transducer Amplifier, World Precision Instruments, Sarasota, FL, United States) (amp-b01, emka Technologies Inc., Falls Church, VA, United States) etc. 2. O2/CO2 (5/95%) gas and regulator. 3. Labware for aortic tissue preparation: microscissors, microtweezers, vials (B20 mL scale), etc. 11.3.1.3 Reagents Analytical grade chemicals and distilled water: 1. Physiological saline solution (PSS) [pH 7.4, composition (mM): NaCl 145, KCl 5, Na2HPO4 1, CaCl2 2.5, MgSO4 0.5, glucose 10, and HEPES 5]. 2. Chemicals as vascular contraction and relaxation stimulators:

260 Chapter 11 Phenylephrine (Wako Pure Chemical Industries, Osaka, Japan); acetylcholine (Wako Pure Chemical Industries); Ang II, AT1R antagonists, and Ca21 channel blockers such as ( 6 )-verapamil (Sigma
Aldrich Co., St. Louis, MO, United States), nifedipine (Nacalai Tesque Inc., Kyoto, Japan), etc.

11.3.2 Measurement of intracellular Ca21 concentration [Ca21]i 11.3.2.1 Materials VSMCs from animals (rat, human, etc.). 11.3.2.2 Equipments 1. CO2 incubator 2. Clean bench 3. Fluorescence spectrophotometer (RF-5300PC, Shimadzu Co., Kyoto, Japan) with a magnetic stirrer 4. Super ion probe software ver. 1.0 (Shimadzu) 5. Three mL fluorescence quartz cuvette 6. Microstirring bar 7. Glass syringe (250 μL) 11.3.2.3 Reagents Analytical grade chemicals and distilled water: 1. PSS. 2. Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, United States). 3. Fura-2/AM (Dojindo, Kumamoto, Japan), Ang II (Sigma
Aldrich), PD 123177 (Sigma
Aldrich), 2-aminoethoxydiphenyl borate (2-APB; Sigma
Aldrich), m-3M3FBS (Sigma
Aldrich), U-73122 (Merck, Darmstadt, Germany), Triton X100 (Nacalai Tesque), ethylene glycol tetraacetic acid (EGTA, Dojindo), etc.

11.3.3 Assay for Ca21
CaM complex formation 11.3.3.1 Materials 96-well, half-area, flat-bottom black microplate (Greiner Bio-One Ltd., Stonehouse, United Kingdom). Note: All apparatuses should be thoroughly washed with 0.2 M EGTA and Chelex-treated Milli-Q water prior to experiments.

Methodologies for investigating the vasorelaxation action of peptides 261 11.3.3.2 Equipments Fluorescent microplate reader (Wallac ARVO SX 1420 Multilabel Counter, Perkin-Elmer Life Sciences, Tokyo, Japan) with an excitation (Ex) wavelength of 492 nm and an emission (Em) wavelength of 520 nm. If the filters are not available, use the filter with approximately 492 nm/520 nm. 11.3.3.3 Reagents Analytical grade chemicals and distilled water: CaM from bovine brain (Genway, San Diego, CA), Stains-all (Sigma
Aldrich), Chelex 100 (Sigma
Aldrich), Fluo-4 (Invitrogen, Eugene, OR), N-(2-hydroxyethyl)piperazine-N0 -2ethanesulfonic acid (HEPES) (Dojindo), and EGTA (Dojindo), etc. Note: All solvents used should be treated with the addition of Chelex 100 to completely remove any contaminating free Ca21 in solution.

11.4 Protocols 11.4.1 Measurement of vascular tension 11.4.1.1 Preparation of aortic rings from rats 1. Sacrifice rats [e.g., 8
11-week-old Sprague-Dawley males (SD, SPF/VAF Crj:SD; Charles River Japan, Kanagawa, Japan)] by exsanguination from the abdominal aorta under anesthesia [e.g., inhalation of sevoflurane (Maruishi Pharmaceutical Co., Osaka, Japan)]. 2. Cut breastbone from the thoracic diaphragm to just below the throat to make a viewing field of the whole heart. 3. Cut all tracts between the heart and throat, including the respiratory tract, esophagus, arteries, and veins. 4. Pinch the thoracic aorta where it connects to the heart and carefully stretch in an abdominal direction. 5. Carefully remove the thoracic aorta by cutting connective tissues between the aorta and backbone. 6. Wash the inside and outside of the isolated thoracic aorta carefully with PSS. 7. Maintain the rings at 37 C for 45 minutes under gentle bubbling with 95% O2/5% CO2 gas. (Fig. 11.4 shows the overall steps for the preparation of aortic ring segments). 8. Remove adhering fat and connective tissues surrounding the thoracic aorta. (During aortic ring preparation, PSS should be frequently soused over the aortic tissue to prevent it from drying). 9. Cut the cleaned thoracic aorta into 2
3 mm wide ring segments.

262 Chapter 11

Figure 11.4: Preparation of ring segments for vascular tension measurements.

10. Mount the rings between two stainless-steel wires in a 5 mL-jacketed organ bath filled with modified PSS buffer. 11. Stretch the mounted rings progressively to preload a resting tension of 2 g (20 mN) for 45 minutes until stabilized. 12. Measure the contractive responses (isometric tension, in g or mN) using a force transducer coupled with a data acquisition system. 11.4.1.2 Measurement of vasorelaxation tension in contracted rat aortic rings 1. Add contractive stimulant (e.g., 1 μM phenylephrine) to organ bath to contract aortic ring after a 45 minutes equilibration. 2. When the plateau tension is achieved, add a sample solution to the bath at 10 minutes intervals, consistently, to assess the vasorelaxant action in a cumulative manner. Notes •

•

The addition of amino acids corresponding to peptides may be useful as a negative control to emphasize the peptide-induced vasorelaxation not caused by the constituent amino acids and to eliminate the risk of unexpected responses from any fatigue of the aortic ring segments (Fig. 11.5A). For endothelium denuded-contractile experiments, the endothelial layer is mechanically removed from intact aortic rings by inserting a stainless-steel wire or thin rolled filter paper into the lumen and gently rolling the ring back and forth on a filter paper wetted with PSS buffer (Akpaffiong & Taylor, 1998).

Methodologies for investigating the vasorelaxation action of peptides 263

Figure 11.5: Representative records of vasoactive peptides in vascular tension measurements to evaluate vasorelaxation effect of His-Arg-Trp (B and C) and corresponding amino acid mixture (A) in rat thoracic aorta rings with endothelial layer (A and B) or without endothelial layer (C). (A) Change in PE-contracted tension in the case of amino acids and (B and C) peptides. Effect of endothelial layer on vascular tension is also displayed: (B) presence of endothelial layer; (C) absence of endothelial layer. ACh, Acetylcholine; PE, phenylephrine.

•

•

To verify the complete removal of the endothelial layer, experiments should be performed with 100 μM acetylcholine in 1 μM phenylephrine-contracted aortic rings to confirm the absence of vasorelaxation (Fig. 11.5C). For inhibitor (or antagonist) experiments, aortic rings must be treated with the inhibitor for 15 minutes before the addition of peptide solution.

11.4.2 Measurement of [Ca21]i 11.4.2.1 Cell culture In this section, the isolation protocol of VSMCs from the rat thoracic aorta is outlined. 1. Carefully remove the thoracic aorta, as mentioned earlier (see Section 11.4.1.1), with a sterile microscissor and a microtweezer. (The following operations must be performed aseptically on a clean bench).

264 Chapter 11 2. Gently remove the fat and connective tissue from the thoracic aorta in sterile PSS buffer. 3. Cut a cleaned aortic tube in the direction of the blood flow. 4. Denude the endothelium by rubbing the luminal surface with sterile gauze in PSS. 5. Incubate the vessels in DMEM containing 315 units/mL collagenase and 10 units/mL elastase for 30 minutes at 37 C. 6. Remove the adventitia of the aorta by rubbing with sterile gauze in DMEM. 7. Repeat step 5. 8. Terminate the digestion by adding DMEM containing 20% fetal bovine serum (FBS; Invitrogen). 9. Centrifuge the cells (1 minute at 200 3 g). 10. Seed the collected VSMCs at 1 3 104 cells/cm2 in DMEM containing 20% FBS, 2 mM L-glutamine, 100 units/mL penicillin (Meiji Seika, Tokyo, Japan), and 100 μg/mL streptomycin (Nacalai Tesque) in a culture flask. 11. Reduce the FBS concentration to 10% gradually after two or three passages. 12. Culture the cells at 37 C in a humidified 5% CO2 incubator. The cultured VSMCs are used at passage numbers 5
7 in this study. 11.4.2.2 Measurement of [Ca21]i in vascular smooth muscle cells Measurement of [Ca21]i is performed using a Ca21-sensitive probe (Fura-2/AM). 1. Culture VSMCs with serum-free DMEM for 24 hours after reaching a semiconfluent condition (70%
80% of confluent) to synchronize the cell cycle to a quiescent stage. 2. Harvest the quiescent VSMCs using trypsin-EDTA solution. 3. Terminate the digestion by adding trypsin inhibitor solution. 4. Centrifuge the VSMCs. 5. Incubate the collected cells with 1 μM Fura-2/AM containing 0.1% dimethyl sulfoxide and 0.04% Cremophor EL (Nacalai Tesque) in PSS buffer for 60 minutes at 37 C. 6. Add the 2 mL cell suspension (1 3 105 cells/mL) into a 3 mL fluorescent quartz cuvette. 7. Monitor fluorescence intensities at a dual excitation wavelength of 340/380 nm and emission wavelength of 500 nm using a fluorescence spectrophotometer. 8. Incubate the cells in the presence or absence of a peptide and/or inhibitor for 10 minutes at 37 C. 9. Inject PSS containing a [Ca21]i stimulator (10 μM Ang II, 50 μM BayK8644, or 20 μM m-3M3FBS, etc.) into the cuvette. 10. Add 1% Triton X100 to lyse VSMCs in buffer containing Ca21 at the end of the experiment, when the loaded Fura-2 is liberated from the cells and saturated with Ca21 to obtain the maximum fluorescence ratio (Rmax).

Methodologies for investigating the vasorelaxation action of peptides 265 11. Add 4 mM EGTA in 40 mM NaOH to completely chelate Ca21 bound to Fura-2, where the minimum fluorescence intensity ratio (Rmin) is obtained. (Fig. 11.6A shows a representative Fura-2 fluorescent [Ca21]i measurement in VSMCs). Notes •

•

Cremophor EL (or Pluronic F-127) is a nonionic surfactant commonly used to improve the dispersion of Fura-2/AM in aqueous solution. It should be mixed with Fura-2/AM before addition to the aqueous buffer. In Ang II-stimulation experiments (Wang et al., 2010), 1.0 μM PD 123177 [an antagonist of angiotensin type 2 receptor (AT2R)] is added 10 minutes before the addition of Ang II

Figure 11.6: Representative records of [Ca21]i profile in VSMCs from a Fura-2 fluorescence Ca21 assay. 21 (A) Representative records of [Ca ]i from a dual (ex 340 nm/380 nm) fluorescence Fura-2 assay. (B) Evaluation of [Ca21]i from intracellular Ca21 stores [endoplasmic reticulum/sarcoplasmic reticulum (ER/SR)]. (C) Evaluation of [Ca21]i from extracellular Ca21 influx, and [Ca21]i inhibition by peptides. VSMC, Vascular smooth muscle cell.

266 Chapter 11

•

•

to avoid activation of vasorelaxation signaling pathways via AT2R or any unexpected attenuation of [Ca21]i. To focus on [Ca21]i release from intracellular Ca21 stores (i.e., the endoplasmic reticulum/sarcoplasmic reticulum) through the IP3 receptor, the probe-loaded cells are added to Ca21-free PSS treated with 0.1 mM EGTA and 1 μM PD 123177. After the treatment, preincubate for 3 minutes with or without a peptide, followed by stimulation with Ang II (10 μM) (Fig. 11.6B). To focus on [Ca21]i increase from extracellular Ca21 influx into VSMCs, the probeloaded cells are added to Ca21-free PSS treated with 0.1 mM EGTA, followed by the addition of 1 μM PD 123177 and 2-APB (200 μM) as an inositol triphosphate (IP3)receptor antagonist (Facemire & Arendshorst, 2005), 10 minutes before the addition of Ang II (10 μM). After confirming no increase in [Ca21]i with the addition of Ang II, preincubate for 3 minutes with or without a peptide followed by stimulation with 2.5 mM CaCl2 (Fig. 11.6C).

11.4.3 Assay for Ca21
CaM complex formation 1. Add an aliquot (10 μL) of a given sample (including Stains-all as a positive control) in 10 mM HEPES buffer (pH 7.5) to 10 μL of 2.5 μM CaM solution dissolved in Chelextreated Milli-Q water in a 96-well, half-area, flat-bottom black microplate. 2. Incubate at 37 C for 15 minutes. 3. Add 10 μL of 12 μM CaCl2 solution to the solution. 4. Incubate at 37 C for 15 minutes. 5. Add 10 μL of 20 μM Fluo-4 solution (in Chelex-treated Milli-Q water) to the solution. 6. Measure the resulting fluorescence intensity (F) using a fluorescent microplate reader at an excitation (Ex) wavelength of 492 nm and an emission (Em) wavelength of 520 nm. A solution without a sample is used as a control, whereas a solution without CaM is used as a blank. Stains-all (a calcium-mimetic dye that specifically binds to the Ca21 binding sites of CaM) and Gly-Gly are recommended as positive and negative controls, respectively.

11.5 Analysis and statistics 11.5.1 Measurement of vascular tension The relaxation power of a peptide can be expressed as the percentage change of stimulator (e.g., phenylephrine and KCl)-induced maximal contraction (Fig. 11.2): Relaxationð%Þ 5 ΔT=T0 ðphenylephrine contractionÞ 3 100

(11.1)

Methodologies for investigating the vasorelaxation action of peptides 267 EC50 is defined as the half-maximal effective concentration of a peptide producing 50% vasorelaxation of the maximal contractive response.

11.5.2 Measurement of [Ca21]i Fura-2 is a representative dual Ca21 indicator (Grynkiewicz, Poenie, & Tsien, 1985). This dye possesses an excitation spectrum at 380 nm (emission at 500 nm) as a Ca21-free form. When the dye binds to Ca21, the excitation is shifted to 340 nm (with the same emission wavelength at 500 nm). Thus the increase in [Ca21]i excites the dye in the Ca21-binding form at 340 nm, along with a decrease in fluorescence intensity at 380 nm from the free form of the dye. [Ca21]i can be calculated by the dual fluorescent intensity (F) at an excitation wavelength of 340/380 nm and emission wavelength of 500 nm, according to the following equation (Grynkiewicz et al., 1985):  21 
Ca i 5 Kd ðR 2 Rmin Þ=ðRmax 2 RÞ R380 nm =R340 nm (11.2) where R 5 F340 nm/F380 nm. Rmax represents F340 nm max/F380 nm min obtained by the addition of 1% Triton X100 to lyse VSMCs, in buffer containing Ca21, at the end of the experiment, where loaded Fura-2 is liberated from the cells and saturated with Ca21. Subsequently, Rmin (namely, F340 nm min/F380 nm max) is calculated by the addition of an excess amount of EGTA (4 mM) to chelate Ca21 bound to Fura-2 (Fig. 11.6A).

11.5.3 Percentage of Ca21
CaM complex formation The ability of a peptide to bind to CaM or inhibit Ca21
CaM complex formation can be indirectly evaluated by comparing the difference in the fluorescence intensity (F) of the peptide (ΔFsample: Fsample blank 2 Fsample) with that of the control (ΔFcontrol: Fcontrol blank 2 Fcontrol) using the following equation:
Ca21
CaM complexð%Þ 5 Fsample blank
Fsample =ðFcontrol blank
Fcontrol Þ 3 100 (11.3) where Fcontrol blank, Fcontrol, and Fsample represent the F of Ca21-Fluo-4 products either in the absence of both the peptide and CaM, in the absence of peptide but in the presence of CaM, or in the presence of both peptide and CaM, respectively (Fig. 11.3). Fsample blank, representing F in the absence of CaM, is used to account for nonspecific effects on Ca21-Fluo-4 fluorescence by the peptide. Thus, in the case that the peptide does not work for the inhibition of Ca21
CaM complex formation, the ratio of the Ca21
CaM complex must be 100%. In addition, as was previously shown, the ΔFcontrol value obtained with 20 μM Fluo-4 (Ca21
CaM complex in the absence of a peptide but in the presence of 2.5 μM CaM) increased with increasing Ca21 concentration (4
12 μM), indicating that under the assay conditions, 12 μM Ca21 with a maximal ΔFcontrol value of 5.4 3 104 would be useful in

268 Chapter 11 evaluating the inhibition power of a peptide on Ca21
CaM complex formation (Kumrungsee et al., 2014).

11.5.4 The Hill-plot analysis To understand the kinetic interaction of a peptide with CaM, the Hill-plot analysis is applicable for the fluorescence assay; the Hill coefficient (nHill) is calculated from the Hill plot using the following equation (Boschek, Squier, & Bigelow, 2007):  
(11.4) log Y=ð1
Y Þ 5 nlog Ca21 free
nlog K where n is the Hill coefficient, K is the macroscopic dissociation constant, which represents the sum of microscopic equilibrium-binding constants for homotropic cooperativity (Boschek et al., 2007), Y is the fractional saturation of CaM, and [Ca21]free is the free Ca21 concentration in solution. To construct the Hill plot using Eq. (11.4), [Ca21]free and Y are obtained from Eqs. (11.5) and (11.6), as follows:  21   
(11.5) Ca free 5 Ca21 total 3 Fsample =Fsample blank where [Ca21]total is the total amount of Ca21 added. Y is calculated as follows:   Y 5 Ca21 bound =4½CaM

(11.6)

where [Ca21]bound is the concentration of CaM-bound Ca21, which is calculated as [Ca21]bound 5 [Ca21]total 2 [Ca21]free, 4 is the total number of Ca21-binding sites of CaM (Schaub & Heizmann, 2008), and [CaM] is the CaM concentration. Thus Y and [Ca21]free are fitted to Eq. (11.4) to construct the Hill plots and calculate nHill. For the assay shown in Fig. 11.7 (Kumrungsee et al., 2014), 10 μL of 600 μM Trp-His (150 μM, final concentration) in 10 mM HEPES buffer (pH 7.5) is added to 10 μL of 2.5 μM CaM solution (CaM dissolved in Chelex-treated Milli-Q water), followed by incubation at 37 C for 15 minutes. Then, 10 μL of 40 μM CaCl2 solution in 10 mM HEPES buffer (pH 7.5) is added to the mixture. After 15 minutes of incubation, 10 μL of 20 μM Fluo-4 in Chelex-treated Milli-Q water is added to the mixture, followed by fluorescence measurement at 492 nm (Ex) and 520 nm (Em). Data can be fitted with GraphPad Prism version 5 software (La Jolla, CA).

11.6 Safety considerations and standards 11.6.1 Animal ethics The use of experimental animals is needed to obtain aortic tissues for vascular tension measurement and VSMC culture. Thus all animal experiments must be carried out under

Methodologies for investigating the vasorelaxation action of peptides 269

Figure 11.7: Hill-plot prediction of the number of peptides in the pocket of CaM by a Fluo-4 fluorescence assay in VSMCs. VSMC, Vascular smooth muscle cell.

the appropriate guidelines. Below are representative example ethical statements for experiments in published articles. 11.6.1.1 Ethical statement “All animal experiments were carried out under the Guidance for Animal Experiments in the Faculty of Agriculture in the Graduate Course of Kyushu University and in accordance with Law (No. 105, 1973) and Notification (No. 6, 1980 of the Prime Minister’s Office) of the Japanese government. All experiments were reviewed and approved by the Animal Care and Use Committee of Kyushu University (Permit Number: A24
051).” (from Zhao et al., 2014). 11.6.1.2 Protocol for euthanasia “Rats were sacrificed by exsanguination from the abdominal aorta under anesthesia by inhaling sevoflurane.” Inclusion of the permission number of the protocols from the institute in which the experiments are carried out, and methods for painless management (anesthesia) and euthanasia, is recommended.

270 Chapter 11

11.7 Pros and cons 11.7.1 Measurement of vascular tension Pros

Cons

Real physical response of aortic tissue including endothelial and smooth muscle layers can be evaluated.

The use of experimental animals (e.g., rats) is essential. Skillful techniques for animal experimentation and tissue handling are needed. Testing the concentration of peptides that could be achieved after the administration of the peptide should be considered. Specific instruments required to measure the vascular tone (e.g., microtissue organ bath with a pair of stainless wires coupled with a force transducer) are essential.

11.7.2 Measurement of [Ca21]i Pros

Cons 21

Cytosolic mechanism(s) involved in [Ca ]i regulation can be evaluated using various inhibitor (s) in VSMCs This measurement could be used as an alternative method to reduce the use of experimental animals for evaluating the mechanism(s) involved in vascular tone regulation.

Testing concentration of peptides that could be achieved after the administration of the peptide should be considered.

11.7.3 Assay for Ca21
CaM complex formation Pros An exclusive method to evaluate the binding of a peptide to CaM at Ca21 binding site.

Cons Testing concentration of peptides that could be achieved after the administration of the peptide should be considered.

11.8 Alternative methods/procedures 11.8.1 Measurement of vascular tension using rat mesenteric arteries Mesenteric arteries, that is, small resistance arteries, are used for vascular tension measurement as well as the thoracic aorta. The direct evaluation of the vascular functions

Methodologies for investigating the vasorelaxation action of peptides 271 of resistance arteries that significantly contribute to the resistance of blood flow or regulation of blood pressure seems to be important, since vascular response or sensitivity appears to be different depending on the artery, that is, whether it is the thoracic aorta, carotid artery, mesenteric artery, or renal artery. Indeed, vasorelaxation reagents (e.g., ( 6 )-1-[(3aR ,4S ,9bS )-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3Hcyclopenta[c]quinolin-8-yl]-ethanone (so-called G1) and 4,40 ,4v-(4-propyl-[1H]-pyrazole1,3,5-triyl)-tris-phenol) have been reported to induce higher vasorelaxation in mesenteric arteries than in the thoracic aorta (Mata et al., 2015). Skillful techniques, such as dissection of third-generation resistance vessels under an operating microscope (Olympus, SZ40) (Zeng et al., 2004), are needed for the preparation of arterial rings from mesenteric arteries.

11.8.2 The patch clamp test The patch clamp test is useful for observing electrophysiological events in cells. Whole-cell recording can perform a real-time measurement of the macroscopic behavior of [Ca21]i in various types of cells, including smooth muscle cells, skeletal cells, and neurons. This technique allows for an evaluation of the voltage-dependent gating behavior of Ca21 channels. However, the patch clamp experiments have limitations, such as refined recording conditions and channel blockade by divalent ions (e.g., cadmium, cobalt, and nickel), and require expert skills for gentle insertion of the microelectrode (pipette) on the surface of a single cell under the control of a micromanipulator. In addition, a series of specific instruments is also essential. Details of the practical protocols are outlined in Brown et al. (2013).

11.9 Troubleshooting and optimization 11.9.1 Measurement of vascular tension Problem Unexpected response following the addition of synthetic peptides in the form of trifluoroacetate salt is observed. Poor stimulation of agonist or unexpected contractive response is observed during an equilibration period.

Solution To eliminate the influence of trifluoroacetate salt in synthetic peptides (by Fmoc solid phase synthesis), the peptides should be neutralized by NH4OHaq followed by lyophilization, before the experiments. When unexpected contractive response is observed during equilibration period, aortic tissues cause the damage. Before the experiment, viability of the rings should be verified by testing a 1 μM phenylephrine-contracted response [more than 0.3 g in isometric tension in our experimental condition (Tanaka et al., 2008), Fig. 11.5]. (Continued)

272 Chapter 11 (Continued) Problem

Solution

Unexpected contractive response is observed during equilibration period.Poor reproducibility of endothelium-dependent vasorelaxation is observed.

Any physical stress, in particular stretching, on aortic tissue must be avoided throughout the procedures, because aortic tissues are sensitive to shear stress, which often induces an unintentional contractive response, such as increasing tension and rhythmic contraction. Since the endothelial layer is susceptible to damage, aortic tissue should be carefully and gently handled throughout the procedures.Endothelial integrity should be confirmed using 100 μM acetylcholine-induced relaxation in 1 μM phenylephrine-contracted aorta rings, as shown in Fig. 11.5.

11.9.2 Measurement of [Ca21]i Problem

Solution 21

Poor [Ca ]i stimulation of agonist.

When the fluorescence intensity is not high enough, the issue is derived from poor loading of the Ca21 dye. As noted earlier (see Section 11.4.2.2), Fura-2/AM should be dissolved in Cremophor EL (or pluronic F197) prior to dilution in aqueous buffer. In addition, mixing with sonication at each dilution step may be effective. If the fluorescence intensity is high enough, the issue may be derived from leakage of the dye from cells. The addition of probenecid, which is an inhibitor of organic anion transporters, may solve the issue when Fura-2 is loaded.

11.10 Summary To understand the in vivo evidence of the antihypertensive effects of dietary peptides, the mechanism(s) involved must be elucidated. Although angiotensin I-converting enzyme inhibition is known to be a prominent mechanism of antihypertensive action in peptides, the vascular action of peptides toward the improvement of impaired vascular tone is now addressed as an alternative antihypertensive action. The methodologies outlined in this chapter, measurements of contractive vascular tension, [Ca21]i, and Ca21
CaM complex formation, can serve as reliable scientific tools for evaluating the vasoactive potential of peptides and for understanding their intracellular events in relation to vasorelaxation.

Methodologies for investigating the vasorelaxation action of peptides 273

References Akpaffiong, M. J., & Taylor, A. A. (1998). Antihypertensive and vasodilator actions of antioxidants in spontaneously hypertensive rats. American Journal of Hypertension: Journal of the American Society of Hypertension, 11, 1450
1460. Aluko, R. E. (2015). Antihypertensive peptides from food proteins. Annual Review of Food Science and Technology, 6, 235
262. Boschek, C. B., Squier, T. C., & Bigelow, D. J. (2007). Disruption of interdomain interactions via partial calcium occupancy of calmodulin. Biochemistry, 46, 4580
4588. Brown, J., Hainsworth, A. H., Stefani, A., et al. (2013). Whole-cell patch-clamp recording of voltage-sensitive Ca21 channel currents in single cells: heterologous expression systems and neurones. In D. G. Lambert, & R. D. Rainbow (Eds.), Calcium signaling protocols. Methods in molecular biology (methods and protocols) (pp. 123
148). Totowa, NJ: Humana Press. Burton, A. C., & Stinson, R. H. (1960). The measurement of tension in vascular smooth muscle. The Journal of Physiology, 153, 290
305. Caday, C. G., & Steiner, R. F. (1985). The interaction of calmodulin with the carbocyanine dye (Stains-all). The Journal of Biological Chemistry, 260, 5985
5990. Erdmann, K., Grosser, N., Schipporeit, K., et al. (2006). The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. The Journal of Nutrition, 136, 2148
2152. Facemire, C. S., & Arendshorst, W. J. (2005). Calmodulin mediates norepinephrine-induced receptor-operated calcium entry in preglomerular resistance arteries. American Journal of Physiology—Renal Physiology, 289, F127
F136. Foltz, M., van der Pijl, P. C., & Duchateau, G. S. M. J. E. (2010). Current in vitro testing of bioactive peptides is not valuable. The Journal of Nutrition, 140, 117
118. Grynkiewicz, G., Poenie, M., & Tsien, R. Y. (1985). A new generation of Ca21 indicators with greatly improved fluorescence properties. The Journal of Biological Chemistry, 260, 3440
3450. Kobayashi, Y., Fukuda, T., Tanaka, M., et al. (2012). The anti-atherosclerotic di-peptide, Trp-His, inhibits the phosphorylation of voltage-dependent L-type Ca21 channels in rat vascular smooth muscle cells. FEBS Open Bio, 2, 83
88. Kumrungsee, T., Saiki, T., Akiyama, S., et al. (2014). Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses. Biochimica et Biophysica Acta, General Subjects, 1840, 3073
3078. Majumder, K., Chakrabarti, S., Morton, J. S., et al. (2013). Egg-derived tri-peptide IRW exerts antihypertensive effects in spontaneously hypertensive rats. PLoS One, 8, e82829. Mata, K. M., Li, W., Reslan, O. M., et al. (2015). Adaptive increases in expression and vasodilator activity of estrogen receptor subtypes in a blood vessel-specific pattern during pregnancy. American Journal of Physiology—Heart and Circulatory Physiology, 309, H1679
H1696. Matsui, T. (2018). Are peptides absorbable compounds? Journal of Agricultural and Food Chemistry, 66, 393
394. Matsui, T., Wang, Z., & Tanaka, M. (2011). Vascular regulation by small peptides. In G. Brahmachari (Ed.), Bioactive natural products (pp. 201
221). World Scientific Publishing Co Pte Ltd. Miguel, M., Manso, M. A., Lo´pez-Fandin˜o, R., et al. (2007). Vascular effects and antihypertensive properties of κ-casein macropeptide. International Dairy Journal, 17, 1473
1477. Schaub, M. C., & Heizmann, C. W. (2008). Calcium, troponin, calmodulin, S100 proteins: From myocardial basics to new therapeutic strategies. Biochemical and Biophysical Research Communications, 369, 247
264. Shen, W., & Matsui, T. (2017). Current knowledge of intestinal absorption of bioactive peptides. Food & Function, 8, 4306
4314.

274 Chapter 11 Tanaka, M., Matsui, T., Ushida, Y., et al. (2006). Vasodilating effect of di-peptides in thoracic aortas from spontaneously hypertensive rats. Bioscience, Biotechnology, and Biochemistry, 70, 2292
2295. Tanaka, M., Tokuyasu, M., Matsui, T., et al. (2008). Endothelium-independent vasodilation effect of di- and tri-peptides in thoracic aorta of Sprague-Dawley rats. Life Sciences, 82, 869
875. Villain, M., Jackson, P. L., Manion, M. K., et al. (2000). De novo design of peptides targeted to the EF hands of calmodulin. The Journal of Biological Chemistry, 275, 2676
2685. Wang, Z., Watanabe, S., Kobayashi, Y., et al. (2010). Trp-His, a vasorelaxant di-peptide, can inhibit extracellular Ca21 entry to rat vascular smooth muscle cells through blockade of dihydropyridine-like L-type Ca21 channels. Peptides, 31, 2060
2066. Zeng, C., Wang, D., Yang, Z., et al. (2004). Dopamine D1 receptor augmentation of D3 receptor action in rat aortic or mesenteric vascular smooth muscles. Hypertension (Dallas, TX 1979), 43, 673
679. Zhao, J., Suyama, A., Tanaka, M., et al. (2014). Ferulic acid enhances the vasorelaxant effect of epigallocatechin gallate in tumor necrosis factor-alpha-induced inflammatory rat aorta. The Journal of Nutritional Biochemistry, 25, 807
814.

CHAPTER 12

Methodologies for studying mechanisms of action of bioactive peptides: a multiomic approach Hua Zhang1 and Yoshinori Mine2 1

Department of Food Nutrition and Safety, College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, P.R. China, 2Department of Food Science, University of Guelph, Guelph, ON, Canada

12.1 Introduction Plenty of dietary peptides and amino acids (AAs) are released from food proteins through digestion process. AAs are subsequently utilized by cells as basic building blocks of proteins that are nutrient and energy constraints for the benefit of maintaining living systems. Nonetheless, peptides composed by a sequence of AAs are inactive within their parent protein molecules derived from food resources. Peptides are commonly generated by gastrointestinal digestion, fermentation with proteolytic starter culture, or hydrolysis (Korhonen & Pihlanto, 2006). The therapeutic proteins derived from functional foods attribute to the specific sequence and molecular derivatives liberated during the metabolism or hydrolysis. Emerging findings suggest that dietary peptides liberated from a variety of their protein resources, including plants, sea algae, and animals, have specific bioactivities (e.g., antimicrobial, immunoregulatory, antihypertensive, and antioxidant) and exert beneficial effects and therapeutic potential on body, beyond their nutritional value (Agyei & Danquah, 2012; Wang, Huang, Kong, & Xu, 2018). In recent decades, researchers, consumers, and food manufacturers have been drawn to their health-promoting claims and functionalities (Agyei & Danquah, 2012). In this context the dietary peptides have been incorporated as ingredients in functional foods, nutraceuticals, and pharmaceuticals are used for diseases’ prevention. However, nowadays, some existing limitations impede the exploitation of therapeutic dietary peptides to enhance human health. Despite the significant therapeutic effects of dietary peptides being identified in cell or animal studies, there is still a lack of in-depth

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00026-1 © 2021 Elsevier Inc. All rights reserved.

275

276 Chapter 12 mechanistic demonstration of the way those dietary peptides interact with the molecular target to possess their regulatory action. Hajfathalian summarizes the bioactivity and functionality of dietary peptides derived from various food resources (Hajfathalian, Ghelichi, Garcı´a-Moreno, Moltke Sørensen, & Jacobsen, 2018). To illustrate it, different dietary peptides have been determined with the high ACE inhibitory activity, but the structure
activity relationship has not been fully defined. Moreover, the immunomodulatory effects of dietary peptides are found to promote lymphocyte activity and downregulate pro-inflammatory cytokine secretion, inhibit microbial growth, and inactivate virus by interfering in the reproductive cycle, whereas the related molecular basis underlying the above actions are unknown. It is necessary for the full exploitation of dietary peptides to enhance human nutrition and health. In recent decades, numerous omics approaches (e.g., trancriptomics, proteomics, and metabolomics) have been widely used in the research of nutrition and dietary intervention. Recently, scientists devote themselves to exploiting an integrated omics research through combining multiple omics analysis, to understand the molecular basis of health-promoting role of food components. Based on that, the concept of nutrigenomics is established to explore the epigenomic modification of nutrients or food bioactivities on host genome (Kato, Takahashi, & Saito, 2011). Since the exact mechanisms of regulatory actions of dietary peptides are largely unknown, it is difficult to predict the fate of these peptides in the host as well as potential toxic side effects. This review aims to unravel the functionality of dietary peptides on the disease condition through the integrative analysis approaches such as molecular biology and omics approaches.

12.2 Investigation of the regulatory properties of dietary peptides in cellular signaling events Various dietary patterns, functional foods, nutrients, and bioactive components have been shown to modulate inflammatory processes within the context of disease risk and progression (Siriwardhana et al., 2013). Among them, proteins derived from foods, not only being essential nutrients but also resources for bioactive peptides, have been shown to modulate cellular signaling events such as inflammatory processes. The role of proteins naturally derived from raw food materials has noticeable physiological functions such as directly regulating humoral or cell-mediated immune functions (Andersen, 2015). Some of the above functions were triggered upon cellular sensing the external signal. In this context, the external signals can be a bioactive protein or peptide sequence derived from it which interacts with a molecular target to trigger the subsequent signaling cascade. Unlike the parent protein, uptake of the small peptides derived from enzymic hydrolysate occurs in the intestine via intestinal peptide transporters, and these peptides subsequently enter the circulation system to exert physiological actions.

Studying mechanisms of action of bioactive peptides 277

12.2.1 In silico approach for characterizing bioactive peptides Thus it is important to identify bioactive peptides from their parent food protein materials. In the digestion process, the enzyme hydrolysis attributes to the modification of the physicochemical properties of resulting peptides. The structure and biological function are primarily affected by the liberated primary sequence, thus determining the biological properties of peptide products (Korhonen & Pihlanto, 2006). The in silico peptide research is the prerequisite for predicting the functional peptide from food protein materials. In this book, the application of in silico approach in bioactive peptides sequences has been well demonstrated. In addition to the conventional protein sequence database, the BIOPEP-UWM database has gained attention from research for the study of bioactive peptides. The BIOPEP-UWM can help to characterize the presence of bioactive fragments in the protein sequences and provide information on the functionality of a given dietary peptide. Moreover, BIOPEP-UWM becomes a tool for converting amino acid sequences into simplified molecular input line entry system (SMILES) code, which is a chemical notation allowing to study the structures and properties of peptides and peptidomimetics (Minkiewicz, Iwaniak, & Darewicz, 2019). For the in silico study, SMILES code of a detected peptide like an intermediate that implements further bioinformatic analyses for understudy the potential interaction with therapeutic target. Several tools have been applied to predict the therapeutic target based on the chemical property such as Swiss Target Prediction (STITCH), therapeutic target database, and DisGeNET which is a platform for studying the molecular target related with human disease at a gene level. The outcome of database or platforms is to generate a chemical language based on the biological sequences that are available to conduct an integrated omics analysis.

12.2.2 In silico approach for investigation of the interaction between bioactive peptides and molecular target The SMILES code of a dietary peptide received from above can be implemented to investigate the interaction with a potential protein target via molecular docking. To validate a peptide-mediated signaling transduction, the interactive activation or binding between the tested peptide and molecular target must be confirmed. The molecular docking is an in silico approach used to quantitatively study a structure
activity relationship between predicted targets and drugs by evaluating the binding energy, which helps to determine their binding potential and agonistic efficacy for evoking a target signaling cascade at a cellular level, as summarized in Table 12.1 (Jiang, Yan, He, & Ma, 2018; Wang, Chen, Fu, Li, & Wei, 2017). The stereochemical structure of selected molecular target needs to be obtained from www.rcsb.org, to process the molecular docking study. Recently, a novel ACE inhibitory peptide Tyr-Ser-Lys from rice bran protein has been identified via molecular docking study (Wang et al., 2017). Furthermore, the molecular docking approach was applied to reveal that two milk protein
derived Ile-Pro-Ile and Trp-Pro inhibit the activity

278 Chapter 12 Table 12.1: Summary of identified dietary peptides interacting with molecular target by molecular docking. Food product

Protein fraction

Soybean

Fraction C-III-2a

Ruditapes philippinarum Bovine milk Soy and lupin

Chlorella vulgaris Cyclina sinensis Phaseolus vulgaris L. Rice Bovine milk

Oncorhynchus mykiss nebulin Camel milk protein Smoothhound viscera proteins C. vulgaris Fermented camel milk

Casein

CSH-I Nondigestible fraction RBPH hydrolysae Fraction G2-2

Hydrolysate A9 Fraction IV and V

Fraction ,3 kDa

Identified peptide

Molecular target

Ref.

Gly-Ser-Arg, Glu-Ala-Lys

α-Glucosidase

Jiang et al. (2018)

AGDDAPR, LAPSTM, FAGDDAPRA, and FLMESH VPYPQ IAVPTGVA, YVVNPDNNEN, LTFPGSAED, LILPKHSDAD, GQEQSHQDEGVIVR VPW and IPR

DPP-IV

Liu et al. (2017)

DPP-IV DPP-IV

Zheng et al. (2019) Lammi, Zanoni, Arnoldi, and Vistoli (2016) Zhu et al. (2017)

DPP-IV

TPMGP GLTSK and GEGSGA

ACE ACE

TSL

ACE

VLPVPQ and VAPFPE

ACE

EGF, HGR, and VDF

ACE

WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR IAGPPGSAGPAG, VVPFEGAV, PLPKRE, and PTVPKRPSPT

Cholesterol esterase ACE

TTP and VHP MVPYPQR

ACE ACE

Yu, Zhang et al. (2018) Gao, Gong, and Mao (2020) Wang et al. (2017) Chen, Shangguan, Bao, Shu, and Chen (2020) Yu, Fan et al. (2018) Mudgil et al. (2019) Abdelhedi et al. (2017)

Xie et al. (2018) Soleymanzadeh, Mirdamadi, Mirzaei, and Kianirad (2019)

DPP, Dipeptidyl peptidase; RBPH, rice bran protein hydrolysate; CSH, Cyclina sinensis.

of dipeptidyl peptidase IV (DPP-IV) (Nongonierma, Mooney, Shields, & FitzGerald, 2014). Recently, the molecular docking analysis was exploited to screen and reveal the peptides with α-glucosidase inhibitory activity from the soybean protein hydrolysate, and those peptides may be novel functional food ingredient owing to potential hypoglycemic efficacy (Jiang et al., 2018). The emerging evidence clearly explicates that the molecular docking is an effective computational and experimental approach to select peptides acting as a molecular activator or inhibitor based on their structure
activity relationship. The in-depth study of allosteric mechanisms of regulating cellular signaling transduction upon the dietary peptide activation is the prerequisite for understanding of structure
activity relationship of dietary peptide acting as a molecular activator or inhibitor rather than evaluating intracellular signaling downstream.

Studying mechanisms of action of bioactive peptides 279

12.2.3 Exploration of the molecular basis of the dietary peptide modulating cellular signaling transduction via an integrated approach It is important to reveal the role of dietary peptides in regulation of cellular transduction to validate the functionality of dietary peptides on restoration of cellular homeostasis. As aforementioned, the stereochemistry of peptides likely plays a role in the regulation of cellular signaling events via the allosteric binding with the active site of a molecular target in the cell. To illustrate it, findings of our previous studies will be exploited to demonstrate how to reveal the regulatory role of dietary peptides at a cellular level through omics research incorporated with molecular approaches as shown in Fig. 12.1. To illustrate this point, we will use the results of previous studies to reveal the regulatory role of dietary peptides at the cellular level through omics studies and molecular approaches. A series of glutamate-based di- or tri-peptides, recognized as taste enhancer peptides, are capable of activating calcium-sensing receptor (CaSR) by allosteric binding (Maruyama, Yasuda, Kuroda, & Eto, 2012; Ohsu et al., 2010). Among them, the γ-glutamyl cysteine (EC) was supplemented in a dextran sulfate sodium (DSS)-treated mouse, and results of this study indicated that γ-EC mitigated the DSS-induced colitis damage and improved the colonic barrier integrity. The microarray was subsequently subjected to explore the epigenomic modification caused by γ-EC supplementation on DSS-treated mouse model. The findings of biological network analysis detected the core molecular targets were regulated by γ-EC were c-Jun N-terminal kinases, mitogen-activated protein kinase p38 and nuclear factor

In-silico

Molecular mechanism analysis

Characterizing bioactive peptides

Bioactivity

Bioactive peptide sequencing database

Cell model

SMILES code

Molecular target

Molecular target Signaling transduction Molecular docking

Figure 12.1 Flow diagram for employing the incorporation of multiomics and molecular biology approaches to explore the functionality and cellular modulatory action of dietary bioactive peptides.

280 Chapter 12 kappa-B (NF-κB), suggesting the γ-EC mediated CaSR intracellular signaling cascades potentially block the inflammatory signaling pathway thereby suppressing inflammatory response (Fig. 12.2). These γ-glutamyl peptides were identified as novel dietary peptides, exerting notable immune-regulatory or antiinflammatory effects on the human gut system. Thereafter, the molecular basis was the prerequisite for understanding the γ-glutamyl dipeptides allosteric mechanism to interfere with the inflammatory signaling transduction in the intestinal epithelial cells. Tissue such as intestine can develop the inflammatory response to either external (e.g., pathogens and endotoxins) or host-derived (e.g., TNF-α) cues. The latter one was found a main attributor for pathogenesis of IBD (Papadakis & Targan, 2000). As γ-glutamyl dipeptides being allosteric agonists to CaSR, it is necessary to investigate the possible crosstalk between TNF-α and CaSR-mediated pathways. CaSR is a prototypical class C G protein
coupled receptor (GPCR) that maintains the cellular calcium homeostasis. It has been found GPCRs display diverse role in shaping immune responses (Sun & Richard, 2012). Upon treatment of γ-glutamyl dipeptides, an increase of releasing intracellular calcium was identified in the intestinal Caco-2 cells that validated the agonistic activity of γ-glutamyl peptides to CaSR. The subsequent finding indicated that β-arrestin-2 recruitment resulted from the allosteric activation of CaSR in response to γ-glutamyl peptides stimulation. β-Arrestins belong to scaffold proteins capable of bridging different signaling pathways to collaboratively regulate the cellular signaling network. Owing to dietary γ-glutamyl dipeptides activating CaSR in the inflamed intestinal cells, β-arrestin-2 was recruited to interact with transforming growth factor (TGF)-β kinase (TAK)-1 binding protein (TAB)-1, therefore blocking the TNF-α-activated transduction of pro-inflammatory NF-κB pathway (Fig. 12.2) (Zhang, Kovacs-Nolan, Kodera, Eto, & Mine, 2015). Nonetheless, intestinal uptake of γ-glutamyl valine (EV) was recently reported, and after entering the gut, γ-EV triggered CaSR-mediated signaling cascade to suppress TNF-α-induced inflammation in endothelial cells (Guha, Paul, Alvarez, Mine, & Majumder, 2020). Being proteolytic-resistant peptides, intestinal uptake of dietary γ-glutamyl dipeptides exert potential therapeutic action, after being delivered by the circulation and distributed to different tissues. According to the finding from the above microarray study in DSSinduced mouse model, γ-EC was shown to attribute to Wnt signaling pathway. CaSR has the ability to sense and respond to nutritional cues to trigger the noncanonical Wnt signaling cascades, which suggests a potential therapeutic role in modulating adipocyte function (Bravo-Sagua, Mattar, Dı´az, Lavandero, & Cifuentes, 2016). Wnt signaling is implicated in the integration of nutritional signaling cascades that attributes to metabolic homeostasis, suggesting that a dysregulated Wnt compromises metabolic state to increase the risk of developing chronic metabolic diseases. In this context, the role of γ-glutamyl dipeptides in regulating the core of Wnt controlled metabolic pathways in mouse adipocyte 3T3-L1 cells was explored. Observations of the present study demonstrated

Figure 12.2 Progressive complexity of in silico model and integrated molecular techniques made it feasible to understand the molecular mechanism underlying dietary peptide-based intervention. (A) Supplementation of γ-EC prevent DSSinduced colonic tissue damage by suppressing TNF-α-activated pro-inflammatory signaling events; (B) the molecular docking study provides visualized evidence to demonstrate that γ-EC can activate CaSR-mediated signaling by interacting with CaSR binding pocket; (C) Upon treatment with γ-EC, internalization of β-arrestin-2 occurs to interact with TAB1 to block formation of TAK1
TAB1 complex, leading to mitigation of TNFα-mediated inflammation. CaSR, Calcium-sensing receptor; DSS, dextran sulfate sodium; γ-EC, γ-glutamyl cysteine.

282 Chapter 12 γ-EV-induced CaSR activation not only prevents TNF-α-induced inflammation but also modulates the crosstalk between Wnt and PPARγ pathways in adipocytes, which leads to restore the adipose metabolic homeostasis and insulin sensitivity (Xing, Zhang, Majumder, Zhang, & Mine, 2019). Our finding validated the dietary γ-glutamyl dipeptides being specific allosteric agonist to CaSR, which displays a strong regulatory efficiency to maintain cellular homeostasis through the integrated and coordinated function of signaling crosstalk, leading to preventing metabolic dysfunction. These above researches demonstrate that the integration of omics data and experimental analysis of contemporary molecular biology provides an insight of mechanistic modeling of bioactive peptide interventions.

12.3 Conclusion Taking together, the establishment of an incorporative assay by employing multidisciplinary omics and biochemical approaches is the prerequisites for screening the therapeutic dietary peptides and understanding the molecular basis of their health-promoting actions. Emerging evidence has clearly illustrated the omics approaches, including proteomics, nutrigenomics, and metabolomics, are effective strategies for exploring therapeutic characteristics of novel dietary peptides prepared from food proteins. A variety of molecules widely distributed along the cell membrane or in cytoplasm is able to sense and respond to nutritional cues that are implicated in sustaining fundamental activity of living organisms. These molecules are associated with the plasma membrane and different cellular compartments in addition to modulating signaling cascades. As aforementioned, the integrated pathway analysis based on the result obtained from microarray study provides information on the feature pathway being regulated by a dietary peptide that helps us to identify molecular targets. And then, in silico approaches, mostly at computational aspect, are going to be employed for illustration of the structure
activity relationship between predicted molecular targets and a known dietary peptide. This finding will be further validated at a cellular level. As we illustrated earlier, biochemical experimental approaches need to be incorporated with omics approach for investigating the integrated relationship among multiple signal networks. For instance, a specific antagonist of CaSR NPS-2143 had been used to verify the γ-glutamyl peptide
mediated CaSR activation, and gene silencing approaches were also used to validate an involvement of specific signaling cascade by blocking a key molecule expression. The aims of the sustainable development of novel functional foods or ingredients are not only necessary to determine the functionality of bioactive peptides but also how these peptides involve in the cell-mediated responses. Therefore lack of understanding about role of bioactive peptides in regulation of cellular homeostasis has hindered the in-depth study of health benefits of dietary peptides. Therefore incorporation of omics and molecular biology experimental approaches will contribute to further

Studying mechanisms of action of bioactive peptides 283 insightful research on functionality of dietary peptides and more importantly their molecular basis is the key to integrated evaluation of their therapeutic potency.

References Abdelhedi, O., Nasri, R., Jridi, M., Mora, L., Oseguera-Toledo, M. E., Aristoy, M.-C., . . . Nasri, M. (2017). In silico analysis and antihypertensive effect of ACE-inhibitory peptides from smooth-hound viscera protein hydrolysate: Enzyme-peptide interaction study using molecular docking simulation. Process Biochemistry, 58, 145
159. Agyei, D., & Danquah, M. K. (2012). Rethinking food-derived bioactive peptides for antimicrobial and immunomodulatory activities. Trends in Food Science & Technology, 23(2), 62
69. Andersen, C. J. (2015). Bioactive egg components and inflammation. Nutrients, 7(9), 7889
7913. Bravo-Sagua, R., Mattar, P., Dı´az, X., Lavandero, S., & Cifuentes, M. (2016). Calcium sensing receptor as a novel mediator of adipose tissue dysfunction: Mechanisms and potential clinical implications. Frontiers in Physiology, 7, 395. Chen, L., Shangguan, W., Bao, C., Shu, G., & Chen, H. (2020). Collaborative optimization and molecular docking exploration of novel ACE-inhibitory peptides from bovine milk by complex proteases hydrolysis. Artificial Cells, Nanomedicine, and Biotechnology, 48(1), 180
187. Gao, J., Gong, H., & Mao, X. (2020). Dipeptidyl peptidase-IV inhibitory activity and related molecular mechanism of bovine α-lactalbumin-derived peptides. Molecules (Basel, Switzerland), 25(13), 3009. Guha, S., Paul, C., Alvarez, S., Mine, Y., & Majumder, K. (2020). Dietary γ-glutamyl valine (γ-EV) ameliorates TNF-α induced vascular inflammation via endothelial calcium-sensing receptor (CaSR). Journal of Agricultural and Food Chemistry, 68(34), 9139
9914. Hajfathalian, M., Ghelichi, S., Garcı´a-Moreno, P. J., Moltke Sørensen, A.-D., & Jacobsen, C. (2018). Peptides: Production, bioactivity, functionality, and applications. Critical Reviews in Food Science and Nutrition, 58(18), 3097
3129. Jiang, M., Yan, H., He, R., & Ma, Y. (2018). Purification and a molecular docking study of α-glucosidase-inhibitory peptides from a soybean protein hydrolysate with ultrasonic pretreatment. European Food Research and Technology, 244(11), 1995
2005. Kato, H., Takahashi, S., & Saito, K. (2011). Omics and integrated omics for the promotion of food and nutrition science. Journal of Traditional and Complementary Medicine, 1(1), 25
30. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16(9), 945
960. Lammi, C., Zanoni, C., Arnoldi, A., & Vistoli, G. (2016). Peptides derived from soy and lupin protein as dipeptidyl-peptidase IV inhibitors: In vitro biochemical screening and in silico molecular modeling study. Journal of Agricultural and Food Chemistry, 64(51), 9601
9606. Liu, R., Zhou, L., Zhang, Y., Sheng, N.-J., Wang, Z.-K., Wu, T.-Z., . . . Wu, H. (2017). Rapid identification of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from Ruditapes philippinarum hydrolysate. Molecules (Basel, Switzerland), 22(10), 1714. Maruyama, Y., Yasuda, R., Kuroda, M., & Eto, Y. (2012). Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells. PLoS One, 7(4), e34489. Minkiewicz, P., Iwaniak, A., & Darewicz, M. (2019). BIOPEP-UWM database of bioactive peptides: Current opportunities. International Journal of Molecular Sciences, 20(23), 5978. Mudgil, P., Baby, B., Ngoh, Y.-Y., Vijayan, R., Gan, C.-Y., & Maqsood, S. (2019). Identification and molecular docking study of novel cholesterol esterase inhibitory peptides from camel milk proteins. Journal of Dairy Science, 102(12), 10748
10759. Nongonierma, A. B., Mooney, C., Shields, D. C., & FitzGerald, R. J. (2014). In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors. Peptides, 57, 43
51.

284 Chapter 12 Ohsu, T., Amino, Y., Nagasaki, H., Yamanaka, T., Takeshita, S., Hatanaka, T., . . . Eto, Y. (2010). Involvement of the calcium-sensing receptor in human taste perception. Journal of Biological Chemistry, 285(2), 1016
1022. Papadakis, K. A., & Targan, S. R. (2000). Role of cytokines in the pathogenesis of inflammatory bowel disease. Annual Review of Medicine, 51(1), 289
298. Siriwardhana, N., Kalupahana, N. S., Cekanova, M., LeMieux, M., Greer, B., & Moustaid-Moussa, N. (2013). Modulation of adipose tissue inflammation by bioactive food compounds. The Journal of Nutritional Biochemistry, 24(4), 613
623. Soleymanzadeh, N., Mirdamadi, S., Mirzaei, M., & Kianirad, M. (2019). Novel β-casein derived antioxidant and ACE-inhibitory active peptide from camel milk fermented by Leuconostoc lactis PTCC1899: Identification and molecular docking. International Dairy Journal, 97, 201
208. Sun, L., & Richard, D. Y. (2012). Role of G protein-coupled receptors in inflammation. Acta Pharmacologica Sinica, 33(3), 342
350. Wang, X., Chen, H., Fu, X., Li, S., & Wei, J. (2017). A novel antioxidant and ACE inhibitory peptide from rice bran protein: Biochemical characterization and molecular docking study. LWT, 75, 93
99. Wang, Y., Huang, Q., Kong, D., & Xu, P. (2018). Production and functionality of food-derived bioactive peptides: A review. Mini Reviews in Medicinal Chemistry, 18(18), 1524
1535. Xie, J., Chen, X., Wu, J., Zhang, Y., Zhou, Y., Zhang, L., . . . Wei, D. (2018). Antihypertensive effects, molecular docking study, and isothermal titration calorimetry assay of angiotensin I-converting enzyme inhibitory peptides from Chlorella vulgaris. Journal of Agricultural and Food Chemistry, 66(6), 1359
1368. Xing, L., Zhang, H., Majumder, K., Zhang, W., & Mine, Y. (2019). γ-Glutamylvaline prevents low-grade chronic inflammation via activation of a calcium-sensing receptor pathway in 3T3-L1 mouse adipocytes. Journal of Agricultural and Food Chemistry, 67(30), 8361
8369. Yu, F., Zhang, Z., Luo, L., Zhu, J., Huang, F., Yang, Z., . . . Ding, G. (2018). Identification and molecular docking study of a novel angiotensin-I converting enzyme inhibitory peptide derived from enzymatic hydrolysates of Cyclina sinensis. Marine Drugs, 16(11), 411. Yu, Z., Fan, Y., Zhao, W., Ding, L., Li, J., & Liu, J. (2018). Novel angiotensin-converting enzyme inhibitory peptides derived from Oncorhynchus mykiss nebulin: Virtual screening and in silico molecular docking study. Journal of Food Science, 83(9), 2375
2383. Zhang, H., Kovacs-Nolan, J., Kodera, T., Eto, Y., & Mine, Y. (2015). γ-Glutamyl cysteine and γ-glutamyl valine inhibit TNF-α signaling in intestinal epithelial cells and reduce inflammation in a mouse model of colitis via allosteric activation of the calcium-sensing receptor. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1852(5), 792
804. Zheng, L., Xu, Q., Lin, L., Zeng, X.-A., Sun, B., & Zhao, M. (2019). In vitro metabolic stability of a casein-derived dipeptidyl peptidase-IV (DPP-IV) inhibitory peptide VPYPQ and its controlled release from casein by enzymatic hydrolysis. Journal of Agricultural and Food Chemistry, 67(38), 10604
10613. Zhu, Q., Chen, X., Wu, J., Zhou, Y., Qian, Y., Fang, M., . . . Wei, D. (2017). Dipeptidyl peptidase IV inhibitory peptides from Chlorella vulgaris: In silico gastrointestinal hydrolysis and molecular mechanism. European Food Research and Technology, 243(10), 1739
1748.

CHAPTER 13

CRISPR
Cas systems in bioactive peptide research Khushwant S. Bhullar1,2, Nan Shang1 and Jianping Wu1 1

Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada, 2Department of Pharmacology, University of Alberta, Edmonton, AB, Canada

13.1 Introduction The genome serves as the blueprint of life, setting the stage for all downstream biological activity. Scientists have endeavored to targeted genome manipulation since the advent of the central dogma of life (Jansen, Embden, Gaastra, & Schouls, 2002; Jinek et al., 2012). The precise editing of genetic information is vital to understanding the physiological and pathological function of a given gene (Gasiunas, Barrangou, Horvath, & Siksnys, 2012). During the past decade, a breakthrough in the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein), emerged as a highly efficient and specific genome editing system, has changed the field of genome engineering (Cong et al., 2013; Jinek et al., 2013; Mali, Esvelt, & Church, 2013). Because of the ease and efficiency of the system, it has been widely adopted and developed, leading to the emergence of a powerful molecular toolbox. Arising primarily from Watson
Crick base pairing between its guide RNA and the target DNA site, Cas9 is an RNA-guided nuclease that exhibits high sequence specificity, in addition to direct interaction with a short protospacer adjacent motif (PAM) of DNA (Bolotin, Quinquis, Sorokin, & Ehrlich, 2005; Marraffini & Sontheimer, 2008). An assortment of CRISPR
Cas systems is an evolutionary adaptive immune mechanism present in many bacteria and archaea. It acts as a prokaryotic adaptive immunity mechanism and helps to cleave invading nucleic acids (Hsu, Lander, & Zhang, 2014; Zhang, Wang, Liu, & Li, 2017). CRISPR-containing microorganisms acquire DNA fragments from invading phages and plasmids before transcribing them into CRISPR RNAs (crRNAs) to guide cleavage of the RNA or DNA of the invading species (Barrangou et al., 2007; Zhang et al., 2017). The CRISPR works via the collaboration of many diverse Cas proteins, broadly divided into two major classes, that is, class 1 systems (types I, III, and IV), which use multiple Cas proteins, and the class 2 systems (type II, putative types V), which use single Cas protein (Makarova et al., 2015;

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00015-7 © 2021 Elsevier Inc. All rights reserved.

285

286 Chapter 13 Zetsche et al., 2015). Overall, six CRISPR
Cas types and at least 29 subtypes have been identified. Most Cas proteins bind to nucleic acids; thus the CRISPR system can form the basis of a flexible genomic engineering toolkit (Spilman et al., 2013). Cas target cleavage is managed by a duplex of two RNAs (1) the crRNA that recognizes the invading DNA through an approximately 20 base pair (bp) Watson
Crick base-pairing region and (2) the Cas9 trans-activating CRISPR RNAs (tracrRNAs) which hybridizes with the crRNA and is unique to the type II CRISPR system (Hale et al., 2009). Briefly, the CRISPR gene editing system works in three stages to carry out a full immune response to invading foreign DNA (Bondy-Denomy & Davidson, 2014; Brouns et al., 2008; Garneau et al., 2010; Hsu et al., 2014; Rath, Amlinger, Rath, & Lundgren, 2015). In the acquisition stage, DNA fragments of organisms are unified into the host CRISPR locus as spacers between crRNA repeats. In the next stage, the CRISPR array comprising acquired spacers is transcribed into precursor CRISPR RNA (pre-crRNA) various once the Cas proteins are expressed, and then, the precrRNA is cleaved and processed into mature crRNAs by Cas proteins and host factors (Deltcheva et al., 2011). The fully processed crRNA containing a spacer sequence that targets invading genome allows for the recognition of the crRNA by Cas proteins and other RNA components (Jinek et al., 2012). In the final stage, Cas proteins recognize the appropriate target with the help of the crRNA and mediate the cleavage of the genome (Hsu et al., 2014). It is vital to mention that the action of CRISPR editing systems also depends on the presence of a sequence-specific PAM that is adjacent to the crRNA target site in the invading genome as the absence of this PAM sequence at the CRISPR locus in the host genome abolishes the genome editing (Bolotin et al., 2005; Hsu et al., 2014; Mojica, Dı´ez-Villasen˜or, Garcı´a-Martı´nez, & Almendros, 2009; Shah, Erdmann, Mojica, & Garrett, 2013). CRISPR-based technologies are now being used in assorted ways to advance medicine and offer unique treatments for different pathologies. As the CRISPR
Cas expands, it spurs further research, which continues to provide additional research and development opportunities. Herein, we briefly review CRISPR
Cas systems and the remarkable biotechnological development of CRISPR
Cas systems. The current research on CRISPR systems stands poised to transform many areas of biological research, including mechanistic research and development in the area of nutraceuticals such as bioactive peptides and related molecules.

13.2 Timeline and development of CRISPR
Cas system CRISPR
Cas systems have evolved as the key adaptive bacterial and archaeal immune systems (Makarova et al., 2015). Working through three general phases of operation, that is, adaptation, crRNA processing, and interference, CRISPR
Cas defends microbes from the invasion of bacteriophage genomes (Marraffini & Sontheimer, 2010). However, there are several variations and diversities in the CRISPR theme, including systems target either or both DNA and RNA; cleaving their targets through distinct mechanisms (Jinek et al., 2012;

CRISPR
Cas systems in bioactive peptide research 287 Marraffini & Sontheimer, 2010; Shmakov et al., 2017). The story of CRISPR
Cas begins in 1987 as a series of regularly interspaced repeats of unknown function, that was observed in the genome of E. coli (Ishino, Shinagawa, Makino, Amemura, & Nakata, 1987). In the same year, following the first report of a CRISPR array, targeted gene insertion via homologous recombination in mice was reported as well, initiating the area of gene modification (Doetschman et al., 1987; Thomas & Capecchi, 1987). Moving along, in the year 2002, a bioinformatics study reported the presence of conserved operons, now known as Cas and CRISPR genes (Jansen et al., 2002; Makarova, Aravind, Grishin, Rogozin, & Koonin, 2002). Further, in 2005 the matched sequences in phage genomes and CRISPR repeats suggested that CRISPR arrays could be involved in immunity against the akin phages (Mojica, Garcı´a-Martı´nez, & Soria, 2005; Pourcel, Salvignol, & Vergnaud, 2005). Likewise, a parallel work on Streptococcus thermophilus also found phage-associated sequences now known as Cas9 (Bolotin et al., 2005; Pourcel et al., 2005). However, at this time, despite the known linkage between CRISPR
Cas and phage infection, the specific function of CRISPR
Cas systems remained ambiguous. Later, studies in S. thermophilus established CRISPR
Cas as a microbial adaptive immune system and related the CRISPR array spacers with acquired and specific immunity against phages carrying matching sequences, possibly via RNA interference (RNAi)-like mechanism (Barrangou et al., 2007; Brouns et al., 2008; Makarova, Grishin, Shabalina, Wolf, & Koonin, 2006). Subsequent studies reported that CRISPR
Cas systems (type III-A) targeting DNA or RNA, emphasizing the distinctions between CRISPR and Cas systems, highlighting the substantial mechanistic differences between CRISPR and Cas systems (Deltcheva et al., 2011; Garneau et al., 2010; Hale et al., 2009; Marraffini & Sontheimer, 2008; Zhang, 2019). Successive studies reporting discovery PAM, an essential sequence for Cas9-mediated interference, blunt double-strand breaks (DSBs), tracrRNA (Deveau et al., 2008; Mojica et al., 2009). These groundbreaking studies together proved that the Cas9 system contains three components, that is, Cas9, crRNA, and tracrRNA accompanied by a DNA target site which is required to be flanked by the appropriate PAM. By 2011, it was clear that Cas9 could cleave DNA in bacterial cells when directed by a crRNA (Garneau et al., 2010); however, the extension of this finding remained unexplored in the context of eukaryotic cells. A wellplanned strategy to use a three-component Cas9 system (Cas9, crRNA, and tracrRNA) to achieve genome editing in eukaryotic cells was employed (Cong et al., 2013). This phase of research demonstrated breakthroughs such as the elucidated mechanism of Cas9 in vitro (Jinek et al., 2012), later confirmed in S. thermophilus (Gasiunas et al., 2012), the discovery of multiple isoforms of the tracrRNA (Deltcheva et al., 2011). Following the validation of Cas9-mediated genome editing in eukaryotic cells, the field of genome editing marched ahead at an astonishing rate. Several improvements and extensions of the CRISPR
Cas technology were reported in quick succession. Success was achieved in multiple eukaryotic model organisms in a year or so as multiple eukaryotes including yeast (Zhang, 2019), mice (Wang et al., 2013), Drosophila (Gratz et al., 2013), C. elegans (Friedland et al., 2013), and

288 Chapter 13 nonhuman primates were edited successfully (Niu et al., 2014). Open sharing culture and rapidly available molecular tools of the CRISPR field have fired the field and allowed it to flourish.

13.3 Beyond Cas9 Natural diversity has given unique technologies, such as restriction enzymes and fluorescent proteins (Loenen, Dryden, Raleigh, Wilson, & Murray, 2014; Rodriguez et al., 2017); therefore further exploration of nature’s array has helped expand this unique and elegant system as well. By mining, the microbial novel subtypes of CRISPR
Cas systems such as Staphylococcus aureus Cas9 (SaCas9), Cas12, and Cas13 have been discovered and systematically developed to expand the CRISPR toolbox (Burstein et al., 2017; Harrington et al., 2018; Konermann et al., 2018; Shmakov et al., 2015; Smargon et al., 2017; Yan et al., 2019; Zetsche et al., 2015). The discovery and development of additional Cas systems that recognize different PAM sequences and have diverse modus operandi have provided a greater choice of target sites. First, Cas9 ortholog from Staphylococcus aureus (SaCas9, PAM 50 -NNGRRT) is a unique subset of Cas machinery. It has demonstrated the highest levels of activity in human cells and can be delivered along with a guide RNA, on a single-adeno-associated virus (AAV) vector for in vivo application (Ran et al., 2015). It is vital to mention that SaCas9 is now being applied as the first in vivo genome editing medicine for humans and other trials are focused on electroporation of patient cells ex vivo (NCT03872479, NCT03745287, and NCT03655678). Second, the next discovery marked the emergence of a new type of class 2 CRISPR
Cas system, Cas12a, a distinct enzyme unrelated to Cas9, in the genomes of Prevotella and Francisella and contained a large protein of unknown function (Schunder, Rydzewski, Grunow, & Heuner, 2013; Vestergaard, Garrett, & Shah, 2014). Experiments showed that expression of CRISPR
Cas12a locus in E. coli led to the obstruction of plasmid DNA transformation, establishing it as a novel CRISPR
Cas (Zetsche et al., 2015). Unlike Cas9, the Cas12a is a more diverse family, devoid of tracrRNA and has RNase activity, which may be beneficial for the editing/ introduction of new sequences (Fonfara, Richter, Bratoviˇc, Le Rhun, & Charpentier, 2016; Zetsche et al., 2015). Interestingly, it is appropriate for multiplex genetic manipulation as multiple guide RNAs (gRNAs) can be easily expressed as a single transcript and subsequently processed into individual guide RNAs by Cas12a itself (Zetsche et al., 2017). The next subset of the Cas12b effectors target DNA, but contrary to Cas12a, are dual-RNA guided, require a tracrRNA, and exhibit reduced off-target activity, compared to SpCas9 (Strecker et al., 2019; Teng et al., 2018). Extensive research in Cas12b systems has revealed two systems with robust genome editing activity in human cells (Strecker et al., 2019; Teng et al., 2018). Other members of the Cas12 family include Cas12c, Cas12d, Cas12e, three Cas12f subtypes, two Cas12e orthologs (DpbCasX and PlmCasX), and other functionally diverse type V CRISPR
Cas systems (Burstein et al., 2017; Harrington et al.,

CRISPR
Cas systems in bioactive peptide research 289 2018; Liu et al., 2019; Shmakov et al., 2015; Yan et al., 2019). Third, the subsequent expansion of the search to use CRISPR repeats led to the identification of Cas 13 and its subtypes, including Cas13b, Cas13c, and Cas13d systems, and adapted for use in mammalian cells to mediate targeted RNA knockdown (Cox et al., 2017; Shmakov et al., 2017; Smargon et al., 2017; Yan et al., 2018). Overall, there is a tremendous variation and diversity in the CRISPR
Cas systems and behavior of Cas effectors owing to disparities among biochemical, bacterial, and mammalian environments.

13.4 Advancing biological research The advent of genome editing via CRISPR
Cas has drastically altered the biological research. This system has helped to create knockout (KO) cell types, including mammalian cell lines, induced pluripotent stem cells, cancer-specific organoids, and primary immune cells (Grobarczyk, Franco, Hanon, & Malgrange, 2015; Matano et al., 2015; Schumann et al., 2015). The CRISPR
Cas system has helped researchers to test the hypothesis of synthetic lethality and edit genomes in the cell type that is most appropriate for the disease of interest (Kasap, Elemento, & Kapoor, 2014; Shi et al., 2015). Similarly, “knocking in” mutant alleles by homology-directed repair (HDR) allows investigators to assess the effects of disease-associated mutations in an isogenic background (Fig. 13.1) (Gratz et al., 2014). Cas plays a vital role in the success of HDR system as it requires delivery of the Cas9
single guide RNA (sgRNA) complex in the form of a viral vector or plasmid (that encodes Cas9 and the sgRNA), or Cas9
sgRNA ribonucleoprotein complexes (comprising Cas9 protein and the sgRNA) along with a DNA repair template (Paix et al., 2014; Paix, Schmidt, & Seydoux, 2016). Despite CRISPR
Cas’s high efficiency in nearly any cell, rates of HDR can differ across cell types, especially in nonmitotic human cells, such as neurons. These barriers are particularly frustrating, use of nonhomologous or microhomology-mediated integration of cassettes offer routes to bypass this difficulty (Nakade et al., 2014; Sakuma, Nakade, Sakane, Suzuki, & Yamamoto, 2016). Precise engineering of Cas enzymes with additional functionalities to enable exact and specific mutations by direct alteration of target bases have been adopted as well. Further, owing to the simplicity of designing potent sgRNAs in the CRISPR
Cas system, it has gained an edge over RNAi screens or insertional mutagenesis screens to study and screen systematic loss-of-function studies (Cheung et al., 2011; Deans et al., 2016; Fellmann & Lowe, 2014; Wang et al., 2015). Three types of CRISPR screens also aid biological research via creating phenotype specific KOs. In CRISPR nuclease (CRISPRn) screens, stably expressed Cas9
sgRNA complexes continue to operate on a target site until it is ablated and can therefore generate homozygous KO phenotypes at high frequency in most cell types (Hart et al., 2015). Similar to RNAi, CRISPR interference (CRISPRi) screens, independent of frameshifting, offer certain advantages over CRISPRn as it mimics the effects of a

290 Chapter 13

Figure 13.1 A schematic representation of the CRISPR
Cas system for gene editing. In the CRISPR genome editing system, a guide RNA hybridizes a 20-nucleotide DNA sequence immediately preceding a Protospacer Adjacent Motif sequence (PAM, NGG), resulting in a double-strand break by Cas9 nuclease 3
4 nucleotides upstream of the PAM site. The double-stranded DNA breaks (DSB) become substrates for DNA repair machinery in the cell that either catalyzes a nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair pathway, repairing DSB via a “paired end complex” which facilitates ligation of DNA breaks, whereas HDR requires homologous DNA strand template for high-precision DSB repair.

small-molecule inhibitor more closely than does whole gene ablation (Mandegar et al., 2016). Finally, CRISPR activation (CRISPRa) screens, which evaluate gene targets whose overexpression leads to a particular phenotype, are an emerging and particularly exciting area of CRISPR
Cas development. Unlike, labor-intensive use of cDNA screening, owing to the complex nature of cDNAs, CRISPRa screens, like CRISPRn or CRISPRi, are much easier to perform (Gilbert et al., 2014; Konermann et al., 2015). However, CRISPRa screens have a drawback as they are subject to their own set of false negatives in case of loss-of-function mutations or missing in the cell line of interest (Kampmann, 2018). Another challenge in the application of CRISPRa screening is the activation of highly repressed genes, which can be overcome via recruitment of multiple and/or diverse transcriptional activation domains to increase the potency of gene activation (Gilbert et al., 2014; Konermann et al., 2015; Nishida et al., 2016). Overall, CRISPR screens have been used chiefly to look for growth advantages and disadvantages, leading to the identification of genes that are essential for proliferation or resistance and to identify candidate drug targets (Sidik et al., 2016).

CRISPR
Cas systems in bioactive peptide research 291 Apart from the cell-based applications, CRISPR
Cas has dramatically transformed the ability of researchers to generate animal models of disease. Indeed, shortly after their initial development, CRISPR
Cas tools have helped “go” or “no-go” decisions in a drug based on results from rapidly created modified disease model animals (Wang et al., 2013). In general, CRISPR
Cas can be employed via microinjection or simple electroporation of zygotes instead of proceeding through traditional embryonic stem cell manipulation (Chen, Lee, Lee, Modzelewski, & He, 2016; Qin et al., 2015; Wang et al., 2016). This is a vital development in two ways (1) in a single step, multiple targets can be targeted leading to the generation of double and triple mutant mice without the necessity for crossing single mutant strains and (2) genome editing in zygotes eliminates the requirement to program cells, which slows the production of mutants and acts a major barrier in research and development (Beard, Hochedlinger, Plath, Wutz, & Jaenisch, 2006; Premsrirut et al., 2011). CRISPR
Cas holds the potential to revolutionize animal model studies and drug discovery by reducing the time that is necessary to generate targeted models from years to months or even weeks. Apart from whole body genetic changes, a combination of CRISPR
Cas with viral or transposon-based vectors can introduce gene editing in, tissues, such as muscle and kidney tissues, in adult animals. This CRISPR
Cas methodology can help create disease models of particular interest and are efficiently equated with xenograft models that require immunosuppressed recipients (Sa´nchez-Rivera & Jacks, 2015; Sa´nchez-Rivera et al., 2014; Xue et al., 2014). The genetic changes with CRISPR
Cas allow investigators to precisely recapitulate disease pathogenesis in native microenvironment in vivo and tissue structure settings. This capability is and will further be transformative for drug discovery, also in preclinical models other than mice such as rats, dogs, and cynomolgus monkeys (DuPage & Jacks, 2013; Li et al., 2013; Niu et al., 2014; Sa´nchez-Rivera & Jacks, 2015; Zou et al., 2015). Of particular interest is the CRISPR
Cas guided disease models in primates, such as rhesus monkeys, further highlights how gene editing fast-track biological development but also test the efficacy and safety of therapeutic compounds (Chen et al., 2015). Another key and deeply impacted area of CRISPR
Cas technology is research in plant science and agriculture as evidenced by a revolution in plant breeding giving rise to new genotypes in a single generation (Brooks, Nekrasov, Lippman, & Van Eck, 2014; Feng et al., 2013; Xu et al., 2016; Zhang et al., 2014). Particularly tailored Cas such as Cas12a and Cas13a have proven to be particularly effective in plants (Aman et al., 2018; Zaidi, Mahfouz, & Mansoor, 2017). Complex genomes such as wheat and woody plants have been successfully edited by CRISPR
Cas, where traditional genetic editing is problematic (Bewg, Ci, & Tsai, 2018). Other successful examples include barley, citrus trees, cucumber, grape, lettuce, maize, mushroom, potato, rice, soy, tomato, and watermelon. CRISPR
Cas is being used to achieve unique traits such as drought resistance, improve yield, pathogen resistance, and efficient ripening (Schindele, Wolter, & Puchta, 2018). Together, CRISPR
Cas is changing biological research, particularly in plant biology studies, and

292 Chapter 13 holds the substantial potential to contribute to developing novel plant plants to address global food security.

13.5 Bioactive peptides and CRISPR
Cas9 Following the discovery of CRISPR
Cas9, numerous interdisciplinary approaches have aimed to adapt the CRISPR approach to different applications including food resources and products with enhanced properties. The food, nutrition, and agricultural sciences have demonstrated significant interest to utilize this technology. In this aspect, CRISPR
Cas holds strong potential for natural product development, and strong evidence is already emerging from plants, bacterial, and fungal products (Tong, Weber, & Lee, 2019). Although CRISPR
Cas has attracted much interest during the past several years, its use in natural product research is still in its infancy. This holds true in the area of bioactive peptides and related protein research as well. We already know that tangible and impactful improvements in human nutrition by bioactive peptides have been guided by advancements in molecular/genetic methodologies. One of the key challenges to the commercialization of bioactive peptide is the lack of understanding of the underlying mechanisms. Genetic KO of target genes with the RNA-guided nuclease Cas9 can provide a new and powerful tool to interrogate molecular mechanisms of bioactive peptides. However, no study has been conducted to reveal pharmacological peptide targets of bioactive peptides. Interestingly, the CRISPR system has already been successfully used in the biosynthesis of bioactives such as benzylisoquinoline alkaloids (e.g., morphine) (Alagoz, Gurkok, Zhang, & Unver, 2016), γ-aminobutyric acid (Cho et al., 2017), flavonoids (Wu, Du, Chen, & Zhou, 2015), and terpenoids (Kim et al., 2016). Bioactive peptides certainly hold strong promise as valuable functional foods/nutraceuticals in healthy diets. The novel genesis of nextgeneration bioactive peptides with enhanced quality and health-promoting functionalities is possible via the CRISPR
Cas application.

13.5.1 Generating CRISPR-guided targets for peptide-based studies in mammalian cells Bioactive peptide-based biological studies can enormously benefit from CRISPR technology as the field of the bioactive peptide(s) remains largely unexplored for their underlying mechanisms. We have recently explored CRISPR technology to investigate the role of the low-density lipoprotein receptor-related protein 1 (LRP1) in the pharmacological action(s) of ovotransferrin in osteoblasts (Shang, Bhullar, & Wu, 2020). Creating LRP2/2 KO cells helped to unveil the interaction of the protein, ovotransferrin, with LRP1 toward the proliferation of bone cells. Likewise, numerous bioactive peptides with known pharmacological efficacy can extensively benefit from CRISPR technology for the

CRISPR
Cas systems in bioactive peptide research 293 elucidation of underlying cellular mechanisms. Herein, we describe a detailed methodology for creating CRISPR
Cas9 guided KO cells.

13.6 Materials, equipment, and reagents 1. The first requirement is the sets of primers that amplify the B400
1200 bp genomic region of comprising either the Cas9 on-target site or an off-target site being assessed in the experiment. Note: For an efficient outcome, the design of the primer must ensure that the cleavage site is situated asymmetrically inside the amplified region of the genome to expedite the efficient resolution of the cleaved fragments following treatment with T7 endonuclease I. Other consumables required for the T7 endonuclease assay include: i. PCR tubes (200 μL), ii. 50 TAE buffer: 42 g Tris base in double-distilled H2O; 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA solution (pH 8.0) Adjust volume to 1 L, iii. Agarose, iv. DNA ladder, v. dNTP mix (10 mM), vi. Ethidium bromide solution (10 mg/mL), vii. Gel electrophoresis unit and related supplies, viii. MinElute PCR Purification Kit (Qiagen), ix. NanoDrop, x. PCR Purification Kit, xi. PCR thermocycler, xii. Q5 High Fidelity DNA Polymerase (B280 times higher efficacy than Taq) or similar, xiii. Sample loading buffer: formamide and glycerol (9:1), xiv. Software for densitometry analysis, xv. T7 endonuclease I, and xvi. UV imager. 2. Other additional consumables required for the Guide-Seq Analysis include: i. 6- or 12-well cell culture plate, ii. AMPure XP beads, iii. Cas9-expressing cell line, iv. Cell counter, v. Cell culture incubator, vi. Cell dissociation reagent, vii. Cell nucleofector Kit, viii. Centrifuge,

294 Chapter 13 ix. x. xi. xii. xiii. xiv. xv. xvi. xvii. xviii. xix. xx. xxi. xxii. xxiii. xxiv.

crRNA, DNeasy Blood & Tissue Kit (Qiagen), dNTP mix (10 mM), End-Repair Mixture, Lonza 4D Nucleofector Core and X-Unit, NdeI endonuclease, NEBNext Library Quant Kit for Illumina, Oligonucleotides for integration and library preparation, PBS (pH 7.4), Platinum Taq DNA Polymerase, Qubit fluorometer, T4 DNA Ligase, Taq DNA Polymerase, TE buffer, TMAC (5 M), and tracrRNA.

13.7 Protocols T7 endonuclease assay: This assay is used to get a first assessment of whether our targeting can be successful or not. As a DNA endonuclease, it recognizes and cleaves structural deformities in DNA heteroduplexes. It catalyzes the cleavage of DNA mismatches and nonβ DNA structures including Holliday junctions and cruciform leaving 30 -OH and 50 -phosphate. T7 endonuclease assay is widely used because it is easy and fast and does not need any specialized equipment. The cleaved products are run on an agarose gel and resolve to full length and/or cleavage products. The assay is sensitive to experimental conditions with reproducibility. T7 Endonuclease I assay can be performed using the following protocol: i. Gene-specific primers containing Illumina adapter overhangs diluted to 10 μM for working concentration. ii. Initiate PCR with the appropriate primers and then purify the PCR product. iii. After purification of PCR product, prepare reactions for T7 endonuclease I digestion on ice. Briefly, mix 2 μL of 10 3 NEBuffer 2 and 200 ng of purified PCR product (quantified by nanodrop) in a 200 μL PCR tube and raise the total volume to 19 μL with molecular biology grade water. iv. After annealing the DNA in PCR thermocycler, initiate reaction by adding 1 μL of T7 endonuclease I (10U) to PCR product and incubate 37 C for 15 min, followed by purification of the digested DNA using the MinElute PCR Purification Kit. v. Then, add 2 3 loading buffer to the purified DNA (1:1) and run on an agarose gel for 1 h.

CRISPR
Cas systems in bioactive peptide research 295 vi. Following running, image the digestion products using a UV imager and quantify the band intensities by ImageJ or similar software. CRISPR cloning in mammalian cells can be performed using the following protocol: i. The initial step is to anneal and phosphorylate the oligos. Resuspend the oligos at a working concentration of 100 μM in molecular grade water. ii. After preparing the reaction mixture, anneal in a PCR thermocycler under the following conditions: 37 C for 30 min; 95 C for 5 min, and then ramp down to 25 C at 5 C/min. iii. Following annealing, dilute product 1:10 in molecular grade water to yield a concentration of working concentration of 1 μM. iv. Next, ligate annealed oligos to an appropriate vector (e.g., pX330) by making 25 μL reaction mix: 50 ng circular vector, 0.5 μL annealed oligos, 2.5 μL restriction enzyme buffer [10 3 ], 2.0 μL (20 U) BbsI restriction enzyme (5000 U/mL), 2.5 μL ATP (10 mM), 0.125 μL (5 μg) BSA (20 mg/mL), 0.187 μL (750 U) T4 DNA ligase (2,000,000 U/mL), and H2O to final volume of 25 μL Then run mixture in a PCR thermocycler under following conditions: Cycles 1
20 (37 C for 5 min, 20 C for 5 min); Cycle 21 (80 C for 20 min). v. Next, transform 10 μL of DH5α E. coli cells with 1 μL of the reaction mixture and plate agar plate with 100 μg/mL ampicillin and incubate overnight at 37 C. vi. The next day, if transformation is successful, pick 3
4 colonies and inoculate into a mini-prep culture, followed by maxi-prep of the sequence-verified colony. vii. For transfection of CRISPRs into cells of interest, grow a minimum of 2 3 106 cells per CRISPR pair. Then, add 5 μg of each CRISPR/Cas9 construct (10 μg total) along with 0.5 μg of GFP expression construct. viii. Initiate transfection by choosing an appropriate method such as electroporation or a reagent-based method and incubate cells at 37 C for 72 h. ix. Using Fluorescence-Activated Cell Sorting (FACS), sort the top 2%
3% of GFP positive cells, possibly with the highest efficacy of CRISPR/Cas9 transfection. Plate the selected KO cells in 96-well round-bottom plates with 100 μL cell culture media per well.

13.8 Analysis and quality control i. Once, cells grow in the appropriate medium, validate intended genomic deletion using PCR. A typical reaction will start with the extraction of DNA and preparation of mixture: 10 μL PCR with the following components: 5 μL 2 3 PCR master mix, 0.25 μL forward primer (10 μM), 0.25 μL reverse primer (10 μM), 50 ng gDNA, and molecular grade H2O up to 10 μL. It is vital to optimize the PCR conditions for each primer pair.

296 Chapter 13 ii. Next, run samples on 2% agarose gel using TAE buffer and examine for the deletion bands. iii. Further evaluation can be conducted using an immunoblot against the relevant downstream protein or perform RT-qPCR for gene expression of the relevant gene using WT cells as control. Note: This protocol is adapted and modified according to the earlier reports (Cromwell; Guschin et al., 2010; Shang et al., 2020). Various commercial CRISPR gene KO kits such as HDR- or KN2.0-based kits are available for every human and mouse gene (some suppliers include: Origene, Genscript, Sigma, ThermoFisher, Biorad, and Addgene). For CRISPR-based animal KO models, single-step zygote injection of the CRISPR/Cas system can efficiently generate single or multiple target genome-modified animals such as pigs, mice, and monkeys (Hai, Teng, Guo, Li, & Zhou, 2014; Wang et al., 2016; Zuo et al., 2017). The step-wise protocols for zygote are available elsewhere (Doe, Brown, & Boroviak, 2018; Tro¨der et al., 2018) and can be adapted for the generation of animal KO models for bioactive peptides or related bio-medical research. As the proverbial CRISPR toolbox continues to expand, its advantages over previously used methods such as zinc-finger nucleases and transcription activator-like effector nucleases systems are now established. These include target design simplicity, rapid and flexible genetic manipulation, high efficiency (Hanna & Doench, 2020), and the ability to induce multiple mutations in multiple genes at the same time by using several gRNAs (Wang et al., 2013). The three phased use of CRISPR in “editing,” “knockout,” and “recruitment” experiments have revolutionized genomic engineering and extensively aided biological and medical research.

13.9 Ethical reflections The amazing power of CRISPR
Cas9 has impacted all areas of biology today but also raised ethical concerns, mostly with the possibility of generating heritable by germline gene editing. There are three different main ways in which CRISPR
Cas gene editing can be applied to human health research. First, as a basic research tool for use in cells or animals to help comprehend normal development, a model human disease in animal models and aid drug discovery. Second, for either ex vivo or in vivo genomic editing in somatic cells to treat or avoid pathologies. Third, for gene editing in gametes or embryos with the aim of correcting disease-causing mutations in the next generation, termed as germline gene editing (Rossant, 2018). Ever since the first genetically edited mice were made via exogenous DNA injection into zygotes, there have been thoughts around the ethics of germline alteration in humans. However, these were rather theoretical and philosophical in nature; however, in November 2018 a scientist announced the use of Cas9 to edit human embryos, creating at least two-genetically modified babies (Cyranoski, 2019).

CRISPR
Cas systems in bioactive peptide research 297 This scandalous newsflash emphasized the far-reaching ethical challenges that CRISPRbased technologies present for human society (Baltimore et al., 2015). To advance the understanding of ethical considerations, CRISPR
Cas specialists from seven countries called for a 5-year halt on the use of clinical germline editing (Lander et al., 2019). Clinical germline editing, mainly categorized as genetic correction and genetic enhancement, with genetic correction changing a rare disease-causing variant to a common variant that is nonpathological, can be described as beneficial. However, genetic enhancement relies on information about rare variants in the human population and such changes to the genome can lead to unpredictable consequences. Further, the moral dilemmas are numerous regarding the ethical considerations surrounding CRISPR-based technologies, as it enhances non-Mendelian transmission and their rapid surge. Gene drives to combat diseases by insect vectors such as malaria and Lyme disease also fall under such considerations as modified organisms may cause irreversible ecological changes (Akbari et al., 2015; CourtierOrgogozo, Morizot, & Boe¨te, 2017; Gantz et al., 2015; Hammond et al., 2016). Even in the future, when substantial improvements in our understanding of genomic editing become adequate to forecast its consequences, whether our society all together should or will adopt germline editing, still needs to be robustly discussed.

13.10 Future directions Indeed, many major leaps forward in science are arrived at tangentially, and this encourages interdisciplinary research and information analysis, that can move research in a particular field not only forward, but also sideways. Likewise, CRISPR
Cas discovery can be termed as a serendipitous encounter, and even since the imagination of the biological research community has been captured by this elegant mechanism of gene editing. The accumulation of microbial genomic sequences initially fueled the discovery of CRISPR
Cas systems, which are the backbone of this nature’s toolbox. CRISPR
Cas tools have revolutionized research and inquiry in previously intractable cells, and many model organisms ranging from simple cells to nonhuman primates and even malarial parasite. This genome editing system has accelerated functional and applied genomics to elucidate novel cellular mechanisms and accelerate drug discovery. Application of CRISPR
Cas editing to animal models has led to improved models of human disease and aided rapid stratification and accelerated development of treatments for patients. The successful development of customized treatments, including cancer-targeting T cells and reprogrammed stem cells, are attributed to CRISPR
Cas guided rapid gene editing. CRISPR
Cas mediated drug discovery allows the expansion of molecular biology protocols without the time-consuming improvement procedures. Also, the pharmacological potential of CRISPR
Cas is also tantalizing, by virtue of being adaptive, it is reprogrammable for the identification of diverse drug target substrates. Exploiting such reprogrammable editing systems can likely offer many new drug discovery platforms via recognition of metabolites, proteins, or

298 Chapter 13 patterns of protein modifications. Thus we say that CRISPR
Cas will be essential to the innovation and success of the next generation of scientific discovery including but not limited to transformational therapies and treatment paradigms. Although, CRISPR
Cas is an exciting area of unique opportunities, yet the specificity of CRISPR systems is under discussion. While CRISPR-based tools are easily programmed to target the desired sequence, they can lead to low rates of off-target editing or sequenceindependent cell cycle arrest (Aguirre et al., 2016; Munoz et al., 2016; Wang et al., 2015). Sequence-dependent off-target inclinations of CRISPR
Cas systems are best known for the Streptococcus pyogenes Cas9 (SpyCas9) enzyme, for which multiple unbiased experiments have begun to shed light on potential liabilities (Crosetto et al., 2013; Kuscu, Arslan, Singh, Thorpe, & Adli, 2014; Tsai et al., 2015). In the functioning of the SpyCas9 enzyme, the 8
10 nucleotides neighboring PAM are strictly recognized, whereas one or two mismatches may occur in the residual nucleotides (Doench et al., 2016; Hsu et al., 2013; Lin et al., 2014). As the off-target Cas sites are determined by the nuclease and the sgRNA sequence, hence, numerous algorithms have been built to calculate sgRNA efficiency and risky sgRNAs can be identified (Bae, Park, & Kim, 2014; Doench et al., 2016; Prykhozhij, Rajan, & Berman, 2016). An alternative strategy to reduce off-target events relies on sgRNA or protein engineering to enforce higher specificity, for example, includes SpyCas9 engineering to reduce off-target edits (Fu, Sander, Reyon, Cascio, & Joung, 2014; Kleinstiver et al., 2015). Other strategies to counter off-target effects may include control over the amount of active Cas9 in cells (Davis, Pattanayak, Thompson, Zuris, & Liu, 2015), the use of multiple sgRNAs, confirmation by CRISPRi or RNAi, and cDNA or CRISPRa studies. Another tactic requires two Cas9 nickases such as Cas9-D10A or Cas9-H840A2, which cleave or “nick” only a single DNA strand, thus boosting the effective target stringency (Mali et al., 2013; Ran et al., 2013). For nontherapeutic usage, such off-target effects can be compensated for with appropriate controls, but the detailed comprehension of off-target events requires to be understood for clinical application. Considering these reports, it is easy to predict how innovative engineering methodologies, combined with a comprehensive grasp of the conformational mechanisms will certainly advance editing precision. Further, the CRISPR
Cas systems throw light at the enormous potential of nature’s own evolutionary armory. Considering the biological, particularly microbial diversity, it is wise to say that current knowledge is solely a scratch on the surface of possible cellular diversity. For example, the discovery of CRISPR
Cas systems and their assortment, which themselves are possibly only a sliver defense system that exists in nature. A large diversity of auxiliary proteins associated with CRISPR also supports the argument. Examples include the sophisticated and evolved arsenal of microbial warfare, numerous anti-Cas9, and antiCas12 proteins along with novel antiphage systems (Doron et al., 2018; Pawluk et al., 2016; Rauch et al., 2017). It is also possible that some bacteria or archaea have developed

CRISPR
Cas systems in bioactive peptide research 299 protein-based adaptive immune or gene editing systems that employ unique defense mechanisms. These findings and possibilities also give intimation to what may lie in the diversity of immune systems in animals, such as variable lymphocyte receptors, indicating adaptive immune systems in higher organisms as well (Han, Herrin, Cooper, & Wilson, 2008). Unique efforts, such as the Earth Microbiome Project (earthmicrobiome.org), are ongoing to thoroughly sample genomes across microbial taxa to achieve a more thorough understanding and prevalence of prokaryotic natural diversity, including gene editing systems. Further elucidation of CRISPR
Cas like systems will be steered by the idea of “guilt by association,” where genes located within an operon are postulated to be related to each other. This hypothesis may be extended to the idea that there is a certain syntax in the organization of bacterial genomes and CRISPR
Cas like systems act as natural defense language. Every discovery in the natural system(s) entices us to explore it in new ways, determining the mechanism of CRISPR
Cas systems has opened new opportunities to tinker, ultimately leading to a minor biological revolution. Still, our understanding of the detailed molecular mechanisms and diversity of CRISPR
Cas9 is still in its infancy, and a lot needs to be studied about the immunological “arms race” between the phage and its bacterial host. The subject of insufficient indel and HDR efficiency is a prospective challenge as well. One key element that allowed the rapid acceptance of CRISPR
Cas technology is its unparalleled indel efficiency, equated to the other programmable nucleases. Some studies have even presented indel values almost reaching perfect efficiency of 100%; however, some target sites show unusually low indel efficiency (Chen et al., 2016; Fu et al., 2014). Additionally, a neat understanding of such “target to target” variations is missing. Efforts to improve indel efficiency have been made by engineering either Cas or gRNA or by using artificial intelligence-based deep learning algorithms (Kim et al., 2018; Moon, Ko, Kim, & Kim, 2019; Strecker et al., 2019). In summary, applying CRISPR
Cas systems in eukaryotic cells and animals has transformed biological and medical research. Even with the extensive use of type II CRISPR
Cas systems, continued discovery, and development of CRISPR systems from prokaryotic species have resulted in new, beneficial technologies, such as Cas13a-based RNA targeting tools. Combining dCas9 to the plethora of effectors will continue to increase the prospects for targeted epigenetic modulation. Further, the multiplexed targeting potential of CRISPR
Cas systems will lead to the sophisticated exploitation of cellular processes. In addition to targeting the genome, CRISPR
Cas-based RNA-targeting tools are being utilized for medical research and drug discovery. Also, of our particular interest, the CRISPR
Cas system can pave the way for the development and acceptance of peptide-based nutraceuticals (Donohoue, Barrangou, & May, 2018). CRISPR
Cas system can amend a major drawback in bioactive peptide research, that is, the lack of understanding of the molecular mode of action. Now CRISPR
Cas-based therapeutics have entered clinical settings and have already exhibited great promise for

300 Chapter 13 rectifying genetic diseases and improving cell therapies. For further exploration of promising preclinical results, safety, and efficacy, alongside mitigation of off-target effects are required to be monitored closely. Collectively, the CRISPR
Cas advances are considerably enhancing our knowledge of biological processes and are propelling its clinical application.

13.11 Conclusions Genome editing took a dramatic turn with the discovery and application of CRISPR
Cas systems, which rely on Watson
Crick base pairing for target specificity. The concept of “gene drive” and “gene editing” is becoming realized by CRISPR
Cas systems. With a few promising triumphs and multiple clinical trials taking place at present, we may soon witness a plethora of clinical applications using CRISPR
Cas tools. However, it is sensible to emphasize that each application necessitates distinct levels of functionality of the CRISPR
Cas system. As described in this chapter, CRISPR
Cas technology still has numerous technical challenges. The off-target concern has become a pillar in efforts to apply the CRISPR system, particularly for clinical uses. Further, the area of peptides and bioactive compounds can benefit enormously from the use of CRISPR
Cas systems. This biotechnological approach can help us understand the underlying mechanisms contributing to the therapeutic efficacy of such compounds. Based on its scope and range, if elegantly refined, CRISPR technology can harness the process of DNA rewriting, which can trigger pioneering applications in natural product chemistry, nutraceutical development, pharmacology, diagnostics, and biotechnology.

References Aguirre, A. J., Meyers, R. M., Weir, B. A., Vazquez, F., Zhang, C.-Z., Ben-David, U., . . . Doshi, M. B. (2016). Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discovery, 6(8), 914
929. Akbari, O. S., Bellen, H. J., Bier, E., Bullock, S. L., Burt, A., Church, G. M., . . . Esvelt, K. M. (2015). Safeguarding gene drive experiments in the laboratory. Science (New York, N.Y.), 349(6251), 927
929. Alagoz, Y., Gurkok, T., Zhang, B., & Unver, T. (2016). Manipulating the biosynthesis of bioactive compound alkaloids for next-generation metabolic engineering in opium poppy using CRISPR-Cas 9 genome editing technology. Scientific Reports, 6, 30910. Aman, R., Ali, Z., Butt, H., Mahas, A., Aljedaani, F., Khan, M. Z., . . . Mahfouz, M. (2018). RNA virus interference via CRISPR/Cas13a system in plants. Genome Biology, 19(1), 1. Bae, S., Park, J., & Kim, J.-S. (2014). Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics (Oxford, England), 30(10), 1473
1475. Baltimore, D., Berg, P., Botchan, M., Carroll, D., Charo, R. A., Church, G., . . . Fenner, M. (2015). A prudent path forward for genomic engineering and germline gene modification. Science (New York, N.Y.), 348 (6230), 36
38. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., . . . Fenner, M. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science (New York, N.Y.), 315(5819), 1709
1712.

CRISPR
Cas systems in bioactive peptide research 301 Beard, C., Hochedlinger, K., Plath, K., Wutz, A., & Jaenisch, R. (2006). Efficient method to generate singlecopy transgenic mice by site-specific integration in embryonic stem cells. Genesis (New York, N.Y.: 2000), 44(1), 23
28. Bewg, W. P., Ci, D., & Tsai, C.-J. (2018). Genome editing in trees: from multiple repair pathways to long-term stability. Frontiers in Plant Science, 9, 1732. Bolotin, A., Quinquis, B., Sorokin, A., & Ehrlich, S. D. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology (Reading, England), 151(8), 2551
2561. Bondy-Denomy, J., & Davidson, A. R. (2014). To acquire or resist: the complex biological effects of CRISPR
Cas systems. Trends in Microbiology, 22(4), 218
225. Brooks, C., Nekrasov, V., Lippman, Z. B., & Van Eck, J. (2014). Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiology, 166(3), 1292
1297. Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., . . . Van Der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science (New York, N.Y.), 321(5891), 960
964. Burstein, D., Harrington, L. B., Strutt, S. C., Probst, A. J., Anantharaman, K., Thomas, B. C., . . . Banfield, J. F. (2017). New CRISPR
Cas systems from uncultivated microbes. Nature, 542(7640), 237
241. Chen, S., Lee, B., Lee, A. Y.-F., Modzelewski, A. J., & He, L. (2016). Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. Journal of Biological Chemistry, 291(28), 14457
14467. Chen, Y., Zheng, Y., Kang, Y., Yang, W., Niu, Y., Guo, X., . . . Xing, R. (2015). Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Human Molecular Genetics, 24(13), 3764
3774. Cheung, H. W., Cowley, G. S., Weir, B. A., Boehm, J. S., Rusin, S., Scott, J. A., . . . Wong, T. C. (2011). Systematic investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific dependencies in ovarian cancer. Proceedings of the National Academy of Sciences, 108(30), 12372
12377. Cho, J. S., Choi, K. R., Prabowo, C. P. S., Shin, J. H., Yang, D., Jang, J., & Lee, S. Y. (2017). CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum. Metabolic Engineering, 42, 157
167. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., . . . Marraffini, L. A. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science (New York, N.Y.), 339(6121), 819
823. Courtier-Orgogozo, V., Morizot, B., & Boe¨te, C. (2017). Agricultural pest control with CRISPR-based gene drive: time for public debate. EMBO Reports, 18(6), 878
880. Cox, D. B., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing with CRISPR-Cas13. Science (New York, N.Y.), 358(6366), 1019
1027. Cromwell, C. R., Jovel, J., & Hubbard, B. P. (2020). Methods for measuring CRISPR/Cas9 DNA cleavage in cells. CRISPR Guide RNA Design (pp. 197
213). Springer. Crosetto, N., Mitra, A., Silva, M. J., Bienko, M., Dojer, N., Wang, Q., . . . Ginalski, K. (2013). Nucleotideresolution DNA double-strand break mapping by next-generation sequencing. Nature Methods, 10(4), 361. Cyranoski, D. (2019). What’s next for the CRISPR babies? Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., & Liu, D. R. (2015). Small molecule
triggered Cas9 protein with improved genome-editing specificity. Nature Chemical Biology, 11(5), 316
318. ¨ kesli, A., Pillay, S., Horlbeck, M. A., Kampmann, M., . . . Smith, M. (2016). Deans, R. M., Morgens, D. W., O Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification. Nature Chemical Biology, 12(5), 361. Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., . . . Charpentier, E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 471(7340), 602
607. Deveau, H., Barrangou, R., Garneau, J. E., Labonte´, J., Fremaux, C., Boyaval, P., . . . Moineau, S. (2008). Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology, 190(4), 1390
1400.

302 Chapter 13 Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., . . . Orchard, R. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology, 34(2), 184. Doetschman, T., Gregg, R. G., Maeda, N., Hooper, M. L., Melton, D. W., Thompson, S., & Smithies, O. (1987). Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature, 330(6148), 576
578. Doe, B., Brown, E., & Boroviak, K. (2018). Generating CRISPR/Cas9-derived mutant mice by zygote cytoplasmic injection using an automatic microinjector. Methods and Protocols, 1(1), 5. Donohoue, P. D., Barrangou, R., & May, A. P. (2018). Advances in industrial biotechnology using CRISPR-Cas systems. Trends in Biotechnology, 36(2), 134
146. Doron, S., Melamed, S., Ofir, G., Leavitt, A., Lopatina, A., Keren, M., . . . Sorek, R. (2018). Systematic discovery of antiphage defense systems in the microbial pangenome. Science (New York, N.Y.), 359, eaar4120. DuPage, M., & Jacks, T. (2013). Genetically engineered mouse models of cancer reveal new insights about the antitumor immune response. Current Opinion in Immunology, 25(2), 192
199. Fellmann, C., & Lowe, S. W. (2014). Stable RNA interference rules for silencing. Nature Cell Biology, 16(1), 10
18. Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, D.-L., Wei, P., . . . Mao, Y. (2013). Efficient genome editing in plants using a CRISPR/Cas system. Cell Research, 23(10), 1229
1232. Fonfara, I., Richter, H., Bratoviˇc, M., Le Rhun, A., & Charpentier, E. (2016). The CRISPR-associated DNAcleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature, 532(7600), 517
521. Friedland, A. E., Tzur, Y. B., Esvelt, K. M., Colaia´covo, M. P., Church, G. M., & Calarco, J. A. (2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nature Methods, 10(8), 741. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology, 32(3), 279. Gantz, V. M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V. M., Bier, E., & James, A. A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proceedings of the National Academy of Sciences, 112(49), E6736
E6743. Garneau, J. E., Dupuis, M.-E`., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., . . . Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), 67
71. Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9
crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences, 109(39), E2579
E2586. Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., . . . Bassik, M. C. (2014). Genome-scale CRISPR-mediated control of gene repression and activation. Cell., 159(3), 647
661. Gratz, S. J., Cummings, A. M., Nguyen, J. N., Hamm, D. C., Donohue, L. K., Harrison, M. M., . . . O’Connor-Giles, K. M. (2013). Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics, 194(4), 1029
1035. Gratz, S. J., Ukken, F. P., Rubinstein, C. D., Thiede, G., Donohue, L. K., Cummings, A. M., & O’Connor-Giles, K. M. (2014). Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics, 196(4), 961
971. Grobarczyk, B., Franco, B., Hanon, K., & Malgrange, B. (2015). Generation of isogenic human iPS cell line precisely corrected by genome editing using the CRISPR/Cas9 system. Stem Cell Reviews and Reports, 11 (5), 774
787. Guschin, D. Y., Waite, A. J., Katibah, G. E., Miller, J. C., Holmes, M. C., & Rebar, E. J. (2010). A rapid and general assay for monitoring endogenous gene modification. Engineered zinc finger proteins (pp. 247
256). Springer. Hai, T., Teng, F., Guo, R., Li, W., & Zhou, Q. (2014). One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research, 24(3), 372
375.

CRISPR
Cas systems in bioactive peptide research 303 Hale, C. R., Zhao, P., Olson, S., Duff, M. O., Graveley, B. R., Wells, L., . . . Terns, M. P. (2009). RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell., 139(5), 945
956. Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., . . . Russell, S. (2016). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology, 34(1), 78
83. Hanna, R. E., & Doench, J. G. (2020). Design and analysis of CRISPR
Cas experiments. Nature Biotechnology, 1
11. Han, B. W., Herrin, B. R., Cooper, M. D., & Wilson, I. A. (2008). Antigen recognition by variable lymphocyte receptors. Science (New York, N.Y.), 321(5897), 1834
1837. Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino, D., Ma, E., Witte, I. P., . . . Doudna, J. A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science (New York, N.Y.), 362 (6416), 839
842. Hart, T., Chandrashekhar, M., Aregger, M., Steinhart, Z., Brown, K. R., MacLeod, G., . . . Sun, S. (2015). Highresolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell, 163(6), 1515
1526. Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262
1278. Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., . . . Shalem, O. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 31(9), 827. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169(12), 5429
5433. Jansen, R., Embden, J. Dv, Gaastra, W., & Schouls, L. M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology, 43(6), 1565
1575. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dualRNA
guided DNA endonuclease in adaptive bacterial immunity. Science (New York, N.Y.), 337(6096), 816
821. Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013). RNA-programmed genome editing in human cells. elife., 2, e00471. Kampmann, M. (2018). CRISPRi and CRISPRa screens in mammalian cells for precision biology and medicine. ACS Chemical Biology, 13(2), 406
416. Kasap, C., Elemento, O., & Kapoor, T. M. (2014). DrugTargetSeqR: A genomics-and CRISPR-Cas9
based method to analyze drug targets. Nature Chemical Biology, 10(8), 626. Kim, H. K., Min, S., Song, M., Jung, S., Choi, J. W., Kim, Y., . . . Kim, H. H. (2018). Deep learning improves prediction of CRISPR
Cpf1 guide RNA activity. Nature Biotechnology, 36(3), 239. Kim, S. K., Han, G. H., Seong, W., Kim, H., Kim, S.-W., Lee, D.-H., & Lee, S.-G. (2016). CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production. Metabolic Engineering, 38, 228
240. Kleinstiver, B. P., Prew, M. S., Tsai, S. Q., Topkar, V. V., Nguyen, N. T., Zheng, Z., . . . Yeh, J.-R. J. (2015). Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature, 523(7561), 481
485. Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., . . . Nishimasu, H. (2015). Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, 517 (7536), 583. Konermann, S., Lotfy, P., Brideau, N. J., Oki, J., Shokhirev, M. N., & Hsu, P. D. (2018). Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell, 173(3), 665
676, e14. Kuscu, C., Arslan, S., Singh, R., Thorpe, J., & Adli, M. (2014). Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature Biotechnology, 32(7), 677. Lander, E. S., Baylis, F., Zhang, F., Charpentier, E., Berg, P., Bourgain, C., . . . Winnacker, E.-L. (2019). Adopt a moratorium on heritable genome editing. Nature Publishing Group. Li, D., Qiu, Z., Shao, Y., Chen, Y., Guan, Y., Liu, M., . . . Lu, X. (2013). Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nature Biotechnology, 31(8), 681.

304 Chapter 13 Lin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., . . . Bao, G. (2014). CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research, 42(11), 7473
7485. Liu, J.-J., Orlova, N., Oakes, B. L., Ma, E., Spinner, H. B., Baney, K. L., . . . Harrington, L. B. (2019). CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature, 566(7743), 218
223. Loenen, W. A., Dryden, D. T., Raleigh, E. A., Wilson, G. G., & Murray, N. E. (2014). Highlights of the DNA cutters: A short history of the restriction enzymes. Nucleic Acids Research, 42(1), 3
19. Makarova, K. S., Aravind, L., Grishin, N. V., Rogozin, I. B., & Koonin, E. V. A. (2002). DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Research, 30(2), 482
496. Makarova, K. S., Grishin, N. V., Shabalina, S. A., Wolf, Y. I., & Koonin, E. V. (2006). A putative RNAinterference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology Direct, 1(1), 7. Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., . . . Haft, D. H. (2015). An updated evolutionary classification of CRISPR
Cas systems. Nature Reviews. Microbiology, 13(11), 722
736. Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., . . . Church, G. M. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology, 31(9), 833. Mali, P., Esvelt, K. M., & Church, G. M. (2013). Cas9 as a versatile tool for engineering biology. Nature Methods, 10(10), 957
963. Mandegar, M. A., Huebsch, N., Frolov, E. B., Shin, E., Truong, A., Olvera, M. P., . . . Spencer, C. I. (2016). CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell, 18(4), 541
553. Marraffini, L. A., & Sontheimer, E. J. (2008). CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science (New York, N.Y.), 322(5909), 1843
1845. Marraffini, L. A., & Sontheimer, E. J. (2010). CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Reviews. Genetics, 11(3), 181
190. Matano, M., Date, S., Shimokawa, M., Takano, A., Fujii, M., Ohta, Y., . . . Sato, T. (2015). Modeling colorectal cancer using CRISPR-Cas9
mediated engineering of human intestinal organoids. Nature Medicine, 21(3), 256. Mojica, F. J., Dı´ez-Villasen˜or, C., Garcı´a-Martı´nez, J., & Almendros, C. (2009). Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading, England), 155 (3), 733
740. Mojica, F. J., Garcı´a-Martı´nez, J., & Soria, E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution, 60(2), 174
182. Moon, S. B., Ko, J.-H., Kim, J.-S., & Kim, Y.-S. (2019). Improving CRISPR genome editing by engineering guide RNAs. Trends in Biotechnology. Munoz, D. M., Cassiani, P. J., Li, L., Billy, E., Korn, J. M., Jones, M. D., . . . McAllister, G. (2016). CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer Discovery, 6(8), 900
913. Nakade, S., Tsubota, T., Sakane, Y., Kume, S., Sakamoto, N., Obara, M., . . . Sakuma, T. (2014). Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nature Communications, 5(1), 1
8. Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., . . . Hara, K. Y. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science (New York, N.Y.), 353(6305), aaf8729. Niu, Y., Shen, B., Cui, Y., Chen, Y., Wang, J., Wang, L., . . . Li, W. (2014). Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 156(4), 836
843.

CRISPR
Cas systems in bioactive peptide research 305 Paix, A., Schmidt, H., & Seydoux, G. (2016). Cas9-assisted recombineering in C. elegans: Genome editing using in vivo assembly of linear DNAs. Nucleic Acids Research, 44(15), e128. Paix, A., Wang, Y., Smith, H. E., Lee, C.-Y. S., Calidas, D., Lu, T., . . . Seydoux, G. (2014). Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 sites in Caenorhabditis elegans. Genetics, 198(4), 1347
1356. Pawluk, A., Amrani, N., Zhang, Y., Garcia, B., Hidalgo-Reyes, Y., Lee, J., . . . Maxwell, K. L. (2016). Naturally occurring off-switches for CRISPR-Cas9. Cell, 167(7), 1829
1838, e9. Pourcel, C., Salvignol, G., & Vergnaud, G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading, England), 151(3), 653
663. Premsrirut, P. K., Dow, L. E., Kim, S. Y., Camiolo, M., Malone, C. D., Miething, C., . . . Kogan, S. C. (2011). A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell, 145(1), 145
158. Prykhozhij, S. V., Rajan, V., & Berman, J. N. (2016). A guide to computational tools and design strategies for genome editing experiments in zebrafish using CRISPR/Cas9. Zebrafish, 13(1), 70
73. Qin, W., Dion, S. L., Kutny, P. M., Zhang, Y., Cheng, A. W., Jillette, N. L., . . . Wang, H. (2015). Efficient CRISPR/ Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics, 200(2), 423
430. Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., . . . Makarova, K. S. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186
191. Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., . . . Zhang, Y. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154(6), 1380
1389. Rath, D., Amlinger, L., Rath, A., & Lundgren, M. (2015). The CRISPR-Cas immune system: Biology, mechanisms and applications. Biochimie, 117, 119
128. Rauch, B. J., Silvis, M. R., Hultquist, J. F., Waters, C. S., McGregor, M. J., Krogan, N. J., & Zhang, Y. (2017). Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell, 168(1-2), 150
158, e10. Rodriguez, E. A., Campbell, R. E., Lin, J. Y., Lin, M. Z., Miyawaki, A., Palmer, A. E., . . . Tsien, R. Y. (2017). The growing and glowing toolbox of fluorescent and photoactive proteins. Trends in Biochemical Sciences, 42(2), 111
129. Rossant, J. (2018). Gene editing in human development: Ethical concerns and practical applications. Development (Cambridge, England), 145(16), dev150888. Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K.-I. T., & Yamamoto, T. (2016). MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nature Protocols, 11(1), 118. Sa´nchez-Rivera, F. J., & Jacks, T. (2015). Applications of the CRISPR
Cas9 system in cancer biology. Nature Reviews. Cancer, 15(7), 387
395. Sa´nchez-Rivera, F. J., Papagiannakopoulos, T., Romero, R., Tammela, T., Bauer, M. R., Bhutkar, A., . . . Xue, W. (2014). Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature, 516(7531), 428
431. Schindele, P., Wolter, F., & Puchta, H. (2018). Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13. FEBS Letters, 592(12), 1954
1967. Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., . . . Doudna, J. A. (2015). Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proceedings of the National Academy of Sciences, 112(33), 10437
10442. Schunder, E., Rydzewski, K., Grunow, R., & Heuner, K. (2013). First indication for a functional CRISPR/Cas system in Francisella tularensis. International Journal of Medical Microbiology, 303(2), 51
60. Shah, S. A., Erdmann, S., Mojica, F. J., & Garrett, R. A. (2013). Protospacer recognition motifs: Mixed identities and functional diversity. RNA Biology, 10(5), 891
899. Shang, N., Bhullar, K. S., & Wu, J. (2020). Ovotransferrin exhibits osteogenic activity partially via low-density lipoprotein receptor-related protein 1 (LRP1) activation in MC3T3-E1 cells. Journal of Agricultural and Food Chemistry, 68(35), 9427
9435.

306 Chapter 13 Shi, J., Wang, E., Milazzo, J. P., Wang, Z., Kinney, J. B., & Vakoc, C. R. (2015). Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nature Biotechnology, 33(6), 661. Shmakov, S., Abudayyeh, O. O., Makarova, K. S., Wolf, Y. I., Gootenberg, J. S., Semenova, E., . . . Severinov, K. (2015). Discovery and functional characterization of diverse class 2 CRISPR-Cas systems. Molecular Cell, 60(3), 385
397. Shmakov, S., Smargon, A., Scott, D., Cox, D., Pyzocha, N., Yan, W., . . . Wolf, Y. I. (2017). Diversity and evolution of class 2 CRISPR
Cas systems. Nature Reviews. Microbiology, 15(3), 169. Sidik, S. M., Huet, D., Ganesan, S. M., Huynh, M.-H., Wang, T., Nasamu, A. S., . . . Niles, J. C. (2016). A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes. Cell, 166(6), 1423
1435, e12. Smargon, A. A., Cox, D. B., Pyzocha, N. K., Zheng, K., Slaymaker, I. M., Gootenberg, J. S., . . . Makarova, K. S. (2017). Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Molecular Cell, 65(4), 618
630, e7. Spilman, M., Cocozaki, A., Hale, C., Shao, Y., Ramia, N., Terns, R., . . . Stagg, S. (2013). Structure of an RNA silencing complex of the CRISPR-Cas immune system. Molecular Cell, 52(1), 146
152. Strecker, J., Jones, S., Koopal, B., Schmid-Burgk, J., Zetsche, B., Gao, L., . . . Zhang, F. (2019). Engineering of CRISPR-Cas12b for human genome editing. Nature Communications, 10(1), 1
8. Teng, F., Cui, T., Feng, G., Guo, L., Xu, K., Gao, Q., . . . Li, W. (2018). Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discovery, 4(1), 1
15. Thomas, K. R., & Capecchi, M. R. (1987). Site-directed mutagenesis by gene targeting in mouse embryoderived stem cells. Cell, 51(3), 503
512. Tong, Y., Weber, T., & Lee, S. Y. (2019). CRISPR/Cas-based genome engineering in natural product discovery. Natural Product Reports, 36(9), 1262
1280. Tro¨der, S. E., Ebert, L. K., Butt, L., Assenmacher, S., Schermer, B., & Zevnik, B. (2018). An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS One, 13(5), e0196891. Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., . . . Le, L. P. (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nature Biotechnology, 33(2), 187. Vestergaard, G., Garrett, R. A., & Shah, S. A. (2014). CRISPR adaptive immune systems of archaea. RNA Biology, 11(2), 156
167. Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., & Jaenisch, R. (2013). Onestep generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153(4), 910
918. Wang, T., Birsoy, K., Hughes, N. W., Krupczak, K. M., Post, Y., Wei, J. J., . . . Sabatini, D. M. (2015). Identification and characterization of essential genes in the human genome. Science (New York, N.Y.), 350 (6264), 1096
1101. Wang, W., Kutny, P. M., Byers, S. L., Longstaff, C. J., DaCosta, M. J., Pang, C., . . . Wang, H. (2016). Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. Journal of Genetics and Genomics, 43(5), 319
327. Wang, X., Cao, C., Huang, J., Yao, J., Hai, T., Zheng, Q., . . . Cheng, J. (2016). One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system. Scientific Reports, 6, 20620. Wu, J., Du, G., Chen, J., & Zhou, J. (2015). Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli. Scientific Reports, 5, 13477. Xu, X., Tao, Y., Gao, X., Zhang, L., Li, X., Zou, W., . . . Hu, R. (2016). A CRISPR-based approach for targeted DNA demethylation. Cell Discovery, 2, 16009, PMID: 27462456. 2016. Xue, W., Chen, S., Yin, H., Tammela, T., Papagiannakopoulos, T., Joshi, N. S., . . . Crowley, D. G. (2014). CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature, 514(7522), 380
384.

CRISPR
Cas systems in bioactive peptide research 307 Yan, W. X., Chong, S., Zhang, H., Makarova, K. S., Koonin, E. V., Cheng, D. R., & Scott, D. A. (2018). Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domaincontaining accessory protein. Molecular Cell, 70(2), 327
339, e5. Yan, W. X., Hunnewell, P., Alfonse, L. E., Carte, J. M., Keston-Smith, E., Sothiselvam, S., . . . Koonin, E. V. (2019). Functionally diverse type V CRISPR-Cas systems. Science (New York, N.Y.), 363(6422), 88
91. Zaidi, S. S.-e-A., Mahfouz, M. M., & Mansoor, S. (2017). CRISPR-Cpf1: A new tool for plant genome editing. Trends in Plant Science, 22(7), 550
553. Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., . . . Regev, A. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163 (3), 759
771. Zetsche, B., Heidenreich, M., Mohanraju, P., Fedorova, I., Kneppers, J., DeGennaro, E. M., . . . Gootenberg, J. S. (2017). Multiplex gene editing by CRISPR
Cpf1 using a single crRNA array. Nature Biotechnology, 35(1), 31. Zhang, F. (2019). Development of CRISPR-Cas systems for genome editing and beyond. Quarterly Reviews of Biophysics, 52. Zhang, H., Zhang, J., Wei, P., Zhang, B., Gou, F., Feng, Z., . . . Xu, N. (2014). The CRISPR/C as9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnology Journal, 12(6), 797
807. Zhang, X., Wang, L., Liu, M., & Li, D. (2017). CRISPR/Cas9 system: A powerful technology for in vivo and ex vivo gene therapy. Science China Life Sciences, 60(5), 468
475. Zou, Q., Wang, X., Liu, Y., Ouyang, Z., Long, H., Wei, S., . . . Shen, J. (2015). Generation of gene-target dogs using CRISPR/Cas9 system. Journal of Molecular Cell Biology, 7(6), 580
583. Zuo, E., Cai, Y.-J., Li, K., Wei, Y., Wang, B.-A., Sun, Y., . . . Wei, W. (2017). One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Research, 27(7), 933
945.

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CHAPTER 14

Databases of bioactive peptides Anna Iwaniak, Małgorzata Darewicz and Piotr Minkiewicz Department of Food Biochemistry, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

14.1 Introduction Looking back on the past times, men have always tried to collect and store the information concerning different aspects of their lives (Berg, Seymour, & Goel, 2013). According to Date (2003), the idea of collecting, categorizing, and storing information has its beginning in library, business, and medical records. The question “what to do with such an amount of data and how to index it” was a natural inspiration to coin a “database” definition, which is briefly “a collection of data” (Berg et al., 2013). Once computers had been invented, people have realized that they can store data for later retrieval. Moreover, this way of data storage and indexing was more space-effective and less costly, compared to those applied so far. In addition, the popularity of WWW, that is, the World Wide Web (Internet), has enabled the computer users’ easy and remote access to data (Date, 2003). According to Rozin, Fischler, Imada, Sarubin, and Wrzesniewski (1999), food is “a critical contributor to physical well-being, a major source of pleasure, worry and stress, a major occupant of waking time and, across the world, the single greatest category of expenditures.” Its health potential results from variety of its macronutrients that regulate body functions, like proteins, fats, carbohydrates, and vitamins (Carreiro et al., 2016). Loads of data on molecules derived from foods can be stored in databases (Iwaniak, Darewicz, Mogut, & Minkiewicz, 2019), due to the progress in the development of information technologies that took place in biology. Huge amount of data was generated in this discipline which triggered the development of many databases enabling the storage of information about genes, proteins, metabolites, molecules, etc. (Hall, Shan, Lushington, & Visvanathan, 2013). This trend can also be observed in food science owing to the need of collecting data concerning, for example, nutritional characteristics of individual foods, meals, diets, dishes as well as food components (Marconi et al., 2018). Peptides derived from food proteins, which due to their biological and functional properties, such as ability to reduce blood pressure, glucose, and cholesterol levels (Sa´nchez & Va´zquez, 2017),

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00025-X © 2021 Elsevier Inc. All rights reserved.

309

310 Chapter 14 affect i.a. the taste of foods (Li-Chan, 2015), are the molecules considered as valuable health-beneficial and functional food components (Sa´nchez & Va´zquez, 2017). The interest of scientists working on bioactive peptides is reflected in the number of articles published between 1900 and 2020. According to the Web of Science database, 1982 papers containing such query words as “bioactive peptides; food; food proteins” had been published within the above time span. Within the last 5 years alone, 1029 articles were published, including 74 papers that were released since the beginning of 2019 to date (data accessed on March 27, 2020). This brief statistical overview shows the growth of interests as well as the development of the knowledge in the field of bioactive peptides. The latter has also its consequences in the need of collecting and storing data on biopeptides in internet archives, that is, in databases. The aim of this chapter is to present some fundamental knowledge about categories of databases and sources of information about peptide of interest, and finally to describe some databases of bioactive peptide sequences as well as some examples of their usage in peptide analyses.

14.2 General overview of databases and their classification When thinking about databases, it is hard not to mention a definition of bioinformatics. According to Wani et al. (2018), bioinformatics (briefly) is a discipline that combines biology, mathematics, and computer science to analyze biological data. Its components are shown in Fig. 14.1.

Figure 14.1 Disciplines being the components of bioinformatics according to Wani et al., 2018.

Databases of bioactive peptides 311 Technology is a knowledge about techniques and methods, whereas computers allow making some computations and performing the work according to a specific technique and a method. The task of both is to make the life/work easier. Databases are understood as tools for organizing, storing, and managing the datasets. “Algorithms and statistics” means developing some tools helpful in analyzing the relationships between large datasets. Computer sciences include the aspects of the scientific and practical approach to computations. The mechanization of procedures helps in the acquisition, representation, processing, storage, communication of, and access to information. “Analysis and data interpretation,” as its name says, first enables data analysis and then the interpretation of results using the knowledge about information to be analyzed. Computational biology covers a wide spectrum of biological areas of research (e.g., genomics, biophysics, and biochemistry) and uses all quantitative methodologies for data analysis. Finally, genomics (shortly, a discipline that aims at the characteristics of genes) uses bioinformatics to analyze the structure and the function of a genome (Wani et al., 2018). Databases (i.e., biological) are the core of bioinformatics and they contain information about sequences of genes, their descriptions, classifications according to ontology categories, and tabular data. In addition, data found in such databases can be presented as tables, some records, XML structures, or pictures. Information about a single molecule (i.e., record) possesses an accession number and may be cross-referenced with another database (Nishant, Kumar, Sathish Kumar, & Vijaya Shanti, 2011). According to Nishant et al. (2011), the content of any database should be easily accessed, managed, and updated. Moreover, data found in such a repository should be easily collected by a user in a user-friendly form and be available to a multiuser system (Nishant et al., 2011). Such an idea of organization of biological databases as well as their advantages might also refer to databases of biomolecules, like peptides. There are several criteria of database classification (Zou, Ma, Yu, & Zhang, 2015). According to Nishant et al. (2011), biological databases can be divided into primary, secondary, and composite ones. The first category includes the information about the structure and/or the sequence of a molecule. The second one contains data from the primary database and shows the information about, for example, conserved regions of a protein and active sites of the residues of the protein families that were derived from the multiple alignment analysis of protein families (Nishant et al., 2011). Composite databases merge a variety of primary databases to make the search efficient, comprehensive, and nonredundant (Bleasby, Akrigg, & Attwood, 1994). It is very difficult to eliminate errors found in a database especially when they were propagated into other databases. To overcome this problem, a new profession called “a database curator” was introduced (Lesk, 2008). According to Odell, Lazo, Woodhouse, Hane, and Sen (2017), a curator usually possesses computer skills as well as biological

312 Chapter 14 education and is involved in the selection of the outcome information to provide reliable, reusable, and accessible data. Taking into account the way of database curation, two types of databases can be distinguished: expert-curator and community-curated databases. The first concerns a curation of data on a specific field, whereas the second enables the curators to work in integrated and collaborating group of professionals (Zou et al., 2015). Considering the type of information collected in databases, Nishant et al. (2011) classified them as sequence, structure, and pathway databases. The first concerns sequences of nucleotides and proteins, and the second one can be referred only to proteins (Nishant et al., 2011). Zou et al. (2015) included a more broad spectrum of database categorization, namely: RNA, DNA, protein, expression, pathway, disease, nomenclature, literature, and ontology databases. While the “big” molecules, like proteins and/or nucleic acids, are in the focus of interest of bioinformatics, the small molecules, for example, amino acids and sugars, have attracted the attention of chemists. It was a consequence of the progress in the “omics” technologies contributing to the detection, identification, and characteristics of lots of small molecules which led to the development of a new discipline called cheminformatics (Wishart, 2012). According to Wishart (2012), this scientific area aims at the employment of computer technologies to “facilitate the collection, storage, analysis, and manipulation of large quantities of chemical data.” This chemical data is collected in chemical databases (Iwaniak, Darewicz, et al., 2019). Although there is a similarity between the definitions of bio- and cheminformatics, they both provide data into databases but differ in data searching. In bioinformatics, the search is sequence-driven, whereas in cheminformatics, it is structure- and/or picture-driven (Minkiewicz et al., 2016; Minkiewicz, Turło, Iwaniak, & Darewicz, 2019; Wishart, 2012). Both disciplines utilize the text-based search. According to Wishart (2012), chemical databases can be classified into small molecule (or metabolic) pathway, metabolite or metabolomic, drug, and toxin/toxic substance databases. Some examples of databases assigned to the categories described earlier are shown in Table 14.1. Classification of an individual database is quite flexible, that is, it can be ascribed to few categories. For example, when looking at the categories presented in Table 14.1, the UniProt database is primary, sequence, and protein database.

14.3 Biological and chemical information on peptides in brief According to Brusic and Zeleznikow (1999), the huge amount of data that is found in databases may contribute to the discovery of new concepts, help speed up the studies, and be complementary to the experiment. Such an approach is related to the KDD, that is, Knowledge Discovery from Databases, defined shortly as the process acquiring useful knowledge from databases. The crucial term is a word “data,” meaning the set of raw facts stored in a file or a database (Brusic & Zeleznikow, 1999).

Databases of bioactive peptides 313 Table 14.1: Some examples of databases associated with the specific category. Category

Database

Web address

Content

Reference

Apweiler et al. (2004) Sigrist et al. (2013)

Biological databases Primary

UniProt

https://www.uniprot.org/

Protein sequences

Secondary

PROSITE

www.expasy.ch/prosite/

Composite

PIR

Expert-Curated

TAIR

https:// proteininformationresource. org/ http://arabidopsis.org

Protein families, domains, and functional sites Integrated protein resources

LncRNAWiki

http://lncrna.big.ac.cn

Sequence

UniProt

https://www.uniprot.org/

Genome database for Arabidopsis thaliana Human long noncoding RNAs Protein sequences

Structure

CATH/Gene3D

http://cath.biochem.ucl.ac. uk/

Protein structure classification

Pathway

MetaCyc

https://metacyc.org/

RNA

RNA central

https://rnacentral.org/

DNA

GenBank

Protein

UniProt

https://www.ncbi.nlm.nih. gov/genbank/ https://www.uniprot.org/

Metabolic pathway database Noncoding RNA sequences Nucleotide sequences

Expression

TiGER

Disease

ICGC

Nomenclature

HGNC

Community-curated

Literature

PubMed

Ontology

GO

Proteolytic enzymes

MEROPS

http://bioinfo.wilmer.jhu. edu/tiger/ https://icgc.org/ https://www.genenames. org/ http://www.ncbi.nlm.nih. gov/pubmed http://geneontology.org https://www.ebi.ac.uk/ merops/

Protein sequences Tissue-specific gene expression profiles Genomes of 25,000 untreated cancers Approved human gene nomenclature Biomedical literature database Gene ontology Proteolytic enzymes

Barker et al. (2000)

Lamesch et al. (2012) Ma et al. (2015) Apweiler et al. (2004) Dawson et al. (2017), Lewis et al. (2018) Caspi et al. (2018) The RNA Central Consortium (2019) Benson et al. (2014) Apweiler et al. (2004) Liu, Yu, Zack, Zhu, and Qian (2008) Hudson et al. (2010) Braschi et al. (2019) Lu (2011) The Gene Ontology Consortium (2019) Rawlings et al. (2018)

Chemical databases Small molecules

PubChem

Small molecules

ChemSpider

Small molecules (or metabolic pathways) Small molecules

https://pubchem.ncbi.nlm. nih.gov/ http://www.chemspider. com/Default.aspx

ZINC

http://zinc.docking.org/

ChEBI

https://www.ebi.ac.uk/ chebi/

General database of small molecules Website containing links to over 500 databases of small molecules Commercially available small compounds Chemical compounds and associated ontologies

Kim et al. (2019) Williams and Tkachenko (2014)

Sterling and Irwin (2015) Hastings et al. (2016)

(Continued)

314 Chapter 14 Table 14.1: (Continued) Category

Database

Web address

Content

Descriptors characterizing structure and physical properties Amino acids BioMagResBank http://www.bmrb.wisc.edu/ Secondary structure ref_info/aadata.dat propensity and physicochemical properties Metabolite or HMDB http://www.hmdb.ca/ Small molecule metabolomic metabolites found in body Enzymes and metabolic BRENDA https://www.brendaEnzymes, their ligands pathways enzymes.org/ and metabolic pathways Amino acids

Drug Toxin/toxic substance

AAIndex

https://www.genome.jp/ aaindex/

DrugBank

https://www.drugbank.ca/

T3DB

www.t3db.ca

Knowledgebase for drugs Environmental toxic compounds

Reference Kawashima et al. (2008) Ulrich et al. (2008)

Wishart et al. (2018)

Jeske, Placzek, Schomburg, Chang, and Schomburg (2019) Wishart et al. (2008) Wishart et al. (2015)

Programs Conversion between different chemical codes Commercial, downloadable molecule editor Prediction of physicochemical properties Prediction of secondary structure and physicochemical properties Proteolysis simulation

Open Babel

http://openbabel.org/wiki/ Main_Page

Marvin Sketch

https://chemaxon.com/ products

Peptide Property Calculator ProtScale

https://pepcalc.com/

PeptideCutter

O’Boyle et al. (2011)

https://web.expasy.org/ protscale/

Gasteiger et al. (2005)

https://web.expasy.org/ peptide_cutter/

Gasteiger et al. (2005)

Data searching in databases relies mostly on the exact match. By knowing the key of the search, the user obtains a list of records comparable to the query (Cha´vez & Gonzalo, 2005). The exact match enables getting the information if the compound of interest is present in the database as well as to extend the knowledge about the molecules by using its structure as a query in another database (Miller, 2002). Biological data found in databases mostly refer to DNA and RNA sequences, genes, proteins (Bouadjenek, Zobel, & Verspoor, 2019), and others such as graphs, geometric information, scalar and vector fields, patterns of organization, constraints, images, scientific prose, as well as biological hypotheses and evidence (Wooley & Lin, 2005). In the case of bioactive peptides, their sequences as well as the sequences of proteins being their sources

Databases of bioactive peptides 315 provide crucial biological information to the scientists. Acquisition of data about peptides and proteins is ensured by databases in different ways; however, they are mainly focused on the sequences written in a one-letter code. Other queries involve text searching options like the name (if any), origin, bioactivity, and length of a protein/peptide chain (Iwaniak, Darewicz, et al., 2019). As it can be seen from the earlier overview, the bioinformatics focuses on the sequences. Chemical data is represented by a structure of a molecule that can be described by specific codes (Wegner et al., 2012). Thus, if the information concerning a peptide provided in biological databases is lacking or insufficient, a user can acquire it from chemical databases using one of the three following chemical codes: SMILES (Simplified Molecular Input-Line Entry System), InChI (The IUPAC International Chemical Identifier), and InChIKey (a hashed version of full InChI). The first two codes describe the structure of a molecule, whereas the third one (InChIKey) is composed of 27 characters and can simply be pasted to Google as a query (Minkiewicz, Iwaniak, & Darewicz, 2017). For example, FF has a bitter taste (BIOPEP-UWM database of sensory peptides and amino acids, peptide ID: 9) as well as acts as an inhibitor of angiotensin-converting enzyme (EC 3.4.15.1) (enzyme MEROPS ID: M04.005) (according to AHTPDB), an inhibitor of pseudolysin (EC 3.4.24.26) (enzyme MEROPS ID: M04.005) (according to BRENDA), and an inhibitor of peptide-transporting ATP-ase (EC 3.6.3.43) (according to BRENDA). The FF sequence translated into chemical codes looks as follows: c1ccc(cc1)C(C@@H)(C(5O)N(C@@H)(Cc1ccccc1)C(5O)O)N (SMILES), 1S/C18H20N2O3/c19-15(11
13-7-3-1
4-8
13)17(21)20-16(18(22)23)12
14-95-2
6-10
14/h1
10,15
16H,11
12,19H2, (H,20,21)(H,22,23)/t15-,16-/m0/s1 (InChI) and GKZIWHRNKRBEOH-HOTGVXAUSA-N (InChIKey). Based on these codes, additional information can be found about FF in, for example, ChemSpider (ID: 5361193), ChEBI (ID: 72723), and PuBChem (CID: 6993090), which concerns its physicochemical properties, its role in biological systems, its chemical role, and its additional bioactivity (i.e., binding affinity to human PepT2 in SKTP cells and displacement function of (3H)SP1
7 from NK1 receptor in Sprague-Dawley rat spinal cord membrane) (based on data derived from the aforementioned cheminformatic tools; accessed on October 2019). How to deal with the conversion of a biological code (i.e., peptide sequence) into a chemical one? For example, the conversion of peptide sequences into SMILES is feasible using, for example, an application available at the website of the BIOPEP-UWM database (Minkiewicz, Iwaniak, & Darewicz, 2019). In contrast, such programs as Open Babel (O’Boyle et al., 2011), Marvin Sketch or Marvin JS molecule editor, available at, for example, HMDB website, enable converting SMILES into other chemical codes. To avoid some potential errors when translating the biological code into the chemical code, a database user needs to possess the knowledge about stereochemistry of amino acids in a peptide sequence, and know how to correct the molecule with missing atoms or containing inappropriate structures of functional groups (Minkiewicz et al., 2017).

316 Chapter 14 A term “data” (both biological and chemical) is associated with data mining (DM) (Raza, 2012). Briefly, DM is defined as the usage of specific tools for pattern discovery and extraction. Pattern is understood as an expression to describe a subset of the data or the model that may be suitable to this subset. Pattern extraction includes the following aspects of DM: fitting model to the data, getting the structure from the data, and acquiring the high-level of data description (Brusic & Zeleznikow, 1999). According to Raza (2012), employment and development of DM are one of the main interests of the bioinformatics to understand the biological phenomena. Due to the considerable progress in the development of new biological and chemical data sources, the scientists postulate the integration of DM to get a better understanding of the complex nature of biological systems resulting from the action of chemical compounds on these systems. It can be managed when both biological and chemical data sources are connected in a specific way and language (Chen et al., 2010). An example of such a connection was introduced by Chen et al. (2010), who developed a web resource called Chem2Bio2RDF to integrate biological and chemical information on small molecules, targets, genes, pathways, and drugs that were derived from different repositories. Moreover, the SPARQL was presented as an example of a query language for cheminformatics and bioinformatics. Both Chem2Bio2RDF and SPARQL were successfully employed in polypharmacology, pathway inhibition, and adverse drug reaction analysis (Chen et al., 2010). To recapitulate, there are loads of information on bioactive molecules provided in different databases. However, according to Wegner et al. (2012), using the data found in different databases might pose a challenge to potential users due to the differences in identifiers, curation, synchronization, error-correcting mechanisms, and the need to provide the tools for efficient substructure and data similarity search. Thus, before solving the problem, critical and multidisciplinary knowledge is required to filter, systemize, and organize the biomolecule (e.g., bioactive peptides) data set for further processing (Wegner et al., 2012).

14.4 Some databases of bioactive peptide sequences According to the scientific reports, three strategies are used to study bioactive peptides: classical, in silico (bioinformatic-assisted), and integrated (hybrid). They were fully described by Iwaniak, Darewicz, et al. (2019). The latter two involve the computer-aided methodologies including peptide searching in different databases and/or application of predictive, and simulation tools for the analysis of the physicochemical nature of peptides (Iwaniak, Darewicz, et al., 2019). Due to the remarkable progress in the development of knowledge about bioactive peptides, the data about them has been collected and stored in databases. Such on-line available catalogs are currently an important source of information about the sequences in

Databases of bioactive peptides 317 terms of their identification in some protein sources as well as their further investigations (Panyayai et al., 2019). When looking into literature and web addresses of databases of biologically active peptides, they can be divided into two basic categories: “thematic” and general databases. The first category includes databases storing information about peptides with a specific activity, while the second one represents databases of peptide sequences with various bioactivities. This categorization of bioactive peptide databases is shown in Table 14.2. The third category (“other databases”) includes also peptides with a specific bioactivity, the bioactivity of which is represented in one or maximum two repositories. All the abovementioned databases are organized in a different way, including the way of peptide search. The most important one is the sequence search (exact and/or nonexact match) available in all databases. When thinking about the databases of bioactive peptide, some other options of finding the peptide of interest can be based on text query like the name of peptide (if any), its accession number (ID), biological activity (function), and reference paper (or link to reference or literature database). Such criteria of peptide search are available in BIOPEP-UWM, EROP-Moscow, and PepBank databases. The BioPepDB enables searching peptides using all the above options except reference. Peptides in this database can also be found by their source. The StraPep includes the following criteria of search: family, UniProt ID, PDB ID, name, and peptide ID. In turn, the MBPDB search tool relies on uploading the TSV (i.e., tab-separated values) formatted file to obtain the results. Other criteria of searching peptides in databases include peptide molecular mass, number of amino acids in a peptide chain, and chemical code, like InChIKey. Such a sequence search is feasible using the BIOPEP-UWM database (all options mentioned earlier). Additional options of peptide search in the EROP-Moscow database include source (eukaryote, kingdom, phylum, class, division, organism, taxon, organ/secreted), physicochemical properties (peptide length, charge, isoelectric point), function (bioactivity selected from a toolbar or queried using own keywords), and references. Peptides acting as ACE (angiotensin converting enzyme, EC 3.4.15.1) inhibitors are potential food ingredients that may reduce blood pressure and are present in variety of food proteins (Iwaniak, Minkiewicz, & Darewicz, 2014). Compared to synthetic drugs, peptides with the ACE-inhibiting effect are considered as milder, nontoxic, and safer (Kumar, Kumar, Sharma, & Baruwa, 2010). Thus food-derived ACE inhibitors attract the attention of scientists and so far have been the best known group of bioactive peptides compared to the sequences representing other bioactivities (Wu, Liao, & Udenigwe, 2017). The AHTPDB is a unique database of antihypertensive peptides developed by Kumar et al. (2015). Its entry page shows ACE mechanism of action as well as percentage distribution of ACE inhibitors found in food sources. According to the information provided at AHTPDB website, it contains 6000 entries

318 Chapter 14 Table 14.2: Some databases of biopeptides based on their content. Database of:

Name

Website

Reference

Antimicrobial peptides

APD BaAMPs

http://aps.unmc.edu/AP/main.html http://www.baamps.it/

CAMPR3

http://www.camp.bicnirrh.res.in/

DBAASP MilkAMP

https://dbaasp.org/ http://milkampdb.org/home.php

YADAMP

http://yadamp.unisa.it/about.aspx

AHTPDB

http://crdd.osdd.net/raghava/ahtpdb/

Wang, Li, and Wang (2016) Di Luca, Maccari, Maisetta, and Batoni (2015) Waghu, Barai, Gurung, and Idicula-Thomas (2015) Pirtskhalava et al. (2016) The´olier, Fliss, Jean, and Hammami (2014) Piotto, Sessa, Concilio, and Ianelli (2012) Kumar et al. (2015)

BIOPEPUWM BioPepDB

http://www.uwm.edu.pl/biochemia

Antihypertensive peptides Bioactive peptides

EROPMoscow

Other

http://bis.zju.edu.cn/biopepdbr/index. php http://erop.inbi.ras.ru/

MBPDB

http://mbpdb.nws.oregonstate.edu/

PepBank

http://pepbank.mgh.harvard.edu/

StraPep AntiTbPdb

http://isyslab.info/StraPep/ http://webs.iiitd.edu.in/raghava/ antitbpdb (antitubercular and myobacterial peptides) http://crdd.osdd.net/servers/avpdb/ (antiviral peptides) http://www.uwm.edu.pl/biochemia (sensory peptides and amino acids)

AVPdb BIOPEPUWM Brainpeps CancerPPD

CPPSite 2.0 Hemolytik NeuroPep Quorumpeps

Minkiewicz, Iwaniak, et al. (2019) Li et al. (2018) Zamyatnin, Borchikov, Vladimirov, and Voronina (2006) Nielsen, Beverly, Qu, and Dallas (2017) Shtatland, Guettler, Kossodo, Pivovarov, and Weissleder (2007) Wang et al. (2018) Usmani, Kumar, Kumar, Singh, and Raghava (2018)

Qureshi, Thakur, Himani, and Kumar (2013) Iwaniak, Minkiewicz, Darewicz, Sieniawski, et al. (2016) http://brainpeps.ugent.be/ (blood
brain Van Dorpe et al. (2012) barrier passing peptides) http://crdd.osdd.net/raghava/ Tyagi et al. (2015) cancerppd/index.php (anticancer peptides and proteins) http://crdd.osdd.net/raghava/cppsite/ Agrawal et al. (2015) (cell-penetrating peptides) http://crdd.osdd.net/raghava/hemolytik/ Gautam et al. (2014) (hemolytic and nonhemolytic peptides) http://isyslab.info/NeuroPep/ Wang et al. (2015) (neuropeptides) http://quorumpeps.ugent.be/ (quorum sensing signaling peptides)

Wynendaele et al. (2013)

(Continued)

Databases of bioactive peptides 319 Table 14.2: (Continued) Database of:

Name

Website

Reference

SATPdb

http://crdd.osdd.net/raghava/satpdb/ links.php (a metabase of therapeutic peptides) http://crdd.osdd.net/raghava/thpdb/ index.html (FDA-approved therapeutic peptides) http://crdd.osdd.net/raghava/ tumorhope/ (tumor homing peptides)

Singh et al. (2016)

THPdb

TumorHoPe

Usmani et al. (2017)

Kapoor et al. (2012)

All web addresses were verified in November 2019.

and 1600 peptides acting as ACE inhibitors/antihypertensive agents (as of November 2019). Criteria of searching for peptides in AHTPDB include sequence and SMILES. Apart from them, a user can apply an advanced search option relying on a conditional search by applying mathematical conditions, that is, “like,” “ 5 ,” “.,”“ , ,” “and,” “or,” “not.” Apart from such data as the sequence of a peptide, its length, molecular weight, reference data, additional information about the peptide of interest that can be obtained from AHTPDB may include, for example, IC50, pI, bitterness, assay, source, purification technique, and extent of systolic pressure decrease (for details see tab called “BASIC SEARCH” provided in AHTPDB). Several databases are dedicated to antimicrobial peptides (see Table 14.2). Peptides exhibiting this activity are in the area of food science that aims to search for the novel methods of food biopreservation being the alternative to replace chemical preservatives such as nitrates, benzoates, sulfites, sorbates, and formaldehyde (Ben said, Fliss, Offret, & Beaulieu, 2019). In majority of antimicrobial databases, the search of peptides includes the following options: name, microorganism, experimental methods, administration, model, activity (expressed in different measures), biofilm stage, target source, and organism group (e.g., BaAMPs, CAMPR3). Additional search criteria available in the antimicrobial peptide databases include UniProt ID, PDB ID, PubMed ID (CAMPR3), sequence length, chemical structure, complexity (DBAASP) and sequence length, mass, net charge, pI, Boman index, and hydropathy index (MilkAMP). The YADAMP database characterizes expanded criteria for peptide search. Apart from “typical” options, like sequence or its length, this database enables finding the peptides of interest by, for example, helicity, flexibility, 3D structures, Boman index, mean hydrophobic moment, and MIC (i.e., minimum inhibitory concentration) values for different microorganisms, etc. In turn, the APD3 database enables finding peptides according to their antimicrobial activity, antiGram(1) and antiGram(2) nature, and percentage of hydrophobic residues. When acquiring the information on peptides via search tools, some of the abovementioned databases operate via logical expressions like “or/and” “yes/no.” When presenting the search options provided in antimicrobial peptide databases, we showed some query examples as they were exactly worded.

320 Chapter 14 Databases assigned to the “other” category (see Table 14.1) may also be a useful source of information about bioactive sequences. For example, the BIOPEP-UWM database is divided into “bioactive peptides database” (see earlier) as well as “sensory peptides and amino acids” (Iwaniak, Minkiewicz, Darewicz, Sieniawski, & Starowicz, 2016). It is well-known that the bioactivity of food protein hydrolysates is often correlated with taste, especially with bitterness resulting from the presence of peptides composed of certain amino acids. Hence, data found in databases can provide a practical option to, for example, associate bioactivity of a peptide with a taste property (Iwaniak, Minkiewicz, Darewicz, & Hrynkiewicz, 2016). To enable this, the BIOPEP-UWM database of sensory peptides is identically organized as its twin database of bioactive peptides, including the search options. The tab called “Additional information” found in both databases contains the information on the additional function of the bioactive/tastant peptide (if any). The search options provided in databases and developed at the Bioinformatics Centre, CSIR-Institute of Microbial Technology, Chandigarh, India, that is, AntiTbPdb, CancerPPD, CPPSite 2.0, Hemolytik, SATPdb, THPdb, and TumorHoPe, are similar. They include, for example, the mechanism of peptide action, its C- and N-terminal modifications, chemical modifications, its nature (cyclic/linear), and its cytotoxicity. Depending on the “thematic” content of a database, the search criteria may include the list of available methods for determinations of the function of peptides and their response (BrainPeps), cell line (AVPdb), and the list of known receptors (Quorumpeps). To recapitulate, we showed some exemplary options enabling the search of peptides using other criteria than the sequence match (as of November 2019). These options are dependent on the “thematic” content of each database. When presenting the peptide search options characteristic for a specific database, we did not mention all of them. We rather tried to show the ones that were distinguishing one thematic database from another (e.g., MilkAMP and YADAMP). The criteria of peptide search described earlier were also presented using the exact words provided in the individual database search tool. Thus, to find out more details concerning this issue, we recommend visiting a particular database website. Our suggestion refers to all databases presented in this chapter.

14.5 Using bioinformatic databases for the analysis of food proteins and peptides Research strategies involving the application of in silico methods can include different areas for studying biopeptides using databases. Databases of peptide sequences can be used for (1) the creation of datasets to analyze the relationships between the structure and the bioactivity of peptides and (2) positive and negative selection of the sequences released from proteins due to the action of enzymes. The first research concerns the QSAR (quantitative structure
activity

Databases of bioactive peptides 321 relationships) approach, whereas the second is related to the situation when the identified peptide is found in the database (positive selection) or not (negative selection). The positive selection is a useful strategy when discovering peptides with known bioactivity present in new protein sources, while the negative selection allows discovering a new peptide in both known and unknown protein sources (Iwaniak, Darewicz, et al., 2019). Iwaniak, Hrynkiewicz, Bucholska, Darewicz, and Minkiewicz (2018) and Iwaniak, Hrynkiewicz, Bucholska, Minkiewicz, and Darewicz (2019) used the BIOPEP-UWM database of sensory peptides and amino acids to create data matrices consisting of 51 bitter di- and tripeptides each. The variables described some physicochemical properties typical of each amino acid being a part of bitter peptides. They were derived from on-line available tools such as AAindex database, ProtScale, and Biological Magnetic Resonance Data Bank. The bitter taste of peptides was expressed in Rcaf. values, that is, bitterness relative to that of 1 mM caffeine solution (Rcaf. 5 1.0). Based on multilinear regression and principal component analysis, it was found that the bitter taste of peptides depended on the presence of amino acids containing branched side chain or ring. Such properties of peptides referred to the presence of L, I, V, Y, and F in a peptide sequence. Moreover, hydrophobicity and bulkiness had also an impact on bitter taste of the analyzed peptides (Iwaniak et al., 2018; Iwaniak, Hrynkiewicz, et al., 2019). Maestri, Pavlicevic, Montorsi, and Marmiroli (2018) proposed a universal protocol involving a database of bioactive peptide sequences (BIOPEP-UWM) to analyze the impact of the type and location of an amino acid in a peptide chain on the bioactivity of peptides derived from animal food sources. Briefly, this protocol relied on the compilation of peptide sequences found in the database (BIOPEP-UWM) as well as in literature to create “own collection” of peptides, then the creation of data matrices differing in the bioactivity of peptides, statistical analysis to compare the frequencies of amino acids, and finally scoring them according to the position of amino acids in a peptide chain. It was reported that opioid peptides derived from animal sources were rich in aromatic residues, whereas antibacterial peptides abundant in positively charged amino acids. Moreover, it was found that the activity of these peptides depended on the location of the residue in a peptide sequence. The biopeptides with the highest bioactivity contained aromatic/positively charged amino acid in the first or penultimate position. In addition, peptides whose effect was confirmed in vivo were rich in proline; however, this amino acid was located in different positions related to the peptide chain length (Maestri et al., 2018). The above examples show the usefulness and universal character of applying databases of bioactive peptide sequences combined with other programs containing biological data to create models helping to better understanding the relationships between the chemical nature of biopeptides and their experimentally determined bioactivity. Gallego, Mora, and Toldra´ (2019) used the BIOPEP-UWM database tools to find sequential fragments of dry-cured ham-originating proteins associated with the biological activity and

322 Chapter 14 taste. According to in silico predictions, some protein sequences of dry-cured ham were reported as potentially good sources of peptides with dual, that is, ACE and DPP-IV, inhibitory activity: AA, AG, AY, EI, EK, GP, HP, IL, IP, KA, KP, LA, LR, PL, PP, QG, RG, RL, VE, VF, VK, VY, and YV. Some of them were also associated with the bitter taste-like peptides AA and EI. Such an example of predicting the “peptide profile” of a food protein can show some composition similarities between peptides with multifunctional activities as well as correlations between bioactivity and taste; however, further empirical studies are required to confirm the results of computer simulations (Gallego et al., 2019). According to Gallego et al. (2019), in silico studies concerning tastant peptides can be a supportive tool in the sensoproteomics approach combining sensory evaluation with instrumental analysis in peptide identification and in monitoring their transformations during food processing. The production of bioactive peptides involves the following steps: protein and enzyme selection, protein hydrolysis, purification of peptide and then its identification, and finally bioactivity assay (in vitro and/or in vivo). It is costly and time-consuming; thus computer simulation of protein hydrolysis may offer the initial step to indicate peptide candidates potentially released from the protein (Anekthanakul, Hongsthong, Senachak, & Ruengjitchatchawalya, 2018). Anekthanakul et al. (2018) developed a platform called SpirPep (http://spirpep.sbi.kmutt.ac.th) helping to discover putative peptides in silico released from proteins. The potentially released peptides were searched using an in-house database collected from 13 databases including APD, BACTIBASE, BAGEL3, CAMP, Defensins knowledgebase, EROP-Moscow, Hmrbase, PenBase (not available now), PeptideDB, PhytAMP, RAPD, AcePepDB (not available now), and BIOPEP-UWM. The SpirPep enables selecting up to 15 enzymes for protein hydrolysis, whereas similar tools for an in silico digestion involve a maximum of 3 (BIOPEP-UWM) to 29 (PeptideCutter) proteases (Anekthanakul et al., 2018). The comparison of results of protein hydrolysis using different in silico tools yielded different outcomes. They resulted from the “specificity” of a program. For example, the BIOPEP-UWM “Enzyme action” tool is based on cut and recognition sequences (Minkiewicz, Iwaniak, et al., 2019), whereas the SpirPep platform works on the same algorithms as PeptideCutter does (Anekthanakul et al., 2018). Thus, for example, in silico hydrolysis of temperature stress
expressed protein by trypsin revealed 418 (SpirPep) and 371 (BIOPEP-UWM) biopeptides released (Anekthanakul et al., 2018). An example of the positive selection was presented by Parmar, Hati, and Sakure (2018) who analyzed fermented goat milk (new proteins source) toward the presence of ACE inhibitors known from their biological effect and present in AHTPDB. They reported that goat milk fermented with LF (Lactobacillus fermentum) and NK9 (Lactobacillus casei KR732325) was the source of 26 peptides identified with RP-LC/MS. All of them matched the ACE-inhibiting fragments listed in AHTPDB. However, they were mostly the fragments

Databases of bioactive peptides 323 of longer chain ACE inhibitors identified in bovine and sheep milk, and their products (e.g., cheese). Among them, one sequence (i.e., AFPEHK) matched the ACE-inhibiting motifs originally derived from goat proteins according to AHTPDB (Parmar et al., 2018). The purpose of the negative selection is not to find the peptides of interest in databases. The peptide not found in databases is then considered as newly discovered (Iwaniak, Darewicz, et al., 2019). Such an approach was employed for the hydrolysis of oat protein isolates with papain and ficin, identification of peptides in the hydrolysates, and synthesis of peptides (not found in BIOPEP-UWM database) to determine their ACE- and DPP-IV inhibitory effect. The following peptides were reported as novel: FFG, IFFFL, PFL, WWK, WCY, FPIL, CPA, FLLA, and FEPL. Among them, FEPL (48.9%), IFFFL (53.0%), WWK (95.3%), FLLA (97.0%), and WCY inhibited ACE, whereas CPA and FPIL inhibited DPP-IV (Bleakley, Hayes, O’Shea, Gallagher, & Lafarga, 2017). This chapter presented only few examples for the study of bioactive peptides using databases of biopeptide sequences as a supportive tool. We focused on examples mostly based on creating the own collection of peptides and/or their selection for further studies. In silico analyses of peptides and their parent proteins present a wider spectrum of usage than that presented above. This spectrum includes, for example, molecular docking and DoE (design of experiment) (Iwaniak, Darewicz, et al., 2019). The statement of Tu, Cheng, Lu, and Du (2018) concerning molecular docking in studying bioactive peptides may be applicable to analyze biopeptides using databases of their sequences. Thus, paraphrasing the words of Tu et al. (2018), it can be said that databases are widely used for bioactive peptide analysis as a standard computational tool; however, some predictions must be overcome to increase the accuracy of prediction and the theoretical results must be validated by experiments. The majority of examples concerning the issues described in this chapter involved the BIOPEP-UWM database. It results from its popularity among food scientists since the database has been introduced for the first time in 2003. Ever since, few hundreds of papers were published, in which the BIOPEP-UWM database and its tools were described and employed for the analysis of food proteins and biopeptides. The likely reason of using the BIOPEP-UWM database in research on bioactive peptides may be the fact that, according to Tu et al. (2018), this database “is a tool that not only interlinks databases of protein sequences, bioactive peptides, and sensory peptides, but also is an inbuilt program that aids in the prediction of proteolytic hydrolysates and allergenic peptides.” Biological and chemical databases are recently recommended as tools for student’s education (Atwood et al., 2015; Minkiewicz et al., 2016; Tuvi-Arad & Blonder, 2019). The bioinformatics exercise is included within the courses of biochemistry or organic chemistry with food biochemistry involved in all areas of study offered by the Faculty of Food Science of University of Warmia and Mazury in Olsztyn. Bioinformatic tools used during exercise include BIOPEP-UWM and EROP-Moscow databases as well as PeptideCutter and

324 Chapter 14 Peptide Property Calculator programs. The brief workflow showing how these databases and tools can be used for students’ education is shown in Fig. 14.2. The presented workflow reflects typical research strategies supported by bioinformatics, applied in peptide science (Iwaniak, Darewicz, et al., 2019; Minkiewicz, Turło, et al., 2019; Minkiewicz, Iwaniak, et al., 2019). The exercise includes protein sequence analysis using multiple tools. Such a strategy is recently recommended as the best practice in work concerning bioactive compounds. An example of the experiment involving peptide identification using mass spectrometry and application of few bioinformatic tools for the interpretation of results has recently been published by Carrera, Ezquerra-Brauer, and Aubourg (2020).

Figure 14.2 The example of workflow presenting the application of bioinformatic databases and associated tools for students’ education.

Databases of bioactive peptides 325

14.6 Conclusion We live in Big Data Era, which implies the development of modern technologies that help analyze peptides derived from foods. These technologies include some computations made remotely from the browser on any computer. Databases are one of the solutions that provide information about peptide sequences. The knowledge acquired from databases can be used to create own datasets for the “peptide structure
activity” studies and/or to initially identify peptides that may occur (or not) in food sources. An important question that should be borne in mind when using the databases is “are they trustworthy?” They can be, if they are continuously updated by knowledgeable experts in the field and if the information about peptides of interest can be completed from other databases (chemical codes of peptides are a convenient way to acquire an additional data). Thus it is recommended to use several, curated, thematic, complementary but not competing databases when collecting information about biopeptides. Such a protocol is especially important in studies aimed at discovering peptides in food sources using the negative selection approach.

Acknowledgments Project financially supported by the Minister of Science and Higher Education in the range of the program entitled “Regional Initiative of Excellence” for the years 2019
2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN as well as the funds of the University of Warmia and Mazury in Olsztyn (Project No. 17.610.014-300). The authors do not declare a conflict of interest.

References Agrawal, P., Bhalla, S., Usmani, S. S., Singh, S., Chaudhary, K., Raghava, G. P., & Gautam, A. (2015). CPPsite 2.0: A repository of experimentally validated cell penetrating peptides. Nucleic Acids Research, 44, D1098
D1103. Anekthanakul, K., Hongsthong, A., Senachak, J., & Ruengjitchatchawalya, M. (2018). SpirPep: An in silico digestion-based platform to assist bioactive peptides discovery from a genome-wide database. BMC Bioinformatics, 19, article 149. Apweiler, R., Bairoch, A., Wu, C. H., Barker, W. C., Boeckmann, B., Ferro, S., . . . Yeh, L. S. (2004). UniProt: The Universal Protein knowledgebase. Nucleic Acids Research, 32, D115
D119. Atwood, T. K., Bongcam-Rudloff, E., Brazas, M. E., Corpas, M., Gaudet, P., Lewitter, F., . . . van Gelder, C. W. G. (2015). GOBLET consortium, GOBLET: The global organisation for bioinformatics learning, education and training. PLoS Computational Biology, 11, Article no e1004143. Barker, W. C., Garavelli, J. S., Huang, H., McGarvey, P. B., Orcutt, B. C., Srinivasarao, G. Y., . . . Wu, C. (2000). The protein information resource (PIR). Nucleic Acids Research, 28, 41
44. Ben said, L., Fliss, I., Offret, C., & Beaulieu, L. (2019). Antimicrobial peptides: The new generation of food additives. In L. Melton, F. Shahidi, & P. Varelis (Eds.), Encyclopedia of food chemistry (Vol. 3, pp. 576
582). Amsterdam: Elsevier. Benson, D. A., Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., & Sayers, E. W. (2014). GenBank. Nucleic Acids Research, 42(D1), D32
D37.

326 Chapter 14 Berg, K. L., Seymour, T., & Goel, R. (2013). History of databases. International Journal of Management & Information Systems, 17(1), 29
35. Bleakley, S., Hayes, M., O’Shea, N., Gallagher, E., & Lafarga, T. (2017). Predicted release and analysis of novel ACE-I, renin, and DPP-IV inhibitory peptides from common oat (Avena sativa) protein hydrolysates using in silico analysis. Foods, 6(12), article 108. Bleasby, A. J., Akrigg, D., & Attwood, T. K. (1994). OWL—A non-redundant composite protein sequence database. Nucleic Acids Research, 22, 3574
3577. Bouadjenek, M. R., Zobel, J., & Verspoor, K. (2019). Automated assessment of biological databases assertion using the scientific literature. BMC Bioinformatics, 20, article 216. Braschi, B., Denny, P., Gray, K., Jones, T., Seal, R., Tweedie, S., . . . Bruford, E. (2019). Genenames.org: The HGNC and VGNC resources in 2019. Nucleic Acids Research, 47(D1), D786
D792. Brusic, V., & Zeleznikow, J. (1999). Knowledge discovery and data mining in biological databases. The Knowledge Engineering Review, 14, 257
277. Carreiro, A. L., Dhillon, J., Gordon, S., Higgins, K. A., Jacobs, A. G., McArthur, B. M., . . . Mattes, R. D. (2016). The macronutrients, appetite, and energy intake. Annual Review of Nutrition, 17, 73
103. Carrera, M., Ezquerra-Brauer, J. M., & Aubourg, S. P. (2020). Characterization of the jumbo squid (Dosidicus gigas) skin by-product by shotgun proteomics and protein-based bioinformatics. Marine Drugs, 18, Article No 31. Caspi, R., Billington, R., Fulcher, C. A., Keseler, I. M., Kothari, A., Krummenacker, M., . . . Karp, P. D. (2018). The MetaCyc database of metabolic pathways and enzymes. Nucleic Acids Research, 46(D1), D633
D639. Cha´vez, E., & Gonzalo, N. (2005). Metric databases. In L. C. Rivero, J. H. Doorn, & V. E. Ferraggine (Eds.), Encyclopedia of database technologies and applications (pp. 367
372). Hershey, PA: Idea Group Inc. Chen, B., Dong, X., Jiao, D., Wang, H., Zhu, Q., Ding, Y., & Wild, D. J. (2010). Chem2Bio2RDF: A semantic framework for linking and data mining chemogenomic and systems chemical biology data. BMC Bioinformatics, 11, article 255. Date, C. J. (2003). An introduction to database systems (8th ed.). Boston, MA: Addison Wesley. Dawson, N. L., Lewis, T. E., Das, S., Lees, J. G., Lee, D., Ashford, P., . . . Sillitoe, I. (2017). CATH: An expanded resource to predict protein function through structure and sequence. Nucleic Acids Research, 45 (D1), D289
D295. Di Luca, M., Maccari, G., Maisetta, G., & Batoni, G. (2015). BaAMPs: The database of biofilm-active antimicrobial peptides. Biofouling, 31, 193
199. Gallego, M., Mora, L., & Toldra´, F. (2019). The relevance of dipeptides and tripeptides in the bioactivity and taste of dry-cured ham. Food Production, Processing, and Nutrition, 1, 2. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In J. M. Walker (Ed.), The proteomics protocols handbook (pp. 571
607). Totowa, NJ: Humana Press Inc. Gautam, A., Chaudhary, K., Singh, S., Joshi, A., Anand, P., Tuknait, A., . . . Raghava, G. P. S. (2014). Hemolytik: A database of experimentally determined hemolytic and non-hemolytic peptides. Nucleic Acids Research, 42, D444
D449. Hall, A. S., Shan, Y., Lushington, G., & Visvanathan, M. (2013). An overview of computational life science databases & exchange formats of relevance to chemical biology research. Combinatorial Chemistry & High Throughput Screening, 16, 189
198. Hastings, J., Owen, G., Dekker, A., Ennis, M., Kale, N., Muthukrishnan, V., . . . Steinbeck, C. (2016). ChEBI in 2016: Improved services and an expanding collection of metabolites. Nucleic Acids Research, 44, D1214
D1219. Hudson, T., Anderson, W., Aretz, A., et al. (2010). International network of cancer genome projects. Nature, 464, 993
998. Iwaniak, A., Darewicz, M., Mogut, D., & Minkiewicz, P. (2019). Elucidation of the role of in silico methodologies in approaches to studying bioactive peptides derived from foods. Journal of Functional Foods, 61, article 103486.

Databases of bioactive peptides 327 Iwaniak, A., Hrynkiewicz, M., Bucholska, J., Darewicz, M., & Minkiewicz, P. (2018). Structural characteristics of food protein-originating di- and tripeptides using principal component analysis. European Food Research and Technology, 244(10), 1751
1758. Iwaniak, A., Hrynkiewicz, M., Bucholska, J., Minkiewicz, P., & Darewicz, M. (2019). Understanding the nature of bitter-taste di- and tripeptides derived from food proteins based on chemometric analysis. Journal of Food Biochemistry, 43, e12500. Iwaniak, A., Minkiewicz, P., & Darewicz, M. (2014). Food-originating ACE inhibitors, including antihypertensive peptides, as preventive food components in blood pressure reduction. Comprehensive Reviews in Food Science and Food Safety, 13, 114
134. Iwaniak, A., Minkiewicz, P., Darewicz, M., & Hrynkiewicz, M. (2016). Food protein-originating peptides as tastants
Physiological, technological, sensory, and bioinformatic approaches. Food Research International, 89, 27
38. Iwaniak, A., Minkiewicz, P., Darewicz, M., Sieniawski, K., & Starowicz, P. (2016). BIOPEP database of sensory peptides and amino acids. Food Research International, 85, 155
161. Jeske, L., Placzek, S., Schomburg, I., Chang, A., & Schomburg, D. (2019). BRENDA in 2019: A European ELIXIR core data resource. Nucleic Acids Research, 47, D542
D549. Kapoor, P., Singh, H., Gautam, A., Chaudhary, K., Kumar, R., Raghava, G. P. S., & Xue, B. (2012). TumorHoPe: A database of tumor homing peptides. PLoS One, 7, e35187. Kawashima, S., Pokarowski, P., Pokarowska, M., Kolinski, A., Katayama, T., & Kanehisa, M. (2008). AAindex: Amino acid index database, progress report 2008. Nucleic Acids Research, 36, D202
D205. Kim, S., Chen, J., Cheng, T., Gindulyte, A., He, J., He, S., . . . Bolton, E. E. (2019). PubChem 2019 update: Improved access to chemical data. Nucleic Acids Research, 47, D1102
D1109. Kumar, R., Chaudhary, K., Sharma, M., Nagpal, G., Chauhan., Singh, J., . . . Raghava, G. P. S. (2015). AHTPDB: A comprehensive platform for analysis and presentation of antihypertensive peptides. Nucleic Acids Research, 43, D956
D962. Kumar, R., Kumar, A., Sharma, R., & Baruwa, A. (2010). Pharmacological review on natural ACE inhibitors. Der Pharmacia Lettre, 2, 273
293. Lamesch, P., Berardini, T. Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., . . . Huala, E. (2012). The Arabidopsis Information Resource (TAIR): Improved gene annotation and new tools. Nucleic Acids Research, 40(D1), D1202
D1210. Lesk, A. M. (2008). Databases. Introduction to bioinformatics (3rd ed., pp. 148
161). New York: Oxford University Press Inc. Lewis, T. E., Sillitoe, I., Dawson, N., Lam, S. D., Clarke, T., Orengo, C. A., & Lees, J. G. (2018). Gene3D: Extensive prediction of globular domains in proteins. Nucleic Acids Research, 46(D1), D435
D439. Li, Q., Zhang, C., Chen, H., Xue, J., Liang, M., & Chen, M. (2018). BioPepDB: An integrated data platform for food-derived bioactive peptides. International Journal of Food Sciences and Nutrition, 69, 963
968. Li-Chan, E. C. Y. (2015). Bioactive peptides and protein hydrolysates: Research trends and challenges for application as nutraceuticals and functional food ingredients. Current Opinion in Food Science, 1, 28
37. Liu, X., Yu, X., Zack, D. J., Zhu, H., & Qian, J. (2008). TiGER: A database for tissue-specific gene expression and regulation. BMC Bioinformatics, 9, 271. Lu, Z. (2011). PubMed and beyond: A survey of web tools for searching biomedical literature. Database, 2011, baq036. Ma, L., Li, A., Zou, D., Xu, X., Xia, L., Yu, J., . . . Zhang, Z. (2015). LncRNAWiki: Harnessing community knowledge in collaborative curation of human long non-coding RNAs. Nucleic Acids Research, 43(D1), D187
D192. Maestri, E., Pavlicevic, M., Montorsi, M., & Marmiroli, N. (2018). Meta-analysis for correlating structure of bioactive peptides in foods of animal origin with regard to effect and stability. Comprehensive Reviews in Food Science and Food Safety, 18(1), 3
30. Marconi, S., Durazzo, A., Camilli, E., Lisiciani, S., Gabrielli, P., Aguzzi, A., . . . Marletta, L. (2018). Food composition databases: Consideration about complex food matrices. Foods, 7, 2.

328 Chapter 14 Miller, M. A. (2002). Chemical database techniques in drug discovery. Nature Reviews Drug Discovery, 1, 220
227. Minkiewicz, P., Turło, M., Iwaniak, A., & Darewicz, M. (2019). Free accessible databases as a source of information about food components and other compounds with anticancer activity
brief review. Molecules, 24, article 789. Minkiewicz, P., Iwaniak, A., & Darewicz, M. (2017). Annotation of peptide structures using SMILES and other chemical codes
Practical solutions. Molecules, 22(2075), 1
17. Minkiewicz, P., Iwaniak, A., & Darewicz, M. (2019). BIOPEP-UWM database of bioactive peptides: Current opportunities. International Journal of Molecular Sciences, 20, article 5978. Minkiewicz, P., Darewicz, M., Iwaniak, A., Bucholska, J., Starowicz, P., & Czyrko, E. (2016). Internet databases of the properties, enzymatic reactions, and metabolism of small molecules-search options and applications in food science. International Journal of Molecular Sciences, 17, article 2039. Nielsen, S. D., Beverly, R. L., Qu, Y., & Dallas, D. C. (2017). Milk bioactive peptide database: A comprehensive database of milk protein-derived peptides and novel visualization. Food Chemistry, 232. Nishant, T., Kumar, A., Sathish Kumar, D., & Vijaya Shanti, B. (2011). Biological databases—Integration of life science data. Journal of Computer Science and Systems Biology, 4, 87
92. O’Boyle, N. M., Banck, M., James, C. A., Morley, C., Vandermeersch, T., & Hutchison, G. R. (2011). Open Babel: An open chemical toolbox. Journal of Cheminformatics, 3, article 33. Odell, S. G., Lazo, G. R., Woodhouse, M. R., Hane, D. L., & Sen, T. Z. (2017). The art of curation of biological database: Principles and application. Current Plant Biology, 11-12, 2
11. Panyayai, T., Ngamphiw, C., Tongsima, S., Mhuantong, W., Limsripraphan, W., Choowongkomon, K., & Sawatdichaikul, O. (2019). FeptideDB: A web application for new bioactive peptides from food protein. Heliyon, 5(7), e02076. Parmar, H., Hati, S., & Sakure, A. (2018). In vitro and in silico analysis of novel ACE-inhibitory bioactive peptides derived from fermented goat milk. International Journal of Peptide Research and Therapeutics, 24(3), 441
453. Piotto, S. P., Sessa, L., Concilio, S., & Ianelli, P. (2012). YADAMP: Yet another database of antimicrobial peptides. International Journal of Antimicrobial Agents, 39, 346
351. Pirtskhalava, M., Gabrielian, A., Cruz, P., Griggs, H. L., Squires, R. B., Hurt, D. E., . . . Tartakovsky, M. (2016). DBAASP v.2: An enhanced database of structure and antimicrobial/cytotoxic activity of natural and synthetic peptides. Nucleic Acids Research, 44, D1104
D1112. Qureshi, A., Thakur, N., Himani, T., & Kumar, M. (2013). AVPdb: A database of experimentally validated antiviral peptides targeting medically important viruses. Nucleic Acids Research, 42, D1147
D1153. Rawlings, N. D., Barrett, A. J., Thomas, P. D., Huang, X., Bateman, A., & Finn, R. D. (2018). The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Research, 46, D624
D632. Raza, K. (2012). Application of data mining in bioinformatics. Indian Journal of Computer Science and Engineering, 1, 114
118. Rozin, P., Fischler, C., Imada, S., Sarubin, A., & Wrzesniewski, A. (1999). Attitudes to food and the role of food in life in the U.S.A., Japan, Flemish Belgium and France: Possible implications for the diet
health debate. Appetite, 33, 163
180. Sa´nchez, A., & Va´zquez, A. (2017). Bioactive peptides: A review. Food Quality and Safety, 1, 29
46. Shtatland, T., Guettler, D., Kossodo, M., Pivovarov, M., & Weissleder, R. (2007). PepBank
A database of peptides based on sequence text mining and public peptide data sources. BMC Bioinformatics, 8, e280. Sigrist, C. J. A., de Castro, E., Cerutti, L., Cuche, B. A., Hulo, N., Bridge, A., . . . Xenarios, I. (2013). New and continuing developments at PROSITE. Nucleic Acids Research, 41, D344
D347. Singh, S., Chaudhary, K., Dhanda, S. K., Bhalla, S., Usmani, S. S., Gautam, A., . . . Raghava, G. P. (2016). SATPdb: A database of structurally annotated therapeutic peptides. Nucleic Acids Research, 44, D1119
D1126.

Databases of bioactive peptides 329 Sterling, T., & Irwin, J. J. (2015). ZINC 15
Ligand discovery for everyone. Journal of Chemical Information and Modeling, 55, 2324
2337. The Gene Ontology Consortium. (2019). The gene ontology resource: 20 Years and still going strong. Nucleic Acids Research, 47(D1), D330
D338. The RNA Central Consortium. (2019). RNAcentral: A hub of information for non-coding RNA sequences. Nucleic Acids Research, 47(D1), D221
D229. The´olier, J., Fliss, I., Jean, J., & Hammami, R. (2014). MilkAMP: A comprehensive database of antimicrobial peptides of dairy origin. Dairy Science and Technology, 94, 181
193. Tu, M., Cheng, S., Lu, W., & Du, M. (2018). Advancement and prospects of bioinformatics analysis for studying bioactive peptides from food-derived protein: Sequence, structure, and functions. Trends in Analytical Chemistry, 105, 7
17. Tuvi-Arad, I., & Blonder, R. (2019). Technology in the service of pedagogy: Teaching with chemistry databases. Israel Journal of Chemistry, 59, 572
582. Tyagi, A., Tuknait, A., Anand, P., Gupta, S., Sharma, M., Mathur, D., . . . Raghava, G. P. S. (2015). CancerPPD: A database of anticancer peptides and proteins. Nucleic Acids Research, 43, D837
D843. Ulrich, E. L., Akutsu, H., Doreleijers, J. F., Harano, Y., Ioannidis, Y. E., Lin, J., & Markley, J. L. (2008). BioMagResBank. Nucleic Acids Research, 36, D402
D408. Usmani, S. S., Bedi, G., Samuel, J. S., Singh, S., Kalra, S., Kumar, P., . . . Raghava, G. P. S. (2017). THPdb: Database of FDA-approved peptide and protein therapeutics. PLoS One, 12, e0181748. Usmani, S. S., Kumar, R., Kumar, V., Singh, S., & Raghava, G. P. S. (2018). AntiTbPdb: A knowledgebase of anti-tubercular peptides. Database (Oxford), 2018, bay025. Van Dorpe, S., Bronselaer, A., Nielandt, J., Stalmans, S., Wynendaele, E., Audenaert, K., . . . De Spiegeleer, B. (2012). Brainpeps: The blood-brain barrier peptide database. Brain Structure and Function, 217, 687
718. Waghu, F. H., Barai, R. S., Gurung, P., & Idicula-Thomas, S. (2015). CAMPR3: A database on sequences, structures and signatures of antimicrobial peptides. Nucleic Acids Research, 44, D1094
D1097. Wang, G., Li, X., & Wang, Z. (2016). APD3: The antimicrobial peptide database as a tool for research and education. Nucleic Acids Research, 44, D1087
D1093. Wang, J., Yin, T., Xiao, X., He, D., Xue, Z., Jiang, X., & Wang, Y. (2018). StraPep: A structure database of bioactive peptides. Database (Oxford), 2018, bay038. Wang, Y., Wang, M., Yin, S., Jang, R., Wang, J., Xue, Z., & Xu, T. (2015). NeuroPep: A comprehensive resource of neuropeptides. Database (Oxford), 2015, bav038. Wani, Y. M., Ganie, N. A., Rani, S., Mehraj, S., Mir, M. R., Baqual, M. F., . . . Dar, K. A. (2018). Advances in and applications of bioinformatics in various fields of life. International Journal of Fauna and Biological Studies, 5, 3
10. Wegner, K., Sterling, A., Guha, R., Bender, A., Faulon, J.-L., Hastings, J., . . . Willighagen, E. (2012). Cheminformatics. Communications of the ACM, 55(11), 65
75. Williams, A., & Tkachenko, V. (2014). The Royal Society of Chemistry and the delivery of chemistry data repositories for the community. Journal of Computer Aided Molecular Design, 28, 1023
1030. Wishart, D., Arndt, D., Pon, A., Sajed, T., Guo, A. C., Djoumbou, Y., . . . Rappaport, S. M. (2015). T3DB: The toxic exposome database. Nucleic Acids Research, 43(Database issue), D928
D934. Wishart, D. S. (2012). Small molecules and disease. PLoS Computational Biology, 8, e100. Wishart, D. S., Feunang, Y. D., Marcu, A., Guo, A. C., Liang, K., Va´zquez-Fresno, R., . . . Scalbert, A. (2018). HMDB 4.0: The human metabolome database for 2018. Nucleic Acids Research, 46(D1), D608
D617. Wishart, D. S., Knox, C., Guo, A. C., Cheng, D., Shrivastava, S., Tzur, D., . . . Hassanali, M. (2008). DrugBank: A knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Research, 36(Database issue), D901
D906. Wooley, J. C., & Lin, H. S. (2005). On the nature of biological data. Catalyzing inquiry at the interface of computing and biology (pp. 35
57). Washington, DC: National Academies Press.

330 Chapter 14 Wu, J., Liao, W., & Udenigwe, C. C. (2017). Revisiting the mechanism of ACE inhibitory peptides from food proteins. Trends in Food Science and Food Technology, 69, 214
219. Wynendaele, E., Bronselaer, A., Nielandt, J., D’Hondt, M., Stalmans, S., Bracke, N., . . . De Spiegeleer, B. (2013). Quorumpeps database: Chemical space, microbial origin and functionality of quorum sensing peptides. Nucleic Acids Research, 41, D655
D659. Zamyatnin, A. A., Borchikov, A. S., Vladimirov, M. G., & Voronina, O. L. (2006). The EROP-Moscow oligopeptide database. Nucleic Acids Research, 34, D261
D266. Zou, D., Ma, L., Yu, J., & Zhang, Z. (2015). Biological databases for human research. Genomics Proteomics and Bioinformatics, 13, 55
63.

CHAPTER 15

Encapsulation technology for protection and delivery of bioactive peptides Xiaohong Sun1,2, Ogadimma D. Okagu3 and Chibuike C. Udenigwe1,3 1

School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada, 2College of Food and Biological Engineering, Qiqihar University, Qiqihar, P.R. China, 3 Department of Chemistry and Biomolecular Sciences, Faculty of Science, University of Ottawa, Ottawa, ON, Canada

15.1 Introduction Proteins are one of the main nutrients in foods, especially those from animal origin such as egg, meat, milk, and fish (Toldra´, Reig, Aristoy, & Mora, 2018). Beyond their nutritional property, food proteins serve as a major source of bioactive peptides that have demonstrated beneficial pharmacological properties for promoting human health (Toldra´ et al., 2018; Udenigwe & Aluko, 2012). Generally, bioactive peptides are composed of 2
20 amino acids and most of them are rich in hydrophobic amino acid residues (Chakrabarti, Guha, & Majumder, 2018). To exert their beneficial effects, bioactive peptides must be released from the parent proteins by fermentation, food processing, enzymatic hydrolysis in vitro, or digestive enzymes in vivo (Hartmann & Meisel, 2007; Udenigwe & Aluko, 2012). To date, the identified biological roles of peptides mainly include antihypertensive, antimicrobial, cholesterol-lowering, antithrombotic, anticancer, immunomodulatory, mineral binding, opioid-like, and antioxidant activities (Chakrabarti et al., 2018; Hartmann & Meisel, 2007). The bioactivities of peptides have been investigated using in vitro biochemical assays, cell culture, in vivo animal models, or human clinical trials (Chakrabarti et al., 2018; Toldra´ et al., 2018). However, in vitro bioactivities of the peptides do not generally translate into in vivo pharmacological functions when evaluated in animal models or human subjects. This issue impedes the applications of bioactive peptides in formulating functional foods and nutraceuticals (Mohan, Rajendran, He, Bazinet, & Udenigwe, 2015). For example, milk-derived peptides MAP1 and MAP2 both showed angiotensin-converting enzyme (ACE)-inhibitory activity in vitro but only MAP1 reduced the blood pressure of human subjects with stage 1 hypertension compared to the placebo in a randomized controlled trial (Boelsma & Kloek, 2010). One of the major reasons for this discrepancy is the Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00028-5 © 2021 Elsevier Inc. All rights reserved.

331

332 Chapter 15 susceptibility of peptides to gastrointestinal digestion resulting in a loss of their structural integrity and bioactivities (Picariello et al., 2013; Udenigwe, 2014). When administered orally, peptides are susceptible to hydrolysis by at least 40 different endogenous enzymes in the gastrointestinal tract before reaching systemic circulation (Segura-Campos, Chel-Guerrero, Betancur-Ancona, & Hernandez-Escalante, 2011). The low biostability of peptides often leads to limited bioavailability. Peptide bioavailability is used to indicate their absorption and is defined as the fraction of the intact bioactive peptide that reaches systemic circulation (Fathi, Martı´n, & McClements, 2014). Thus, high biostability and bioavailability are prerequisites for utilizing bioactive peptides as enterally potent health-promoting compounds (Udenigwe & Aluko, 2012). Moreover, bioactive peptides often possess bitter taste and hygroscopicity due to the exposure of their hydrophobic and hygroscopic amino acid residues after hydrolysis, which further hinder their food applications (Mohan et al., 2015). Encapsulation technology has been developed for the protection, sustained release, and delivery of bioactive peptides with the primary goal of preserving their structures and physiological effects as well as improving their sensory properties. Encapsulation can be categorized into microencapsulation and nanoencapsulation (Mohan et al., 2015). In this chapter, peptide delivery systems are classified into microparticulate, hydrogel, and nanoparticulate delivery systems. We first provided an overview of each of these delivery systems followed by a discussion of their recent applications, challenges, and future trends in peptide encapsulation.

15.2 Microparticulate delivery systems In the 1980s, significant research efforts were made to develop the microencapsulation technology. Microencapsulation is a process that uniformly coats a functional compound (core material) with a continuous film of polymer or lipid (shell or matrix material) to fabricate free-flowing microparticles, which are mostly from 1 to 1000 μm in diameter (Suganya & Anuradha, 2017; Ye, Georges, & Selomulya, 2018). Generally, there are two types of the microparticulate system: microspheres and microcapsules, which are different in their internal structure and morphology, as shown in Fig. 15.1 (Herrero-Vanrell, Bravo-Osuna, Andre´s-Guerrero, Vicario-de-la-Torre, & Molina-Martı´nez, 2014). Microspheres are homogeneous dispersion of the active ingredient (core material) in the matrix of polymers (Prajapati, Jani, & Kapadia, 2015; Wong, Al-Salami, & Dass, 2018). Conversely, microcapsules are heterogeneous particles where a membrane shell forms a reservoir to entrap the core material (Lengyel, Ka´llai-Szabo´, Antal, Laki, & Antal, 2019). This section focuses on the carrier materials, fabrication methods of microparticulate delivery systems, and characterization of the sensory properties, hygroscopicity, release profile, gastric stability, and bioavailability of the encapsulated peptides.

Encapsulation technology for protection and delivery of bioactive peptides 333

Figure 15.1 Structure of microparticles: microspheres and microcapsules. Source: Adapted from Herrero-Vanrell, R., Bravo-Osuna, I., Andre´s-Guerrero, V., Vicario-de-la-Torre, M., & Molina-Martı´nez, I. T. (2014). The potential of using biodegradable microspheres in retinal diseases and other intraocular pathologies. Progress in Retinal and Eye Research, 42, 27
43.

15.2.1 Food-grade microparticulate carrier materials Biodegradable polymers, including natural and synthetic polymers, are extensively utilized as microparticle carrier materials in the pharmaceutical, biomedical, cosmetic, and plastic industries (Prajapati et al., 2015). Only natural polymers are approved to be applied as carrier matrices in the food industry, and the materials must be edible, biodegradable, biocompatible, nontoxic, and inexpensive. The carrier materials should also be structurally stable in food product matrices during processing and storage (Fathi et al., 2014). Food-grade microparticle carrier materials are classified into three categories: polysaccharides, proteins, and lipids (Mohan et al., 2015). 15.2.1.1 Polysaccharide-based carriers Polysaccharides are natural polymers of monosaccharides that vary in the number, type, distribution, and linkage of their monomer units (Fathi et al., 2014). Polysaccharides, mostly derived from plant, marine, algal, or microbial origins, are considered as one of the ideal choices of carrier matrices for preparing biodegradable delivery systems because they are readily available, inexpensive, rich in reactive functional groups, and structurally stable (Fathi et al., 2014). A variety of polysaccharides have been used as carriers of food compounds during production and processing, including starch, pectin, cellulose, dextrin, gum, alginate, chitosan, cyclodextrin, and their chemically modified derivatives (Fathi et al., 2014). For example, natural starch and hydrophobic starch derivatives have been used for the encapsulation of insulin and polyunsaturated fatty acids (Go¨kmen et al., 2011; Jain, Khar,

334 Chapter 15 Ahmed, & Diwan, 2008; Lesmes, Cohen, Shener, & Shimoni, 2009). Food-derived bioactive peptides have been successfully encapsulated in polysaccharide-based carriers. For instance, maltodextrin, maltodextrin/β-cyclodextrin, and maltodextrin/gum Arabic have been designed as shell materials to coat the peptides in casein hydrolysate, whey protein hydrolysate, and chicken meat protein hydrolysate (Kurozawa, Park, & Hubinger, 2009; Rao, Bajaj, Mann, Arora, & Tomar, 2016; Rocha, Trindade, Netto, & Favaro-Trindade, 2009; Yang et al., 2012). However, polysaccharides are prone to react with encapsulated peptides under extreme conditions. For example, Maillard reaction may occur at high temperature and result in the loss of bioactive peptides and production of potentially toxic products (Mohan et al., 2015). Thus, relatively inert polysaccharide carriers have been developed by the modification of their reactive functional groups by processes such as carboxymethylation (Ruiz, Campos, Ancona, & Guerrero, 2016). 15.2.1.2 Protein-based carriers Proteins have tremendous potential as encapsulation carriers because of their functional properties, such as solubility, water binding capacity, film formation, foaming, gelation, and emulsification (Mohan et al., 2015; Ye et al., 2018). Besides, protein-based carriers have additional nutritional benefits because they can provide essential amino acids after hydrolysis in the digestive tract (Mohan et al., 2015). Food proteins have been demonstrated to be effective carriers of hydrophobic compounds, such as flavonoids, β-carotene, fatty acids, and vitamins (Tavares, Croguennec, Carvalho, & Bouhallab, 2014). In contrast, the application of protein carriers in peptide encapsulation is challenging due to their structural similarity, which may lead to instability issues; only microencapsulation has been achieved to date (Mohan et al., 2015; Ye et al., 2018). Specifically, soybean protein-based carriers, including soybean protein isolate (SPI), SPI/gelatin and SPI/pectin, have been used to encapsulate casein hydrolysate to reduce its bitterness and hygroscopicity (Favaro-Trindade, Santana, Monterrey-Quintero, Trindade, & Netto, 2010; Mendanha et al., 2009; Molina Ortiz et al., 2009). Likewise, whey protein concentrate hydrolysate has been coated with its parent protein or protein-alginate blend by spray drying or freeze drying (Ma et al., 2014). Using spray drying, corn zein was used as a carrier to encapsulate antimicrobial peptide, nisin (mixed with thymol), to enhance their activity against Listeria monocytogenes during storage of a milk product (Xiao, Davidson, & Zhong, 2011). Chemical and physical modifications have been used to improve the functional properties of proteins to make them more suitable for peptide microencapsulation by spray drying. For instance, compared to native rapeseed protein isolate, acylated and high pressure-treated proteins showed higher encapsulation efficiencies, whereas the hydrolysate resulting from enzymatic modification had lower encapsulation efficiency when used as the wall material. Loss of the protein secondary structures after enzymatic modification caused the decrease in encapsulation efficiency (Wang, Ju, He, Yuan, & Wang, 2015).

Encapsulation technology for protection and delivery of bioactive peptides 335 15.2.1.3 Lipid-based carriers The two types of lipid-based carriers used for the encapsulation of bioactive peptides and protein hydrolysates are lipospheres and liposomes (Mohan et al., 2015). The focus herein would be on lipospheres; liposomes will be discussed in Section 15.4. Lipospheres consist of a hydrophobic matrix inner layer that contains the carrier fatty acids and the encapsulated core material, and a hydrophilic outer layer from the hydrophilic moiety of the carrier fatty acids or phospholipids (Ye et al., 2018). Because of this arrangement, lipospheres are suitable for encapsulating hydrophobic peptides, which associate with the carrier via hydrophobic interaction (Barbosa et al., 2004). Lipospheres have been fabricated from a mixture of stearic acid and soybean phosphatidylcholine to encapsulate different casein hydrolysate fractions using the melting process, resulting in encapsulation efficiencies of 50%
83%. The casein hydrolysate fraction with the strongest hydrophobicity showed the highest encapsulation efficiency, and hydrophobicity of the casein hydrolysates decreased after incorporation into the lipospheres (Barbosa et al., 2004). Other parameters may have also affected the encapsulation efficiency of peptides in the lipospheres since two peptide fractions with similar hydrophobicity had significantly different encapsulation efficiency (Barbosa et al., 2004). Casein hydrolysate has also been encapsulated in lipospheres consisting of 80% stearic acid, 20% cupuacu butter, and 4% polysorbate 80, resulting in an encapsulation efficiency of 74% (Pinho & da Silva, 2013). Casein hydrolysate loaded in lipospheres showed good oxidative stability over 60 days of storage; this may be because lipospheres are predominantly composed of saturated stearic acid and are devoid of oxidatively labile unsaturated fatty acids (Barbosa et al., 2004). However, the saturated fatty acid content of lipospheres raises health concerns, which limits their application in food products.

15.2.2 Techniques for fabricating microparticles Many techniques have been proposed for the preparation of biodegradable microparticles, such as coacervation, ionotropic gelation, solvent evaporation, spray drying, emulsification/ solidification, and extrusion (Prajapati et al., 2015). This section discusses the two most widely used approaches for the microencapsulation of bioactive peptides: spray drying and coacervation. 15.2.2.1 Spray drying Spray drying is often adapted to achieve the microencapsulation of both the protein and polysaccharide-based carriers owing to the low processing cost and ease of use of the technique (Mohan et al., 2015). Generally, the wall material is dispersed in a volatile solvent and mixed with the core material by high-speed homogenization. The resulting solution or dispersion is atomized in a stream of heated air to generate microparticles.

336 Chapter 15 The microparticle size typically ranges from 1 to 100 μm, depending on the atomizing conditions (Prajapati et al., 2015). The spray drying technique is only capable of producing microspheres with the core material homogeneously distributed in the matrix rather than having a clearly defined shell and core (Favaro-Trindade et al., 2010; Prajapati et al., 2015). The microspheres fabricated by spray drying have good stability and mostly show concavities on the surfaces resulting from the rapid solvent evaporation (Prajapati et al., 2015; Rocha et al., 2009). Different kinds of bioactive peptides, such as nisin, and hydrolysates-derived from casein, whey protein or chicken meat protein, have been successfully encapsulated in carriers by spray drying (Favaro-Trindade et al., 2010; Kurozawa et al., 2009; Ma et al., 2014; Molina Ortiz et al., 2009; Xiao et al., 2011). The drawbacks of this technique include low yield of the product and the likelihood of inducing peptide or protein denaturation and aggregation due to the high temperature applied during spray drying (Mohan et al., 2015; Prajapati et al., 2015). 15.2.2.2 Coacervation Coacervation is an effective method for the generation of microspheres and microcapsules through liquid
liquid phase separation (Prajapati et al., 2015). During the coacervation process, the core material is dispersed in the wall-forming polymer solution and the phases are subsequently separated to achieve encapsulation by changing environmental factors, such as ionic strength, pH and temperature (Lengyel et al., 2019). This technique is classified into simple coacervation and complex coacervation. Simple coacervation is mostly caused by salting out to give rise to incompatibilities between the polymers. The rationale behind complex coacervation is to form an insoluble complex, through electrostatic attraction, between polyelectrolyte polymers with opposite charges, which simultaneously encapsulates the core material. The pH of the mixture plays an important role in complex coacervation due to its influence on the isoelectric point of the polymers (Lengyel et al., 2019). Coacervation is extensively utilized for encapsulating hydrophobic compounds, especially flavor oils (Gouin, 2004; Lengyel et al., 2019). The most well-characterized complex coacervate microcapsule is composed of gelatin and gum acacia as the wall materials (Rabiˇskova´ & Vala´sˇkova´, 1998). Casein hydrolysate has been successfully encapsulated by complex coacervation with SPI and pectin as wall materials, and the encapsulation efficiency reached 92% (Mendanha et al., 2009). The major limitations of producing coacervated food ingredients are the high cost, complicated process, and potential health concerns due to the chemical cross-linkers used in the process (Gouin, 2004).

15.2.3 Bitter taste and hygroscopicity of microencapsulated peptides 15.2.3.1 Bitter taste Bitter taste of bioactive peptides hinders their applications in food products. Bitter taste of peptides is commonly evaluated by a sensory panel or taste dilution analysis

Encapsulation technology for protection and delivery of bioactive peptides 337 (Ma et al., 2014; Rocha et al., 2009). The bitterness intensity of encapsulated whey protein concentrate hydrolysate was reduced compared with nonencapsulated sample (Ma et al., 2014; Yang et al., 2012). Spray drying has a stronger bitterness-attenuating effect than lyophilization and mechanical blend techniques (Ma et al., 2014; Yang et al., 2012). Whey protein concentration, whey protein concentration/sodium alginate mixture, maltodextrin and maltodextrin/β-cyclodextrin mixture as wall materials have exhibited potent bitternessmasking effects (Ma et al., 2014; Yang et al., 2012). The bitterness intensity of peptides is positively correlated with the amounts of exposed hydrophobic amino acid groups (Rocha et al., 2009). Accordingly, it was suggested that the special cylinder-shaped hydrophobic cavity of β-cyclodextrin is beneficial for the encapsulation of bitter peptides via hydrophobic interaction (Yang et al., 2012). Microencapsulation of casein hydrolysate by spray drying to mask bitterness has been widely studied for potential applications in food products such as protein bars (Rocha et al., 2009). The decrease in bitterness was attributed to reduced exposure of hydrophobic groups of peptides due to their hydrophobic interactions with the wall material, such as maltodextrin/gum Arabic, gelatin/soy protein isolate and maltodextrin, during the encapsulation process (Favaro-Trindade et al., 2010; Molina Ortiz et al., 2009; Rao et al., 2016; Rocha et al., 2009). The bitter-masking effect may also be associated with low rate of dissolution in water of peptides in encapsulated hydrolysates (Favaro-Trindade et al., 2010). Similarly, the complex coacervation process suppressed the bitter taste of casein hydrolysate, and the encapsulation efficiency did not affect the bitter-masking effect (Mendanha et al., 2009). The encapsulation of casein hydrolysate in lipospheres slightly reduced bitterness but no significant difference was observed compared to the unencapsulated counterpart; the authors indicated that phospholipid astringency may have interfered with the sensory evaluation (Barbosa et al., 2004; Morais et al., 2004). 15.2.3.2 Hygroscopicity High hygroscopicity is not favorable for the storage, handling, and applications of bioactive peptides. Microencapsulation by spray drying is considered a promising technique for attenuating peptide hygroscopicity. For example, encapsulation of whey protein hydrolysate in whey protein concentrate/sodium alginate, maltodextrin, or maltodextrin/β-cyclodextrin carriers reduced hygroscopicity compared to the free hydrolysate (Ma et al., 2014; Yang et al., 2012). A similar result was obtained when the chicken meat protein hydrolysate was coated with maltodextrin/gum Arabic as the wall material (Kurozawa et al., 2009). The molecular weight of wall materials was suggested to significantly affect the hygroscopicity of encapsulated peptides (Yang et al., 2012). Likewise, the hygroscopicity of casein hydrolysate-loaded gelatin/soy protein isolate or maltodextrin device was significantly lower than that of the unencapsulated form (Favaro-Trindade et al., 2010; Rocha et al., 2009). On the other hand, encapsulated casein hydrolysate was more hygroscopic than the empty carriers (Favaro-Trindade et al., 2010).

338 Chapter 15 In contrast, the increased hygroscopicity of casein hydrolysate was also reported after encapsulation (Molina Ortiz et al., 2009). This variation could be attributed to the possible physical and structural changes of proteins during encapsulation (Mohan et al., 2015). Besides spray drying, complex coacervation was utilized to prepare encapsulated casein hydrolysate with SPI/pectin to reduce the hygroscopicity and it was indicated that the hygroscopicity was negatively correlated to encapsulation efficiency (Mendanha et al., 2009).

15.2.4 Release characteristics, gastric stability, and bioavailability of microencapsulated peptides Sustained release, good biostability and high bioavailability of encapsulated bioactive peptides are desirable for them to exert their physiological activities and functions as functional foods or nutraceuticals since the peptides need to reach their biological targets in an active form (Mohan et al., 2015). In vitro simulated gastrointestinal digestion model is commonly used to analyze the release characteristics and gastric stability of encapsulated active ingredients (Segura-Campos et al., 2011). It was reported that peptides encapsulated in microparticles (high pressure-treated rapeseed protein isolate as wall material) achieved sustained release in a simulated intestinal digestion system without an initial burst and showed good stability in a simulated gastric environment (Wang, Ju, He, Yuan, & Aluko, 2015). Active component release from microparticles involves several mechanisms, such as diffusion and swelling from nondegraded delivery systems or release through the degraded and erosive wall material (Sinha & Trehan, 2003; Wang, Ju, He, Yuan, & Aluko, 2015). High-pressure treatment was believed to produce a more rigid wall material to resist gastric digestion and be slowly degraded under simulated intestinal conditions, thus exhibiting good biostability and controlled release (Wang, Ju, He, Yuan, & Aluko, 2015). Additionally, the in vitro nisin release from alginate/resistant starch microparticles showed a sustained-release profile that was in accordance with a Fickian diffusion mechanism (Hosseini et al., 2014). When casein-derived peptides were encapsulated in water-in-oil-in-water double emulsions, the release pattern in the gastrointestinal environment was closely associated with the composition and hydrophobicity of the oil phase. It was indicated that mineral oil strongly retarded peptide release due to its indigestibility (Giroux, Robitaille, & Britten, 2016). Moreover, the release behavior of peptides was influenced by the microencapsulation methods. For example, the membrane emulsification technique was more conducive than the encapsulator and needle extrusion methods to achieve sustained release of encapsulated peptides (Huang, Xiao, Hao, & Yang, 2017). Encapsulation matrices also play a vital role in the release characteristics and gastric stability of bioactive peptides, so the most appropriate carrier should be selected (Go´mez-Mascaraque, Miralles, Recio, & Lopez-Rubio, 2016). It should be noted that the released peptides from microparticles in small intestine are still susceptible to further degradation before entering into enterocytes and subsequently into

Encapsulation technology for protection and delivery of bioactive peptides 339 circulation (Mohan et al., 2015). Hence, bioavailability of released peptides is paramount to the efficacy of some bioactivities, such as antioxidant, anti-inflammatory, and antihypertensive activities. Caco-2 cell monolayer is a widely used in vitro model for evaluating peptide transport and estimating their bioavailability. Huang et al. (2017) demonstrated that the microencapsulation of angiotensin 1-converting enzyme-inhibitory peptide by membrane emulsification did not affect the peptide transport in Caco-2 cell culture model compared with the peptide in its free form. Overall, there is a paucity of research work in understanding the release kinetics, biostability, and bioavailability of encapsulated bioactive peptides.

15.3 Hydrogel delivery systems Hydrogel beads (microgels) are colloidal particles containing a network of one or more types of biopolymer cross-linked by physical and/or chemical bonds (McClements, 2018). Microgels are promising delivery systems for encapsulation, protection, and release of bioactive peptides (Perry & McClements, 2020). Generally, the size of microgels ranges from a few hundred nanometers to a few millimeters, and is determined by the biopolymer composition and preparation methods (McClements, 2017). Protein and/or polysaccharides are widely used as food-grade biopolymers to manufacture microgels in food applications (Perry & McClements, 2020). Bioactive peptides can be trapped inside microgels either before or after microgel formation using different fabrication approaches (McClements, 2018). This section provides detailed information on the methods for producing bioactive peptide-loaded microgels. The encapsulation efficiency, release behavior, and bioactive activities of encapsulated peptides in microgels are also discussed.

15.3.1 Fabrication of bioactive peptide-loaded microgels Typically, the process of fabricating bioactive compound-loaded microgels involves two steps: particle formation and particle gelation, as shown in Fig. 15.2A. First, the bioactive compound and biopolymer are mixed in a solution to generate small particles with diameters from 100 nm to 1000 μm. Next, the biopolymers within the first-step small particles are cross-linked with each other by physical and/or chemical bonds giving rise to the bioactive compound-loaded microgels (McClements, 2017; McClements, 2018). In the first step, two distinct mixing approaches are usually employed depending on the physicochemical properties of the encapsulated compounds. To be specific, hydrophilic compounds are generally mixed directly with the biopolymer solution, whereas hydrophobic compounds are loaded into emulsions or nanoemulsions prior to blending with the biopolymer solution (McClements, 2017). Commonly used methods for fabricating microgels include emulsion templating, injection
gelation, coacervation, antisolvent precipitation, molding, and thermodynamic

(Continued)

Encapsulation technology for protection and delivery of bioactive peptides 341 incompatibility methods (McClements, 2017, 2018; Perry & McClements, 2020). The most appropriate method should be selected for specific applications because each method is unique in different aspects, such as cost, scalability, simplicity, throughput, and ability to prepare microgels with different structures and features (McClements, 2017, 2018). Two commonly used methods for producing bioactive peptide-incorporated microgels are summarized below. 15.3.1.1 Injection
gelation method

L

The common procedure of this method is to inject small aliquots of the loaded peptide and biopolymer solution into another solution to achieve biopolymer gelation, as illustrated in Fig. 15.2B (McClements, 2017). Mineral ions, enzymes, acids and bases in the gelling solution are extensively used to promote the cross-linking of biopolymer molecules. For instance, multivalent cations in the gelling solution, such as calcium, are commonly used to cross-link anionic polysaccharides (such as pectic, alginate, and carrageenan) and proteins (such as caseins at a pH above their isoelectric point). Likewise, transglutaminase and laccase are often utilized to enzymatically cross-link proteins (such as casein and whey protein) and some polysaccharides (such as beet pectin), respectively (Guo, Zhang, & Yang, 2012; McClements, 2017; Zeeb, Fischer, & Weiss, 2014). Alternatively, some biopolymers can be cross-linked by heat-set or cold-set gelation by controlling the temperature. For instance, gelatin microgels can be produced by injecting a hot-gelatin solution into a cold solution because gelatin adopts a random coil conformation at high temperature but changes to helical regions in cold environment that facilitates gelatin cross-linking through hydrogen bonding (Karim & Bhat, 2008). The other way around, globular protein microgels can be generated by heat denaturation that induces protein unfolding and subsequently results in protein cross-linking via hydrophobic interaction and disulfide bond (Foegeding, 2006). For example, Phaseolus lunatus protein hydrolysate has been successfully entrapped in the Delonix regia carboxymethylated gum/sodium alginate microgels that were cross-linked by calcium chloride (Ruiz et al., 2016). Similarly, casein hydrolysate was encapsulated in chitosan microgels using polyphosphoric acid (PPA) as a gelling agent (Yuan, Jacquier, & O’Riordan, 2018).

Figure 15.2 (A) Fabrication of bioactive compound-loaded microgels, including the two major steps: particle formation and particle gelation. (B) Preparation process of the injection
gelation method. The common procedure is to inject small aliquots of the compound and biopolymer solution into another solution to achieve biopolymer gelation. (C) Preparation process of the emulsion templating method. This technique consists of three major steps: particle formation, particle gelation, and oil removal.

342 Chapter 15 In particular, whey protein microgels that are produced using the cold-set gelation method are demonstrated to be promising encapsulation systems (O’Neill, Egan, Jacquier, O’Sullivan, & O’Riordan, 2014). However, it has been indicated that diffusional losses during cross-linking and washing of the microgels may happen when using the common procedure (as shown in Fig. 15.2B), that is, dropping the blend of encapsulated compound and whey protein solution into the gelling solution. Accordingly, higher encapsulation efficiency was obtained when blank microgels were first prepared and utilized as sorbents for immobilization of bioactive compounds, such as riboflavin and peptides (O’Neill et al., 2014; O’Neill, Egan, Jacquier, O’Sullivan, & O’Riordan, 2015). This method was reported to be efficient and have promising scale-up potential (O’Neill et al., 2015). 15.3.1.2 Emulsion templating This method includes three major steps: particle formation, particle gelation, and oil removal. The preparation process is depicted in Fig. 15.2C. First, a water-in-oil (W/O) emulsion is formed by homogenization of a compound-biopolymer aqueous solution with an oil phase containing surfactants. The internal structure of this emulsion is biopolymer-rich water droplets suspended in an oil phase. Second, the gelation of the biopolymer chains inside the water droplets can be accomplished by changing the system temperature or addition of chemical cross-linking agents such as calcium ions, acids, or bases, which is similar to the injection
gelation method. Finally, the oil phase is removed to obtain the hydrogel beads by centrifugation, filtration and/or solvent extraction (McClements, 2017). As a bioactive peptide model, insulin has been successfully encapsulated within different biopolymer microgels with this emulsification
gelation technique using alginate and chitosan, hydroxypropyl cellulose and poly(L-glutamic acid), alginate and dextran sulfate, and calcium alginate-based microgels (Bai et al., 2012; Goswami, Bajpai, & Bajpai, 2014; Lopes et al., 2015; Zhang, Wei, Lv, Wang, & Ma, 2011). Nonetheless, if insulin and alginate are mixed in the solution directly as illustrated in Fig. 15.2C, aggregation would occur immediately due to electrostatic interactions. Therefore, insulin should be loaded in alginate
chitosan microgels by other ways. For example, insulin can be dissolved in calcium chloride solution or chitosan solution or their combination (Zhang et al., 2011). This approach produces small hydrogel beads (,1 μm) with well-defined sizes, which is considered to be one of its major advantages (Goswami et al., 2014). However, the process is tedious, complex and time-consuming, and may not be appropriated for functional food applications due to the use of nonfood grade chemicals and organic solvents (Goswami et al., 2014; McClements, 2017, 2018; Zhang et al., 2011).

15.3.2 Encapsulation efficiency of bioactive peptides in microgels The maximum encapsulation efficiency obtained for pentapeptide (Leu-Trp-Met-Arg-Phe), dipeptide (Phe-Trp), riboflavin, and tryptophan in whey protein microgels were 95%, 56%,

Encapsulation technology for protection and delivery of bioactive peptides 343 57%, and 45%, respectively. These results indicated that encapsulation efficiency may increase with increase in hydrophobicity of the compounds (O’Neill et al., 2014). However, this study did not consider the possible effect of net charge and other structural properties on encapsulation efficiency (Mohan et al., 2015). When proteins and peptides were entrapped in chitosan
PPA microgels, high encapsulation efficiencies ( . 95%) were obtained for all the proteins, including bovine serum albumin (BSA), whey protein isolate and insulin, whereas casein hydrolysate only achieved B50% encapsulation efficiency (Yuan et al., 2018). Using BSA as a model protein to investigate the underlying reasons, it was suggested that the PPA concentration played a paramount role in encapsulation within the chitosan
PPA microgels. Specifically, the encapsulation efficiency of BSA remarkably increased from 53% to 96% with the PPA concentration increasing from 0.25% to 1% (w/w) (Yuan et al., 2018). It is possible that PPA at high concentrations forms a complex coating with chitosan on the surface of the microgels, thus preventing BSA from leaking out from the microgels. In contrast, the microgels were fragile and easily damaged during fabrication at low PPA concentrations. Hence, the high encapsulation efficiency of proteins may be attributed to the coated matrix type structure of microgels and the interaction between protein and PPA. For casein hydrolysate, the low molecular weight and lack of defined secondary structures may have caused the entrapped peptides to diffuse from the microgels more easily compared to the proteins, resulting in low encapsulation efficiency. Low encapsulation efficiency is one of the major challenges of manufacturing peptide-loaded chitosan
PPA microgels (Yuan et al., 2018).

15.3.3 Release behavior and bioactive properties of encapsulated peptides in microgels Three main release mechanisms of bioactive compounds from microgels include simple diffusion, swelling-controlled diffusion, and disintegration; the mechanisms are influenced by the nature of the biopolymers (McClements, 2017). The release profiles of insulin from hydroxypropyl cellulose- and poly (L-glutamic acid)-based microgels in simulated gastrointestinal conditions were found to mainly involve swelling-controlled diffusion mechanism (Bai et al., 2012). Specifically, limited amount of insulin was released in simulated gastric fluid (pH 1.2) because shrinkage of the microgels, resulting from strong H-bonding and hydrophobic interaction, impeded the insulin release. Three stages were observed during the process of insulin release in simulated intestinal conditions (pH 6.8). First, a burst release (B50%) occurred in 25 minutes possibly due to the release of insulin present at the microgel surface. Second, a sustained release (8%
10%) was achieved in 425 minutes, which was attributed to the swelling-controlled diffusion mechanism. Finally, a small amount of insulin was released because of an equilibrium of insulin concentration between the microgels and surrounding medium, and the interaction between residual insulin and the microgel structure (Bai et al., 2012). To maintain its hypoglycemic activity, insulin is

344 Chapter 15 expected to be released from microgels in a sustained manner at the intestinal phase. Therefore, the insulin-encapsulated microgels discussed above need to be improved. Entrapment in alginate
chitosan microgels was demonstrated to protect insulin from gastric degradation and realize a sustained release in the small intestine phase (Zhang et al., 2011). The release characteristics of entrapped peptides are affected by many factors, such as peptide hydrophobicity and cross-linking agent concentration. It was suggested that the highly hydrophobic peptides had lower release rate owing to the hydrophobic interaction between the peptides and microgels (O’Neill et al., 2015). In addition, the concentration of cross-linker CaCl2 was indicated as the main parameter that influences the release of protein hydrolysate from carboxymethylated gum/sodium alginate microgels. Low CaCl2 concentration (1.0 mM) led to a small amount (16.4%) of hydrolysate release in the gastric phase and an increased hydrolysate release (60.1%) in the intestinal digestion phase. The released hydrolysates in the simulated intestinal condition exerted potent ACE-inhibitory activity (Ruiz et al., 2016). The insulin released from alginate and chitosan-based microgel showed a good stability after digestion in the simulated gastric fluid (Zhang et al., 2011). In addition, the hypoglycemic effect of insulin-loaded microgels has been validated in vivo using diabetic rats (Lopes et al., 2015; Zhang et al., 2011). For instance, the blood glucose level of diabetic rats was significantly decreased and maintained for 60 hours after oral administration of the insulin-encapsulated alginate
chitosan microgels (Zhang et al., 2011). Therefore, these microgels may be utilized as insulin carriers for functional food applications.

15.4 Nanoparticulate delivery systems for bioactive peptides Most biological processes occur at nanoscale, which makes the application of nanotechnology in bioactive peptide encapsulation and delivery promising. The use of nanoencapsulation technologies has demonstrated significant potential in protecting bioactive peptides from changes in physical and chemical properties of the environment (Lemes et al., 2016). This technique limits the interaction of peptides with the environment and improves their aqueous solubility, bioavailability, circulation time, and cellular uptake (Alle´mann, Leroux, & Gurny, 1998; Lemes et al., 2016). Bioactive peptide is protected from degradation, transported, and discharged at the target site with the desired nutritional properties and decreased toxicity (Brandelli, 2012; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2013). The bioactive peptide can be encapsulated into a nanosphere where the encapsulating agent is completely homogenized with the bioactive compound such that the various nucleus cannot be distinguished. Nanocapsules with identifiable distinct cores could also be fabricated through nanoencapsulation where a polymeric material encloses the peptide thus protecting it from environmental effects (Lemes et al., 2016).

Encapsulation technology for protection and delivery of bioactive peptides 345 Bioactive peptides may be degraded in vivo (Maeno, Yamamoto, & Takano, 1996), which may not always be overcome through increasing the peptide dosage (Taylor, Bruce, Weiss, & Davidson, 2008). Preserving the peptides through nanoencapsulation technologies has been shown to increase their applicability in food by limiting their interaction with food components, oral and gastrointestinal fluids, and also increases their resistance against endogenous proteases (Laridi et al., 2003). A potential nanodelivery system should have some diverse properties and functions for efficient encapsulation, loading, and release (McClements, Decker, Park, & Weiss, 2009; Wang et al., 2014). Several nanoencapsulation systems (Table 15.1) have been developed for oral delivery of chemically unstable bioactive peptides for food, biomedical and biopharmaceutical applications. Some of the systems with their advantages and shortcomings are described below.

15.4.1 Liposome-based nanoencapsulation system for bioactive peptides Liposomes are made up of phospholipid bilayers containing hydrophobic and hydrophilic nanoorder interfaces. Their amphipathic structural properties enable the binding, transport, and release of both hydrophilic and lipophilic bioactive peptides (Mozafari, Johnson, Hatziantoniou, & Demetzos, 2008). Hydrophobic compounds are entrapped in the bilayer membrane whereas the aqueous center entraps hydrophilic molecules (Hua & Wu, 2013). Phosphatidylcholine nanovesicle-based encapsulation system derived from partially purified soy lecithin enhanced the stability of antimicrobial bacteriocin-like peptide P34 derived from aquatic Bacillus species. The bioactive peptide encapsulated by hydration film method showed antimicrobial activity against L. monocytogenes in both skim and whole milk within 21 days (Malheiros, Sant’Anna, Utpott, & Brandelli, 2012). This microorganism survives extreme environmental conditions that are detrimental to most bacterial and therefore remains active in food causing a number of infections (Cunningham, O’Byrne, & Oliver, 2009). The study demonstrates that peptide P34 encapsulated in lipid nanovesicles have strong potential for application as biopreservative for dairy products (Malheiros et al., 2012). Mohan, McClements, and Udenigwe (2016) also reported a successful encapsulation of whey peptides of various molecular weight into soy lecithin-derived nanoliposomes whose encapsulation efficiency and size were independent of the molecular weight of the various peptide fractions. However, molecular weight affected the inclusion and location of the bioactive peptides in the nanoliposome (Mohan et al., 2016). Conversely, peptide net charge influenced the encapsulation efficiency in nanoliposomes, with lower values reported for anionic peptides compared to cationic peptides; this is likely due to electrostatic repulsion of the anionic peptides with the liposome surface (Mohan, Rajendran, Thibodeau, Bazinet, & Udenigwe, 2018). Liposome-based nanodelivery system has also been reported to increase the stability and resident time of a short-lived appetite-stimulating peptide hormone, ghrelin, in the plasma (Moeller, Holst, Nielsen, Pedersen, & Østergaard, 2010).

Table 15.1: Common nanoencapsulation-based systems for protection and delivery of bioactive peptides. Nanoencapsulation technique

Encapsulated peptide/protein

Source of peptide/protein

Encapsulation/ Association efficiency (%)

Bioactivity

Size of encapsulated system (nm)

Nanoliposomes

Hydration film

P34

Aquatic Bacillus sp.

100

Antilisterial

160

Nanoliposomes

Hydration film

Whey peptides

B90

n.d.

127
189

Nanoliposomes

Hydration film

B80

Antioxidant

263
266

Nanoliposomes

Hydration film

Protein hydrolysates Ghrelin

Whey protein islolate Micropogonias furnieri Commercial

n.d

81
3035

Chitosan/alginate polyelectrolyte complex system Chitosan/pectin polyelectrolyte complex system PEG-graftedtrimethyl-chitosan copolymer-insulin nanocomplex Nanoemulsion

Ionic gelation

Insulin

Human

72.8

Appetitestimulation Glucose regulation

Electrostatic selfassembly

Insulin

Human

B62

Glucose regulation

240
1900

Maciel et al. (2017)

Electrostatic selfassembly

Insulin

Human

90

Glucose regulation

200
400

Mao et al. (2005)

Phase-inversion method Water-in-oil nanoemulsion Coacervation

Bovine serum albumin Insulin

Commercial

.90

None

22

Sun et al. (2012)

Human

B92

Glucose regulation

10
500

Insulin and leuprolide

48.5
89

None

320
522

Warm oil-in-water microemulsion

Cyclosporin

Bovine insulin and commercial leuprolide acetate Commercial

Barbari et al. (2017) Gallarate et al. (2011)

n.d.

Immunosuppression

250
290

Nanoencapsulation system

Nanoemulsion Solid lipid nanoparticles Solid lipid nanoparticles

n.d., Data not available; PEG, polyethylene glycol.

750

Reference Malheiros et al. (2012) Mohan et al. (2016) Mohan et al. (2018) Moeller et al. (2010) Sarmento et al. (2007)

Ugazio et al. (2002)

Encapsulation technology for protection and delivery of bioactive peptides 347 Despite several advantages which liposome-based nanovesicle systems offer, such as selfassembly, amphipathicity, low toxicity, flexibility, protection of compounds from early degradation or inactivation, modifiable biophysical and physicochemical properties, and biocompatibility (Sercombe et al., 2015), there are drawbacks limiting their clinical translation. Liposome-based nanodelivery systems face instability challenges during oral delivery as they are quite sensitive to their environment and often breakdown in food matrices and gastrointestinal environment. This problem is often overcome by using appropriate lipid combination, coating with polymers, double liposome preparation, proliposomes, and incorporation of stabilizing lipids in the structure (Daeihamed, Dadashzadeh, Haeri, & Akhlaghi, 2017). Some studies have also shown that liposome is not as immunologically inactive as once reported and could trigger immune response (Szebeni & Moghimi, 2009), hence leading to the production of antibodies against the encapsulated peptides. Multiple body defense systems treat liposome-based nanovesicles like other foreign nanoparticles. These defense systems are responsible for recognizing, neutralizing, and eliminating foreign invading particles. Liposome-based vesicles are usually cleared by macrophages in the reticuloendothelial system via direct interactions with the phagocytic cells. It has been hypothesized that saturation of macrophages with liposome-based particles could lead to immunosuppression and vulnerability to infectious diseases (Chrai, Murari, & Ahmad, 2002; Sercombe et al., 2015). Liposomes can also be eliminated by opsonization, which is usually dependent on the size and charge of the vesicles. Studies have shown that large unmodified liposome-based nanocarriers have higher propensity of being destroyed by opsonins than small, neutral, or positively charged liposomes (Oku & Namba, 1994).

15.4.2 Polyelectrolyte-based nanoencapsulation system for bioactive peptide delivery Polyelectrolytes are giant electrolyte molecules that dissociate in aqueous solution producing multiple charges. They are classified into polyanions, polycations, or polyampholytes. Polyelectrolyte nanocomplexes of chitosan, alginate, pectin, chondroitin sulfate, dextran, hyaluronic acid, and poly(γ-glutamic acid) have gained increasing attention as potential nanocarriers for bioactive compounds such as peptides and polyphenols. This is due to their low toxicity, biocompatibility, biodegradability, nonimmunogenicity, and modifiable surfaces. They possess multiple negative or positive charges as well as multilayers designed to protect bioactive peptides from degradation during storage or gastric digestion and enable the absorption of physiologically relevant amount of bioactive peptides (Zhao, Skwarczynski, & Toth, 2019). Most commonly applied negatively charged polyelectrolytes include alginate, dextran, hyaluronic acid, and chondroitin sulfate, which have shown immunomodulating properties and could be used as adjuvants (Petrovsky & Cooper, 2011). Chitosans are equally known to have adjuvant properties, eliciting the

348 Chapter 15 activation of macrophages and triggering the production of inflammatory cytokines (Carroll et al., 2016). The polyelectrolytes (alginates, heparin, dextran, and hyaluronic acid) have been approved by regulatory bodies for several applications (Zhao et al., 2019). Polyelectrolyte nanocomplexes are nanoparticles comprising oppositely charged polyelectrolytes held together by electrostatic interactions. They are formed by assembling the primary complex through electrostatic interaction, followed by conformational changes in the polyelectrolyte chains driven by hydrogen bonding, and finally aggregation of secondary structures induced by hydrophobic effect (Zhao et al., 2019). Various techniques such as ionic gelation, jet mixing, polyelectrolyte titration, and self-assembly have been reported for the preparation of polyelectrolyte nanocomplexes (Kulkarni et al., 2016). Chitosan and negatively charge polyelectrolyte counterions have been widely used in the delivery of bioactive peptides. Polyelectrolyte complex of insulin-loaded chitosan/alginate of mean diameter of B750 nm was prepared by interacting insulin with alginate followed by inducing ionotropic pregel with calcium counterions before polyelectrolyte complexation with chitosan. The nanocomplex showed an association efficiency and loading capacity of 72.8% and 9.9%, respectively. The release study under simulated gastrointestinal conditions showed that encapsulated insulin was released in a pH-dependent manner with higher bioavailability compared to free insulin, and subsequently lowered basal serum glucose levels by over 40% and sustained hypoglycemia for more than 18 hours (Sarmento et al., 2007). The insulin-loaded polyelectrolyte complex demonstrated clear adhesion to rat intestinal epithelium and insulin internalization in the intestinal mucosa (Sarmento et al., 2007). The findings indicate that polyelectrolyte complex nanoparticles could enhance the oral absorption and bioactivity of insulin. Spherically shaped insulin-loaded chitosan/pectin nanocomplex prepared by electrostatic self-assembly showed improved gastric stability and limited burst release than insulin-loaded chitosan/alginate. Only 13% of insulin was released after 120 minutes of exposure in the gastric phase at pH 1.2 and 90% was released in the simulated intestinal fluid. This pattern was attributed to weak electrostatic interaction between the highly positively charged insulin and the polyelectrolytes on the surface of the nanoparticles (Maciel, Yoshida, Pereira, Goycoolea, & Franco, 2017). A major challenge with chitosan-based polyelectrolyte complexes as a nanodelivery system is the low aqueous solubility of chitosan. Acetic acid is often used during the preparation of polyelectrolyte complexes of chitosan. This solution is unfavorable for bioactive peptides as it may lead to hydrolysis. A modified chitosan, known as trimethyl-chitosan, has better aqueous solubility but recent results indicate the need for optimization. For instance, Kissel et al. prepared positively charged polyethylene glycol (PEG)-grafted-trimethyl-chitosan copolymer
insulin nanocomplex of 230 nm diameter, which enhanced in vitro insulin uptake by Caco-2 cells but did not significantly improve hypoglycemic effect in diabetic rats after

Encapsulation technology for protection and delivery of bioactive peptides 349 intranasal administration compared to insulin (Mao et al., 2005). Moreover, preparation of desirable polyelectrolyte nanocomplexes could be tricky due to the charge-to-charge stoichiometry, charge density, polyelectrolyte concentration, pH, ionic strength, mode of mixing polyelectrolyte solutions, order of addition, mixing ratio, and salt concentration, which may affect the formation and stability of the complexes (Kulkarni et al., 2016).

15.4.3 Nanoemulsion-based delivery system for bioactive peptides delivery Nanoemulsions are oil, water, and surfactant-based small and uniformly sized droplets of high kinetic stability and low viscosity commonly used in the nanoencapsulation of lipophilic and hydrophilic bioactive ingredients for food, medical, and pharmaceutical applications (McClements, 2012). Like microemulsion, nanoemulsion-based delivery systems have been extensively applied in the oral delivery of bioactive compounds and they are prepared by microfluidization, ultrasonication, solvent diffusion, homogenization, or phase-inversion temperature method (Lim et al., 2011; McClements, Henson, Popplewell, Decker, & Choi, 2012). The major advantage of these systems is their high kinetic stability, which reduces sedimentation, phase separation, or creaming (Costa, Basri, Shamsudin, & Basri, 2014). They also improve the stability, aqueous solubility and bioavailability of bioactive compounds, increase the proportion of the compounds reaching a target, and reduce toxicity associated with off-target bioactives. This system can be modified to ensure target specificity and could deliver more than one bioactive compound at a time (Ma et al., 2014). Nanoemulsion oral-delivery system for BSA showed high encapsulation efficiency of over 90% and improved the stability, specificity, and bioactivity of the loaded BSA (Sun et al., 2012). A monodispersed and water-dispersible nanoparticle from chitosan, cross-linked with PEG of about 92% encapsulation efficiency was obtained through W/O nanoemulsion-based technique (Barbari et al., 2017). The surface of the nanoparticle was grafted with a novel cell-penetration peptide, KWFKIQMQIRRWKNKR which is an analog of well-known penetratin, RQIKIWFQNRRMKWKK. The nanoparticle showed an extensive swelling property in aqueous solution, maintained structural integrity at a wide range of pH and tight junction opening properties. The oral delivery of insulin loaded into this nanoparticle was found to improve the transport of insulin across the monolayer of Caco-2 cell line by 15%
19% (Barbari et al., 2017). Even though nanoemulsions-based delivery systems have proven to have higher loading capacity and improved bioavailability (due to higher surface area-to-volume ratio) for lipophilic bioactive compounds than microemulsions, the significant amount of energy required for their formation makes them thermodynamically less stable than microemulsion. Nanoemulsion is susceptible to instability challenges such as Oswald ripening, creaming, and flocculation often common with all emulsion-based systems. Oswald ripening is usually overcome by the addition of large quantities of ripening inhibitors to the lipid phase before the formation of nanoemulsion-based

350 Chapter 15 delivery systems (Lim et al., 2011; McClements et al., 2012). Nanoemulsion-based delivery system often experiences structural modification in the gastrointestinal tract leading to flocculation or coalescence of droplets (Singh, Ye, & Horne, 2009).

15.4.4 Solid lipid nanoparticles for bioactive peptide delivery Solid lipid nanodelivery system is one of the most extensively used nanoencapsulation system for peptides and proteins. They are made up of lipid core of fatty acids, waxes, steroids, triglycerides or partial glycerides, and an emulsifier that proffers stability in aqueous solution. Peptide-loaded solid lipid nanoparticles are fabricated by either coacervation, solvent emulsification evaporation, microemulsion, solvent emulsification diffusion, supercritical fluid technology or high-pressure homogenization (Almeida & Souto, 2007; Gallarate, Battaglia, Peira, & Trotta, 2011). Similar to liposome and nanoemulsion-based delivery systems, solid lipid delivery systems are stable nontoxic amphipathic delivery systems that can encapsulate, protect and deliver both hydrophilic and lipophilic bioactive peptides (Almeida & Souto, 2007). The design of peptide-loaded solid lipid nanoparticles, even with some limitations, seems less complicated than other nano-based encapsulation systems (Almeida & Souto, 2007). Peptide drugs, insulin, and leuprolide loaded into stearic acid-derived solid lipid nanoparticles after hydrophobic ion pairing with anionic surfactant were reported by Gallarate et al. (2011). The peptide-loaded solid lipid nanoparticle was the first kind to be prepared by the coacervation technique. The solid lipid nanocomplex maintained the chemical integrity of the encapsulated drugs after the coacervation process and ensured sustained release (Gallarate et al., 2011). An extremely hydrophobic cyclic undecapeptide cyclosporin, known for its immunosuppressive properties, was encapsulated into solid lipid nanoparticles by warm oil-in-water microemulsion technique. The nanocomplex of 250
290 nm diameter improved the solubility and bioavailability of the peptide and showed slow release when administered orally (Ugazio, Cavalli, & Gasco, 2002). Several factors can cause instability problem for solid lipid nanoparticles in the delivery of bioactive compounds. For instance, an increase in particle size has been reported during prolonged storage of the particles. Unpredictable gelation tendency and unexpected dynamics of polymeric transition are also major challenges associated with solid lipid nanoparticles as delivery vehicles. For instance, in aqueous solution, there are reported cases of formation of other colloidal structures such as liposomes, nanocrystals, or micelles. Instability challenges could also result from the physical state complexity of the lipid (Ekambaram, Sathali, & Priyanka, 2012). At high concentration of the emulsifier, solid lipid nanoparticles have shown some toxicological concerns attributed to preservatives and nonionic emulsifiers present in the aqueous phase (Schubert & Mu¨ller-Goymann, 2005). Also, there are limitations in the encapsulation of hydrophilic peptides using this system (Kovalainen et al., 2015).

Encapsulation technology for protection and delivery of bioactive peptides 351

15.5 Conclusion and future perspectives Encapsulation can be considered a promising technology for the protection, controlled release and delivery of bioactive peptides. This chapter discussed the advances on bioactive peptide encapsulation in different delivery systems, including microparticulate, hydrogel, and nanoparticle carriers. Microencapsulation of bioactive peptides offers tremendous potentials for industrial commercialization of functional foods, especially using the spray drying method. The protein and dietary fiber used as the wall materials of microparticles can serve as supplemental nutrients and bioactive materials. The encapsulation efficiency, sensory property, and storage stability (hygroscopicity) of microencapsulated peptides have been well studied, whereas the release kinetics, biostability, and bioavailability remain poorly understood. Two commonly applied approaches for fabrication of bioactive peptide-incorporated microgels are injection
gelation and emulsion templating. A potential drawback of encapsulating bioactive peptides in microgels is the low encapsulation efficiency due to their relatively large pores compared to the peptide size, resulting in peptide diffusion. This challenge can be overcome by establishing interactions between bioactive peptides and the biopolymer network of microgels. Further research is needed on large-scale fabrication of peptide-loaded microgels from food-grade ingredients and investigation of the bioavailability and bioactivity of encapsulated peptides in vivo using animal models and human subjects. The most common nanoencapsulation-based systems for the delivery of bioactive peptides include nanoemulsions, polyelectrolyte complex nanoparticles, liposomes, and solid lipid nanoparticles. The choice of these systems depends on the nature of the peptide and encapsulation matrices, such as stability, solubility, storage time, biocompatibility, delivery route, method of preparation, and intended application. Nanoencapsulation-based systems have demonstrated wider interests and applications in food, medical and pharmaceutical industry owing to their higher surface area-to-volume ratio, which has resulted in improved stability, bioavailability, bioaccessibility, permeability, and residence time. However, future studies should investigate the interaction of the nanodelivery systems with biological structures (nano
bio interactions) to validate the safety of these systems. Overall, micro- and nanoencapsulation offer promising opportunities for utilizing bioactive peptides as functional foods and food ingredients because these technologies can improve the physicochemical and sensory properties, enhance biostability and bioavailability, achieve controlled release, and maintain potent biological activities of peptides. However, encapsulation is yet to be scaled up in the industrial production of bioactive peptides. Future research needs to address the current challenges associated with the cost of finished encapsulated bioactive peptide products.

352 Chapter 15

References Alle´mann, E., Leroux, J. C., & Gurny, R. (1998). Polymeric nano-and microparticles for the oral delivery of peptides and peptidomimetics. Advanced Drug Delivery Reviews, 34(2
3), 171
189. Almeida, A. J., & Souto, E. (2007). Solid lipid nanoparticles as a drug delivery system for peptides and proteins. Advanced Drug Delivery Reviews, 59(6), 478
490. Bai, Y., Zhang, Z., Zhang, A., Chen, L., He, C., Zhuang, X., & Chen, X. (2012). Novel thermo-and pH-responsive hydroxypropyl cellulose-and poly (l-glutamic acid)-based microgels for oral insulin controlled release. Carbohydrate Polymers, 89(4), 1207
1214. Barbari, G. R., Dorkoosh, F. A., Amini, M., Sharifzadeh, M., Atyabi, F., Balalaie, S., . . . Tehrani, M. R. (2017). A novel nanoemulsion-based method to produce ultrasmall, water-dispersible nanoparticles from chitosan, surface modified with cell-penetrating peptide for oral delivery of proteins and peptides. International Journal of Nanomedicine, 12, 3471. Barbosa, C. M., Morais, H. A., Delvivo, F. M., Mansur, H. S., De Oliveira, M. C., & Silvestre, M. P. (2004). Papain hydrolysates of casein: molecular weight profile and encapsulation in lipospheres. Journal of the Science of Food and Agriculture, 84(14), 1891
1900. Boelsma, E., & Kloek, J. (2010). IPP-rich milk protein hydrolysate lowers blood pressure in subjects with stage 1 hypertension, a randomized controlled trial. Nutrition Journal, 9(1), 52. Brandelli, A. (2012). Nanostructures as promising tools for delivery of antimicrobial peptides. Mini Reviews in Medicinal Chemistry, 12(8), 731
741. Carroll, E. C., Jin, L., Mori, A., Mun˜oz-Wolf, N., Oleszycka, E., Moran, H. B. T., . . . Lavelle, E. C. (2016). The vaccine adjuvant chitosan promotes cellular immunity via DNA sensor cGAS-STING-dependent induction of type I interferons. Immunity, 44(3), 597
608. Chakrabarti, S., Guha, S., & Majumder, K. (2018). Food-derived bioactive peptides in human health: Challenges and opportunities. Nutrients, 10(11), 1738. Chrai, S. S., Murari, R., & Ahmad, I. (2002). Liposomes (a review). Part two: Drug delivery systems. BioPharm, 15(1). Costa, S. D., Basri, M., Shamsudin, N., & Basri, H. B. (2014). Formation of stable palm kernel oil esters nanoemulsion system containing hydrocortisone. Asian Journal of Chemistry, 26(10). Cunningham, E., O’Byrne, C., & Oliver, J. D. (2009). Effect of weak acids on Listeria monocytogenes survival: Evidence for a viable but nonculturable state in response to low pH. Food Control, 20(12), 1141
1144. Daeihamed, M., Dadashzadeh, S., Haeri, A., & Akhlaghi, M. F. (2017). Potential of liposomes for enhancement of oral drug absorption. Current Drug Delivery, 14(2), 289
303. Ekambaram, P., Sathali, A. A. H., & Priyanka, K. (2012). Solid lipid nanoparticles: A review. Science Reviews Chemical Communications, 2(1), 80
102. ´ ., & McClements, D. J. (2014). Nanoencapsulation of food ingredients using carbohydrate Fathi, M., Martı´n, A based delivery systems. Trends in Food Science & Technology, 39(1), 18
39. Favaro-Trindade, C. S., Santana, A. S., Monterrey-Quintero, E. S., Trindade, M. A., & Netto, F. M. (2010). The use of spray drying technology to reduce bitter taste of casein hydrolysate. Food Hydrocolloids, 24(4), 336
340. Foegeding, E. A. (2006). Food biophysics of protein gels: A challenge of nano and macroscopic proportions. Food Biophysics, 1(1), 41
50. Gallarate, M., Battaglia, L., Peira, E., & Trotta, M. (2011). Peptide-loaded solid lipid nanoparticles prepared through coacervation technique. International Journal of Chemical Engineering, 2011, 132435. Giroux, H. J., Robitaille, G., & Britten, M. (2016). Controlled release of casein-derived peptides in the gastrointestinal environment by encapsulation in water-in-oil-in-water double emulsions. LWT-Food Science and Technology, 69, 225
232. Go¨kmen, V., Mogol, B. A., Lumaga, R. B., Fogliano, V., Kaplun, Z., & Shimoni, E. (2011). Development of functional bread containing nanoencapsulated omega-3 fatty acids. Journal of Food Engineering, 105(4), 585
591.

Encapsulation technology for protection and delivery of bioactive peptides 353 Go´mez-Mascaraque, L. G., Miralles, B., Recio, I., & Lopez-Rubio, A. (2016). Microencapsulation of a whey protein hydrolysate within micro-hydrogels: Impact on gastrointestinal stability and potential for functional yoghurt development. Journal of Functional Foods, 26, 290
300. Goswami, S., Bajpai, J., & Bajpai, A. K. (2014). Calcium alginate nanocarriers as possible vehicles for oral delivery of insulin. Journal of Experimental Nanoscience, 9(4), 337
356. Gouin, S. (2004). Microencapsulation: Industrial appraisal of existing technologies and trends. Trends in Food Science & Technology, 15, 330
347. Guo, J., Zhang, Y., & Yang, X. Q. (2012). A novel enzyme cross-linked gelation method for preparing food globular protein-based transparent hydrogel. Food Hydrocolloids, 26(1), 277
285. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: From research to food applications. Current Opinion in Biotechnology, 18(2), 163
169. Herrero-Vanrell, R., Bravo-Osuna, I., Andre´s-Guerrero, V., Vicario-de-la-Torre, M., & Molina-Martı´nez, I. T. (2014). The potential of using biodegradable microspheres in retinal diseases and other intraocular pathologies. Progress in Retinal and Eye Research, 42, 27
43. Hosseini, S. M., Hosseini, H., Mohammadifar, M. A., German, J. B., Mortazavian, A. M., Mohammadi, A., . . . Khaksar, R. (2014). Preparation and characterization of alginate and alginate-resistant starch microparticles containing nisin. Carbohydrate Polymers, 103, 573
580. Hua, S., & Wu, S. Y. (2013). The use of lipid-based nanocarriers for targeted pain therapies. Frontiers in Pharmacology, 4, 143. Huang, G. Q., Xiao, J. X., Hao, L. Q., & Yang, J. (2017). Microencapsulation of an angiotensin I-converting enzyme inhibitory peptide VLPVP by membrane emulsification. Food and Bioprocess Technology, 10(11), 2005
2012. Jain, A. K., Khar, R. K., Ahmed, F. J., & Diwan, P. V. (2008). Effective insulin delivery using starch nanoparticles as a potential trans-nasal mucoadhesive carrier. European Journal of Pharmaceutics and Biopharmaceutics, 69(2), 426
435. Karim, A. A., & Bhat, R. (2008). Gelatin alternatives for the food industry: Recent developments, challenges and prospects. Trends in Food Science & Technology, 19(12), 644
656. Kovalainen, M., Mo¨nka¨re, J., Riikonen, J., Pesonen, U., Vlasova, M., Salonen, J., . . . Herzig, K. H. (2015). Novel delivery systems for improving the clinical use of peptides. Pharmacological Reviews, 67(3), 541
561. Kulkarni, A. D., Vanjari, Y. H., Sancheti, K. H., Patel, H. M., Belgamwar, V. S., Surana, S. J., & Pardeshi, C. V. (2016). Polyelectrolyte complexes: Mechanisms, critical experimental aspects, and applications. Artificial Cells, Nanomedicine, and Biotechnology, 44(7), 1615
1625. Kurozawa, L. E., Park, K. J., & Hubinger, M. D. (2009). Effect of carrier agents on the physicochemical properties of a spray dried chicken meat protein hydrolysate. Journal of Food Engineering, 94(3
4), 326
333. Laridi, R., Kheadr, E. E., Benech, R. O., Vuillemard, J. C., Lacroix, C., & Fliss, I. (2003). Liposome encapsulated nisin Z: Optimization, stability and release during milk fermentation. International Dairy Journal, 13(4), 325
336. Lemes, A. C., Sala, L., Ores, J. D. C., Braga, A. R. C., Egea, M. B., & Fernandes, K. F. (2016). A review of the latest advances in encrypted bioactive peptides from protein-rich waste. International Journal of Molecular Sciences, 17(6), 950. Lengyel, M., Ka´llai-Szabo´, N., Antal, V., Laki, A. J., & Antal, I. (2019). Microparticles, microspheres, and microcapsules for advanced drug delivery. Scientia Pharmaceutica, 87(3), 20. Lesmes, U., Cohen, S. H., Shener, Y., & Shimoni, E. (2009). Effects of long chain fatty acid unsaturation on the structure and controlled release properties of amylose complexes. Food Hydrocolloids, 23(3), 667
675. Lim, S. S., Baik, M. Y., Decker, E. A., Henson, L., Popplewell, L. M., McClements, D. J., & Choi, S. J. (2011). Stabilization of orange oil-in-water emulsions: A new role for ester gum as an Ostwald ripening inhibitor. Food Chemistry, 128(4), 1023
1028. Lopes, M. A., Abrahim-Vieira, B., Oliveira, C., Fonte, P., Souza, A. M. T., Lira, T., . . . Ribeiro, A. J. (2015). Probing insulin bioactivity in oral nanoparticles produced by ultrasonication-assisted emulsification/internal gelation. International Journal of Nanomedicine, 10, 5865.

354 Chapter 15 Ma, J. J., Mao, X. Y., Wang, Q., Yang, S., Zhang, D., Chen, S.-W., & Li, Y. H. (2014). Effect of spray drying and freeze drying on the immunomodulatory activity, bitter taste and hygroscopicity of hydrolysate derived from whey protein concentrate. LWT - Food Science and Technology, 56(2), 296
302. Ma, Y., Liu, D., Wang, D., Wang, Y., Fu, Q., Fallon, J. K., . . . Liu, F. (2014). Combinational delivery of hydrophobic and hydrophilic anticancer drugs in single nanoemulsions to treat MDR in cancer. Molecular Pharmaceutics, 11(8), 2623
2630. Maciel, V. B., Yoshida, C. M. P., Pereira, S. M. S. S., Goycoolea, F. M., & Franco, T. T. (2017). Electrostatic self-assembled chitosan-pectin nano-and microparticles for insulin delivery. Molecules, 22(10), 1707. Maeno, M., Yamamoto, N., & Takano, T. (1996). Identification of an antihypertensive peptide from casein hydrolysate produced by a proteinase from Lactobacillus helveticus. Journal of Dairy Science, 79(8), 1316
CP1321. Malheiros, P. S., Sant’Anna, V., Utpott, M., & Brandelli, A. (2012). Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk. Food Control, 23(1), 42
47. Mao, S., Germershaus, O., Fischer, D., Linn, T., Schnepf, R., & Kissel, T. (2005). Uptake and transport of PEG-graft-trimethyl-chitosan copolymer
insulin nanocomplexes by epithelial cells. Pharmaceutical Research, 22(12), 2058
2068. McClements, D. J. (2012). Nanoemulsions versus microemulsions: Terminology, differences, and similarities. Soft Matter, 8(6), 1719
1729. McClements, D. J. (2017). Recent progress in hydrogel delivery systems for improving nutraceutical bioavailability. Food Hydrocolloids, 68, 238
245. McClements, D. J. (2018). Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems: A review. Advances in Colloid and Interface Science, 253, 1
22. McClements, D. J., Decker, E. A., Park, Y., & Weiss, J. (2009). Structural design principles for delivery of bioactive components in nutraceuticals and functional foods. Critical Reviews in Food Science and Nutrition, 49(6), 577
606. McClements, D. J., Henson, L., Popplewell, L. M., Decker, E. A., & Choi, S. J. (2012). Inhibition of Ostwald ripening in model beverage emulsions by addition of poorly water soluble triglyceride oils. Journal of Food Science, 77(1), C33
C38. Mendanha, D. V., Molina Ortiz, S. E., Favaro-Trindade, C. S., Mauri, A., Monterrey-Quintero, E. S., & Thomazini, M. (2009). Microencapsulation of casein hydrolysate by complex coacervation with SPI/pectin. Food Research International, 42(8), 1099
1104. Moeller, E. H., Holst, B., Nielsen, L. H., Pedersen, P. S., & Østergaard, J. (2010). Stability, liposome interaction, and in vivo pharmacology of ghrelin in liposomal suspensions. International Journal of Pharmaceutics, 390(1), 13
18. Mohan, A., McClements, D. J., & Udenigwe, C. C. (2016). Encapsulation of bioactive whey peptides in soy lecithin-derived nanoliposomes: Influence of peptide molecular weight. Food Chemistry, 213, 143
148. Mohan, A., Rajendran, S. R., Thibodeau, J., Bazinet, L., & Udenigwe, C. C. (2018). Liposome encapsulation of anionic and cationic whey peptides: Influence of peptide net charge on properties of the nanovesicles. LWT, 87, 40
46. Mohan, A., Rajendran, S. R. C. K., He, Q. S., Bazinet, L., & Udenigwe, C. C. (2015). Encapsulation of food protein hydrolysates and peptides: A review. RSC Advances, 5(97), 79270
79278. Molina Ortiz, S. E., Mauri, A., Monterrey-Quintero, E. S., Trindade, M. A., Santana, A. S., & Favaro-Trindade, C. S. (2009). Production and properties of casein hydrolysate microencapsulated by spray drying with soybean protein isolate. LWT - Food Science and Technology, 42(5), 919
923. Morais, H. A., Da Silva Barbosa, C. M., Delvivo, F. M., Mansur, H. S., Oliveira, M. C. D., & Silvestre, M. P. C. (2004). Comparative study of microencapsulation of casein hydrolysates in lipospheres and liposomes. Journal of Food Biochemistry, 28(1), 21
41. Mozafari, M. R., Johnson, C., Hatziantoniou, S., & Demetzos, C. (2008). Nanoliposomes and their applications in food nanotechnology. Journal of Liposome Research, 18(4), 309
327. O’Neill, G. J., Egan, T., Jacquier, J. C., O’Sullivan, M., & O’Riordan, E. D. (2014). Whey microbeads as a matrix for the encapsulation and immobilisation of riboflavin and peptides. Food Chemistry, 160, 46
52.

Encapsulation technology for protection and delivery of bioactive peptides 355 O’Neill, G. J., Egan, T., Jacquier, J. C., O’Sullivan, M., & O’Riordan, E. D. (2015). Kinetics of immobilisation and release of tryptophan, riboflavin and peptides from whey protein microbeads. Food Chemistry, 180, 150
155. Oku, N., & Namba, Y. (1994). Long-circulating liposomes. Critical Reviews in Therapeutic Drug Carrier Systems, 11(4), 231
270. Perry, S. L., & McClements, D. J. (2020). Recent advances in encapsulation, protection, and oral delivery of bioactive proteins and peptides using colloidal systems. Molecules, 25(5), 1161. Petrovsky, N., & Cooper, P. D. (2011). Carbohydrate-based immune adjuvants. Expert Review of Vaccines, 10(4), 523
537. Picariello, G., Iacomino, G., Mamone, G., Ferranti, P., Fierro, O., Gianfrani, C., . . . Addeo, F. (2013). Transport across Caco-2 monolayers of peptides arising from in vitro digestion of bovine milk proteins. Food Chemistry, 139, 203
212. Pinho, S. C., & da Silva, J. C. (2013). Viability of the microencapsulation of a casein hydrolysate in lipid microparticles of cupuacu butter and stearic acid. International Journal of Food Studies, 2(1), 48
59. Prajapati, V. D., Jani, G. K., & Kapadia, J. R. (2015). Current knowledge on biodegradable microspheres in drug delivery. Expert Opinion on Drug Delivery, 12(8), 1283
1299. Rabiˇskova´, M., & Vala´sˇkova´, J. (1998). The influence of HLB on the encapsulation of oils by complex coacervation. Journal of Microencapsulation, 15, 747
751. Rao, P. S., Bajaj, R. K., Mann, B., Arora, S., & Tomar, S. K. (2016). Encapsulation of antioxidant peptide enriched casein hydrolysate using maltodextrin
gum arabic blend. Journal of Food Science and Technology, 53(10), 3834
3843. Rocha, G. A., Trindade, M. A., Netto, F. M., & Favaro-Trindade, C. S. (2009). Microcapsules of a casein hydrolysate: Production, characterization, and application in protein bars. Food Science and Technology International, 15(4), 407
413. Ruiz, J. C. R., Campos, M. R. S., Ancona, D. A. B., & Guerrero, L. A. C. (2016). Encapsulation of Phaseolus lunatus protein hydrolysate with angiotensin-converting enzyme inhibitory activity. ISRN Biotechnology, 2013, 341974. Sarmento, B., Ribeiro, A., Veiga, F., Sampaio, P., Neufeld, R., & Ferreira, D. (2007). Alginate/chitosan nanoparticles are effective for oral insulin delivery. Pharmaceutical Research, 24(12), 2198
2206. Schubert, M. A., & Mu¨ller-Goymann, C. C. (2005). Characterisation of surface-modified solid lipid nanoparticles (SLN): Influence of lecithin and nonionic emulsifier. European Journal of Pharmaceutics and Biopharmaceutics, 61(1
2), 77
86. Segura-Campos, M., Chel-Guerrero, L., Betancur-Ancona, D., & Hernandez-Escalante, V. M. (2011). Bioavailability of bioactive peptides. Food Reviews International, 27(3), 213
226. Sercombe, L., Veerati, T., Moheimani, F., Wu, S. Y., Sood, A. K., & Hua, S. (2015). Advances and challenges of liposome assisted drug delivery. Frontiers in Pharmacology, 6, 286. Singh, H., Ye, A., & Horne, D. (2009). Structuring food emulsions in the gastrointestinal tract to modify lipid digestion. Progress in Lipid Research, 48(2), 92
100. Sinha, V. R., & Trehan, A. (2003). Biodegradable microspheres for protein delivery. Journal of Controlled Release, 90(3), 261
280. Suganya, V., & Anuradha, V. (2017). Microencapsulation and nanoencapsulation: A review. International Journal of Pharmaceutical and Clinical Research, 9(3), 233
239. Sun, H., Liu, K., Liu, W., Wang, W., Guo, C., Tang, B., . . . Zeng, H. (2012). Development and characterization of a novel nanoemulsion drug-delivery system for potential application in oral delivery of protein drugs. International Journal of Nanomedicine, 7, 5529. Szebeni, J., & Moghimi, S. M. (2009). Liposome triggering of innate immune responses: a perspective on benefits and adverse reactions: Biological recognition and interactions of liposomes. Journal of Liposome Research, 19(2), 85
90. Tamjidi, F., Shahedi, M., Varshosaz, J., & Nasirpour, A. (2013). Nanostructured lipid carriers (NLC): A potential delivery system for bioactive food molecules. Innovative Food Science & Emerging Technologies, 19, 29
43.

356 Chapter 15 Tavares, G. M., Croguennec, T., Carvalho, A. F., & Bouhallab, S. (2014). Milk proteins as encapsulation devices and delivery vehicles: Applications and trends. Trends in Food Science & Technology, 37(1), 5
20. Taylor, T. M., Bruce, B. D., Weiss, J., & Davidson, P. M. (2008). Listeria Monocytogenes and Escherichia coli O157: H7 inhibition in vitro by liposome-encapsulated nisin and ethylene diaminetetraacetic acid. Journal of Food Safety, 28(2), 183
197. Toldra´, F., Reig, M., Aristoy, M. C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404. Udenigwe, C. C. (2014). Bioinformatics approaches, prospects and challenges of food bioactive peptide research. Trends in Food Science & Technology, 36(2), 137
143. Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides: Production, processing, and potential health benefits. Journal of Food Science, 77(1), R11
R24. Ugazio, E., Cavalli, R., & Gasco, M. R. (2002). Incorporation of cyclosporin A in solid lipid nanoparticles (SLN). International Journal of Pharmaceutics, 241(2), 341
344. Wang, S., Su, R., Nie, S., Sun, M., Zhang, J., Wu, D., & Moustaid-Moussa, N. (2014). Application of nanotechnology in improving bioavailability and bioactivity of diet-derived phytochemicals. Journal of Nutritional Biochemistry, 25(4), 363
376. Wang, Z., Ju, X., He, R., Yuan, J., & Aluko, R. E. (2015). Effect of high pressure treatment on rapeseed protein microparticle properties and gastrointestinal release behavior of the encapsulated peptides. Food Research International, 77, 549
555. Wang, Z., Ju, X., He, R., Yuan, J., & Wang, L. (2015). The effect of rapeseed protein structural modification on microstructural properties of peptide microcapsules. Food and Bioprocess Technology, 8(6), 1305
1318. Wong, C. Y., Al-Salami, H., & Dass, C. R. (2018). Microparticles, microcapsules and microspheres: A review of recent developments and prospects for oral delivery of insulin. International Journal of Pharmaceutics, 537, 223
244. Xiao, D., Davidson, P. M., & Zhong, Q. (2011). Spray-dried zein capsules with coencapsulated nisin and thymol as antimicrobial delivery system for enhanced antilisterial properties. Journal of Agricultural and Food Chemistry, 59(13), 7393
7404. Yang, S., Mao, X. Y., Li, F. F., Zhang, D., Leng, X. J., Ren, F. Z., & Teng, G. X. (2012). The improving effect of spray-drying encapsulation process on the bitter taste and stability of whey protein hydrolysate. European Food Research and Technology, 235(1), 91
97. Ye, Q., Georges, N., & Selomulya, C. (2018). Microencapsulation of active ingredients in functional foods: From research stage to commercial food products. Trends in Food Science & Technology, 78, 167
179. Yuan, D., Jacquier, J. C., & O’Riordan, E. D. (2018). Entrapment of proteins and peptides in chitosanpolyphosphoric acid hydrogel beads: A new approach to achieve both high entrapment efficiency and controlled in vitro release. Food Chemistry, 239, 1200
1209. Zeeb, B., Fischer, L., & Weiss, J. (2014). Stabilization of food dispersions by enzymes. Food & Function, 5(2), 198
213. Zhang, Y., Wei, W., Lv, P., Wang, L., & Ma, G. (2011). Preparation and evaluation of alginate
chitosan microspheres for oral delivery of insulin. European Journal of Pharmaceutics and Biopharmaceutics, 77(1), 1
19. Zhao, L., Skwarczynski, M., & Toth, I. (2019). Polyelectrolyte-based platforms for the delivery of peptides and proteins. ACS Biomaterials Science & Engineering, 5(10), 4937
4950.

CHAPTER 16

Plant sources of bioactive peptides Vermont P. Dia Department of Food Science, The University of Tennessee, Knoxville, TN, United States

16.1 Introduction Proteins are nitrogenous-containing substances that are polymers of amino acids. It performs numerous functions not only in the living system but also in foods and food products. In the living system, proteins are sources of nitrogen and essential amino acid, serve as regulators of body processes (enzymes and hormones), assist in defense in the forms of antibodies and toxins, provide structure and flexibility, and serve as storage in seeds and egg proteins. In food science, proteins accomplish important functionality without which food products’ characteristics and desirability would be impossible. These include the ability of proteins to be hydrated, to absorb oil, to stabilize interfacial tensions (emulsions and foams), to form three-dimensional structures, to bind flavor and texturize important in meat analogs, and to provide color and flavor through Maillard browning. In case of wheat proteins, the ability of gluten to form a viscoelastic dough is responsible for the quality characteristics of baked products. In the past few decades, it becomes eminent that in addition to nutrition and functionality, proteins are also sources of biologically active peptides that can positively affect one’s health. Due to vastness of proteins in the plant kingdom ranging from ,1% (fruits and vegetables) to B45% (legumes), plants are important sources of diverse bioactive peptides. Bioactive peptides in plants can be naturally occurring or products of food processing. Edible plant tissues such as cereal grains, legumes (nuts, seeds, and pulses), fruits, and vegetables contain parent proteins which upon hydrolysis (either through enzymatic action, chemical action, or process-induced) led to the production of peptides that can modify processes in the body potentially promoting overall human health. For instance, tryptophancontaining dipeptides (IW, LW, KW, FW, and VW) enriched from plant hydrolysates (soy, rice, wheat, and pea) by butanol extraction showed potent inhibition of the angiotensin I-converting enzyme (ACE) with 50% inhibitory concentration ranging from 1.1 to 10 μM (Rudolph, Lunow, Kaiser, & Henle, 2017). ACE causes vasoconstriction of blood vessels leading to hypertension as such these tryptophan-containing dipeptides can be used in regulating high blood pressure. In addition to products of protein hydrolysis, plants also Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00003-0 © 2021 Elsevier Inc. All rights reserved.

357

358 Chapter 16 contain naturally occurring bioactive peptides and proteins. For instance, inhibitors of protease Bowman
Birk inhibitor (BBI) and Kunitz-type inhibitor (KTI) are found naturally in soybean. BBI and KTI have shown a wide range of bioactivities including antioxidant, anti-inflammatory, anticancer, antiviral among others (Fang, Wong, & Ng, 2010; Ma et al., 2016; Sadeghalvad, Mohammadi-Motlagh, Karaji, & Mostafaie, 2019). Fruits and vegetables contain less amount of proteins, edible basis, than cereals, and legumes are also good sources of bioactive peptides. Kissper, a 39-amino acid peptide originally isolated from kiwifruits, showed anti-inflammatory properties in in vitro, ex vivo human intestinal, and celiac disease mucosa models (Ciacci et al., 2014; Ciardiello et al., 2008; Russo et al., 2019). Fermentation of fruits and vegetables also leads to the production of diverse group of bioactive molecules including bioactive peptides. This chapter on plant sources of bioactive peptides focuses on the classification, isolation, and extraction of plant proteins. A general approach on the preparation and production of biologically active peptides from edible plant tissues is also presented. In addition, examples of biological activities and mechanisms of action from selected plant-derived bioactive peptides are discussed. Current challenges and future directions conclude this chapter on plant sources of bioactive peptides.

16.2 Plant proteins classification and isolation and extraction methods Proteins play a critical role in plant as they serve multiple functions. They are important catalysts of plant metabolism via their enzymatic actions in the different compartments of plant cells. Moreover, plant proteins serve as regulators of processes in plants (hormones) and provide structural integrity (cell wall proteins) and source of nutrients in the form of storage proteins (SPs). Plant protein intake of the human population depends on the region and economic status. In general, low- to middle-income countries used plants as their major source of dietary proteins, whereas population from high-income countries continue to utilize animals as sources of dietary proteins (Kim, Rebholz, Caulfield, Ramsing, & Nachman, 2018; Rampal, 2018). With the increasing incidence of chronic diseases associated with animal protein intake as well as concerns on the contribution of livestock in climate change, plant proteins are considered the most accepted alternative source of proteins for health promotion and sustainability (Grasso, Hung, Olthof, Verbeke, & Brouwer, 2019). Among the different types of proteins present in plant tissues, SPs are considered the major source in human diets. SPs play an important role in plants as they serve as reservoirs for amino acids and elemental nutrients (sulfur, carbon, nitrogen) needed by the developing plants. As such they supply the majority of nutrients from plant-based diet. SP in plants can be classified based on location in the plant tissues: seed SPs are found in the seed and vegetative SPs are found in the vegetative tissues of the plants such as tubers, stems, roots, and leaves

Plant sources of bioactive peptides 359 (Mouzo, Bernal, Lo´pez-Pedrouso, Franco, & Zapata, 2018). Osborne classification of SPs based on solubility in different solvents (Osborne, 1908) is the most widely used technique in differentiating SPs. They are grouped into four groups namely albumins (water-soluble), globulins (dilute salt-soluble), prolamins (aqueous alcohol-soluble), and glutelins (dilute acid/ base-soluble). As SPs account for a large percentage of plant proteins, they are also considered major sources of bioactive peptides. Table 16.1 shows some of SPs in common edible plants. Most of the SPs in edible plants belong to either water-soluble albumins, dilute salt-soluble globulins, and alcohol-soluble prolamins. In addition to difference in solubility of these proteins, plant proteins are also distinguished by their sedimentation coefficients such as 2S, 7S, and 11S. Table 16.1 also shows the diverse biological activities of peptides derived from edible plant proteins such as antioxidant, hypotensive, antidiabetic, anti-inflammatory and anticancer. Due to differences in solubility, plant proteins are usually extracted and isolated based on this property. Proteins are known to be highly negative in alkaline pH as such solubilized and can be precipitated at their isoelectric pH (pI). Fig. 16.1 shows the commonly used extraction procedure for plant proteins. Another typical isolation and fraction method for plant proteins is based on the use of different solvents. As an example, for this type of protein extraction, an alcohol-soluble protein (prolamin) from sorghum known as kafirin was selected. Sequential extraction process is the most commonly used method for the isolation of kafirin from sorghum. In this process, water-soluble albumins and salt-soluble globulins are removed first. Due to higher hydrophobicity of kafirin, a less polar solvent is usually used as opposed to 70%
80% ethanol used for extraction of corn prolamin zein. Commonly used solvents are 60% t-butanol and 60% isopropanol (Sullivan, Pangloli, & Dia, 2018a; Xiao, Chen, & Huang, 2017). Modification of extraction conditions to increase yield has been reported such as the use of elevated temperature, addition of reducing agents, application of ultrasonic, and addition of pepsin and sodium tetraborate (Xiao et al., 2017). Sorghum kafirins are differentiated based on their molecular weight as α-kafirin (22
26 kDa), β-kafirin (19 kDa), and γ-kafirin (22
26 kDa), and a fourth kafirin known as δ-kafirin (11 kDa) has been reported as minor constituent in some sorghum varieties (Cremer et al., 2014). Kafirin is considered one of the most hydrophobic proteins associated with at least 60% of its amino acid being lipophilic. In addition, the presence of sulfur-containing amino acids, cysteine, and methionine, in β- and γ-kafirins led to its propensity in forming intermolecular disulfide linkages and protein aggregates (Belton, Delgadillo, Halford, & Shewry, 2006). To date, the study on the bioactivity of kafirin as well as peptides derived from its enzymatic hydrolysates is limited. A mixture of kafirin has been reported to reduce inflammation in THP-1 human macrophages by reducing intracellular reactive oxygen species (Sullivan, Pangloli, & Dia, 2018b) and supplementation of kafirin in the diet of hyperlipidemic rats resulted in improved lipid metabolism and serum antioxidant potential (Ortı´z Cruz et al., 2015). Alcalase hydrolysates of kafirin have been reported to possess

Table 16.1: Storage proteins in edible plant tissues and their representative bioactive peptides. Plant source

Storage proteins and characteristics

Example of bioactive peptides

References

Oilseeds and nuts 1.

Soybean

2.

Peanut

3.

Rapeseed

4.

Coconut

5.

Sunflower

6.

Walnut

β-Conglycinin (trimer, B190 kDa) Glycinin (hexamer, 300
380 kDa) [Globulins] Cupin (Vicilin Ara h 1 conarachin and legumin Ara h 3 arachin) [Globulins] Napin (10
15 kDa) [Albumin] Cruciferin (B300 to 350 kDa) [Globulin] 55-kDa albumin and globulin SESA-2 albumins (13
15 kDa) Helianthinin (20
35 kDa) [Globulin] Vicilin Jug R 4 [Globulin] Jug R 1 [Albumin]

IAVPGEVA, IAVPTGVA, LPYP as modulators Lammi, Zanoni, and Arnoldi (2015); Mojica, of glucose metabolism; VRIRLLQRFNKRS as Dia, and de Mejia (2015); Nishi, Hara, appetite suppressor Asano, and Tomita (2003) IEY and KLYMRP as modulators of hypertension by inhibiting ACE

Jimsheena and Gowda (2010); Mills et al. (2002); Ozias-Akins and Breiteneder (2019); Shi et al. (2014) LY, RALP, and GHS as modulators of reninDefaix et al. (2019); Gacek, Bartkowiakangiotensin system pathway; DHNNPQIR as Broda, and Batley (2018); He et al. (2019); antioxidant against metabolic disorders Zhao et al. (2019) KAQYPYV, KIIIYN, and KILIYG as Benjakul and Patil (2019); Li, Zheng, Zhang, antioxidants and ACE inhibitor Xu, and Gao (2018) RW, PG, and AF as inhibitors of ACE Franke, Colgrave, Mylne, and Rosengren (antihypertensive) and DPP-IV (antidiabetic) (2016); Gonza´lez-Pe´rez et al. (2005); Han, Maycock, Murray, and Boesch (2019) PPKNW as promoter of cognitive function; Blankestijn et al. (2018); Downs et al. VEGNLQVLRPR and LAGNPHQQQQN as (2014); Sheng et al. (2019); Wang et al. antioxidants (2019) Cereals and grains

1.

Wheat

Gliadins (30
80 kDa) Glutenins (130
150 kDa) [Prolamins and Glutelins]

YPG, YYPG, and YIPP with opioid-like activities; MDATALHYENQK as antioxidant; IGGIGTVPVGR as ACE inhibitor

2.

Rice

20
40 kDa Glutelins

3.

Corn

Zeins (10
50 kDa) [Prolamin]

FYNEGDAPVVAL as immunomodulator; PGLTIGDTVPNLEL and DSTHGKIRIH as inhibitors of α-glucosidase (antidiabetic) LAP, LSP, and LQP as ACE inhibitors; IIGGAL, PPYLSP, and FLPPVTSMG as antiinflammatory peptides

Garg, Apostolopoulos, Nurgali, and Mishra (2018); Karami, Peighambardoust, Hesari, Akbari-Adergani, and Andreu (2019); Ortega, Moure, Gonza´lez, and Alconada (2019) Rein et al. (2019); Uraipong and Zhao (2016); Zhang et al. (2017) Jiao et al. (2018); Liang, Chalamaiah, Ren, Ma, and Wu (2018); Miyoshi, Kaneko, Ishikawa, Tanaka, and Maruyama (1995) (Continued)

Table 16.1: (Continued) Plant source

Storage proteins and characteristics

Example of bioactive peptides

References

4.

Barley

Hordeins (30
75 kDa) [Prolamin]

IL, LL, PG, YP, and ILLPGAQDGL as DPP-IV inhibitors

5.

Oats

Avenins (20
38 kDa) [Prolamin] Avenalins (19
43 kDa) [Globulin]

FFG, IFFFL, PFL, WWK, WCY, FPIL, CPA FLLA, and FEPL as DPP-IV inhibitors; EFLLAGNNKR as antithrombotic

Connolly, O’Keefe, Nongonierma, Piggott, and Fitzgerald (2017); Lacroix and Li-Chan (2012); Tanner, Colgrave, Blundell, Howitt, and Bacic (2019) Bleakley, Hayes, O’ Shea, Gallagher, and Lafarga (2017); Jing, Yang, and Zhang (2016); Tanner et al. (2019); Yu, Wang, Zhang, and Fan (2016) Bae et al. (2006); Choi et al. (2007); Koyama et al. (2013); Li, Matsui, Matsumoto, Yamasaki, and Kawasaki (2002); Zhou et al. (2019) Hu et al. (2011); Leung, Venus, Zeng, and Tsopmo (2018); Ribeiro et al. (2012); Udechukwu et al. (2017) Burrieza, Rizzo, Moura Vale, Silveira, and Maldonado (2019); Shi, Hao, Teng, Yao, and Ren (2019)

6.

Buckwheat 13S globulin (24
56 kDa) 2S GPP, YQY, PSY, LGI, and ITP as ACE albumin (19 kDa) inhibitors; DVWY, FQ, VVG, VAE, and WTFR as antihypertensive peptides

7.

Rye

Secalins (45
75 kDa) [Prolamin]

8.

Quinoa

7S, 11S, and 13S globulins (39
97 kDa)

9.

Sorghum

10.

Amaranth

Kafirins (19
50 kDa) [Prolamin] 7S vicilin (16
66 kDa) [Globulin] 11S amarantin (24
59 kDa) [Globulin] Globulin P (280 kDa)

CQV, QCA as antioxidants and inhibitors of TNF-α converting enzyme; VOO, IPP, LQP, and LLP as ACE inhibitors FGVSEDIAEKLQAKQDERGNIVL, AEGGLTEVWDTQDQQF, YIEQGNGISGLMIPG, AVVKQAGEEGFEW, and HGSDGNVF as anti-adipogenic (antiobesity) VAQNMP, VQSVVQ, QPQCSP as Cremer et al. (2014); Wu, Du, Jia, and antioxidants;TLS as ACE inhibitor Kuang (2016); Xu, Shen, Chen, et al. (2019) ´n ˜o VIKP as hypotensive peptide; FLISCLL, Aphalo, Castellani, Martinez, and An ´n-Cruz, Napier, SVFDEELS and DFIILE as ACE and (2004); Osuna-Castro, Rasco α-amylase inhibitors Fido, and Shewry (2000); Quiroga, ´n ˜o Martı´nez, Rogniaux, Geairon, and An ´n, and ˜o (2010); Sua´rez, Aphalo, Rinaldi, An Quiroga (2020); Vilcacundo, Martı´nezVillaluenga, Miralles, and Herna´ndezLedesma (2019) (Continued)

Table 16.1: (Continued) Plant source

Storage proteins and characteristics

Example of bioactive peptides

References

Pulses 1.

Common bean

Phaseolins (43
53 kDa) [Globulin]

2.

Pigeon pea

3.

Chickpea

7S vicilin (47
64 kDa) 11S legumin (17
44 kDa) [Globulin] 7S Vicilin-type (50
70 kDa) 11S Legumin-type (20 kDa) [Globulin]

4.

Mungbean

5.

Lentil

6.

Lupin

Conglutins (49
69 kDa) [Globulin]

7.

Faba bean

11S legumin-like (19
51 kDa) 7S vicilin-like (31
66 kDa) [Globulin]

7S (16
28 kDa), 8S (vicilintype, 26
60 kDa) and 11S (legumin-type, 24
40 kDa) [Globulin] Convicilin (60
67 kDa) [Globulin]

KTYGL, KKSSG, CPGNK, GGGLHK as antioxidants and inhibitors of ACE, DPP-IV and α-glucosidase; GLTSK, LSGNK, GEGSGA, MPACGSS and MTEEY as antiproliferative peptides VVSLSIPR as ACE inhibitor

Emani and Hall (2008); Luna Vital, Gonza´lez ˜a (2014); de Mejı´a, Dia, and Loarca-Pin Mojica, Luna-Vital, Gonza´lez, and de Mejı´a (2017)

LHQNIGSSSSPDIYNPQAGR, LHQNIGSSSSPDIYNPQAGRIK, QQSQETDVIVK, and VLLEEQEQ-KPK as anti-inflammatory peptides PLGSPLYEYSR, AVKPEPAR, GCGLPVR, and HNVAMER as antioxidant peptides

Bar-El Dadon et al. (2013); Mila´n-Noris et al. (2018)

LLSGTQNQPSFLSGF, NSLTLPILRYL, and TLEPNSVFLPVLLH as antioxidants and ACE inhibitors GQEQSHQDEGVIVR as inhibitor of HMGCoA reductase (hypocholesterolemic effect); LTFPGSAED as inhibitor of DPP-IV IR and YLR as antioxidant and anticancer peptides; DALEPDNRIESEGGLIETWNPNNRQ, FEEPQQSEQGEGR and GSRQEEDEDEDE as antioxidants and ACE inhibitors

Boachie et al. (2019); Nawaz et al. (2017)

Lapsongphon and Yongsawatdigul (2013); Mendoza, Adachi, Bernardo, and Utsumi (2001) Garcı´a-Mora et al. (2017); Xia, Pittelli, ´n (2016) Church, and Colo Czubinski and Feder (2019); Lammi, Zanoni, Arnoldi, and Vistoli (2016); Lammi, Bollati, Lecca, Abbracchio, and Arnoldi (2019) ´n-Espinosa Karas, Zlotek, et al. (2019); Leo et al. (2016); Warsame, O’Sullivan, and Tosi (2018)

(Continued)

Table 16.1: (Continued) Plant source

Storage proteins and characteristics

Example of bioactive peptides

References

Roots and tubers 1.

Potato

Patatin (40 kDa)

2.

Sweet potato

Sporamins (22
31 kDa)

3.

Yam

Dioscorin (28
32 kDa)

4.

Taro

Tarin G1 globulin (12.5 kDa) G2 globulin (22
24 kDa)

1.

Tomato seeds

19
48 kDa albumins and globulins

2.

Kiwi

3.

Pumpkin seeds

7S (10
150 kDa) and 11S (6
100 kDa) globulins 2S (,6 kDa) albumin Cucurbitin (11 , 20
35 kDa) [Globulin] 2S albumin (14.3 kDa)

DIKTNKPVIF as an antidiabetic and antiinflammatory peptide AIWGAGGGGLR, NVGRFKDPMLRTTR, YMVSAIWGAGGGGLR and YCQF as antioxidant peptides RRDY, RL, and DPF as inhibitor of DPP-IV and regulator of glucose metabolism; KTCGNGME and KTCGY as antioxidant peptides TDY and NHK as antioxidant peptides; HPDGR and GPSVF as antimicrobials; DGSTVW as anticancer peptides (in silico analysis)

Dumeus et al. (2018); Marthandam Asokan, Wang, Su, and Lin (2019); Shewry (2003) Shewry (2003); Zhang, Huang, and Mu (2019b) Han, Liu, Fang, and Hou (2013); Lin et al. (2016); Shewry (2003)

Bester et al. (2019); Shewry (2003)

Fruits HTQHQFFHG, THPDVPGEPT, STTTKKHHPQYL, and GVSLIRHVIQ as antioxidant peptides; DGVVYY as ACE inhibitor Kissper (39-amino acid peptide) as antioxidant and anti-inflammatory peptide LR as DPP-IV inhibitor (in silico analysis)

Meshginfar et al. (2018); Sarkar, Kamaruddin, Bentley, and Wang (2016); Moayedi et al. (2018) Ciacci et al. (2014); Ciardiello et al. (2008); Nilsson et al. (2015); Russo et al. (2019) Redhu, Dhanda, Singh, and Khaket (2015); Rezig et al. (2013); Rudakova, Rudakov, Kakhovskaya, and Shutov (2014)

In bioactive peptide sequence each letter corresponds to single letter code for each amino acid; ACE, angiotensin-I-converting enzyme; DPP-IV, dipeptidyl peptidase IV.

364 Chapter 16

Figure 16.1 Diagram for the most commonly used isolation procedure of proteins from edible plants.

antioxidant potential as evaluated by different in vitro methods (Xu, Shen, Chen, Bean, & Li, 2019; Xu, Shen, & Li, 2019). Fig. 16.2 depicts the general method for the isolation and extraction of kafirin from sorghum.

16.3 Sources and production of bioactive plant peptides Bioactive peptides from plants can be naturally occurring, produced during processing such as curing or fermentation or produced using controlled hydrolytic processes by employing different proteases.

16.3.1 Naturally occurring bioactive peptides in plants Diversity in the plant kingdom offers us tremendous amounts of molecules that are present in nature with different biological properties. One of these properties is health promotion and management as such it is not surprising that majority of pharmaceutical drugs are inspired by compounds found in nature. One group of these compounds with healthpromoting properties are the naturally occurring bioactive peptides. 1. Plant protease inhibitors (PPIs) are one large group of naturally occurring peptides in plants. They are known to inhibit the activity of digestive enzymes (trypsin and chymotrypsin) and are considered as antinutritional factors. PPIs are most commonly found in legume and seeds and include the well-studied BBI and KTI. BBI originally

Plant sources of bioactive peptides 365

Figure 16.2 Schematic diagram for the isolation of kafirin, an alcohol-soluble protein (prolamin), from sorghum using sequential solvent extraction method.

isolated from soybean has both trypsin and chymotrypsin inhibitory sites (Odani & Ikenaka, 1973). Soybean BBI is possibly the most studied in terms of bioactivity as it has gone through all the testing from in vitro and in vivo tests to clinical trials.

366 Chapter 16 In the form of soybean BBI concentrate, a clinical trial was performed to see the chemopreventive effects on oral cancer by the University of California Irvine. In a phase I clinical trial, a dose-escalation administration ranging from 25 to 800 chymotrypsin inhibitory units (CIU) resulted in no clinically observed toxicities (Armstrong, Kennedy, Wan, Atiba, et al., 2000). In a phase IIa clinical trial, 32 patients received BBI concentrate from 200 to 1066 CIU twice per day for 1 month (Armstrong, Kennedy, Wan, Taylor, et al., 2000). The study concluded that BBI concentrate demonstrated clinical activity, measured by reduction in total lesion area, when given orally to patients with oral leukoplakia and suggested that BBI concentrate be investigated in a randomized clinical trial (Armstrong, Kennedy, Wan, Taylor, et al., 2000). Follow-up phase IIb randomized, placebo-controlled clinical trial led to a unfavorable outcome showing that reduction in total lesion area as well as clinical responses was not statistically different between the BBIC-treatment arm and placebotreatment arm (Armstrong et al., 2013). The anti-inflammatory effect of soybean BBI has also been reported leading to studies on the potential effect of BBI on human immunodeficiency virus (HIV). BBI inhibited the replication of HIV in macrophages and blocked the entry of HIV into macrophages (Ma et al., 2016; Ma et al., 2018). In addition to its own bioactivity, soybean BBI also showed potential to prevent degradation of other bioactive peptides through its ability to inhibit trypsin and chymotrypsin. For instance, BBI protected the bioactive peptide lunasin from digestive proteases (Price, Pangloli, Krishnan, & Dia, 2016) leading to antiproliferative property of lunasin against colon cancer cells (Cruz-Huerta et al., 2015) and induction of cell death in breast tumor sections (Hsieh, Herna´ndez-Ledesma, Jeong, Park, & de Lumen, 2010). These studies further support the idea of complementarity as well as potential synergistic effects when using food-derived bioactive components in chemoprevention studies. In addition to BBI from soybean, numerous BBIs have been discovered in many plant species with various array of biological activities. BBI from the seeds of Luetzelburgia auriculate showed antimicrobial activity against Staphylococcus aureus (Martins et al., 2018), Clitoria fairchildiana demonstrated insecticidal potential (Dantzger et al., 2015), Vigna unguiculata enhanced the action of bradykinin-related ´ lvares et al., 2014), and chickpea inhibited the growth of peptides (da Cunha Morales A breast and prostate cancer cells (Magee, Owusu-Apenten, McCann, Gill, & Rowland, 2012). In addition to BBI, other PPI exhibited different biological activities. For instance, a trypsin inhibitor from the seeds of Momordica charantia called BG-4 exhibited antiproliferative and anti-inflammatory properties (Dia & Krishnan, 2016; Jones, Pangloli, Krishnan, & Dia, 2018; Nieto-Veloza, Wang, Zhong, Krishnan, & Dia, 2019). Kunitz trypsin inhibitors from Cassia leiandra showed antifungal and insecticidal activities (Arau´jo et al., 2019; Dias et al., 2017), tamarind improved metabolic alterations in obese and diabetic rats (Carvalho et al., 2019), and Erythrina velutina

Plant sources of bioactive peptides 367 seeds demonstrated anticoagulant and anti-inflammatory properties during sepsis (Machado et al., 2013). Other protease inhibitors from plants have shown potential in affecting innate immunity (serine protease inhibitors from plant seeds), ameliorating thrombosis (carboxypeptidase from potato), modulating inflammation and enhancing osteogenicity (phytocystatin from citrus), inhibiting growth rate of Helicoverpa armigera moth (bifunctional α-amylase/trypsin inhibitor from pigeon pea seeds), and potentiating the effect of antiretroviral drugs (cyclotide from leaves of sweet violet) (Gadge et al., 2015; Gerlach et al., 2019; Laparra & Haros, 2019; Leguizamo´n et al., 2019; Wang, Smith, Hsu, Ogletree, & Schumacher, 2006). These studies showed the diversity of both types and biological activities of PPI. 2. Lunasin, a 43-amino acid peptide originally isolated from soybean, has shown multiple biological activities in different in vitro and in vivo studies. Its chemopreventive properties are associated with unique amino acid sequence embedded on its primary structure as shown in Fig. 16.3. The primary structure entails three regions with unique putative functions responsible for its reported biological activities. The helical region corresponding to amino acid sequence 23
31 was reported to be a chromatin-binding domain responsible for immunomodulatory activities of lunasin, the RGD domain known to interact with integrins associated with lunasin internatilization and modification of integrin signaling and the polyaspartic acid tail that affects histone acetylation through interaction with core H3 and H4 histones (Cam, Sivaguru, & Gonzalez de Mejia, 2013; Chang et al., 2014; Dia & Gonzalez de Mejia, 2011b; Galvez & de Lumen, 1999; Galvez, Chen, Macasieb, & de Lumen, 2001; Herna´ndez-Ledesma, Hsieh, & de Lumen, 2011; Inaba, McConnell, & Davis, 2014; Jeong, Jeong, Kim, & de Lumen, 2007; Tung et al., 2014). These putative functions are potentially responsible for the reported anti-inflammatory and chemopreventive properties of lunasin against the cancer of the breast, colon, lung, and skin. As soy protein consumption is associated with a reduction of cardiometabolic risk, studies on the role of lunasin on cholesterol metabolism and obesity have been reported. Mechanistic studies in in vitro and in vivo models have shown the promising role of lunasin in mitigating cardiometabolic risk factors. Lunasin enhanced the uptake of low-density lipoprotein cholesterol (LDL-C) by increasing the expression of LDL receptor (Gu et al., 2017; Gu et al., 2019), alleviated

Figure 16.3 Primary sequence of soybean bioactive peptide lunasin.

368 Chapter 16 liver damage in a model of nonalcoholic steatohepatitis (Drori, Rotnemer-Golinkin, Zolotarov, & Ilan, 2017), and attenuated obesity-associated inflammation and metastasis (Hsieh, Wang, & Huang, 2016; Hsieh, Chou, & Wang, 2017). In a triple-blind, placebocontrolled 2 3 2 cross-over randomized clinical trial, administration of four lunasin capsules (each capsule contains 84 mg lunasin-enriched soy extract, 40 mg soy protein isolate, and 186 mg Soy Fibrim, DuPont) per day for 8 weeks resulted in a nonsignificant reduction of cardiometabolic factors (Haddad Tabrizi et al., 2019). The authors concluded that future studies should focus on higher dose, longer treatment time, and larger sample size to delineate the role of lunasin in reducing cardiometabolic risk factors (Haddad Tabrizi et al., 2019). 3. Dioscorin is one of the soluble proteins in yams with storage functions and a monomeric molecular weight of about 31 kDa (Lu, Chia, Liu, & Hou, 2012). Gene characterization study suggested that it plays in yam sprouting, regrowth, tuberization, and response to environmental stressors (Liu et al., 2017). Several biological activities of dioscorin have been reported using different in vitro and in vivo studies. Immunomodulatory activities have been reported associated with the ability of dioscorin to restore tight junction proteins, increase natural killer cells and their cytotoxic activity, stimulate phagocytic activity of lymphoid cells, and suppress allergic reactions induce by ovalbumin by promoting Th1 cells hence modulating Th1/Th2 immune response balance. In addition, administration of dioscorin also showed promise in preventing metabolic syndrome by lowering blood pressure, improving impaired glucose tolerance, and reducing total cholesterol, LDL-C, and total visceral lipid content (Fu et al., 2009; Hsu, Weng, Lin, & Lin, 2013; Lin, Lin, Weng, & Lin, 2009; Liu et al., 2009; Shih, Lin, Lin, & Hou, 2015; Wu, Lin, Liang, & Hou, 2018; Yang & Lin, 2014). Moreover, synthetic peptides derived from hydrolysis of dioscorins showed biological activities including amelioration of metabolic syndrome (NW dipeptide) (Wu et al., 2018), inhibition of dipeptidyl peptidases-IV (DPP-IV) (RRDY, RL, DPF, and MGSF peptides) (Lin, Han, Lin, & Hou, 2016), improvement in oral glucose tolerance test (RRDY and RL peptides) (Lin et al., 2016), and vasodilation in phenylephrineinduced tensions leading to blood pressure regulation (KTCGY and KRIHF pentapeptides) (Lin, Lu, Wang, Liang, & Hou, 2014). 4. Sporamins and patatins are SPs of sweet potato and potato, respectively. They are known to play a role in storage, defense, and regulation of endogenous proteases in tubers (Shewry, 2003). Sporamin showed anticancer properties in both in vitro and in vivo models of carcinogenesis. In vitro studies showed the ability of sporamin to induce apoptosis and suppress growth of cancer cells by inhibiting nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB) signaling, glycogen synthase-3 kinase signaling, and mitogen-activated protein kinase signaling (Qian, Chen, et al., 2017; Qian, Qi, Chen, Zeng, & Yao, 2017; Yao & Qian, 2011). In the xenograft model of colorectal carcinoma, intragastric infusion of sporamin led to the suppression of tumor

Plant sources of bioactive peptides 369 growth which could be associated with downregulation of β-catenin and vascular endothelial growth factor (Yang, Zhang, Zhang, Xiao, & Li, 2019). On the other hand, potato SP patatin demonstrated different antioxidant activities in vitro including radical scavenging activity, antihuman LDL peroxidation, and protection from hydroxy radicalinduced DNA damage (Liu, Han, Lee, Hsu, & Hou, 2003; Sun, Jiang, & Wei, 2013).

16.3.2 Plant-derived bioactive peptides through enzymatic hydrolysis Biologically active peptides are usually embedded in the primary structure of the parent protein. The bioactivity of these peptides is dependent on amino acid composition and amino acid sequence as such the use of enzymes with correct specificity toward parent protein as substrate is very important. Table 16.2 lists the properties of some of the commercially available enzymes that are currently used for the production of plant-derived bioactive peptides. In addition, examples of plant proteins and bioactive peptide sequences derived from the action of these enzymes are provided. Enzymatic treatment is one the most common ways of releasing bioactive peptides that are originally buried in the threedimensional structure of the parent protein. These enzymes are collectively known as proteases with the ability to hydrolyze peptide bonds present in parent proteins and low molecular weight peptides. Fig. 16.4 shows the general approach in the production of plantderived bioactive peptides using proteases as well as the techniques used to analyze the released peptides. Particle size reduction through grinding and homogenization assures uniformity of the material for efficient protein extraction. Protein from the plant material can then be extracted by either alkaline solubilization or solvent extraction. The extracted/ ground sample is suspended in water or appropriate buffer, adjusted and equilibrated to the optimum conditions for the enzyme (pH, T), enzyme addition, and hydrolysis for predetermined amount of time. More recently, assisted enzymatic hydrolysis has been employed to effectively and efficiently produce plant-derived bioactive peptides. For instance, the use of ultrasonication during enzymatic hydrolysis of walnut meal protein led to efficient trypsin hydrolysis as evidenced by improved kinetic and thermodynamic parameters as well as increased porosity and possible unfolding of walnut meal protein (Golly et al., 2019). The same effect of ultrasonic treatment was reported on the hydrolysis of corn protein zein where ultrasonication improved the solubility of zein leading to higher rate of hydrolysis and more efficient production of ACE-I inhibitory peptides (Ren, Liang, & Ma, 2018). Microwave treatment and autoclaving of sweet potato proteins led to improved hydrolysis and antioxidant activities of peptides released during gastrointestinal digestion (Zhang, Huang, & Mu, 2019a) while a combined ultrasonication and microwave treatment resulted in protein hydrolysates from chia seed with improved bioactivity and functionality (Urbizo-Reyes, San Martin-Gonzalez, Garcia-Bravo, Vigil, & Liceaga, 2019). These studies clearly support the idea of using food processing technologies in enzymatic hydrolysis of plant proteins to improve the extraction and production of plant-derived

370 Chapter 16 Table 16.2: Commercially available proteases for the production of bioactive peptides from different plant sources. Enzyme Alcalase

Optimum conditions

Protein substrate

pH: 6.5
8.5; T: 45 C
60 C

Soybean glutelin Sweet potato protein Mungbean vicilin Peanut protein isolate Cherry seed protein

Lupine protein isolate Soybean pH: 5.0
8.5; T: β-conglycinin 50 C
60 C Rice glutelin

Bioactive peptide/activity

References

IILLSF, RDILKDL, SIKFGSF, RSPEDSIIF, and AGTGLPIDRF as ACE inhibitors VSAIW, AIWGA, FVIKP, VVMPSTF, and FHDPMLR as ACE inhibitors Hydrolysate with antioxidant, anticancer, and ACE-I inhibition properties SWAQL, GNHEAGE, CFNEYE with antithrombotic properties

Zhang et al. (2019)

DGDPLLDQ, NLPLL, NGDPLLDQ, and ESGAVTE as antioxidants and ACE inhibitors Hydrolysate as inhibitor of enzymes involved in inflammation

Nazir, Mu, and Zhang (2019) Gupta, Srivasta, and Bhagyawant (2018) Zhang (2016)

Garcia et al. (2015)

Milla´n-Linares Mdel, Yust Mdel, Alcaide-Hidalgo, Milla´n, Pedroche (2014) Sufian et al. (2011)

Hydrolysate as suppressor of appetite by inducing cholecystokinin release SPFWNINAHS, MPVDVIANAYR, Selamassakul, Laohakunjit, VVYFDQTQAQA, and Kerdchoechuen, Yang, Maier VEVGGGARAP as antioxidants (2018) Rice bran KVDHFPL with antilisterial and Pu and Tang (2017) protein antibiofilm properties Black bean Hydrolysate with antioxidant Ficin pH: Zheng, Li, Li, Sun, Liu protein capacity 6.0
8.0; T: (2019) 40 C
70 C Oat avenin IFFFL and PFL as ACE-I and Zheng et al. (2019) DPP-IV inhibitors Oat 11S FPIL as ACE and DPP-IV Bleakley et al. (2017) gloubulin inhibitors Flaxseed Hydrolysate with antioxidant and Udenigwe, Lu, Han, How, meal protein anti-inflammatory properties Aluko (2009) Flavourzyme pH: 5.0; T: Esfandi, Willmore, Tsopmo Oat bran Hydrolysates antioxidant and 50  C (2009) protein antiapoptotic Soy protein VVHV as lipolysis stimulant, Marthandam Asokan, Hung, isolate regulator of proteins involved in Chiang, and Lin (2018); lipid metabolism leading to Tsou, Kao, Lu, Kao, and ameliorated muscle atrophy, Chiang (2013) antiobesity Pumpkin Hydrolysate with antioxidant Venuste et al. (2013) meal properties Corn protein Hydrolysate with bile acid Kongo-Dia-Moukala, Zhang, binding capacity and Irazoke (2011) Bromelain

(Continued)

Plant sources of bioactive peptides 371 Table 16.2: (Continued) Enzyme

Optimum conditions

Protein substrate

Bioactive peptide/activity

References

Hydrolysate with antiproliferative Carrilo, Gomez-Ruiz, Ruiz, Walnut property against melanoma cells and Cavallo (2017) protein isolate Denatured Hydrolysate with antioxidant and Xu, Zhao, Qu, and Ye (2015) soybean anti-fatigue effect properties meal Cottonseed Hydrolysate fraction rich in F, H, Gao, Cao, and Li (2010) meal protein P, M, I, C with antioxidant isolate property Potato Hydrolysate with antioxidant and Pihlanto, Akkanen, and protein and ACE-I inhibition properties Korkonen (2008) by-products Papain pH: Cotabarren et al. (2019) Chia expeller Hydrolysate with antioxidant 6.0
7.0; T: activity 65 C Rice bran SSEYYGGEGSSSEQGYYGEG as Kubglomsong et al. (2018) albumin tyrosinase inhibitor and copper chelator LPKF, ALRYFM, NFLARF, and Barley Gangopadhyay et al. (2016) GFPTLKIF as ACE-I inhibitors protein concentrate Sicklepod FHAPWK as ACE-I inhibitor Thermolysin pH: Shih, Chen, Wang, and Hsu seeds 5.0
8.5; T: (2019) 65 C
85 C Wheat germ WV and WI as vasoconstrictive Kumrungsee, Akiyama, Guo, enzyme inhibitor Tanaka, and Matsui (2016) Bitter melon VSGAGRY and VDSDVVKG with Priyanto et al. (2015) hypotensive and ACE-I inhibition seeds properties protein Trypsin pH: 7.8, T: Perilla seed Kim, Liceaga, and Yoon ISPRILSYNLR as antioxidant 37 C (2019) meal Finger millet TSSSLNMAVRGGLTR and Agrawal, Joshi, and Gupta protein STTVGLGISMRSASVR as (2019) antioxidant Rice protein Hydrolysate with Fang et al. (2017) immunomodulatory effect Wheat IPALLKR and Assaran Darban, Sharegi, gluten AQQLAAQLPAMCR as ACE-I Asoodeh and Chamani inhibitors (2017) Neutrase

pH: 7; T: 40 C
50 C

In bioactive peptide sequence each letter corresponds to single letter code for each amino acid; ACE, angiotensin-Iconverting enzyme; DPP-IV, dipeptidyl peptidase IV.

bioactive peptides. After hydrolysis, enzymes are either deactivated (mostly by heat or pH adjustment) or recycled for further use in case of continuous reactors. The hydrolysates are then separated from the insoluble material by centrifugation and then characterized. The extent of enzymatic hydrolysis is measured by degree of hydrolysis which is the percentage

372 Chapter 16

Figure 16.4 Typical schematic for the production and analysis of bioactive peptides from plant sources using enzymatic hydrolysis.

of peptide bond in the parent protein that was cleaved by the enzyme. In most cases, the hydrolysates are fractionated based on molecular weight by using filtration (ultrafiltration, ultracentrifugation) and chromatographic (size-exclusion) techniques. The fractions are further analyzed to confirm molecular weight distribution, amino acid content, as well as amino acid sequences for fractions with high bioactivity. After sequencing, the bioactivity is validated by synthesizing peptides and testing. In addition to single enzyme hydrolysis, studies have also shown that the use of two or more enzymes can lead to the production of highly bioactive peptides from plant proteins. Enzymatic production of plant-derived

Plant sources of bioactive peptides 373 bioactive peptides can be time-consuming but offer advantages associated with the specificity and selectivity of different proteases available as such certain process can be tailored for the production of multifunctional bioactive peptides.

16.3.3 Plant-derived bioactive peptides through fermentation Fermentation is the use of microbial enzymatic system to hydrolyze parent proteins for the production of peptides with potential bioactivity. Several health-promoting properties such as immunomodulation, hypotensive effect, and scavenging of oxidative reactive substances have been attributed to fermented foods partly due to peptides released during fermentation. Most studies on the generation of bioactive peptides used lactic acid bacteria (LAB) as starter culture which by themselves are highly proteolytic. As different LAB possesses diverse enzymatic system, various peptides with multifunctional activities can be generated from plant proteins. The proteolytic machinery of LAB is composed of a cell wall bound proteinase, specific transport systems for oligopeptides, and numerous peptidases responsible for further proteolysis (Liu, Bayjanov, Renckens, Nauta, & Siezen, 2010; Pessione & Cirrincione, 2016; Rodrı´guez-Serrano et al., 2018). In addition to LAB, certain molds such as Rhizopus oligosporus and Aspergillus oryzae have been used for the production of bioactive peptides from plant proteins. Table 16.3 shows some of the plantderived bioactive peptides generated through fermentation. It is not surprising to see that peptides from fermented products exhibited higher bioactivity than corresponding raw materials. This observation may be attributed to size and sequence of the peptides released as well as the potential production of amino acids attributed to microbial growth which could ultimately lead to changes in amino acid composition. In addition, microbial fermentation can also lead to the production of free amino acids and other bioactives (aside from peptides) contributing to overall increase in the biological and health-promoting properties of hydrolysates. For instance, sourdough fermentation of Triticum dicoccum (spelt) led to time-dependent increase in concentrations of total phenolics and the following amino acids: alanine, phenylalanine, methionine, tyrosine, tryptophan, valine, isoleucine, and leucine (Colosimo et al., 2020). As shown in the previous tables, it is known that branched-chain amino acids as well as tyrosine and tryptophan residues are components of peptides with enhanced bioactivities. In case of Mediterranean faba bean accessions, fermentation with Lactobacillus plantarum DPPMAB24W increased the concentrations of free amino acids, organic acids, and aromatic compounds with potential antioxidant activities (Verni, De Mastro, De Cillis, Gobbetti, & Rizzello, 2019). Another important effect of fermentation is on the digestibility of the raw materials. For instance, a mixed starter fermentation with Lactobacillus plantarum MRS1 and Lactobacillus brevis MRS4 of legume flours (red and yellow lentils, white and black beans, chickpeas and peas flours) for both raw and gelatinized states led to increase in vitro protein digestibility (IVPD) of the flour (De Pasquale, Pontonio, Gobbetti, & Rizzello, 2019). The same effect of fermentation

374 Chapter 16 Table 16.3: Bioactives generated by fermentation of plant-based materials. Plant source Soybean

Starter culture/conditions

Bioactive/activity

References

Bacillus subtilis followed by thermolysin hydrolysis; ultrasonication B. subtilis KN12C; Bacillus amyloliquefaciens KN2G and Bacillus licheniformis KN13C and B. licheniformis KN13C Solid-state, 42 C for 24 h Tane koji (riched in Aspergillus oryzae), 45 C for 5 days

QC, GPANV, and PANV with vasorelaxation activity; QC and CQ as ACE-I inhibitors Hydrolysates with high total polyphenol content, trichloroacetic acid soluble protein, and antioxidant activity

Wang et al. (2017)

Lactobacillus plantarum Lp6, 37 C for 72 h

Wheat

Lactobacillus brevis CECT 8183; 30 C for 48 h

Quinoa

Bean (Phaseolus sp)

Peptide fraction (,3 kDa) with ACE-I inhibitory property and antioxidant activity from sourdough

Nakahara et al. (2010) Amadou, Gbadamosi, Shi, Kamara, and Jin (2010) ˜as, Diana, Pen Frias, Quı´lez, Martı´nezVillaluenga (2015) Ayyash, Johnson, Liu, Al-Mheiri, and Abushelaibi (2018) Galli et al. (2018)

Hydrolysate with antioxidant, antiproliferative (MCF-7 cells) and inhibitory activity against ACE-I, α-amylase, and α-glucosidase Low molecular weight extracts from water-soluble extract of sourdough with anti-inflammatory activities in lipopolysaccharide-induced inflammation in macrophages B. subtilis, Bacillus pumilis, and Dei Piu’ et al. KSYQDVYNVAESS, FQQQYYPGLSN, Bacillus licheniformis; 37 C for 48 h (2014) QYKSYQDVYN among others with antioxidant activity L. plantarum, L. rossiae, Pediococcus Rizzelo et al. VGFGI, FTLIIN, IVLVQEG, LFRPEN, and pentosaceus; 37 C for 24 h (2017) LENSGDKKY as antioxidants and has protective effect against H2O2 damage Lactobacillus casei; 42 C for 8 h LAHMIVAGA and VAHPVF as inhibitors Obaroakpo et al. of ACE-I and α-glucosidase activity (2019) Cordyceps militaris (L.) Fr.; 25 C for Flour hydrolysates with improved ACE- Xiao et al. (2018) 7 days I inhibition INEGSLLLPH and FVVAEQAGNEEGFE Jakubczyk, Kara´s, L. plantarum 299 V; 22 C, 30 C, as ACE-I and α-amylase inhibitors; and 37 C for 3 h, 3 and 7 days Złotek, and INEGSLLLPH, SGGGGGGVAGAATASR, Szymanowska followed by pepsin
pancreatin among others as α-amylase inhibitors (2017) digestion (PPD) Hydrolysates with improved ACE-I Lactobacillus bulgaricus (37 C for Rui et al. (2015) inhibitory properties 5 h); L. plantarum B1
6 (37 C for 3 h); L. plantarum 70810 (31 C for 2 h) followed by pepsin, trypsin and α-chymotrypsin digestion Bifidobacterium animals DSM10140; Bifidobacterium breve DSM20213; Bifidobacterium longum DSM20097; 37 C for up to 72 h L. brevis, Lactobacillus farciminis, Lactobacillus plantarum, Lactobacillus rossiae, and Lactobacillus sanfranciscensis; 30 C for 24 h

Rice

AW, GW, AY, SY, and GY from fermented soy sauce with ACE-I inhibitory activity FNDHVE, FNHLDH, VIAGH, VLAGH as antioxidants

Rai et al. (2017)

(Continued)

Plant sources of bioactive peptides 375 Table 16.3: (Continued) Plant source

Starter culture/conditions

Bioactive/activity

References

Rye

Pool of Lactobacillus alimentarius 15M, L. brevis 14G, L. sanfranciscensis 7A, and Lactobacillus hilgardii 51B; 37 C for 24 h

Coda. Rizzello, Pinto, and Gobbetti (2012)

Amaranth

L. casei Shirota (37 C for 36 h); Streptococcus thermophilus (37 C for 36 h) and combination L. plantarum with Savinase (37 C, pH 8.5 for 15 h) followed by PPD

PAEMVAAALDR, KVALMSAGSMH, DLADIPQQQRLMAGLALVVATVIFLK among others with antioxidant activity and protective effect against H2O2induced oxidative stress Hydrolysates with antioxidant activity and inhibitory property against ACE-I and thrombin SGREKWERKEDEEKVVEEEEGEWRGS, FNTEYEEIEKVLLEEQ, NTEYEEIEKVLL among others with antioxidant activity and inhibitory properties against ACE-I, lipase and α-glucosidase

Lentil

˜o et al. Ayala-Nin (2019) ´sito Bautista-Expo et al. (2018)

In bioactive peptide sequence each letter corresponds to single letter code for each amino acid; ACE, angiotensin-Iconverting enzyme; DPP-IV, dipeptidyl peptidase IV.

with Lactobacillus plantarum VTT E-133328 was found on the IVPD of bread made from faba bean flour (Sozer et al., 2019). It is also possible that the increase in the digestibility of plant proteins after microbial fermentation could contribute to the increased production of bioactive peptides. Hence, in addition to direct effect on the production of plant-derived bioactive peptides, fermentation itself affects other properties of the plant material which can contribute to the overall increased biological activities of the resulting hydrolysates.

16.3.4 Unique aspects of plant proteins and preparing bioactive peptides from plant sources Fruits, vegetables, cereals, and legumes are good sources of protein for human nutrition. Combination of these sources will lead to a balanced diet providing all essential amino acids which are commonly provided just by consuming one animal protein. For instance, consumption of rice and soybean practice in many Asian countries provides all the essential amino acids needed by the individual to perform bodily functions. One unique aspect of plant proteins as sources of bioactive peptides is how plant proteins can be easily differentiated, and therefore be isolated from the parent material, by virtue of their solubility and sedimentation coefficient. For instance, soy proteins are mixtures of globular proteins glycinin and β-conglycinin and can be separated to 7S and 11S globulins according to their molecular weight and sedimentation coefficients (Cho, Rhee, & Wiss, 2004; Subirade, Kelly, Gueguen, & Pezolet, 1998). On the other hand, at least 80% of wheat proteins are composed of the alcohol-soluble gliadin and alkali-soluble glutelins (Kinsella, 1982; Kirsten, Peggy, & Phoebe, 2009) while the major protein in sunflower is called

376 Chapter 16 helianthinin, an 11S oligomeric protein with molecular weight as high as 350 kDa (Gonzalez-Perez & Vereijken, 2007). As such, due to the specificity of plant proteins, it is easier to define what particular protein source is responsible for the generation of plantderived bioactive peptides. The processing of plant materials including fruits, vegetables, and cereals generates a significant amount of waste coproducts. These coproducts are used mostly in animal feed and can be used as sources of bioactive plant peptides. Rice bran, a coproduct of white rice processing, is an inexpensive source of high-quality proteins that have been used in the production of bioactive peptides. Pepsin
pancreatin hydrolysis of rice bran protein led to production of three fractions (mass ranges of 120
743 m/z, 141.96
903.42 m/z, and 153.94
741.83 m/z) with modulatory effect on cholesterol synthesis (Kumar, Kurup, & Tiku, 2020). The mechanism involved was through binding of bile salts, inhibiting HMGCoA reductase activity, and lowering micellar lipid-carrying capacity (Kumar et al., 2020). Alcalase hydrolysates of rice bran protein with a molecular weight less than 3 kDa showed blood pressure-lowering activity through antioxidant property and inhibition of ACE (Piotrowicz et al., 2020). Stones from fruit processing industry can also be used as source of plant-derived bioactive peptides. Alcalase, thermolysin, flavourzyme, and gastrointestional digestion of apricot, plum, olive, and peach seed demonstrated antioxidant and cholesterol-lowering properties (Garcı´a, Gonza´lez-Garcı´a, Va´squez-Villanueva, & Marina, 2016). Alcalase and thermolysin treatment of cherry seeds led to production of peptides with antioxidant and ACE-inhibitory properties (Garcı´a, Endermann, Gonza´lezGarcı´a, & Marina, 2015). These coproducts are usually of lower economical value and contribute to environmental pollution. Hence, converting them to bioactive peptides that can promote overall human health is a way for their utilization increasing their value. Another unique aspect of bioactive plant peptide generation is through germination. Germination, also known as sprouting, of legumes caused differential metabolic activities within the seeds resulting in hydrolysis of seed proteins and starch (Maleki, & Razavi, 2020 Jul, 14). Germination of soybean followed by gastrointestinal digestion led to the production of peptides with antidiabetic properties through inhibition of enzymes involved in postprandial glycemic response (Gonza´lez-Montoya, Herna´ndez-Ledesma, MoraEscobedo, & Martı´nez-Villaluenga, 2018). Digests from germinated chickpeas demonstrated higher anti-inflammatory properties than those from cooked chickpeas associated with a higher concentration of bioactive peptides and isoflavones (Mila´n-Noris, Gutie´rrez-Uribe, Santacruz, Serna-Saldı´var, & Martı´nez-Villaluenga, 2018). The most bioactive peptide digest fraction featured 24 different peptides derived mainly from legumin and vicillin (Mila´n-Noris et al., 2018). Germination of cowpea for 24 hour followed by alcalase hydrolysis led to the production of peptides with higher antioxidant and inhibitory activity against DPP-IV (de Souza Rocha, Hernandez, Chang, & de Mejı´a, 2014). One of the identified peptides, TTAGLLE, was reported to interact with the S2 (Glu205-Glu206) and S3

Plant sources of bioactive peptides 377 (Ser209-Arg358-Phe357) pockets of DPP-IV with expectation of blocking the active site of DPP-IV (de Souza Rocha et al., 2014). Hence, the production of bioactive peptides from plant sources entails unique aspects that can be attributed to the different physicochemical properties of plant proteins, sources (processing coproducts), and production (germination).

16.4 Mechanistic insights on the biological activities of bioactive peptides from plants The vastness of plant proteins results in numerous plant-derived peptides with various biological activities. In addition, the number of enzymes and microorganisms, that can be used in the production of plant-derived bioactive peptides, contributes to a large number of mechanistic potentials by which these peptides can exert their biological activities. These biological activities involved either a single target to modify certain signaling pathways or multiple biomarkers to exert biological effects and may include pathways involved in inflammation and immunomodulation, carcinogenesis (initiation, progression, and metastasis) as well as those involved in metabolic syndrome (hypertension, weight management, imbalanced cholesterol, and high triglycerides). This part discusses some of the mechanistic insights by which plant-derived bioactive peptides are involved in the aforementioned pathways and targets.

16.4.1 The role of plant-derived peptides in inflammation and immunomodulation Inflammation is part of the cellular defense system against external and internal stimuli that can cause infection and injury. However, uncontrolled and chronic inflammation has been associated with the development of several diseases in humans including obesity, type 2 diabetes, rheumatoid arthritis, and neurodegenerative disease (Garcia, Hartkamp, et al., 2016; Lepedda et al., 2016; Lykhmus et al., 2016; Zheng et al., 2016). It is tightly regulated by macrophages which are part of our immune system responsible for the production of an array of molecules including reactive oxygen and nitrogen species and pro-inflammatory cytokines that act as signaling molecules. One of the well-studied signaling pathways involved in inflammation and immunomodulation and their associated malignancies is the NF-κB signaling. NF-κB transcription factor is a central mediator of inflammation and immunity with multiple links to various human diseases. It is mostly located in the cytosol bound to its inhibitor known as inhibitor of kappa B (IκB), this inhibitor can be ubiquitinated leading to proteasomal degradation and nuclear translocation of NF-κB. A variety of molecules can lead to the activation of this pathway such as lipopolysaccharide (LPS), viruses, and pro-inflammatory cytokines such as IL-1β and TNF-α. For instance, binding of LPS to its receptor TLR-4 can cause phosphorylation of IκB (IKKα and IKKβ)

378 Chapter 16 leading to proteasomal degradation of IκB and activation of NF-κB (Mussbacher et al., 2019). This pathway, known as the canonical NF-κB signaling, is only one of the cascades by which NF-κB can be activated. NF-κB is important in immunomodulation but aberrant NF-κB signaling can lead to initiation and progression of diseases. Compounds that can inhibit uncontrolled NF-κB signaling have been studied and are still currently being investigated. For instance, a novel 9-amino acid residue derived from fructokinase-1 of corn silk extract and hydrolyzed by trypsin was able to inhibit NF-κB signaling in LPS-induced inflammation in HepG2/NF-κB recombinant cells and BALB/C mice (Ho, Li, Lo, Chen, & Hsiang, 2017). In their study, the 9-amino acid peptide composed of TMKLLLVTL was able to interact on the ATP-binding pocket in the N-terminal kinase domain of IKKβ as well as to its catalytic site involving Asp145 (Ho et al., 2017). Mechanistic investigations showed that TMKLLLVTL from trypsin-digest of corn silk was able to reduce phosphorylation of IκB, relative IKKβ, and NF-κB activities in HepG2/NF-κB recombinant cells (Ho et al., 2017). Moreover, the level of IL-1β in the sera, as well as expression of p65 and IL-1β in the small intestine of BALB/C mice given 1 mg/kg LPS, was reduced when TMKLLLVTL was orally administered at a dose of 1 mg/kg (Ho et al., 2017). In another study, simulated gastrointestinal digest of amaranth seeds produced anticoagulant peptides coming from the surface of amaranth 11S globulins and agglutinin (Sabbione, Luna-Vital, Scilingo, An˜o´n, Gonza´lez de Mejı´a, 2018). Cytokine array analysis of THP-1 cells treated with LPS with or without amaranth anticoagulant peptide fraction showed that the peptide fraction was able to reduce the expression of cytokines involved in NF-κB signaling (Sabbione et al., 2018). Another important role of NF-κB signaling is immunomodulation involving allergic responses. For instance, treatment of β-lactoglobulin and α-lactalbumin, two whey components suspected as major allergens in milk, was able to mimic the effect of LPS in RAW 264.7 macrophages associated with NF-κB activation and increased production of pro-inflammatory cytokines IL-6 and TNF-α. This effect can be ameliorated with some of the plant-derived bioactive peptides. For instance, the antiinflammatory peptide derived from amaranth seeds with a sequence of SSEDIKE was able to modulate IgE-mediated food allergy (Moronta, Smaldini, Fossati, Anon, & Docena, 2016). In this study, cow milk protein served as the allergen and SSEDIKE was given at a dose of 100 μg per mouse, and result showed that SSEDIKE administration was able to lower the clinical allergy score and mean increase in footpad thickness without itself being allergenic as well as reduce level of IL-5, IL-13, and IFN-γ secreted by isolated splenocytes (Moronta et al., 2016). In addition, SSEDIKE treatment led to a reduction in serum-specific sow milk protein IgE and IgG1 (Moronta et al., 2016). On the other hand, activation of NF-κB signaling is an important factor in the immune response during infection, and studies have shown that plant extracts, proteins, and peptides can have this biological effect. For instance, a glycoprotein from Chinese Yam (Dioscorea opposite Thunb) rich in aspartic acid, glutamic acid, and cysteine was able to activate multiple signaling pathways including NF-κB leading to immunomodulatory effects (Niu et al., 2017).

Plant sources of bioactive peptides 379 They found a dose-dependent effect of yam glycoprotein on splenocyte proliferation, phagocytic activity, and production of inflammatory mediators TNF-α, IL-6, and nitric oxide (Niu et al., 2017). These studies emphasized the balance needed during inflammation and immunomodulation when investigating the biological activities of not only plant-derived bioactive peptides but also other naturally occurring bioactives. The current state of the literature supports the idea that plant proteins can be sources of biologically active peptides with anti-inflammatory properties during uncontrolled inflammation conditions as well as immunomodulatory effects that can enhance the immune system to fight certain diseases.

16.4.2 The anticancer effect of plant-derived peptides: prevention, initiation, and progression Cancer continues to be a leading cause of deaths worldwide second only to cardiovascular diseases. One factor that contributes to increased cancer risk is diet. For instance, high intake of processed and red meat has been shown to increase the rate of certain types of cancers such as gastric, bladder, colorectal among others (Barry et al., 2019; Ferro et al., 2019; Mehta et al., 2019). On the other hand, isocaloric substitution of red and processed meat proteins with plant protein resulted in lower cancer-related mortality (Budhathoki et al., 2019). These population-based studies indicate the importance of plant and plant proteins in the prevention of cancer. Carcinogenesis is a multistep process resulting from the accumulation of successive gene mutations that ultimately lead to uncontrollable cell growth. This aberrant cell growth resulted in hallmarks of cancer as defined by Hanahan and Weinberg including sustaining proliferative signaling, evading growth suppressors, resisting apoptosis (cell death), enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis (Hanahan & Weinberg, 2011). As such these hallmarks have been the target of bioactive molecules including peptides and proteins from plant sources. Numerous studies looking at the anticancer effects of plant proteins and peptides focused on the ability of these molecules to inhibit proliferation and promote apoptosis. For instance, the anticancer property of the bioactive peptide lunasin from soybean has been associated with its ability to cause cytotoxicity and promote programmed cell death to a variety of immortalized human cancer cell lines including colon cancer cell lines HT-29, HCT-116 and KM12L4, breast cancer cell lines MDA-MB-231 and MCF-7, and skin cancer cell lines A375 and SK-MEL-28 (Dia & Gonzalez de Mejia, 2011a; Dia & Mejia, 2010; Hao et al., 2019; Jiang et al., 2016; Montales, Simmen, Ferreira, Neves, & Simmen, 2015; Pabona et al., 2013; Shidal, Al-Rayyan, Yaddanapudi, & Davis, 2016; Shidal, Inaba, Yaddanapudi, & Davis, 2017). These in vitro anticancer effects have been supported by a number of in vivo anticancer studies utilizing xenograft and orthotopic models of cancer. Most studies on the anticancer effect of soybean lunasin focused on its therapeutic effect indicating the ability of lunasin to promote apoptosis, arrest cell cycle, and potential

380 Chapter 16 targeting of cancer stem cells (Vuyyuri, Shidal, & Davis, 2018). These anticancer effects are associated with the ability of lunasin to bind core histone, inhibit histone acetylation, and antagonize integrin signaling via unique amino acid sequences present in the lunasin primary structure (Vuyyuri et al., 2018). Proteases, enzymes involved in homeostasis, are potential targets for plant-derived bioactive peptides for their anticancer activity. Dysregulation in protease activity and function can lead to carcinogenesis. PPIs have shown anticancer activities in multiple studies. For instance, soybean BBI has chemopreventive effect against colorectal cancer via modulation of the activity of the following serine proteases: proteasome, matriptase, chymase, cathepsin G, duodenase, and elastase (Clemente & Arques Mdel, 2014). These serine proteases are known to be involved in carcinogenesis by promoting inflammatory processes, tumor invasion, and metastasis. Sunflower trypsin inhibitor is the smallest known BBI composed of 14 amino acid residues, a cyclic peptide stabilized by a single disulfide bond, and an intramolecular H-bond (Korsinczky, Clark, & Craik, 2005; Le˛gowska et al., 2010). The anticancer property of sunflower trypsin inhibitor is associated with its ability to inhibit matriptase, a serine protease involved in the degradation of extracellular matrix components which could promote cancer cell metastasis (Yuan et al., 2011). The unique structural properties (small size, complex rigidity, plasticity, and well-defined structure) of sunflower trypsin inhibitor have been explored in numerous drug design studies acting as molecular scaffold for drug discovery (Chen, Kinsler, Macmillan, & Di, 2016; Fittler, Avrutina, Empting, & Kolmar, 2014; Lesner, Łe˛gowska, Wysocka, & Rolka, 2011). Another cyclic peptide with trypsin inhibitory property is Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II is composed of 34 amino acid residues with rigid structure, resistant to thermal and enzymatic degradation, and highly stable in vivo originally isolated from Vietnamese squash (Werle, Kafedjiiski, Kolmar, & Bernkop-Schnu¨rch, 2007). MCoTI-II also showed strong inhibitory property against the serine protease matriptase (Gray et al., 2014). Another cyclic peptide isolated from Viola ignobilis and termed vigno 5 is composed of 29 amino acid residues with three disulfide bridges (Esmaeili et al., 2016). Vigno 5 was able to induced apoptosis in He
La cervical cancer cells characterized by nuclear shrinkage, DNA fragmentation, and cleavage of polyADPribose polymerase (Esmaeili et al., 2016). Novel cyclotides DC1, DC2, and DC3 from Hedyotis diffusa showed antiproliferative properties against prostate cancer cells (Hu, Wang, Chen, & Tao, 2015). The most active cyclotide DC3 (IC50 of 0.21 μM against LNcap prostate cancer cells) showed antiinvasion and antimigratory properties as well as ability to inhibit growth of LNcap xenografts (Hu et al., 2015). Protein isolates and enzymatically derived peptides whether in the form of protein hydrolysates mixture or purified/synthetic peptides from plant proteins have also shown promise as potential anticancer molecules. Alcalase hydrolysates of hempseed cake protein isolate inhibited the growth of cervical cancer HeLa cell line while stimulating the growth

Plant sources of bioactive peptides 381 of normal keratinocytes HaCaT cells (Logaruˇsi´c et al., 2019). Two enzyme hydrolysates of fenugreek protein isolate led to the activation of apoptosis, G1 phase cell cycle arrest, and growth inhibition of undifferentiated Caco-2/TC7 cells giving initial evidence on the role of fenugreek protein as nutraceutical for colorectal cancer (Allaoui et al., 2019). Moreover, plant protein isolates and hydrolysates offer protective effect against cancer in models of chemically induced cancer. Chickpea protein hydrolysate administered in the diet protected colon cancer formation in mice given with the procarcinogenic azoxymethane with more pronounced effect when mice is in hypercaloric diet (Sa´nchez-Chino et al., 2019). Administration of soy protein isolate in mice treated with diethylnitrosamine and alcohol reduced adenoma progression associated with the ability of soy protein isolate to reduce inflammation and hepatocyte proliferation via inhibition of ethanol-induced β-catenin signaling (Mercer et al., 2016; Mercer et al., 2018). Similar protective effect of soy protein isolate on the development of mammary tumors when given in utero and lifetime feeding was observed in mice treated with direct carcinogen N-methyl-N-nitrosourea with increased apoptotic status and expression of phosphatase and tensin homolog deleted on chromosome ten as potential mechanism of action (Su et al., 2007). Rice protein isolate was also able to alter mammary tumor formation in rats treated with the carcinogen 7,12-dimethylbenz [alpha]anthracene irrespective of circulating estrogen level (Morita & Kiriyama, 1996). These studies demonstrate the various mechanisms by which plant-derived bioactive proteins and peptides can be used in the prevention of cancer as well as their potential application in cancer therapeutics.

16.4.3 The role of plant-derived peptides in metabolic syndrome Metabolic syndrome is a collection of metabolic abnormalities, which increases the risk of an individual in developing cardiovascular disease and type 2 diabetes. In 1998 the World Health Organization diabetes research group defined metabolic syndrome as factors (physiological, biochemical, clinical, and metabolic) that increases the risk of cardiovascular disease, type 2 diabetes, and all-cause mortality (Kaur, 2014). It includes abdominal obesity (waist circumference: $ 94 cm for men and $ 80 cm for women), prehypertension or hypertension (blood pressure: $ 130 mm Hg systolic and $ 85 mm Hg diastolic), dyslipidemia (triglycerides: $ 1.7 mmol/L and high-density lipoprotein cholesterol: # 1.03 mmol/L), and prediabetes (impaired fasting glucose or type 2 diabetes) (Duprez & Toleuova, 2013; Gregory, 2019; Pe´rez, Gonza´lez, Martı´nez-Espinosa, Vila, Reig Garcı´a-Galbis, 2019). The beneficial role of proteins in weight management, satiety, and improving cholesterol levels has been reported in the literature. In addition, dietary guidelines emphasize the consumption of plant-based foods as such whole grains, legumes, and pulses can be considered sources of quality plant protein. Population studies on the effect of plant proteins in metabolic syndrome usually evaluate the comparative effect of animal protein when substituted with plant proteins in a whole diet approach. It is important

382 Chapter 16 to note that the presence of other compounds may contribute to the observed effect of plant proteins in metabolic syndrome. A longitudinal study performed in Melbourne, Australia, suggested that higher intake of plant proteins and lower intake of animal proteins may help to prevent metabolic syndrome in healthy community-dwelling adults (Shang et al., 2017). In this study, 452 cases of metabolic syndrome were recorded during a mean follow-up of 11.2 years and found a positive association between metabolic syndrome and chicken and red meat consumption, whereas an inverse association was observed between metabolic syndrome and plant protein consumption (Shang et al., 2017). Moreover, each 5% increment in energy intake from animal protein was associated with increase in waist circumference, increase in systolic blood pressure, and increase in weight (Shang et al., 2017). However, an inverse association was found between plant protein intake and waist circumference and weight (Shang et al., 2017). On the other hand, during a random order cross-over design study (72% compliance) involving overweight and obese adults, plant proteins (soy/legume) do not significantly differ with animal proteins (beef/pork) in affecting appetite control, energy expenditure, and cardiometabolic risk factors during energy-restricted-induced weight loss (Li, Armstrong, & Campbell, 2016). A systematic review of observational and interventional studies on human concluded that soy protein isolate with isoflavones may prevent the onset of hypercholesterolemia and hypertension in humans but was not able to conclude on the positive effect of plant proteins on glucose homeostasis and body composition (Chalvon-Demersay et al., 2017). The positive effects of plant proteins in metabolic syndrome may be associated with the ability of plant-derived peptides to inhibit the activity of enzymes related to hypertension (ACE-I), cholesterol synthesis (HMGCoA reductase), and carbohydrate metabolism (DPP-IV, α-amylase, and α-glucosidase) as shown in Tables 16.1
16.3. A naturally occurring 37-amino acid soybean peptide called aglycin improved glucose tolerance and controlled hyperglycemia when given at 50 mg/kg/d in high-fat diet, streptozocin-induced diabetes in BALB/c mice (Lu, Zeng, et al., 2012). The study suggested that this antidiabetic effect of aglcyin was due to the increased insulin receptor signaling in the skeletal muscle of mice (Lu, Zeng, et al., 2012). Leginsulin, a 4 kDa cysteine-rich peptide which is another 37-amino acid peptide naturally occurring in soybean, has preferential solubility in 50% isopropanol and accumulated in higher amount in Asian soybean cultivars than North American soybean cultivars (Kim, Jang, & Krishnan, 2012). It is a hormone-like peptide and a recombinant leginsulin has been reported to have stronger insulin-like activity in a glucose uptake assay (Hashidume et al., 2018) further increasing the evidence on the important role of soybean peptides in controlling symptoms of metabolic syndrome. In addition to naturally occurring peptides in soybean, soybean peptides derived from enzymatic hydrolysis of soybean proteins have shown the ability to alleviate markers of metabolic syndrome. A diet supplemented with soybean hydrolysate derived from protease hydrolysis of soybean protein isolate led to increased postprandial

Plant sources of bioactive peptides 383 carbohydrate oxidation and energy expenditure in type II KK-Ay diabetic mice induced by high-fat diet (Ishihara et al., 2003). Furthermore, soybean hydrolysate was able to reduce the absorption of dietary lipids with increased lipid excretion (Ishihara et al., 2003). An alcalase hydrolysate of soybean β-conglycinin was able to reduce lipid accumulation, and lipoprotein lipase and fatty acid mRNA expression in 3T3-L1 adipocytes accompanied by anti-inflammatory effects in LPS-induced RAW 264.7 macrophages (Martinez-Villaluenga, Dia, Berhow, Bringe, & Gonzalez de Mejia, 2009). Recent studies have focused on other sources of plant peptides with the ability to manage metabolic syndrome. Gastrointestinal digestion of millet protein fractions after heat treatment showed different abilities to inhibit enzymes involved in metabolic syndrome (Kara´s, Jakubczyk, et al., 2019). Hydrolysates from millet globulin 11S heated at 65 C had the highest potential to inhibit ACE, millet glutelin heated at 100 C was the most potent in inhibiting α-amylase, and millet prolamin heated at 65 C exhibited the highest inhibitory activity against α-glucosidase (Kara´s, Jakubczyk, et al., 2019). A bioactive synthetic peptide from the roots of Astragalus membranaceus called AM-1 with sequence LVPPHA inhibited ACE activity with IC50 of 414.88 μM (Wu, Li, Lo, Hsiang, & Ho, 2020). Moreover, oral administration of AM-1 at 10 μmol/kg in rats led to a reduction of systolic blood pressure by 42 mm Hg, statistically similar to control drug captopril at 10 mg/kg (Wu et al., 2020). As the agricultural and food industries moved toward sustainable sources of proteins to feed the growing population, it is expected that novel food protein ingredients will be studied as sources of potential bioactive peptides capable of controlling metabolic syndrome. Hence, it is important to further study the effect of plant proteins and peptides on metabolic syndrome components.

16.5 Challenges and opportunities in studying the health benefits of plant-derived peptides Proteins as dietary nutrients are being recognized for their potential beneficial role in human health. Some of these proteins are naturally occurring such as PPIs present in many legumes and seeds of various plant species. These proteins are present in small amounts compared to SPs in plant tissues as such applications in human health outcome studies can be limited by difficulty in extraction and purification of these bioactive proteins. For instance, the clinical trials conducted on the role of BBI from soybean in prostate cancer and ulcerative colitis used a concentrated form (Lichtenstein et al., 2008; Malkowicz et al., 2001) which is contaminated by other compounds. The presence of these other compounds may either enhance or diminish the effect of the actual bioactive protein. Others have successfully tried recombinant expression of naturally occurring bioactive proteins and peptides to increase the quantity with the hope of not negatively affecting biological activities. For instance, the bioactive peptide soybean lunasin has been successfully expressed in yeast and bacteria and the previously observed biological activities against

384 Chapter 16 LPS-induced inflammation in macrophages, as well as antiproliferative activities in breast and colon cancer cells were confirmed (Liu & Pan, 2010; Setrerrahmane, Zhang, Dai, Lv, & Tan, 2014; Zhu, Nadia, Yao, Shi, & Ren, 2018). The tuber SP yam dioscorins were successfully expressed in Escherichia coli with retained antioxidant and immunostimulating properties (Jheng et al., 2012). Recombinant expression of these bioactive peptides and proteins, especially those that are naturally present in small quantities, is an effective strategy to enhance the production and thus to leverage the use of plant-derived peptides and proteins in human health studies. The same production challenges are being faced by plant-derived peptides produced by either enzymatic hydrolysis or microbial fermentation. Searching for a suitable parent protein, appropriate enzyme, and microbial starter culture and conditions (temperature, pH, time) of enzymatic hydrolysis and microbial fermentation is critical in obtaining bioactive peptides. Most of the published research works would take one factor at a time such as the use of different enzymes for a particular protein source or the use of the same enzyme for different hydrolysis time. This type of experiment, though the most commonly used, is time-consuming and requires a lot of manpower and resources. The use of optimization techniques such as response surface methodology, design of experiments, fractional factorial design, and high throughput screening must be developed and applied religiously to avoid the time-consuming one-factor-at-a-time approach. For instance, response surface methodology was used to address the time of hydrolysis and enzyme concentration on the production of antioxidant and ACE-I inhibitory peptides from pollen proteins (Maqsoudlou et al., 2019). Following sequence identification and biological activity determination, the cost associated with purification is another challenge. Unlike synthetic and recombinant peptides, the purification of a specific peptide from a mixture of peptides in food protein hydrolysates is cumbersome, costly, and prone to insolubility as most of these plant-derived bioactive peptides are hydrophobic in nature and insoluble at high concentrations. On the other hand, the mixture of peptides in food protein hydrolysates can confer higher biological activities as synergistic interactions among peptides are possible. The consistency of quality from batch-to-batch production is also a challenge as reported studies were conducted in laboratory settings and scaling up to commercial production level may result in reproducibility and quality issues. These issues then pose the question of the actual marketability of these bioactive peptides derived from enzymatic hydrolysis and microbial fermentations of plant proteins and must be addressed by future research works in this field. Oral delivery of plant-derived peptides is another major challenge when using these molecules in human health studies. Like other food nutrients, proteins and peptides are prone to digestion by proteases found in the gastrointestinal tract of the human body. Digestion can have both beneficial and detrimental effects on the biological activities. Numerous studies have shown that mimicking gastrointestinal digestion in vitro using

Plant sources of bioactive peptides 385 pepsin
pancreatin hydrolysis resulted in the production of peptides with improved biological activities. Others have shown that digests of naturally occurring bioactive proteins and peptides have retained bioactivity. It is also possible that after gastrointestinal digestion, the resulting digests (peptides and amino acids) may not have any biological activity. A study on the gastrointestinal digestion of broccoli protein hydrolysate led to the identification of two more potent bioactive peptides with ACE-I inhibitory and hypotensive properties (Dang et al., 2019). This study discovered an ACE-I inhibitor peptide with sequence LVLPGELAK which was further degraded to LVLPGE and LAK following simulated gastrointestinal digestion (Dang et al., 2019). The released peptides demonstrated more potent (at least twice) ACE-I inhibitory and hypotensive properties (Dang et al., 2019). On the other hand, simulated gastrointestinal digestion of bioactive peptide lunasin led to reduced ability to inhibit inflammasomes in THP-1 macrophages (Price, Pangloli, & Dia, 2017). Design of delivery systems for plant-derived peptides is another challenge as they need to be absorbed prior to exerting biological effects in the organism. Traditional belief that only amino acids can be absorbed in the brush border of the small intestines has been challenged and many studies support the notion that intestinal uptake of peptides can happen. Several mechanisms have been proposed on how peptides can cross the brush border of the small intestines and be found in systemic circulation including endocytosis and phagocytosis, paracellular transport, direct penetration of the epithelial cells, and active transport by carrier proteins (Lundquist & Artursson, 2016). After absorption, these peptides are still subject to proteolytic action of the enzymes found in the bloodstream which can negatively affect bioactivity. This then raises a question on the real bioavailability of bioactive peptides. Currently, studies on the bioavailability of plant-derived biologically active peptides and proteins are very scarce. However, some peptides can have local biological effects that do not require absorption. As such, future studies should focus on the design of peptides that are protected from the proteolytic environment found in the body or peptides that can be engineered to specifically target organs and tissues in the body. This can be done with different nanotechnological advancements and labeling techniques. Moreover, focus on determining bioavailability is of utmost importance. Other potential issues and challenges that face plant-derived peptides and proteins use in promoting overall human health are the allergenicity of these molecules as well as the regulatory environment that differs from one country to another. Wheat, soybean, peanuts, and tree nuts (and possibly other legumes) are known to contain allergenic proteins that can trigger the immune system in some individuals, which can be fatal. The effect of commonly used food processing techniques on the allergenicity as well as the production of bioactive peptides is currently being investigated and a vast number of literatures show that daily food processing methods can alter allergenicity depending on the type of food, allergenic components/contents, and processing conditions (Cabanillas & Novak, 2019). The functional food designation and claims regarding novel foods, nutraceuticals, natural, and

386 Chapter 16 supplements are regulatory issues that must be addressed prior to commercialization of plant-derived peptides. The question of whether these compounds or mixtures can be considered as drugs, foods, pharmaceutical, or supplements also poses another layer of regulatory requirement that must be met prior to market introduction. These issues, challenges, and opportunities must then be supported by data obtained by high-quality research in the future to move forward the utilization of plant-derived proteins and peptides for the promotion of overall health.

16.6 Conclusion Plants provide a vast number of different types of proteins that are potential sources of biologically active molecules. These molecules can be naturally present in plants or embedded in the structure of the plant proteins. Bioactive peptides embedded in this structure can be released during food processing, enzymatic hydrolysis, and microbial fermentation of plant tissues thereby increasing the biological benefits of plant proteins. The important role of plant proteins in human malignancies is highlighted by studies supporting the biological activities of plant-derived peptides in the prevention and management of inflammatory-related diseases, cancer, and metabolic syndrome. The mechanism by which these biological benefits are achieved is largely unknown but recent studies gave us a glimpse of how we observed these effects. Challenges on production, stability, allergenicity, and regulatory issues give an important opportunity for future research to advance the commercialization of plant-derived bioactive peptides and proteins.

Acknowledgements The author would like to acknowledge the support from The University of Tennessee Institute of Agriculture Start-up Package and United States Department of Agriculture HATCH Project 1010230.

References Agrawal, H., Joshi, R., & Gupta, M. (2019). Purification, identification and characterization of two novel antioxidant peptides from finger millet (Eleusine coracana) protein hydrolysate. Food Research International, 120, 697
707. Allaoui, A., Gasco´n, S., Benomar, S., Quero, J., Osada, J., Nasri, M., . . . Boualga, A. (2019). Protein hydrolysates from fenugreek (Trigonella foenum graecum) as nutraceutical molecules in colon cancer treatment. Nutrients, 11(4), E724. Amadou, I., Gbadamosi, O. S., Shi, Y., Kamara, M. T., & Jin, S. (2010). Identification of antioxidative peptides from Lactobacillus plantarum Lp6 fermented soybean protein meal. Research Journal of Microbiology, 5, 372
380. Aphalo, P., Castellani, O. F., Martinez, E. N., & An˜o´n, M. C. (2004). Surface physicochemical properties of globulin-P amaranth protein. Journal of Agricultural and Food Chemistry, 52(3), 616
622.

Plant sources of bioactive peptides 387 Arau´jo, N. M. S., Dias, L. P., Costa, H. P. S., Sousa, D. O. B., Vasconcelos, I. M., de Morais, G. A., & Oliveira, J. T. A. (2019). ClTI, a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds, exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis. Biochimica et Biophysica Acta - Biomembranes, 1861(11), 183032. Armstrong, W. B., Kennedy, A. R., Wan, X. S., Atiba, J., McLaren, C. E., & Meyskens, F. L., Jr. (2000). Single-dose administration of Bowman-Birk inhibitor concentrate in patients with oral leukoplakia. Cancer Epidemiology, Biomarkers & Prevention, 9(1), 43
47. Armstrong, W. B., Kennedy, A. R., Wan, X. S., Taylor, T. H., Nguyen, Q. A., Jensen, J., . . . Meyskens, F. L., Jr. (2000). Clinical modulation of oral leukoplakia and protease activity by Bowman-Birk inhibitor concentrate in a phase IIa chemoprevention trial. Clinical Cancer Research, 6(12), 4684
4691. Armstrong, W. B., Taylor, T. H., Kennedy, A. R., Melrose, R. J., Messadi, D. V., Gu, M., . . . Meyskens, F. L., Jr. (2013). Bowman birk inhibitor concentrate and oral leukoplakia: A randomized phase IIb trial. Cancer Prevention Research (Philadelphia, PA), 6(5), 410
418. Assaran Darban, R., Shareghi, B., Asoodeh, A., & Chamani, J. (2017). Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin. Journal of Biomolecular Structure and Dynamics, 35 (16), 3648
3662. Ayala-Nin˜o, A., Rodrı´guez-Serrano, G. M., Jime´nez-Alvarado, R., Bautista-Avila, M., Sa´nchez-Franco, J. A., Gonza´lez-Olivares, L. G., & Cepeda-Saez, A. (2019). Bioactivity of peptides released during lactic fermentation of amaranth proteins with potential cardiovascular protective effect: An in vitro study. Journal of Medicinal Food, 22(10), 976
981. Ayyash, M., Johnson, S. K., Liu, S. Q., Al-Mheiri, A., & Abushelaibi, A. (2018). Cytotoxicity, antihypertensive, antidiabetic and antioxidant activities of solid-state fermented lupi, quinoa and wheat by Bifidobacterium species: In vitro investigations. LWT - Food Science and Technology, 95, 295
302. Bae, I. Y., Lee, H. G., Yang, C. B., Ma, M., Bae, I., Lee, H., & Yang, C. (2006). Purification and identification of angiotensin I-converting enzyme inhibitory peptide from buckwheat (Fagopyrum esculentum Moench). Food Chemistry, 96, 36
42. Bar-El Dadon, S., Pascual, C. Y., Eshel, D., Teper-Bamnolker, P., Iba´n˜ez, M. D., & Reifen, R. (2013). Vicilin and the basic subunit of legumin are putative chickpea allergens. Food Chemistry, 138(1), 13
18. Barry, K. H., Jones, R. R., Cantor, K. P., Freeman, L. E. B., Wheeler, D. C., Baris, D., . . . Ward, M. H. (2019). Ingested nitrate and nitrite and bladder cancer in Northern New England. Epidemiology. Bautista-Expo´sito, S., Pen˜as, E., Duen˜as, M., Silva´n, J. M., Frias, J., & Martı´nez-Villaluenga, C. (2018). Individual contributions of Savinase and Lactobacillus plantarum to lentil functionalization during alkaline pH-controlled fermentation. Food Chemistry, 257, 341
349. Belton, P. S., Delgadillo, I., Halford, N. G., & Shewry, P. R. (2006). Kafirin structure and functionality. Journal of Cereal Science, 44, 272
286. Benjakul, S., & Patil, U. (2019). Characteristics of albumin and globulin from coconut meat and their role in emulsion stability without and with proteolysis. Food Hydrocolloids, 69, 220
228. Bester, J., Neitz, W., Gaspar, A. R. M., Ibrahim, M. A., Bester, M. J., Neitz, A. W., & Gaspar, A. R. M. (2019). Tuber storage proteins as potential precursors of bioactive peptides: An in silico analysis. International Journal of Peptide Research and Therapeutics, 25(2), 437
446. Blankestijn, M. A., den Hartog Jager, C. F., Blom, W. M., Otten, H. G., de Jong, G. A. H., Gaspari, M., . . . Verhoeckx, K. C. M. (2018). A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4. Clinical & Experimental Allergy, 48(9), 1206
1213. Bleakley, S., Hayes, M., O’Shea, N., Gallagher, E., & Lafarga, T. (2017). Predicted release and analysis of novel ACE-I, renin, and DPP-IV inhibitory peptides from common oat (Avena sativa) protein hydrolysates using in silico analysis. Foods, 6(12), E108. Boachie, R. T., Okoro, F. L., Imai, K., Sun, L., Elom, S. O., Nwankwo, J. O., . . . Udenigwe, C. C. (2019). Enzymatic release of dipeptidyl peptidase-4 inhibitors (gliptins) from pigeon pea (Cajanus cajan) nutrient reservoir proteins: In silico and in vitro assessments. Journal of Food Biochemistry, e13071.

388 Chapter 16 Budhathoki, S., Sawada, N., Iwasaki, M., Yamaji, T., Goto, A., Kotemori, A., . . . Tsugane, S.Japan Public Health Center
based Prospective Study Group. (2019). Association of animal and plant protein intake with all-cause and cause-specific mortality in a Japanese cohort. JAMA Internal Medicine, 179(11), 1509
1518. Burrieza, H. P., Rizzo, A. J., Moura Vale, E., Silveira, V., & Maldonado, S. (2019). Shotgun proteomic analysis of quinoa seeds reveals novel lysine-rich seed storage globulins. Food Chemistry, 293, 299
306. Cabanillas, B., & Novak, N. (2019). Effects of daily food processing on allergenicity. Critical Reviews in Food Science and Nutrition, 59(1), 31
42. Cam, A., Sivaguru, M., & Gonzalez de Mejia, E. (2013). Endocytic mechanism of internalization of dietary peptide lunasin into macrophages in inflammatory condition associated with cardiovascular disease. PLoS One, 8(9), e72115. Carrillo, W., Go´mez-Ruiz, J. A., Ruiz, A. L., & Carvalho, J. E. (2017). Antiproliferative activity of walnut (Juglans regia L.) proteins and walnut protein hydrolysates. Journal of Medicinal Food, 20(11), 1063
1067. Carvalho, F. M. C., Lima, V. C. O., Costa, I. S., Luz, A. B. S., Ladd, F. V. L., Serquiz, A. C., . . . Morais, A. H. A. (2019). Anti-TNF-α agent tamarind Kunitz trypsin inhibitor improves lipid profile of Wistar rats presenting dyslipidemia and diet-induced obesity regardless of PPAR-γ induction. Nutrients, 11(3), E512. Chalvon-Demersay, T., Azzout-Marniche, D., Arfsten, J., Egli, L., Gaudichon, C., Karagounis, L. G., & Tome´, D. (2017). A systematic review of the effects of plant compared with animal protein sources on features of metabolic syndrome. Journal of Nutrition, 147(3), 281
292. Chang, H. C., Lewis, D., Tung, C. Y., Han, L., Henriquez, S. M., Voiles, L., . . . Robertson, M. J. (2014). Soypeptide lunasin in cytokine immunotherapy for lymphoma. Cancer Immunology, Immunotherapy, 63(3), 283
295. Chen, W., Kinsler, V. A., Macmillan, D., & Di, W. L. (2016). Tissue kallikrein inhibitors based on the sunflower trypsin inhibitor scaffold - A potential therapeutic intervention for skin diseases. PLoS One, 11 (11), e0166268. Cho, S. Y., Rhee, C., & Wiss, L. (2004). Mechanical properties and water vapor permeability of edible films made from fractionated soy proteins with ultrafiltration. U-Technology, 37, 833
839. Choi, S. Y., Sohn, J. H., Lee, Y. W., Lee, E. K., Hong, C. S., & Park, J. W. (2007). Application of the 16-kDa buckwheat 2S storage albumin protein for diagnosis of clinical reactivity. Annals of Allergy, Asthma & Immunology, 99(3), 254
260. Ciacci, C., Russo, I., Bucci, C., Iovino, P., Pellegrini, L., Giangrieco, I., . . . Ciardiello, M. A. (2014). The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models. Clinical and Experimental Immunology, 175(3), 476
484. Ciardiello, M. A., Meleleo, D., Saviano, G., Crescenzo, R., Carratore, V., Camardella, L., . . . Picone, D. (2008). Tamburrini M. Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features. Journal of Peptide Science, 14(6), 742
754. Clemente, A., & Arques Mdel, C. (2014). Bowman Birk inhibitors from legumes as colorectal chemopreventive agents. World Journal of Gastroenterology, 20, 10305
10315. Coda, R., Rizzello, C. G., Pinto, D., & Gobbetti, M. (2012). Selected lactic acid bacteria synthesize antioxidant peptides during sourdough fermentation of cereal flours. Applied and Environmental Microbiology, 78, 1087
1096. Colosimo, R., Gabriele, M., Cifelli, M., Longo, V., Domenici, V., & Pucci, L. (2020). The effect of sourdough fermentation on Triticum dicoccum from Garfagnana: 1H NMR characterization and analysis of the antioxidant activity. Food Chemistry, 305, 125510. Connolly, A., O’Keefe, M. B., Nongonierma, A. B., Piggott, C. D., & Fitzgerald, R. J. (2017). Isolation of peptides from a novel brewers spent grain protein isolate with potential to modulate glycaemic response. International Journal of Food Science and Technology, 52, 146
153. Cotabarren, J., Rosso, A. M., Tellechea, M., Garcı´a-Pardo, J., Rivera, J. L., Obrego´n, W. D., & Parisi, M. G. (2019). Adding value to the chia (Salvia hispanica L.) expeller: Production of bioactive peptides with antioxidant properties by enzymatic hydrolysis with Papain. Food Chemistry, 274, 848
856. Cremer, J. E., Bean, S. R., Tilley, M. M., Ioerger, B. P., Ohm, J. B., Kaufman, R. C., . . . Godwin, I. D. (2014). Grain sorghum proteomics: Integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants. Journal of Agricultural and Food Chemistry, 62(40), 9819
9831.

Plant sources of bioactive peptides 389 Cruz-Huerta, E., Ferna´ndez-Tome´, S., Arques, M. C., Amigo, L., Recio, I., Clemente, A., & Herna´ndezLedesma, B. (2015). The protective role of the Bowman-Birk protease inhibitor in soybean lunasin digestion: The effect of released peptides on colon cancer growth. Food & Function, 6(8), 2626
2635. Czubinski, J., & Feder, S. (2019). Lupin seeds storage protein composition and their interactions with native flavonoids. Journal of the Science of Food and Agriculture, 99(8), 4011
4018. ´ lvares, A., Schwartz, E. F., Amaral, N. O., Trindade, N. R., Pedrino, G. R., Silva, L. P., & da Cunha Morales A de Freitas, S. M. (2014). Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides. Molecules, 19(11), 17536
17558. Dang, Y., Zhou, T., Hao, L., Cao, J., Sun, Y., & Pan, D. (2019). In vitro and in vivo studies on the angiotensinconverting enzyme inhibitory activity peptides isolated from broccoli protein hydrolysate. Journal of Agricultural and Food Chemistry, 67(24), 6757
6764. Dantzger, M., Vasconcelos, I. M., Scorsato, V., Aparicio, R., Marangoni, S., & Macedo, M. L. (2015). Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential. Phytochemistry, 118, 224
235. De Pasquale, I., Pontonio, E., Gobbetti, M., & Rizzello, C. G. (2019). Nutritional and functional effects of the lactic acid bacteria fermentation on gelatinized legume flours. International Journal of Food Microbiology, 316, 108426. de Souza Rocha, T., Hernandez, L. M. R., Chang, Y. K., & de Mejı´a, E. G. (2014). Impact of germination and enzymatic hydrolysis of cowpea bean (Vigna unguiculata) on the generation of peptides capable of inhibiting dipeptidyl peptidase IV. Food Research International, 64, 799
809. Defaix, C., Aymes, A., Albe Slabi, S., Basselin, M., Mathe´, C., Galet, O., & Kapel, R. (2019). A new sizeexclusion chromatography method for fast rapeseed albumin and globulin quantification. Food Chemistry, 287, 151
159. Dei Piu’, L., Tassoni, A., Serrazanetti, D. I., Ferri, M., Babini, E., Tagliazucchi, D., & Gianotti, A. (2014). Exploitation of starch industry liquid by-product to produce bioactive peptides from rice hydrolyzed proteins. Food Chemistry, 155, 199
206. Dia, V. P., & Gonzalez de Mejia, E. (2011a). Lunasin induces apoptosis and modifies the expression of genes associated with extracellular matrix and cell adhesion in human metastatic colon cancer cells. Molecular Nutrition & Food Research, 55(4), 623
634. Dia, V. P., & Gonzalez de Mejia, E. (2011b). Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α5β1 integrin and suppresses FAK/ERK/NF-κB signaling. Cancer Letter, 313(2), 167
180. Dia, V. P., & Krishnan, H. B. (2016). BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells. Scientific Reports, 6, 33532. Dia, V. P., & Mejia, E. G. (2010). Lunasin promotes apoptosis in human colon cancer cells by mitochondrial pathway activation and induction of nuclear clusterin expression. Cancer Letter, 295(1), 44
53. Dias, L. P., Oliveira, J. T. A., RochaBezerra, L. C. B., Sousa, D. O. B., Costa, H. P. S., Araujo, N. M. S., . . . Vasconcelos, I. M. (2017). A trypsin inhibitor purified from Cassia leiandra seeds has insecticidal activity against Aedes aegypti. Process Biochemistry, 57, 228
238. Downs, M. L., Semic-Jusufagic, A., Simpson, A., Bartra, J., Fernandez-Rivas, M., Rigby, N. M., . . . Mills, E. N. (2014). Characterization of low molecular weight allergens from English walnut (Juglans regia). Journal of Agricultural and Food Chemistry, 62(48), 11767
11775. Drori, A., Rotnemer-Golinkin, D., Zolotarov, L., & Ilan, Y. (2017). Oral administration of CardioAid and lunasin alleviates liver damage in a high-fat diet nonalcoholic steatohepatitis model. Digestion, 96(2), 110
118. Dumeus, S., Shibu, M. A., Lin, W. T., Wang, M. F., Lai, C. H., Shen, C. Y., . . . Huang, C. Y. (2018). Bioactive peptide improves diet-induced hepatic fat deposition and hepatocyte proinflammatory response in SAMP8 ageing mice. Cellular Physiology and Biochemistry, 48(5), 1942
1952. Duprez, D., & Toleuova, A. (2013). Prehypertension and the cardiometabolic syndrome: Pathological and clinical consequences. Expert Review of Cardiovascular Therapy, 11, 1725
1733. Emani, C., & Hall, T. C. (2008). Phaseolin: Structure and evolution. The Open Evolution Journal, 2, 66
74.

390 Chapter 16 Esfandi, R., Willmore, W. G., & Tsopmo, A. (2019). Antioxidant and anti-apoptotic properties of oat bran protein hydrolysates in stressed hepatic cells. Foods, 8(5), E160. Esmaeili, M. A., Abagheri-Mahabadi, N., Hashempour, H., Farhadpour, M., Gruber, C. W., & Ghassempour, A. (2016). Viola plant cyclotide vigno 5 induces mitochondria-mediated apoptosis via cytochrome C release and caspases activation in cervical cancer cells. Fitoterapia, 109, 162
168. Fang, E. F., Wong, J. H., & Ng, T. B. (2010). Thermostable Kunitz trypsin inhibitor with cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory activities from Korean large black soybeans. Journal of Bioscience and Bioengineering, 109(3), 211
217. Fang, Y., Chen, X., Luo, P., Pei, F., Kimatu, B. M., Liu, K., . . . Hu, Q. (2017). The correlation between in vitro antioxidant activity and immunomodulatory activity of enzymatic hydrolysates from selenium-enriched rice protein. Journal of Food Science, 82(2), 517
522. Ferro, A., Rosato, V., Rota, M., Costa, A. R., Morais, S., Pelucchi, C., . . . Lunet, N. (2019). Meat intake and risk of gastric cancer in the Stomach cancer Pooling (StoP) project. International Journal of Cancer. Fittler, H., Avrutina, O., Empting, M., & Kolmar, H. (2014). Potent inhibitors of human matriptase-1 based on the scaffold of sunflower trypsin inhibitor. Journal of Peptide Science, 20(6), 415
420. Franke, B., Colgrave, M. L., Mylne, J. S., & Rosengren, K. J. (2016). Mature forms of the major seed storage albumins in sunflower: A mass spectrometric approach. Journal of Proteomics, 16(147), 177
186. Fu, L. S., Ko, Y. H., Lin, K. W., Hsu, J. Y., Chu, J. J., & Chi, C. S. (2009). Dioscorin protects tight junction protein expression in A549 human airway epithelium cells from dust mite damage. Journal of Microbiology, Immunology and Infection, 42(6), 457
463. Gacek, K., Bartkowiak-Broda, I., & Batley, J. (2018). Genetic and molecular regulation of seed storage proteins (SSPs) to improve protein nutritional value of oilseed rape (Brassica napus L.) seeds. Frontiers in Plant Science, 9, 890. Gadge, P. P., Wagh, S. K., Shaikh, F. K., Tak, R. D., Padul, M. V., & Kachole, M. S. (2015). A bifunctional α-amylase/trypsin inhibitor from pigeonpea seeds: Purification, biochemical characterization and its bioefficacy against Helicoverpa armigera. Pesticide Biochemistry and Physiology, 125, 17
25. Galli, V., Mazzoli, L., Luti, S., Venturi, M., Guerrini, S., Paoli, P., . . . Pazzagli, L. (2018). Effect of selected strains of lactobacilli on the antioxidant and anti-inflammatory properties of sourdough. International Journal of Food Microbiology, 286, 55
65. Galvez, A. F., Chen, N., Macasieb, J., & de Lumen, B. O. (2001). Chemopreventive property of a soybean peptide (lunasin) that binds to deacetylated histones and inhibits acetylation. Cancer Research, 61(20), 7473
7478. Galvez, A. F., & de Lumen, B. O. (1999). A soybean cDNA encoding a chromatin-binding peptide inhibits mitosis of mammalian cells. Nature Biotechnology, 17(5), 495
500. Gangopadhyay, N., Wynne, K., O’Connor, P., Gallagher, E., Brunton, N. P., Rai, D. K., & Hayes, M. (2016). In silico and in vitro analyses of the angiotensin-I converting enzyme inhibitory activity of hydrolysates generated from crude barley (Hordeum vulgare) protein concentrates. Food Chemistry, 203, 367
374. Gao, D., Cao, Y., & Li, H. (2010). Antioxidant activity of peptide fractions derived from cottonseed protein hydrolysate. Journal of the Science of Food and Agriculture, 90(11), 1855
1860. Garcı´a, M. C., Endermann, J., Gonza´lez-Garcı´a, E., & Marina, M. L. (2015). HPLC-Q-TOF-MS identification of antioxidant and antihypertensive peptides recovered from cherry (Prunus cerasus L.) subproducts. Journal of Agricultural and Food Chemistry, 63(5), 1514
1520. Garcı´a, M. C., Gonza´lez-Garcı´a, E., Va´squez-Villanueva, R., & Marina, M. L. (2016). Apricot and other seed stones: Amygdalin content and the potential to obtain antioxidant, angiotensin I converting enzyme inhibitor and hypocholesterolemic peptides. Food & Function, 7(11), 4693
4701. Garcia, S., Hartkamp, L. M., Malvar-Fernandez, B., van Es, I. E., Lin, H., Wong, J., . . . Reedquist, K. A. (2016). Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis. Arthritis Research & Therapy, 18(1), 75. Garcı´a-Mora, P., Martı´n-Martı´nez, M., Angeles Bonache, M., Gonza´lez-Mu´niz, R., Pen˜as, E., Frias, J., & Martinez-Villaluenga, C. (2017). Identification, functional gastrointestinal stability and molecular docking studies of lentil peptides with dual antioxidant and angiotensin I converting enzyme inhibitory activities. Food Chemistry, 221, 464
472.

Plant sources of bioactive peptides 391 Garg, S., Apostolopoulos, V., Nurgali, K., & Mishra, V. (2018). Evaluation of in silico approach for prediction of presence of opioid peptides in wheat. Journal of Functional Foods, 41, 34
40. Gerlach, S. L., Chandra, P. K., Roy, U., Gunasekera, S., Go¨ransson, U., Wimley, W. C., . . . Mondal, D. (2019). The membrane-ctive phytopeptide cycloviolacin O2 simultaneously targets HIV-1-infected cells and infectious viral particles to potentiate the efficacy of antiretroviral drugs. Medicines (Basel), 6(1), E33. Golly, M. K., Ma, H., Yuqing, D., Wu, P., Dabbour, M., Sarpong, F., & Farooq, M. (2019). Enzymolysis of walnut (Juglans regia L.) meal protein: Ultrasonication-assisted alkaline pretreatment impact on kinetics and thermodynamics. Journal of Food Biochemistry, 43(8), e12948. Gonza´lez-Montoya, M., Herna´ndez-Ledesma, B., Mora-Escobedo, R., & Martı´nez-Villaluenga, C. (2018). Bioactive peptides from germinated soybean with anti-diabetic potential by inhibition of dipeptidyl peptidase-IV, α-amylase, and α-glucosidase enzymes. International Journal of Molecular Science, 19(10), 2883. Gonza´lez-Pe´rez, S., van Konigsveld, G. A., Vereijken, J. M., Merck, K. B., Gruppen, H., & Voragen, A. G. (2005). Emulsion properties of sunflower (Helianthus annuus) proteins. Journal of Agricultural and Food Chemistry, 53(6), 2261
2267. Gonzalez-Perez, S., & Vereijken, J. M. (2007). Sunflower proteins: Overview of their physicochemical, structural and functional properties. Journal of the Science of Food and Agriculture, 87, 2173
2191. Grasso, A. C., Hung, Y., Olthof, M. R., Verbeke, W., & Brouwer, I. A. (2019). Older consumers’ readiness to accept alternative, more sustainable protein sources in the European Union. Nutrients, 11(8), pii: E1904. Gray, K., Elghadban, S., Thongyoo, P., Owen, K. A., Szabo, R., Bugge, T. H., . . . Ellis, V. (2014). Potent and specific inhibition of the biological activity of the type-II transmembrane serine protease matriptase by the cyclic microprotein MCoTI-II. Thrombosis and Haemostasis, 112(2), 402
411. Gregory, J. W. (2019). Prevention of obesity and metabolic syndrome in children. Frontiers in Endocrinology (Lausanne), 10, 669. Gu, L., Gong, Y., Zhao, C., Wang, Y., Tian, Q., Lei, G., . . . Tan, S. (2019). Lunasin improves the LDL-C lowering efficacy of simvastatin via inhibiting PCSK9 expression in hepatocytes and ApoE-/- mice. Molecules., 24(22), E4140. Gu, L., Wang, Y., Xu, Y., Tian, Q., Lei, G., Zhao, C., . . . Tan, S. (2017). Lunasin functionally enhances LDL uptake via inhibiting PCSK9 and enhancing LDLR expression in vitro and in vivo. Oncotarget, 8(46), 80826
80840. Gupta, N., Srivastava, N., & Bhagyawant, S. S. (2018). Vicilin-A major storage protein of mungbean exhibits antioxidative potential, antiproliferative effects and ACE inhibitory activity. PLoS One, 13(2), e0191265. Haddad Tabrizi, S., Haddad, E., Rajaram, S., Oda, K., Kaur, A., & Sabate´, J. (2019). The effect of soybean lunasin on cardiometabolic risk factors: A randomized clinical trial. Journal of Dietary Supplements, 7, 1
14. Han, C. H., Liu, J. C., Fang, S. U., & Hou, W. C. (2013). Antioxidant activities of the synthesized thiolcontained peptides derived from computer-aided pepsin hydrolysis of yam tuber storage protein, dioscorin. Food Chemistry, 138(2
3), 923
930. Han, R., Maycock, J., Murray, B. S., & Boesch, C. (2019). Identification of angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory peptides derived from oilseed proteins using two integrated bioinformatic approaches. Food Research International, 115, 283
291. Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: The next generation. Cell, 144(5), 646
674. Hao, Y., Fan, X., Guo, H., Yao, Y., Ren, G., Lv, X., & Yang, X. (2019). Overexpression of the bioactive lunasin peptide in soybean and evaluation of its anti-inflammatory and anti-cancer activities in vitro. Journal of Bioscience and Bioengineering, S1389
1723(19), 30486-4. Hashidume, T., Sakano, T., Mochizuki, A., Ito, K., Ito, S., Kawarasaki, Y., & Miyoshi, N. (2018). Identification of soybean peptide leginsulin variants in different cultivars and their insulin-like activities. Scientific Reports, 8(1), 16847. He, R., Yang, Y. J., Wang, Z., Xing, C. R., Yuan, J., Wang, L. F., . . . Ju, X. R. (2019). Rapeseed proteinderived peptides, LY, RALP, and GHS, modulates key enzymes and intermediate products of reninangiotensin system pathway in spontaneously hypertensive rat. npj Science of Food, 3, 1.

392 Chapter 16 Herna´ndez-Ledesma, B., Hsieh, C. C., & de Lumen, B. O. (2011). Relationship between lunasin’s sequence and its inhibitory activity of histones H3 and H4 acetylation. Molecular Nutrition & Food Research, 55(7), 989
998. Ho, T. Y., Li, C. C., Lo, H. Y., Chen, F. Y., & Hsiang, C. Y. (2017). Corn silk extract and its bioactive peptide ameliorated lipopolysaccharide-induced inflammation in mice via the nuclear factor-κB signaling pathway. Journal of Agricultural and Food Chemistry, 65(4), 759
768. Hsieh, C. C., Chou, M. J., & Wang, C. H. (2017). Lunasin attenuates obesity-related inflammation in RAW264.7 cells and 3T3-L1 adipocytes by inhibiting inflammatory cytokine production. PLoS One, 12(2), e0171969. Hsieh, C. C., Herna´ndez-Ledesma, B., Jeong, H. J., Park, J. H., & de Lumen, B. O. (2010). Complementary roles in cancer prevention: Protease inhibitor makes the cancer preventive peptide lunasin bioavailable. PLoS One, 5(1), e8890. Hsieh, C. C., Wang, C. H., & Huang, Y. S. (2016). Lunasin attenuates obesity-associated metastasis of 4T1 breast cancer cell through anti-Inflammatory property. International Journal of Molecular Sciences, 17(12), E2109. Hsu, Y. J., Weng, C. F., Lin, K. W., & Lin, K. C. (2013). Suppression of allergic reactions in ovalbuminsensitized mice by yam storage proteins dioscorins. Journal of Agricultural and Food Chemistry, 61(47), 11460
11467. Hu, E., Wang, D., Chen, J., & Tao, X. (2015). Novel cyclotides from Hedyotis diffusa induce apoptosis and inhibit proliferation and migration of prostate cancer cells. International Journal of Clinical and Experimental Medicine, 8(3), 4059
4065. Hu, Y., Stromeck, A., Loponen, J., Lopes-Lutz, D., Schieber, A., & Ga¨nzle, M. G. (2011). LC-MS/MS quantification of bioactive angiotensin I-converting enzyme inhibitory peptides in rye malt sourdoughs. Journal of Agricultural and Food Chemistry, 59(22), 11983
11989. Inaba, J., McConnell, E. J., & Davis, K. R. (2014). Lunasin sensitivity in non-small cell lung cancer cells is linked to suppression of integrin signaling and changes in histone acetylation. International Journal of Molecular Sciences, 15(12), 23705
23724. Ishihara, K., Oyaizu, S., Fukuchi, Y., Mizunoya, W., Segawa, K., Takahashi, M., . . . Yasumoto, K. (2003). A soybean peptide isolate diet promotes postprandial carbohydrate oxidation and energy expenditure in type II diabetic mice. Journal of Nutrition, 133(3), 752
757. Jakubczyk, A., Kara´s, M., Złotek, U., & Szymanowska, U. (2017). Identification of potential inhibitory peptides of enzymes involved in the metabolic syndrome obtained by simulated gastrointestinal digestion of fermented bean (Phaseolus vulgaris L.) seeds. Food Research International, 100(Pt 1), 489
496. Jeong, H. J., Jeong, J. B., Kim, D. S., & de Lumen, B. O. (2007). Inhibition of core histone acetylation by the cancer preventive peptide lunasin. Journal of Agricultural and Food Chemistry, 55(3), 632
637. Jheng, Y. J., Tsai, W. Y., Chen, K. H., Lin, K. W., Chyan, C. L., Yang, C. C., & Lin, K. C. (2012). Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities. Protein Expression and Purification, 85(1), 77
85. Jiang, Q., Pan, Y., Cheng, Y., Li, H., Liu, D., & Li, H. (2016). Lunasin suppresses the migration and invasion of breast cancer cells by inhibiting matrix metalloproteinase-2/-9 via the FAK/Akt/ERK and NF-κB signaling pathways. Oncology Reports, 36(1), 253
262. Jiao, Y., Zheng, X., Chang, Y., Li, D., Sun, X., & Liu, X. (2018). Zein-derived peptides as nanocarriers to increase the water solubility and stability of lutein. Food & Function, 9(1), 117
123. Jimsheena, V. K., & Gowda, L. R. (2010). Arachin derived peptides as selective angiotensin I-converting enzyme (ACE) inhibitors: Structure-activity relationship. Peptides, 31(6), 1165
1176. Jing, X., Yang, C., & Zhang, L. (2016). Characterization and analysis of protein structures in oat bran. Journal of Food Science, 81(10), C2337
C2343. Jones, L. D., Pangloli, P., Krishnan, H. B., & Dia, V. P. (2018). BG-4, a novel bioactive peptide from Momordica charantia, inhibits lipopolysaccharide-induced inflammation in THP-1 human macrophages. Phytomedicine, 42, 226
232.

Plant sources of bioactive peptides 393 Karami, Z., Peighambardoust, S. H., Hesari, J., Akbari-Adergani, B., & Andreu, D. (2019). Identification and synthesis of multifunctional peptides from wheat germ hydrolysate fractions obtained by proteinase K digestion. Journal of Food Biochemistry, 43(4), e12800. Kara´s, M., Jakubczyk, A., Szymanowska, U., Je˛derka, K., Lewicki, S., & Złotek, U. (2019). Different temperature treatments of millet grains affect the biological activity of protein hydrolyzates and peptide fractions. Nutrients, 11(3), 550. Karas, M., Zlotek, U., Szymanowska, U., Baraniak, B., Jakubczyk, A., Kara´s, M., . . . Bochnak, J. (2019). Peptides obtained from fermented faba bean seeds (Vicia faba) as potential inhibitors of an enzyme involved in the pathogenesis of metabolic syndrome. LWT - Food Science and Technology, 105, 306
313. Kaur, J. (2014). A comprehensive review on metabolic syndrome. Cardiology Research and Practice, 2014, 943162. Kim, H., Rebholz, C. M., Caulfield, L. E., Ramsing, R., & Nachman, K. E. (2018). Trends in types of protein in US adults: Results from the National Health and Nutrition Examination Survey 1999
2010. Public Health Nutrition, 1
11. Kim, J. M., Liceaga, A. M., & Yoon, K. Y. (2019). Purification and identification of an antioxidant peptide from perilla seed (Perilla frutescens) meal protein hydrolysate. Food Science & Nutrition, 7(5), 1645
1655. Kim, W. S., Jang, S., & Krishnan, H. B. (2012). Accumulation of leginsulin, a hormone-like bioactive peptide, is drastically higher in Asian than in North American soybean accessions. Crop Science, 52, 262
271. Kinsella, J. E. (1982). Relationships between structure and functional properties of food proteins. In Codon Fon (Ed.), Food proteins (pp. 51
104). New York: Springer Publishing. Kirsten, D., Peggy, M. T., & Phoebe, Q. (2009). Structure and function of Protein Based edible films and coatings. Edible Films and Coatings for Food Applications, 25
56. Kongo-Dia-Moukala, J. U., Zhang, H., & Irakoze, P. C. (2011). In vitro binding capacity of bile acids by defatted corn protein hydrolysate. International Journal of Molecular Science, 12(2), 1066
1080. Korsinczky, M. L., Clark, R. J., & Craik, D. J. (2005). Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry, 44(4), 1145
1153. Koyama, M., Naramoto, K., Nakajima, T., Aoyama, T., Watanabe, M., & Nakamura, K. (2013). Purification and identification of antihypertensive peptides from fermented buckwheat sprouts. Journal of Agricultural and Food Chemistry, 61(12), 3013
3021. Kubglomsong, S., Theerakulkait, C., Reed, R. L., Yang, L., Maier, C. S., & Stevens, J. F. (2018). Isolation and identification of tyrosinase-inhibitory and copper-chelating peptides from hydrolyzed rice-bran-derived albumin. Journal of Agricultural and Food Chemistry, 66(31), 8346
8354. Kumar, V., Kurup, L. V., & Tiku, P. K. (2020). The modulatory effect if ccholesterol synthesis by Oryza sativa derived bioactive peptide fractions: An in vitro investigation. International Journal of Peptide Research and Therapeutics, Published online 29 May 2020. Kumrungsee, T., Akiyama, S., Guo, J., Tanaka, M., & Matsui, T. (2016). Identification of peptides in wheat germ hydrolysate that demonstrate calmodulin-dependent protein kinase II inhibitory activity. Food Chemistry, 213, 329
335. Lacroix, I. M., & Li-Chan, E. C. (2012). Evaluation of the potential of dietary proteins as precursors of dipeptidyl peptidase (DPP)-IV inhibitors by an in silico approach. Journal of Functional Foods, 4, 403
422. Lammi, C., Bollati, C., Lecca, D., Abbracchio, M. P., & Arnoldi, A. (2019). Lupin peptide T9 (GQEQSHQDEGVIVR) modulates the mutant PCSK9D374Y pathway: In vitro characterization of its dual hypocholesterolemic behavior. Nutrients, 11(7), E1665. Lammi, C., Zanoni, C., & Arnoldi, A. (2015). Three peptides from soy glycinin modulate glucose metabolism in human hepatic HepG2 cells. International Journal of Molecular Sciences, 16(11), 27362
27370. Lammi, C., Zanoni, C., Arnoldi, A., & Vistoli, G. (2016). Peptides derived from soy and lupin protein as dipeptidyl-peptidase IV inhibitors: In vitro biochemical screening and in silico molecular modeling study. Journal of Agricultural and Food Chemistry, 64(51), 9601
9606.

394 Chapter 16 Laparra, J. M., & Haros, C. M. (2019). Plant seed protease inhibitors differentially affect innate immunity in a tumor microenvironment to control hepatocarcinoma. Food & Function, 10(7), 4210
4219. Lapsongphon, N., & Yongsawatdigul, J. (2013). Production and purification of antioxidant peptides from a mungbean meal hydrolysate by Virgibacillus sp. SK37 proteinase. Food Chemistry, 141(2), 992
999. Le˛gowska, A., De˛bowski, D., Lukajtis, R., Wysocka, M., Czaplewski, C., Lesner, A., & Rolka, K. (2010). Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases. Bioorganic & Medicinal Chemistry, 18(23), 8188
8193. Leguizamo´n, N. D. P., Rodrigues, E. M., de Campos, M. L., Nogueira, A. V. B., Viola, K. S., Schneider, V. K., . . . Cirelli, J. A. (2019). In vivo and in vitro anti-inflammatory and pro-osteogenic effects of citrus cystatin CsinCPI-2. Cytokine, 123, 154760. ´ lvarez-Gonza´lez, R. I., Da´vila-Ortiz, G., Leo´n-Espinosa, E. B., Sa´nchez-Chino, X., Gardun˜o-Siciliano, L., A ´ ´ Madrigal-Bujaidar, E., . . . Jimenez-Martınez, C. (2016). Hypocholesterolemic and anticarcinogenic effect of Vicia faba protein hydrolyzates. Nutrition and Cancer, 68(5), 856
864. Lepedda, A. J., Lobina, O., Rocchiccioli, S., Nieddu, G., Ucciferri, N., De Muro, P., . . . Formato, M. (2016). Identification of differentially expressed plasma proteins in atherosclerotic patients with type 2 diabetes. Journal of Diabetes and its Complications, S1056
8727(16), 30044. Lesner, A., Łe˛gowska, A., Wysocka, M., & Rolka, K. (2011). Sunflower trypsin inhibitor 1 as a molecular scaffold for drug discovery. Current Pharmaceutical Design, 17(38), 4308
4317. Leung, R., Venus, C., Zeng, T., & Tsopmo, A. (2018). Structure-function relationships of hydroxyl radical scavenging and chromium-VI reducing cysteine-tripeptides derived from rye secalin. Food Chemistry, 254, 165
169. Li, C. H., Matsui, T., Matsumoto, K., Yamasaki, R., & Kawasaki, T. (2002). Latent production of angiotensin I-converting enzyme inhibitors from buckwheat protein. Journal of Peptide Science, 8 (6), 267
274. Li, J., Armstrong, C. L., & Campbell, W. W. (2016). Effects of dietary protein source and quantity during weight loss on appetite, energy expenditure, and cardio-metabolic responses. Nutrients., 8(2), 63. Li, Y., Zheng, Y., Zhang, Y., Xu, J., & Gao, G. (2018). Antioxidant activity of coconut (Cocos nucifera L.) protein fractions. Molecules, 23(3), E707. Liang, Q., Chalamaiah, M., Ren, X., Ma, H., & Wu, J. (2018). Identification of new anti-inflammatory peptides from zein hydrolysate after simulated gastrointestinal digestion and transport in Caco-2 cells. Journal of Agricultural and Food Chemistry, 66(5), 1114
1120. Lichtenstein, G. R., Deren, J. J., Katz, S., Lewis, J. D., Kennedy, A. R., & Ware, J. H. (2008). Bowman-Birk inhibitor concentrate: A novel therapeutic agent for patients with active ulcerative colitis. Digestive Diseases and Science, 53(1), 175
180. Lin, P. L., Lin, K. W., Weng, C. F., & Lin, K. C. (2009). Yam storage protein dioscorins from Dioscorea alata and Dioscorea japonica exhibit distinct immunomodulatory activities in mice. Journal of Agricultural and Food Chemistry, 57(11), 4606
4613. Lin, Y. S., Han, C. H., Lin, S. Y., & Hou, W. C. (2016). Synthesized peptides from yam dioscorin hydrolysis in silico exhibit dipeptidyl peptidase-IV inhibitory activities and oral glucose tolerance improvements in normal mice. Journal of Agricultural and Food Chemistry, 64(33), 6451
6458. Lin, Y. S., Lu, Y. L., Wang, G. J., Liang, H. J., & Hou, W. C. (2014). Vasorelaxing and antihypertensive activities of synthesized peptides derived from computer-aided simulation of pepsin hydrolysis of yam dioscorin. Botanical Studies, 55(1), 49. Liu, C. F., & Pan, T. M. (2010). Recombinant expression of bioactive peptide lunasin in Escherichia coli. Applied Microbiology and Biotechnology, 88(1), 177
186. Liu, L., Huang, Y., Huang, X., Yang, J., Wu, W., Xu, Y., . . . Huang, D. (2017). Characterization of the dioscorin gene family in Dioscorea alata reveals a role in tuber development and environmental response. International Journal of Molecular Sciences, 18(7), E1579. Liu, M., Bayjanov, J. R., Renckens, B., Nauta, A., & Siezen, R. J. (2010). The proteolytic system of lactic acid bacteria revisited: A genomic comparison. BMC Genomics, 11, 36.

Plant sources of bioactive peptides 395 Liu, Y. W., Han, C. H., Lee, M. H., Hsu, F. L., & Hou, W. C. (2003). Patatin, the tuber storage protein of potato (Solanum tuberosum L.), exhibits antioxidant activity in vitro. Journal of Agricultural and Food Chemistry, 51(15), 4389
4393. Liu, Y. W., Liu, J. C., Huang, C. Y., Wang, C. K., Shang, H. F., & Hou, W. C. (2009). Effects of oral administration of yam tuber storage protein, dioscorin, to BALB/c mice for 21-days on immune responses. Journal of Agricultural and Food Chemistry, 57(19), 9274
9279. Logaruˇsi´c, M., Slivac, I., Radoˇsevi´c, K., Bagovi´c, M., Redovnikovi´c, I. R., & Srˇcek, V. G. (2019). Hempseed protein hydrolysates’ effects on the proliferation and induced oxidative stress in normal and cancer cell lines. Molecular Biology Reports, 46(6), 6079
6085. Lu, J., Zeng, Y., Hou, W., Zhang, S., Li, L., Luo, X., . . . Xiang, M. (2012). The soybean peptide aglycin regulates glucose homeostasis in type 2 diabetic mice via IR/IRS1 pathway. Journal of Nutritional Biochemistry, 23(11), 1449
1457. Lu, Y. L., Chia, C. Y., Liu, Y. W., & Hou, W. C. (2012). Biological activities and applications of dioscorins, the major tuber storage proteins of yam. Journal of Traditional and Complementary Medicine, 2(1), 41
46. Luna Vital, D. A., Gonza´lez de Mejı´a, E., Dia, V. P., & Loarca-Pin˜a, G. (2014). Peptides in common bean fractions inhibit human colorectal cancer cells. Food Chemistry, 157, 347
355. Lundquist, P., & Artursson, P. (2016). Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues. Advanced Drug Delivery Reviews, 106(Pt B), 256
276. Lykhmus, O., Mishra, N., Koval, L., Kalashnyk, O., Gergalova, G., Uspenska, K., . . . Skok, M. (2016). Molecular mechanisms regulating LPS-induced inflammation in the brain. Frontiers in Molecular Neuroscience, 9, 19. Ma, T. C., Guo, L., Zhou, R. H., Wang, X., Liu, J. B., Li, J. L., . . . Ho, W. Z. (2018). Soybean-derived Bowman-Birk inhibitor (BBI) blocks HIV entry into macrophages. Virology, 513, 91
97. Ma, T. C., Zhou, R. H., Wang, X., Li, J. L., Sang, M., Zhou, L., . . . Ho, W. Z. (2016). Soybean-derived Bowman-Birk inhibitor (BBI) inhibits HIV replication in macrophages. Scientific Reports, 6 34752. Machado, R. J., Monteiro, N. K., Migliolo, L., Silva, O. N., Pinto, M. F., Oliveira, A. S., . . . Santos, E. A. (2013). Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds. PLoS One, 8(5), e63571. Magee, P. J., Owusu-Apenten, R., McCann, M. J., Gill, C. I., & Rowland, I. R. (2012). Chickpea (Cicer arietinum) and other plant-derived protease inhibitor concentrates inhibit breast and prostate cancer cell proliferation in vitro. Nutrition and Cancer, 64(5), 741
748. Maleki, S., & Razavi, S. H. (2020). Pulses’ germination and fermentation: Two bioprocessing against hypertension by releasing ACE inhibitory peptides. Critical Reviews in Food Science and Nutrition, 1
18, Jul. Malkowicz, S. B., McKenna, W. G., Vaughn, D. J., Wan, X. S., Propert, K. J., Rockwell, K., . . . Kennedy, A. R. (2001). Effects of Bowman-Birk inhibitor concentrate (BBIC) in patients with benign prostatic hyperplasia. Prostate, 48(1), 16
28. Maqsoudlou, A., Sadeghi Mahoonak, A., Mora, L., Mohebodini, H., Ghorbani, M., & Toldra´, F. (2019). Controlled enzymatic hydrolysis of pollen protein as promising tool for production of potential bioactive peptides. Journal of Food Biochemistry, 43(5), e12819. Marthandam Asokan, S., Hung, T. H., Chiang, W. D., & Lin, W. T. (2018). Lipolysis-stimulating peptide from soybean protects against high fat diet-induced apoptosis in skeletal muscles. Journal of Medicinal Food, 21 (3), 225
232. Marthandam Asokan, S., Wang, T., Su, W. T., & Lin, W. T. (2019). Antidiabetic effects of a short peptide of potato protein hydrolysate in STZ-induced diabetic mice. Nutrients, 11(4), pii: E779. Martinez-Villaluenga, C., Dia, V. P., Berhow, M., Bringe, N. A., & Gonzalez de Mejia, E. (2009). Protein hydrolysates from beta-conglycinin enriched soybean genotypes inhibit lipid accumulation and inflammation in vitro. Molecular Nutrition & Food Research, 53(8), 1007
1018.

396 Chapter 16 Martins, T. F., Vasconcelos, I. M., Silva, R. G. G., Silva, F. D. A., Souza, P. F. N., Varela, A. L. N., . . . Oliveira, J. T. A. (2018). A Bowman-Birk inhibitor from the seeds of Luetzelburgia auriculata inhibits Staphylococcus aureus growth by promoting severe Cell membrane damage. Journal of Natural Products, 81(7), 1497
1507. Mehta, S. S., Arroyave, W. D., Lunn, R. M., Park, Y. M., Boyd, W. A., & Sandler, D. P. (2019). A prospective analysis of red and processed meat consumption and risk of colorectal cancer in women. Cancer Epidemiology, Biomarkers & Prevention. Mendoza, E. M., Adachi, M., Bernardo, A. E., & Utsumi, S. (2001). Mungbean [Vigna radiata (L.) Wilczek] globulins: Purification and characterization. Journal of Agricultural and Food Chemistry, 49(3), 1552
1558. Mercer, K. E., Pulliam, C., Hennings, L., Lai, K., Cleves, M., Jones, E., . . . Ronis, M. (2016). Soy protein isolate protects against ethanol-mediated tumor progression in diethylnitrosamine-treated male mice. Cancer Prevention Research (Philadelphia, PA), 9(6), 466
475. Mercer, K. E., Pulliam, C. F., Hennings, L., Cleves, M. A., Jones, E. E., Drake, R. R., & Ronis, M. J. J. (2018). Diet supplementation with soy protein isolate, but not the isoflavone genistein, protects against alcoholinduced tumor progression in DEN-treated male mice. Advances in Experimental Medicine and Biology, 1032, 115
126. Meshginfar, N., Sadeghi Mahoonak, A., Hosseinian, F., Ghorbani, M., & Tsopmo, A. (2018). Production of antioxidant peptide fractions from a by-product of tomato processing: Mass spectrometry identification of peptides and stability to gastrointestinal digestion. Journal of Food Science and Technology, 55(9), 3498
3507. Mila´n-Noris, A. K., Gutie´rrez-Uribe, J. A., Santacruz, A., Serna-Saldı´var, S. O., & Martı´nez-Villaluenga, C. (2018). Peptides and isoflavones in gastrointestinal digests contribute to the anti-inflammatory potential of cooked or germinated desi and kabuli chickpea (Cicer arietinum L.). Food Chemistry, 268, 66
76. Milla´n-Linares Mdel, C., Yust Mdel, M., Alcaide-Hidalgo, J. M., & Milla´n, F. (2014). Pedroche J. Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway. Food Chemistry, 151, 141
147. Mills, E. N., Jenkins, J., Marigheto, N., Belton, P. S., Gunning, A. P., & Morris, V. J. (2002). Allergens of the cupin superfamily. Biochemical Society Transactions, 30(Pt 6), 925
929. Miyoshi, S., Kaneko, T., Ishikawa, H., Tanaka, H., & Maruyama, S. (1995). Production of bioactive peptides from corn endosperm proteins by some proteases. Annals of the New York Academy of Sciences, 750, 429
431. Moayedi, A., Mora, L., Aristoy, M. C., Safari, M., Hashemi, M., & Toldra´, F. (2018). Peptidomic analysis of antioxidant and ACE-inhibitory peptides obtained from tomato waste proteins fermented using Bacillus subtilis. Food Chemistry, 250, 180
187. Mojica, L., Dia, V. P., & de Mejia, E. G. (2015). Soy proteins. In Z. Uztunol (Ed.), Applied food protein chemistry. West Sussex, England: John Wiley & Sons Ltd. Mojica, L., Luna-Vital, D. A., Gonza´lez., & de Mejı´a, E. (2017). Characterization of peptides from common bean protein isolates and their potential to inhibit markers of type-2 diabetes, hypertension and oxidative stress. Journal of the Science of Food and Agriculture, 97(8), 2401
2410. Montales, M. T., Simmen, R. C., Ferreira, E. S., Neves, V. A., & Simmen, F. A. (2015). Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells. Genes & Nutrition, 10(6), 49. Morita, T., & Kiriyama, S. (1996). A rice protein isolate alters 7,12-dimethylbenz[alpha]anthracene-induced mammary tumor development in female rats. Journal of Nutritional Science and Vitaminology (Tokyo), 42 (4), 325
337. Moronta, J., Smaldini, P. L., Fossati, C. A., Anon, M. C., & Docena, G. H. (2016). The anti-inflammatory SSEDIKE peptide from Amaranth seeds modulates IgE-mediated food allergy. Journal of Functional Foods., 25, 579
587. Mouzo, D., Bernal, J., Lo´pez-Pedrouso, M., Franco, D., & Zapata, C. (2018). Advances in the biology of seed and vegetative storage proteins based on two-dimensional electrophoresis coupled to mass spectrometry. Molecules, 23(10), E2462.

Plant sources of bioactive peptides 397 Mussbacher, M., Salzmann, M., Brostjan, C., Hoesel, B., Schoergenhofer, C., Datler, H., . . . Schmid, J. A. (2019). Cell type-dpecific roles of NF-κB linking inflammation and thrombosis. Frontiers in Immunology, 10, 85. Nakahara, T., Sano, A., Yamaguchi, H., Sugimoto, K., Chikata, H., Kinoshita, E., & Uchida, R. (2010). Antihypertensive effect of peptide-enriched soy sauce-like seasoning and identification of its angiotensin I-converting enzyme inhibitory substances. J. Agric. Food Chemistry, 58, 821
827, Erratum in: J. Agric. Food Chem., 58, 5858 (2010). Nawaz, K. A. A., David, S. M., Murugesh, E., Thandeeswaran, M., Kiran, K. G., Mahendran, R., . . . Angayarkanni, J. (2017). Identification and in silico characterization of a novel peptide inhibitor of angiotensin converting enzyme from pigeon pea (Cajanus cajan). Phytomedicine, 36, 1
7. Nazir, M. A., Mu, T. H., & Zhang, M. (2019). Preparation and identification of angiotensin I-converting enzyme inhibitory peptides from sweet potato protein by enzymatic hydrolysis under high hydrostatic pressure. International Journal of Food Science & Technology, Jul 15. Nieto-Veloza, A., Wang, Z., Zhong, Q., Krishnan, H. B., & Dia, V. P. (2019). BG-4 from bitter gourd (Momordica charantia) differentially affects inflammation in vitro and in vivo. Antioxidants (Basel), 8(6), E175. Nilsson, C., Brostedt, P., Hidman, J., van Odijk, J., Borres, M. P., Sjo¨lander, S., & Englund, H. (2015). Recognition pattern of kiwi seed storage proteins in kiwifruit-allergic children. Pediatric Allergy and Immunology, 26(8), 817
820. Nishi, T., Hara, H., Asano, K., & Tomita, F. (2003). The soybean beta-conglycinin beta 51
63 fragment suppresses appetite by stimulating cholecystokinin release in rats. Journal of Nutrition, 133(8), 2537
2542. Niu, X., He, Z., Li, W., Wang, X., Zhi, W., Liu, F., & Qi, L. (2017). Immunomodulatory activity of the glycoprotein isolated from the Chinese yam (Dioscorea opposita Thunb). Phytotherapy Research, 31(10), 1557
1563. Obaroakpo, J. U., Liu, L., Zhang, S., Lu, J., Pang, X., & Lv, J. (2019). α-Glucosidase and ACE dual inhibitory protein hydrolysates and peptide fractions of sprouted quinoa yoghurt beverages inoculated with Lactobacillus casei. Food Chemistry, 299, 124985. Odani, S., & Ikenaka, T. (1973). Scission of soybean Bowman-Birk proteinase inhibitor into two small fragments having either trypsin or chymotrypsin inhibitory activity. Journal of Biochemistry, 74, 857
860. Ortega, L. M., Moure, M. C., Gonza´lez, E. M., & Alconada, T. M. (2019). Wheat storage proteins: Changes on the glutenins after wheat infection with different isolates of Fusarium graminearum. International Microbiology, 22(2), 289
296. Ortı´z Cruz, R. A., Ca´rdenas Lo´pez, J. L., Gonza´lez Aguilar, G. A., Astiazara´n Garcı´a, H., Gorinstein, S., Canett Romero, R., . . . Sa´nchez, M. (2015). Influence of sorghum kafirin on serum lipid profile and antioxidant activity in hyperlipidemic rats (in vitro and in vivo studies). BioMed Research International, 2015, 164725. Osborne, T. B. (1908). Our present knowledge of plant proteins. Science, 28(718), 417
427. Osuna-Castro, J. A., Rasco´n-Cruz, Q., Napier, J., Fido, R. J., Shewry, P. R., & Paredes-Lo´pez, O. (2000). Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli. Journal of Agricultural and Food Chemistry, 48(11), 5249
5255. Ozias-Akins, P., & Breiteneder, H. (2019). The functional biology of peanut allergens and possible links to their allergenicity. Allergy, 74(5), 888
898. Pabona, J. M., Dave, B., Su, Y., Montales, M. T., de Lumen, B. O., de Mejia, E. G., . . . Simmen, R. C. (2013). The soybean peptide lunasin promotes apoptosis of mammary epithelial cells via induction of tumor suppressor PTEN: Similarities and distinct actions from soy isoflavone genistein. Genes & Nutrition, 8(1), 79
90. Pen˜as, E., Diana, M., Frias, J., Quı´lez, J., & Martı´nez-Villaluenga, C. (2015). A multistrategic approach in the development of sourdough bread targeted towards blood pressure reduction. Plant Foods for Human Nutrition, 70(1), 97
103. Pe´ rez, E. A., Gonza´lez, M. P., Martı´nez-Espinosa, R. M., Vila, M. D. M., & Reig Garcı´a-Galbis, M. (2019). Practical guidance for interventions in adults with metabolic syndrome: Diet and exercise vs. changes in body composition. International Journal of Environmental Research and Public Health, 16(18), pii: E3481.

398 Chapter 16 Pessione, E., & Cirrincione, S. (2016). Bioactive molecules released in food by lactic acid bacteria: Encrypted peptides and biogenic amines. Frontiers in Microbiology, 7, 876. Pihlanto, A., Akkanen, S., & Korhonen, H. J. (2008). ACE-inhibitory and antioxidant properties of potato (Solanum tuberosum). Food Chemistry, 109(1), 104
112. Piotrowicz, I. B. B., Garce´s-Rimo´n, M., Moreno-Ferna´ndez, S., Aleixandre, A., Salas-Mellado, M., & MiguelCastro, M. (2020). Antioxidant, angiotensin-converting enzyme inhibitory properties and blood-pressurelowering effect of rice bran protein hydrolysates. Foods., 9(6), 812. Price, S. J., Pangloli, P., & Dia, V. P. (2017). Pepsin-pancreatin hydrolysis reduced the ability of lunasinenriched material to inhibit activation of the inflammasomes in THP-1 human macrophages. Food & Function, 8(12), 4449
4458. Price, S. J., Pangloli, P., Krishnan, H. B., & Dia, V. P. (2016). Kunitz trypsin inhibitor in addition to BowmanBirk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis. Food Research International, 90, 205
215. Priyanto, A. D., Doerksen, R. J., Chang, C. I., Sung, W. C., Widjanarko, S. B., Kusnadi, J., . . . Hsu, J. L. (2015). Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. Journal of Proteomics, 128, 424
435. Pu, C., & Tang, W. (2017). The antibacterial and antibiofilm efficacies of a liposomal peptide originating from rice bran protein against Listeria monocytogenes. Food & Function, 8(11), 4159
4169. Qian, C., Chen, X., Qi, Y., Zhong, S., Gao, X., Zheng, W., . . . Yao, J. (2017). Sporamin induces apoptosis and inhibits NF-κB activation in human pancreatic cancer cells. Tumour Biology, 39(7), 1010428317706917. Qian, C. J., Qi, Y. X., Chen, X. Y., Zeng, J. P., & Yao, J. (2017). Sporamin suppresses growth of human esophageal squamous cell carcinoma cells by inhibition of NF-κB via an AKT-independent pathway. Molecular Medicine Reports, 16(6), 9620
9626. Quiroga, A., Martı´nez, E. N., Rogniaux, H., Geairon, A., & An˜o´n, M. C. (2010). Amaranth (Amaranthus hypochondriacus) vicilin subunit structure. Journal of Agricultural and Food Chemistry, 58(24), 12957
12963. Rai, A. K., Sanjukta, S., Chourasia, R., Bhat, I., Bhardwaj, P. K., & Sahoo, D. (2017). Production of bioactive hydrolysate using protease, β-glucosidase and α-amylase of Bacillus spp. isolated from kinema. Bioresource Technology, 235, 358
365. Rampal, P. (2018). An analysis of protein consumption in India through plant and animal sources. Food and Nutrition Bulletin, 39(4), 564
580. Redhu, D., Dhanda, S., Singh, J., & Khaket, T. P. (2015). In silico evaluation of potential DPP-III inhibitor precursors from dietary proteins. International Journal of Food Properties, 18(3), 499
507. Rein, D., Ternes, P., Demin, R., Gierke, J., Helgason, T., & Scho¨n, C. (2019). Artificial intelligence identified peptides modulate inflammation in healthy adults. Food & Function, 10(9), 6030
6041. Ren, X., Liang, Q., & Ma, H. (2018). Effects of sweeping frequency ultrasound pretreatment on the hydrolysis of zein: Angiotensin-converting enzyme inhibitory activity and thermodynamics analysis. Journal of Food Science and Technology, 55(10), 4020
4027. Rezig, L., Chibani, F., Chouaibi, M., Dalgalarrondo, M., Hessini, K., Gue´guen, J., & Hamdi, S. (2013). Pumpkin (Cucurbita maxima) seed proteins: Sequential extraction processing and fraction characterization. Journal of Agricultural and Food Chemistry, 61(32), 7715
7721. Ribeiro, M., Seabra, L., Ramos, A., Santos, S., Pinto-Carnide, O., Carvalho, C., & Igrejas, G. (2012). Polymorphism of the storage proteins in Portuguese rye (Secale cereale L.) populations. Hereditas, 149(2), 72
84. Rizzello, C. G., Lorusso, A., Russo, V., Pinto, D., Marzani, B., & Gobbetti, M. (2017). Improving the antioxidant properties of quinoa flour through fermentation with selected autochthonous lactic acid bacteria. International Journal of Food Microbiology, 241, 252
261. Rodrı´guez-Serrano, G. M., Garcı´a-Garibay, M., Cruz-Guerrero, A. E., Go´mez-Ruiz, L., Ayala-Nin˜o, A., Castan˜eda-Ovando, A., & Gonza´lez-Olivares, L. G. (2018). Proteolytic system of Streptococcus thermophilus. Journal of Microbiology and Biotechnology, 28(10), 1581
1588. Rudakova, A. S., Rudakov, S. V., Kakhovskaya, I. A., & Shutov, A. D. (2014). 11S Storage globulin from pumpkin seeds: regularities of proteolysis by papain. Biochemistry (Mosc), 79(8), 820
825.

Plant sources of bioactive peptides 399 Rudolph, S., Lunow, D., Kaiser, S., & Henle, T. (2017). Identification and quantification of ACE-inhibiting peptides in enzymatic hydrolysates of plant proteins. Food Chemistry, 224, 19
25. Rui, X., Wen, D., Li, W., Chen, X., Jiang, M., & Dong, M. (2015). Enrichment of ACE inhibitory peptides in navy bean (Phaseolus vulgaris) using lactic acid bacteria. Food & Function, 6(2), 622
629. Russo, I., Del Giorno, C., Giangrieco, I., Hajji, N., Ciardiello, M. A., Iovino, P., & Ciacci, C. (2019). A Peptide from kiwifruit exerts anti-inflammatory effects in celiac disease mucosa. Journal of the American College of Nutrition, 38(5), 433
440. Sabbione, A. C., Luna-Vital, D., Scilingo, A., An˜o´n, M. C., & Gonza´lez de Mejı´a, E. (2018). Amaranth peptides decreased the activity and expression of cellular tissue factor on LPS activated THP-1 human monocytes. Food & Function, 9(7), 3823
3834. Sadeghalvad, M., Mohammadi-Motlagh, H. R., Karaji, A. G., & Mostafaie, A. (2019). In vivo anti-inflammatory efficacy of the combined Bowman-Birk trypsin inhibitor and genistein isoflavone, two biological compounds from soybean. Journal of Biochemical and Molecular Toxicology, e22406. ´ lvarez-Gonza´lez, Sa´nchez-Chino, X. M., Jime´nez Martı´nez, C., Leo´n-Espinosa, E. B., Gardun˜o-Siciliano, L., A I., Madrigal-Bujaidar, E., . . . Da´vila-Ortiz, G. (2019). Protective effect of chickpea protein hydrolysates on colon carcinogenesis associated with a hypercaloric diet. Journal of the American College of Nutrition, 38 (2), 162
170. Sarkar, A., Kamaruddin, H., Bentley, A., & Wang, S. (2016). Emulsion stabilization by tomato seed protein isolate: Influence of pH, ionic strength and thermal treatment. Food Hydrocolloids, 57, 160
168. Selamassakul, O., Laohakunjit, N., Kerdchoechuen, O., Yang, L., & Maier, C. S. (2018). Isolation and characterisation of antioxidative peptides from bromelain-hydrolysed brown rice protein by proteomic technique. Process Biochemistry, 70, 179
187. Setrerrahmane, S., Zhang, Y., Dai, G., Lv, J., & Tan, S. (2014). Efficient production of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli. Applied Biochemistry and Biotechnology, 174(2), 612
622. Shang, X., Scott, D., Hodge, A., English, D. R., Giles, G. G., Ebeling, P. R., & Sanders, K. M. (2017). Dietary protein from different food sources, incident metabolic syndrome and changes in its components: An 11-year longitudinal study in healthy community-dwelling adults. Clinical Nutrition, 36(6), 1540
1548. Sheng, J., Yang, X., Chen, J., Peng, T., Yin, X., Liu, W., . . . Yang, X. (2019). Antioxidative effects and mechanism study of bioactive peptides from defatted walnut (Juglans regia L.) meal hydrolysate. Journal of Agricultural and Food Chemistry, 67(12), 3305
3312. Shewry, P. R. (2003). Tuber storage proteins. Annals of Botany, 91(7), 755
769. Shi, A., Liu, H., Liu, L., Hu, H., Wang, Q., & Adhikari, B. (2014). Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One, 9(10), e111188. Shi, Z., Hao, Y., Teng, C., Yao, Y., & Ren, G. (2019). Functional properties and adipogenesis inhibitory activity of protein hydrolysates from quinoa (Chenopodium quinoa Willd.). Food Science & Nutrition, 7(6), 2103
2112. Shidal, C., Al-Rayyan, N., Yaddanapudi, K., & Davis, K. R. (2016). Lunasin is a novel therapeutic agent for targeting melanoma cancer stem cells. Oncotarget, 7(51), 84128
84141. Shidal, C., Inaba, J. I., Yaddanapudi, K., & Davis, K. R. (2017). The soy-derived peptide Lunasin inhibits invasive potential of melanoma initiating cells. Oncotarget, 8(15), 25525
25541. Shih, S. L., Lin, Y. S., Lin, S. Y., & Hou, W. C. (2015). Effects of yam dioscorin interventions on improvements of the metabolic syndrome in high-fat diet-induced obese rats. Botanical Studies, 56(1), 4. Shih, Y. H., Chen, F. A., Wang, L. F., & Hsu, J. L. (2019). Discovery and study of novel antihypertensive peptides derived from Cassia obtusifolia seeds. Journal of Agricultural and Food Chemistry, 67(28), 7810
7820. Sozer, N., Melama, L., Silbir, S., Rizzello, C. G., Flander, L., & Poutanen, K. (2019). Lactic acid fermentation as a pre-treatment process for faba bean flour and its effect on textural, structural and nutritional properties of protein-enriched gluten-free faba bean breads. Foods, 8(10), E431.

400 Chapter 16 Su, Y., Eason, R. R., Geng, Y., Till, S. R., Badger, T. M., & Simmen, R. C. (2007). In utero exposure to maternal diets containing soy protein isolate, but not genistein alone, protects young adult rat offspring from NMU-induced mammary tumorigenesis. Carcinogenesis, 28(5), 1046
1051. Sua´rez, S., Aphalo, P., Rinaldi, G., An˜o´n, M. C., & Quiroga, A. (2020). Effect of amaranth proteins on the RAS system. In vitro, in vivo and ex vivo assays. Food Chemistry, 308, 125601. Subirade, M., Kelly, I., Gueguen, J., & Pezolet, M. (1998). Molecular basis of film formation from a soybean protein: Comparison between the conformation of glycinin in aqueous solution and in Films. International Journal of Biological Macromolecules, 23, 241
249. Sufian, K. N., Hira, T., Nakamori, T., Furuta, H., Asano, K., & Hara, H. (2011). Soybean β-conglycinin bromelain hydrolysate stimulates cholecystokinin secretion by enteroendocrine STC-1 cells to suppress the appetite of rats under meal-feeding conditions. Bioscience, Biotechnology, and Biochemistry, 75(5), 848
853. Sullivan, A. C., Pangloli, P., & Dia, V. P. (2018a). Impact of ultrasonication on the physicochemical properties of sorghum kafirin and in vitro pepsin-pancreatin digestibility of sorghum gluten-like flour. Food Chemistry, 240, 1121
1130. Sullivan, A. C., Pangloli, P., & Dia, V. P. (2018b). Kafirin from Sorghum bicolor inhibition of inflammation in THP-1 human macrophages is associated with reduction of intracellular reactive oxygen species. Food and Chemical Toxicology, 111, 503
510. Sun, Y., Jiang, L., & Wei, D. (2013). Partial characterization, in vitro antioxidant and antiproliferative activities of patatin purified from potato fruit juice. Food & Function, 4(10), 1502
1511. Tanner, G., Juha´sz, A., Florides, C. G., Nye-Wood, M., Be´ke´s, F., Colgrave, M. L., . . . Tye-Din, J. A. (2019). Preparation and characterization of avenin-enriched oat protein by chill precipitation for feeding trials in celiac disease. Frontiers in Nutrition, 6, 162. Tanner, G. J., Colgrave, M. L., Blundell, M. J., Howitt, C. A., & Bacic, A. (2019). Hordein accumulation in developing barley grains. Frontiers in Plant Science, 10, 649. Tsou, M. J., Kao, F. J., Lu, H. C., Kao, H. C., & Chiang, W. D. (2013). Purification and identification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein. Food Chemistry, 138 (2
3), 1454
1460. Tung, C. Y., Lewis, D. E., Han, L., Jaja, M., Yao, S., Li, F., . . . Chang, H. C. (2014). Activation of dendritic cell function by soypeptide lunasin as a novel vaccine adjuvant. Vaccine, 32(42), 5411
5419. Udechukwu, M. C., Tsopmo, A., Mawhinney, H., He, R., Kienesberger, P. C., & Udenigwe, C. C. (2017). Inhibition of ADAM17/TACE activity by zinc-chelating rye secalin-derived tripeptides and analogues. RSC Advances, 7(42), 26361
26369. Udenigwe, C. C., Lu, Y. L., Han, Ch, How, W. C., & Aluko, R. E. (2009). Flaxseed protein-derived peptide fractions: antioxidant properties and inhibition of lipopolysaccharide-induced nitric oxide production in murine macrophages. Food Chemistry, 116, 277
284. Uraipong, C., & Zhao, J. (2016). Rice bran protein hydrolysates exhibit strong in vitro α-amylase, β-glucosidase and ACE-inhibition activities. Journal of the Science of Food and Agriculture, 96(4), 1101
1110. Urbizo-Reyes, U., San Martin-Gonzalez, M. F., Garcia-Bravo, J., Vigil, A. L. M., & Liceaga, A. M. (2019). Physicochemical characteristics of chia seed (Salvia hispanica) protein hydrolysates produced using ultrasonication followed by microwave-assisted hydrolysis. Food Hydrocolloids, 97, UNSP 105187. Venuste, M., Zhang, X., Shoemaker, C. F., Karangwa, E., Abbas, S., & Kamdem, P. E. (2013). Influence of enzymatic hydrolysis and enzyme type on the nutritional and antioxidant properties of pumpkin meal hydrolysates. Food & Function, 4(5), 811
820. Verni, M., De Mastro, G., De Cillis, F., Gobbetti, M., & Rizzello, C. G. (2019). Lactic acid bacteria fermentation to exploit the nutritional potential of Mediterranean faba bean local biotypes. Food Research International, 125, 108571. Vilcacundo, R., Martı´nez-Villaluenga, C., Miralles, B., & Herna´ndez-Ledesma, B. (2019). Release of multifunctional peptides from kiwicha (Amaranthus caudatus) protein under in vitro gastrointestinal digestion. Journal of the Science of Food and Agriculture, 99(3), 1225
1232.

Plant sources of bioactive peptides 401 Vuyyuri, S. B., Shidal, C., & Davis, K. R. (2018). Development of the plant-derived peptide lunasin as an anticancer agent. Current Opinion Pharmacology, 41, 27
33. Wang, M., Amakye, W. K., Guo, L., Gong, C., Zhao, Y., Yao, M., & Ren, J. (2019). Walnut-derived peptide PW5 ameliorates cognitive impairments and alters gut microbiota in APP/PS1 transgenic mice. Molecular Nutrition & Food Research, 63(18), e1900326. Wang, X., Smith, P. L., Hsu, M. Y., Ogletree, M. L., & Schumacher, W. A. (2006). Murine model of ferric chloride-induced vena cava thrombosis: Evidence for effect of potato carboxypeptidase inhibitor. Journal of Thrombosis and Haemostasis, 4(2), 403
410. Wang, Z., Cui, Y., Liu, P., Zhao, Y., Wang, L., Liu, Y., & Xie, J. (2017). Small peptides isolated from enzymatic hydrolyzate of fermented soybean meal promote endothelium-independent vasorelaxation and ACE inhibition. Journal of Agricultural and Food Chemistry, 65(50), 10844
10850. Warsame, A. O., O’Sullivan, D. M., & Tosi, P. (2018). Seed storage proteins of faba bean (Vicia faba L): Current status and prospects for genetic improvement. Journal of Agricultural and Food Chemistry, 66(48), 12617
12626. Werle, M., Kafedjiiski, K., Kolmar, H., & Bernkop-Schnu¨rch, A. (2007). Evaluation and improvement of the properties of the novel cystine-knot microprotein McoEeTI for oral administration. International Journal of Pharmaceutics, 332(1
2), 72
79. Wu, G. C., Lin, S. Y., Liang, H. J., & Hou, W. C. (2018). 135-day interventions of yam dioscorin and the dipeptide Asn-Trp (NW) to reduce weight gains and improve impaired glucose tolerances in high-fat dietinduced C57BL/6 mice. Journal of Agricultural and Food Chemistry, 66(3), 645
652. Wu, J. S., Li, J. M., Lo, H. Y., Hsiang, C. Y., & Ho, T. Y. (2020). Anti-hypertensive and angiotensin-converting enzyme inhibitory effects of Radix Astragali and its bioactive peptide AM-1. Journal of Ethnopharmacology, 254, 112724. Wu, Q., Du, J., Jia, J., & Kuang, C. (2016). Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study. Food Chemistry, 199, 140
149. Xia, K., Pittelli, S., Church, J., & Colo´n, W. (2016). Kinetic stability of proteins in beans and peas: Implications for protein digestibility, seed germination, and plant adaptation. Journal of Agricultural and Food Chemistry, 64(40), 7649
7657. Xiao, J., Chen, Y., & Huang, Q. (2017). Physicochemical properties of kafirin protein and its applications as building blocks of functional delivery systems. Food & Function, 8(4), 1402
1413. Xiao, Y., Sun, M., Zhang, Q., Chen, Y., Miao, J., Rui, X., & Dong, M. (2018). Effects of Cordyceps militaris (L.) Fr. fermentation on the nutritional, physicochemical, functional properties and angiotensin I converting enzyme inhibitory activity of red bean (Phaseolus angularis [Willd.] W.F. Wight.) flour. Journal of Food Science and Technology, 55(4), 1244
1255. Xu, J., Zhao, Q., Qu, Y., & Ye, F. (2015). Antioxidant activity and anti-exercise-fatigue effect of highly denatured soybean meal hydrolysate prepared using neutrase. Journal of Food Science and Technology, 52 (4), 1982
1992. Xu, S., Shen, Y., Chen, G., Bean, S., & Li, Y. (2019). Antioxidant characteristics and identification of peptides from sorghum kafirin hydrolysates. Journal of Food Science, 84(8), 2065
2076. Xu, S., Shen, Y., & Li, Y. (2019). Antioxidant activities of sorghum kafirin alcalase hydrolysates and membrane/gel filtrated fractions. Antioxidants (Basel), 8(5), E131. Yang, C., Zhang, J. J., Zhang, X. P., Xiao, R., & Li, P. G. (2019). Sporamin suppresses growth of xenografted colorectal carcinoma in athymic BALB/c mice by inhibiting liver β-catenin and vascular endothelial growth factor expression. World Journal of Gastroenterology, 25(25), 3196
3206. Yang, C. C., & Lin, K. C. (2014). Class A dioscorins of various yam species suppress ovalbumin-induced allergic reactions. Immunopharmacology and Immunotoxicology, 36(3), 242
249. Yao, J., & Qian, C. (2011). Sporamin induce apoptosis in human tongue carcinoma cells by down-regulating Akt/GSK-3 signaling. Fundamental & Clinical Pharmacology, 25(2), 229
236. Yu, G., Wang, F., Zhang, B., & Fan, J. (2016). In vitro inhibition of platelet aggregation by peptides derived from oat (Avena sativa L.), highland barley (Hordeum vulgare Linn. var. nudum Hook. f.), and buckwheat (Fagopyrum esculentum Moench) proteins. Food Chemistry, 194, 577
586.

402 Chapter 16 Yuan, C., Chen, L., Meehan, E. J., Daly, N., Craik, D. J., Huang, M., & Ngo, J. C. (2011). Structure of catalytic domain of Matriptase in complex with Sunflower trypsin inhibitor-1. BMC Structural Biology, 11, 30. Zhang, M., Huang, T. S., & Mu, T. H. (2019a). Improvement of thermal, microwave and ultrasonication pretreatment on the production of antioxidant peptides from sweet potato protein via invitro gastrointestinal digestion. International Journal of Food Science & Technology, 54, 2338
2345. Zhang, M., Huang, T. S., & Mu, T. H. (2019b). Production and in vitro gastrointestinal digestion of antioxidant peptides from enzymatic hydrolysates of sweet potato protein affected by pretreatment. Plant Foods for Human Nutrition, 74(2), 225
231. Zhang, S. B. (2016). In vitro antithrombotic activities of peanut protein hydrolysates. Food Chemistry, 202, 1
8. Zhang, Y., Zhang, Y., Chen, P., Shu, F., Li, K., Qiao, L., . . . Wang, L. (2019). A novel angiotensin-I converting enzyme inhibitory peptide derived from the glutelin of vinegar soaked black soybean and its antihypertensive effect in spontaneously hypertensive rats. Journal of Biochemistry, 166(3), 223
230. Zhang, W., Sun, J., Zhao, G., Wang, J., Liu, H., Zheng, H., . . . Zou, D. (2017). Association analysis of the glutelin synthesis genes GluA and GluB1 in a Japonica rice collection. Molecular Breeding, 37, 129. Zhao, Q., Xu, H., Hong, S., Song, N., Xie, J., Yan, Z., . . . Jiang, X. (2019). Rapeseed protein-derived antioxidant peptide RAP ameliorates nonalcoholic steatohepatitis and related metabolic disorders in mice. Molecular Pharmaceutics, 16(1), 371
381. Zheng, C., Yang, Q., Cao, J., Xie, N., Liu, K., Shou, P., . . . Shi, Y. (2016). Local proliferation initiates macrophage accumulation in adipose tissue during obesity. Cell Death & Disease, 7, e2167. Zheng, Z. J., Li, J. X., Li, J. W., Sun, H., & Liu, Y. F. (2019). Physicochemical and antioxidative characteristics of black bean protein hydrolysates obtained from different enzymes. Food Hydrocolloids, 97. Zhou, Y., Jiang, Y., Shi, R., Chen, Z., Li, Z., Wei, Y., & Zhou, X. (2019). Structural and antioxidant analysis of tartary buckwheat (Fagopyrum tartaricum Gaertn.) 13S globulin. Journal of the Science of Food and Agriculture. Zhu, Y., Nadia, E., Yao, Y., Shi, Z., & Ren, G. (2018). Tandem repeated expression of lunasin gene in Pichia pastoris and its anti-inflammatory activity in vitro. Journal of Bioscience and Bioengineering, 126(1), 1
8.

CHAPTER 17

Generation of bioactivities from proteins of animal sources by enzymatic hydrolysis and the Maillard reaction Keizo Arihara1, Issei Yokoyama1 and Motoko Ohata2 1

School of Veterinary Medicine, Kitasato University, Towada, Japan, 2College of Bioresource Sciences, Nihon University, Fujisawa, Japan

17.1 Introduction Bioactive peptides, usually between 2 and 20 amino acids, generated from food proteins are representative functional food ingredients, since they contribute to our health beyond a nutritional source of amino acids (Bhandari et al., 2020; Bowman, 2015; Sa´nchez & Va´zquez, 2017). They have been found in hydrolysates of proteins from various food sources. Proteins are one of the main components in foods, especially in those of animal origin. Most animal sources of proteins, such as dairy, meat, poultry, fish, and eggs, contain all amino acids that our body needs. Peptides generated from these proteins by proteolysis consist of different sequences and have diverse bioactivities (Fig. 17.1). They include various types of activities, such as (angiotensin-converting enzyme) ACE inhibitory/antihypertensive, antioxidative, opioid, immunomodulating, antimicrobial, antithrombotic, and hypocholesterolemic. Review articles concerning these bioactive peptides from animal sources are available (Albenzio, Santillo, Caroprese, Malva, & Marino, 2017; Bhat, Kumar, & Bhat, 2015; Maestri, Pavlicevic, Montorsi, & Marmiroli, 2018; Toldra´, Gallego, Reig, Aristoy, & Mora, 2020a). Also, each bioactivity of protein-derived peptides is described in other chapters of this book. Animal by-products are also good sources of bioactive peptides, since significant amounts of animal by-products are produced during the various processing of food industry (Bechaux, Gatellier, Page, Drillet, & Sante-Lhoutellier, 2019). However, most parts of such by-products are discarded as waste or used for low-value products. Since animal/meat by-products are rich in proteins, they are appropriate sources of bioactive peptides by enzymatic treatments. For such reasons, the use of by-products as a source of bioactive peptides has been extensively studied in recent years (Bechaux et al., 2019; Mora, Reig, & Toldra´, 2019; Rezaharsamto & Subroto, 2019; Toldra´, Mora, & Reig, 2016). Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00001-7 © 2021 Elsevier Inc. All rights reserved.

403

404 Chapter 17 Proteins of animal sources Milk, meat, fish, eggs, by-products

Protein hydrolysis Gastrointestinal digestion Aging/storage Fermentation Protease treatment

Bioactive peptides Circulatory system

Nervous system

Antihypertensive peptides

Opioid peptides

Antioxidative peptides

- Opioid agonist

Antithrombotic peptides

Alimentary system Mineral binding peptides

- Opioid antagonist

Immune system Immunomodulating peptides

Antimicrobial peptides

Antimicrobial peptides

Prebiotic peptides

Cytomodulatory peptides

Figure 17.1 Generation of bioactive peptides from proteins of animal sources.

In association with the utilization of bioactive peptides, the Maillard reaction plays a critical role during processing and cooking of foods. This reaction brings numerous chemical changes in foodstuffs and is especially responsible for colors and flavors. Some Maillard reaction products (MRPs) have shown physiologically positive effects. Changes of amino acids or proteins and reduced sugars by the Maillard reaction have been studied but also changes of peptides have been received attention in recent years (Arihara, Yokoyama, & Ohata, 2019; Arihara, Zhou, & Ohata, 2017; Fu, Zhang, Soladoye, & Aluko, 2019). Since food protein-derived peptides and protein hydrolysates are utilized in many functional foods, the changes of peptides by the Maillard reaction in foods during processing or storage deserve concern and research. In this chapter, the generation of bioactive peptides from animal sources and potential benefits of such peptides are briefly discussed, since these topics have been already reviewed in many articles. Special attention is paid to the utilization of animal by-products

Bioactivity generation from proteins of animal sources 405 for generating bioactive peptides. Then, MRPs, particularly their bioactivities, generated from food protein-derived peptides are focused later in this chapter.

17.2 Bioactive peptides from milk 17.2.1 Generation of peptides from milk Milk and dairy products are important protein sources in our diets. The main milk proteins are α-lactoalbumin, β-lactoglobulin, αs-casein, β-casein, and κ-casein (Vargas-Bello-Pe´rez, Ma´rquez-Herna´ndez, & Herna´ndez-Castellano, 2019). These proteins are considered as potential ingredients for the production of health-promoting foods. To date, various bioactive peptides generated from milk proteins by enzymatic hydrolysis have been found. Mellander (1950) first reported bioactive peptides generated from food proteins. He suggested that milk casein-derived phosphorylated peptides enhanced vitamin D-independent bone calcification in rachitic infants. Later, Brantl, Teschemacher, Henschen, and Lottspeich (1979) reported β-casomorphins which are a group of opioid peptides released during gastrointestinal digestion or food processing from the β-casein of milk proteins. Since then, information on bioactivities of peptides generated from food proteins, such as milk and meat proteins, has steadily accumulated (Table 17.1), and numerous peptides exhibiting various bioactivities have been discovered (Bowman, 2015; Hettiarachchy, 2012; Owusu-Apenten, 2010; Vargas-Bello-Pe´rez et al., 2019). Table 17.1: Examples of bioactive peptides generated from milk and meat proteins in early studies. Bioactivity

Sequence

Source

Preparation

References

ACE inhibitory/ antihypertensive

IPP, VPP

Milk

Fermentation

MNPPK, ITTNP

Pork

Thermolysin

RMLGQTPTK YFYPEL DAQEKLE YGLF, YLLF VGPIPY FVAPFPEVFG αs1-casein f(43-58) Casein phosphopeptides IIAEK ELM

Pork Milk Pork Milk Milk Cheese Milk

Pepsin Pepsin Papain Trypsin Trypsin Trypsin Trypsin

Milk

Trypsin

Nakamura, Yamamoto, Sakai, and Takano (1995) Fujita, Yokoyama, and Yoshikawa (2000) Arihara, Nakashima, Mukai, Ishikawa, and Itoh (2001) Katayama et al. (2003) Suetsuna, Ukeda, and Ochi (2000) Saiga, Tanabe, and Nishimura (2003) Chiba, Tani, and Yoshikawa (1989) Fiat et al. (1993) Rizzello et al. (2005) Gagnaire, Pierre, Molle, and Leonil (1996) Cross, Huq, and Reynolds (2005)

Milk Pork

Trypsin Papain

Nagaoka et al. (2001) Arihara, Ishikawa, and Itoh (2006)

IKW

Antioxidative Opioid agonistic Immunomodulating Antimicrobial Mineral-binding Anticarcinogenic Hypocholesteromic Prebiotic

ACE, Angiotensin-converting enzyme.

Chicken Thermolysin

406 Chapter 17 Like any other bioactive peptides, milk protein-derived peptides are inactive within the native proteins (Sa´nchez & Va´zquez, 2017; Toldra´, Reig, Aristoy, & Mora, 2018). Since chemical hydrolysis of proteins by acid and alkali brings racemization and destruction of amino acids and peptides, enzymatic hydrolysis or microbial fermentation is a practical approach to generate peptides from proteins (Fig. 17.1). Therefore sequences of peptides within milk proteins are activated by (1) proteases present in milk, (2) digestive enzymes, (3) enzymes produced by starter cultures, and (4) purified enzymes added to milk proteins (Vargas-Bello-Pe´rez et al., 2019). Antihypertensive/ACE inhibitory activity is the most widely studied properties of milk protein-derived peptides (Ryan, Ross, Bolton, Fitzgerald, & Stanton, 2011). Based on these activities, bioactive peptides from milk proteins have gained attention in the formulation of new functional foods. For example, tripeptides isoleucine
proline
proline and valine
proline
proline, generated by the fermentation with Lactobacillus helveticus have been utilized in several commercial dairy products (Beltra´n-Barrientos, Herna´ndezMendoza, Torres-Llanez, Gonza´lez-Co´rdova, & Vallejo-Co´rdoba, 2016; Garcı´a-Burgos, Moreno-Ferna´ndez, Alfe´rez, Dı´az-Castro, & Lo´pez-Aliaga, 2020).

17.2.2 Utilization of cheese whey for producing peptides Cheese whey is a major by-product of dairy industry. It is usually used as feedstock for animal feeding or to produce ricotta cheese (Zotta, Soloeri, Iacumin, Picozzi, & Gullo, 2020). Major whey proteins are α-lactoalbumin, β-lactoglobulin, serum albumin, immunoglobulin, and lactoferrin. Since they are a rich source of branched-chain amino acids, which are good for sarcopenic cases, whey protein-derived products would present important biological activities. Bioactive peptides can be generated by enzymatic hydrolysis of whey proteins. Whey-derived peptides have displayed several activities, such as antioxidant, antimicrobial, and antihypertensive activities (Brandelli, Daroit, & Correˆa, 2015; Dinika, Verma, Balia, Utama, & Patel, 2020). Recent studies have reported that enzymatic hydrolysis of cheese whey proteins results in different bioactive peptides with antioxidative properties, anticancer, and opioid functions (Lappa et al., 2019). Fermentative processes converting cheese whey into value-added products are expected to allow both to reduce the pollution potential and to bring economic benefit (Zotta et al., 2020). The conversion of cheese whey into functional beverages by fermentation is an attractive approach, since the market of functional beverages gains interest because of the increasing consumer demand for health-enhancing foods. As a recent example of such approaches, fermentation of cheese whey by its native microbiota represents a process to give value to whey for the production of whey-based beverages or functional foods with potential antihypertensive properties by the generation of whey protein-derived peptides (Mazorra-Manzano et al., 2020).

Bioactivity generation from proteins of animal sources 407

17.2.3 Evaluation of milk proteins for bioactive peptides Although dairy food industry has adopted diverse processes for introducing bioactive peptides in products, consumer’s demands seem to be currently shifting to natural and less processed food products (Roman, Sanchez-Siles, & Siegrist, 2017). Recently, Vargas-BelloPe´rez et al. (2019) mentioned in their review article that there is a need for further research to fully evaluate the true potential of milk proteins as a source of bioactive peptides. Animal genetics and animal nutrition play an important role in the relative proportions of milk proteins and could be used to manipulate the concentration of specific bioactive peptides. To date, unfortunately, only a quite limited studies have reported changes in milk protein-derived bioactive peptides associated with animal genetics and nutrition. Animal genetics seems the most effective for increasing the content of milk proteins which are precursors of bioactive peptides. Modification of milk protein genes can be used to change milk protein profile and thus increase the content of precursors for bioactive peptides. Nutrition strategies are another practical way to improve the content of milk proteins. For example, manipulation of ruminal microbiota has been investigated to increase the content of milk protein (Patra & Saxena, 2011). Diets containing high starch levels may be used to increase precursors for milk bioactive peptides (Vargas-Bello-Pe´rez et al., 2019). In future studies, these strategies would bring an increase of milk proteins and their derived bioactive peptides. Regarding the generation of bioactive peptides in milk, it goes without saying that control/regulation of proteolytic enzyme activities is critical for peptide production, although peptides are generated by enzymatic hydrolysis of proteins in the gastrointestinal tract.

17.3 Bioactive peptides from meat Various bioactive peptides can be generated from meat proteins. There is a great possibility of utilizing such components for developing novel functional meat products and food ingredients (Arihara, 2006, 2013, 2014; Arihara & Komiya, 2017; Arihara & Ohata, 2011a, 2011b, 2017; Pathera, Jairath, Singh, & Yadav, 2017; Xing, Liu, Cao, Zhang, & Zhou, 2019). Bioactive peptides identified in hydrolysates of meat proteins and in meat products have been reviewed (Ahhmed & Muguruma, 2010; Arihara, 2006; Arihara & Ohata, 2017; Lafarga & Hayes, 2014; Mora & Toldra´, 2014; Ryan et al., 2011; Udenigwe & Howard, 2013; Wu, Jahandideh, Yu, & Majumder, 2015).

17.3.1 Generation of peptides by gastrointestinal digestion There are several ways in which peptides are generated from meat proteins along the gastrointestinal digestion (Toldra´ et al., 2020a). For example, ACE inhibitory activity was generated from meat proteins (i.e., myosin, actin, tropomyosin, and troponin) by pancreatic

408 Chapter 17 protease treatment (Katayama et al., 2003). ACE inhibitory (antihypertensive) peptides generated from pork were found during in vitro gastrointestinal digestion (Escudero, Aristoy, Nishimura, Arihara, & Toldra´, 2012; Escudero, Sentandreu, Arihara, & Toldra´, 2010). ACE inhibitory peptides were also identified in the hydrolysate of boneless chicken leg meat digested with artificial gastric juice (Terashima et al., 2010). Furthermore, Simonetti, Gambacorta, and Perna (2016) reported the enhancement of the biological activity of pork by in vitro gastrointestinal digestion. Peptides released from cooked meat during in vitro gastric and intestinal digestion were identified and quantified (Sayd, Chambon, & Sante´-Lhoutellier, 2016). In addition to these studies, effects of gastrointestinal digestion on the profile of bioactive peptides generated from meat and meat products were evaluated in recent studies (Gallego, Mauri, Aristoy, Toldra´, & Mora, 2020; Xiao et al., 2020).

17.3.2 Generation of peptides during aging During aging or storage, meat proteins are hydrolyzed by muscle endogenous proteases (Etherington 1984; Koohmaraie 1994). Initially, endopeptidases (e.g., calpains and cathepsins) hydrolyze muscle proteins and generate polypeptides. Then such polypeptides are further hydrolyzed by exopeptidases and dipeptyl peptidases. Enzymatic hydrolysis contributes to improvement of sensory properties of meat (e.g., texture, taste, and flavor). In addition, peptides with bioactivities can be generated in meat during postmortem aging. Bauchart et al. (2006) reported that the content of several presumable bioactive peptides in fresh beef was greatly increased after 14 days of ripening. Arihara & Ohata (2015) identified ACE inhibitory (antihypertensive) peptides (i.e., Gly-Pro-Leu-Lys, Ile-Pro-IleLys, Ile-Pro) in aged pork. Also, they showed an increase in ACE inhibitory activity of beef during cold storage (Arihara & Ohata, 2017). Fu, Young, and Therkildsen (2017) investigated endogenous release of bioactive (e.g., ACE inhibitory) peptides in beef during aging. From these findings, development of healthy beef can be prepared through postmortem aging.

17.3.3 Generation of peptides during fermentation Proteolytic events that occur during fermentation of raw sausages and dry-cured ham have been studied extensively (Ordonez, Hierro, Bruna, & de la Hoz, 1998; Toldra´ & Flores, 1998; Toldra´, 2014). Both muscle endogenous and microbial proteolytic enzymes are involved in the fermentation of meat products, but the former enzymes seem to contribute greatly to protein hydrolysis. During fermentation of meat products, proteins are initially degraded into peptides by endogenous enzymes (cathepsins B, D, H, and L). Although calpains play a central role in the conditioning of meat, they do not exhibit activity at the end of the salting step (Sarraga, Gil, Arnau, Monfort, & Cusso, 1989). Since most bacteria

Bioactivity generation from proteins of animal sources 409 (e.g., lactobacilli) found in fermented meat products have only weak proteolytic activity, the degradation of proteins is not greatly affected by the bacteria (Hierro, de la Hoz, & Ordonez, 1999). Lactic acid bacteria affect protein hydrolysis by causing reduction in pH, which results in increased activity of muscle proteases (Kato et al., 1994). Many small bioactive peptides were identified in dry-cured ham (Mora, Sentandreu, Fraser, Toldra´, & Bramley, 2009; Sentandreu et al., 2003; Toldra´, Gallego, Reig, Aristoy, & Mora, 2020b). A peptidomic approach was applied to characterize proteolysis and low molecular weight peptides of commercial Argentinean fermented sausages (Lo´pez, Bru, Vignolo, & Fedda, 2015). This study represented a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers. Ohata, Uchida, Zhou, and Arihara (2016) identified an antioxidative peptide (Gln-Tyr-Pro) in fermented meat sauce, which was prepared by mixing ground pork, koji (rice fermented with koji mold such as Aspergillus), and salt, and fermenting for 24 weeks.

17.3.4 Generation of peptides by protease treatments The most efficient way for peptide preparation from meat proteins is by utilizing commercial exogenous proteases. Various enzymes from animal, plant, and microbial sources have been applied to food protein solution for the industrial-scale production of bioactive peptides (Agyei & Danquah, 2011; Young & Mine, 2009). Many bioactive peptides have been experimentally generated by treatment with commercial enzymes (Korhonen & Pihlanto, 2003; Pihlanto & Korhonen, 2003; Pihlanto-Leppa¨la¨, 2001; Toldra´ et al., 2020a). Single proteinases from animal, plant, and microbial sources and combinations of them have been used for the digestion of food proteins (Yoshikawa, 1996). In the meat industry, proteolytic enzymes have been used for meat tenderization. The most commonly used enzymes for meat tenderization are the plant enzymes papain, bromelain, and ficin (Dransfield & Etherington, 1981). In meat treated with enzymatic tenderization, peptides having bioactivities could be generated. On the other hand, effects of commercial proteases on protein breakdown and sensory characteristics of dry fermented sausages were investigated (Bruna, Fernandez, Hierro, Ordonez, & de la Hoz, 2000; Diaz, Fernandez, Garcia De Fernando, Hoz, & Ordonez, 1997). Such treatments would also generate bioactive peptides in meat products. Mirdhayati, Hermanianto, Wijaya, Sajuthi, and Arihara (2015) prepared hydrolysates of goat meat proteins by using sequential digestion of commercial endoproteinase and protease complex. Since they successfully obtained hydrolysates with ACE inhibitory and antihypertensive activities, they are trying to develop a functional ingredient utilizing a meat protein hydrolysate. Ryder, Bekhit, McConnell, and Carne (2016) evaluated commercially available food-grade microbial protease preparations for their ability to produce bioactive peptides by hydrolyzing meat myofibrillar and connective tissue protein extracts.

410 Chapter 17

17.4 Bioactive peptides from animal by-products Animal by-products can be defined as entire bodies or parts of animals, products of animal origin, or other products obtained from animals, which are not intended for direct human consumption (Lafarga & Hayes, 2014; Toldra´, Aristoy, Mora, & Reig, 2012, Toldra´ et al., 2016). They are generally recognized as carcasses, skins, bones, meat trimmings, blood, fatty tissues, horns, feet, hoofs, or internal organs. Significant amounts of animal byproducts are produced in slaughterhouses and during the processing of meats. Recently, dry-cured ham by-products (bones) were also suggested as a potential source of peptides with cardioprotective effects (Gallego, Mora, Hayes, Reig, & Toldra´, 2019). Although most parts of animal by-products are discarded as waste or used for low-value products, they can be utilized as high value-added ingredients. Meat by-products, such as trimmings and mechanically recovered meat, collagen, blood are rich in proteins and seem to be promising sources for bioactive peptides (Arihara & Ohata, 2017; Bechaux et al., 2019; Bernardini, Harnedy, Bolton, Kerry, & O’Neil, 2011; Lafarga & Hayes, 2014; Liu, Xing, Fu, Zhou, & Zhang, 2016; Mora & Toldra´, 2014; Mora, Reig, & Toldra´, 2014; Mora et al., 2019; Rezaharsamto & Subroto, 2019; Ryder et al., 2016; Toldra´ et al., 2012, 2016).

17.4.1 Generation of peptides from blood One of the most abundant animal by-products is blood (Pare´s, Saguer, & Carretero, 2011). Large volumes of blood from cattle and pigs are produced in industrial slaughterhouses all over the world. Blood seems to be a good protein source for generating bioactive peptides since approximate protein content of blood is 18% (Bah, Bekhit, Carne, & McConnell, 2013). Antihypertensive, antioxidative, antimicrobial, and opioid peptides have been found in hydrolysates of blood proteins, such as hemoglobin and plasma (Arihara & Ohata, 2017; Bechaux et al., 2019; Mora et al., 2019). For example, ACE inhibitory peptides (Yu, et al., 2006) and antioxidative peptides (Chang, Wu, & Chiang, 2007) corresponding to the sequences of porcine hemoglobin were identified. Biologically active peptides with affinity for opioid receptor were found in the hemoglobin peptic hydrolysate (Nyberg, Sanderson, & Glamsta, 1997; Zhao, Garreau, Sannier, & Piot, 1997). Several antimicrobial peptides have been found in the hydrolysates of hemoglobin (Daoud et al., 2005; Fogaca et al., 1999; Froidevaux et al., 2001; Hu et al., 2011). Przybylski, Firdaous, Chaˆtaigne´, Dhulster, and Nedjar (2016) identified a small antimicrobial peptide (Thr-Ser-Tyr-Arg, fragment of hemoglobin) from pepsin-treated bovine cruor.

17.4.2 Generation of peptides from collagen Collagen is the most abundant protein in mammals (Mora & Toldra´, 2014). The most common motifs in the amino acid sequence of collagen are Gly-Pro-X and Gly-X-Hyp,

Bioactivity generation from proteins of animal sources 411 where X can be any amino acid other than Gly, Pro, or Hyp. Since collagen lacks certain essential amino acids, it has less nutritional value than most other food proteins (GomezGuillen, Gimenez, Lopez-Caballero, & Montero, 2011). Saiga et al. (2003) identified four ACE inhibitory peptides in Aspergillus protease-treated chicken muscle extract. Three of those four peptides possessed a common sequence, Gly-X-X-Gly-X-X-Gly-X-X, which is homologous with that of collagen. Banerjee and Shanthi (2012) identified ACE inhibitory peptides generated from bovine Achilles tendon collagen hydrolysate. Antioxidative peptides were purified from hydrolyzed bovine skin (Kim, Kim, Byun, Park, & Ito, 2001) and porcine skin (Li, Chen, Xang, Ji, & Wu, 2007). Fermentation of by-products from meat processing has been used to generate bioactive compounds (Hayes & Garcı´a-Vaquero, 2016). The goat placenta has long been used in Chinese medicine and is recognized as rich in bioactive components (Chakraborty & Bhattacharyya, 2005). Hou et al. (2014) assayed the antioxidative activity of peptides generated in the goat placenta by fermentation. Ohata et al. (2016) prepared fermented meat sauce and identified an antioxidative peptide. Meat by-products seem to be suitable for the ingredient of such fermented meat products.

17.5 Bioactive peptides from marine sources Marine organisms make up approximately one half of the total global biodiversity. Since the demand for seafood is rising all over the world, enormous amounts of seafood byproducts are discarded during processing. Fish, shellfish, and their waste contain significant levels of high-quality proteins, which represents a source for bioactive peptide mining (Harnedy & FitzGerald, 2012; Pavlicevic, Maestri, & Marmiroli, 2020). Marine by-products are generally either discarded or used as animal feed or fertilizers. Conversion of marine by-products into high-value functional ingredients may provide a solution for economic and environmental problems.

17.5.1 Generation of peptides from seafood and its by-products Some marine organisms such as fish and shellfish are rich sources of protein, thus they are appropriate starting materials for the generation of bioactive peptides by enzymatic hydrolysis. In fact, various bioactive peptides have been found in the enzymatic hydrolysates of proteins from marine organisms. As such peptides, ACE inhibitory, antioxidative, neuroprotective, antidiabetic, dipeptidyl peptidase IV (DPP-IV) inhibitory, immunomodulatory, antibacterial, cholecystokinin release regulating, antiproliferative, and anticancer peptides have been reported (Harnedy & FitzGerald, 2012; Jakubczyk, Karas´, Rybczy´nska-Tkaczyk, Zieli´nska, & Zieli´nski, 2020; Pavlicevic et al., 2020; Ryan et al., 2011). Similar to bioactive peptides generated from other sources, ACE inhibitory and

412 Chapter 17 antioxidant peptides are the most frequent types of peptides found in seafood hydrolysates. Interestingly, peptides from seafood have effective anticancer activity (Ishak & Sarbon, 2018; Nwachukwu & Aluko, 2019; Yaghoubzadeh, Ghadikolaii, Kaboosi, Safari, & Fattahi, 2020). Since the demand for seafood is rising all over the world, enormous amounts of seafood byproducts are discarded during processing. Such by-products (e.g., heads, skin, viscera, bones) contain approximately 60% protein on a dry weight basis. As described above, various bioactive peptides have been found from enzymatic hydrolysis of seafood proteins. Fermentation is one possible way to produce value-added products from seafood byproducts. Fermentation of fish was first introduced for its preservation. Fish sauces and pastes are microbiologically stable diets in some areas, such as Southeast Asia and Scandinavia (Hayes & Garcı´a-Vaquero, 2016). During fermentation of fish, muscle proteins are degraded and bioactive peptides are generated. For example, antioxidative peptides from fermented fish sauce by-product were isolated and characterized (Choksawangkarn, Phiphattananukoon, Jaresitthikunchai, & Roytrakul, 2018). Fish sauce by-product refers to solid waste from fish sauce industry and is composed of nutritionally important biomolecules, such as fish protein hydrolysate. The findings of this study would provide useful information for the development of value-added products from fish by-products.

17.5.2 Commercial development of marine-derived peptides Japan has a long history of using foods with health benefits and Japanese people are wellknown for their longevity (Iwatani & Yamamoto, 2019). In 1991 the government of Japan introduced a functional food regulation called “foods for specified health uses” (FOSHU). After the introduction of the functional food system, many clinically proven FOSHU products with health benefits have been developed and launched in the market. FOSHU refers to foods containing ingredients with functions for health and is officially approved to claim its physiological effects on the human body. FOSHU is intended to be consumed for the maintenance/promotion of health or special health uses by people who wish to control health conditions, including blood pressure or blood cholesterol. To sell a food as FOSHU, the assessment for the safety of the food and effectiveness of the functions for health is required, and the claim must be approved by the government of Japan. Several FOSHU products containing fish protein hydrolysates or peptides as functional ingredients have been approved in Japan. Many of these products claim to be suitable for consumption by individuals with mild hypertension (Harnedy & FitzGerald, 2012; Hayes & Garcı´a-Vaquero, 2016; Jakubczyk et al., 2020). For example, a thermolysin digest of Katsuobushi (dried bonito) was approved as FOSHU in Japan due to its blood pressurelowering properties. However, these products have not yet received the European Food Safety Authority approval for any health claim but do have approval for use as a novel food.

Bioactivity generation from proteins of animal sources 413 After 2007 the Japanese market for FOSHU products was almost saturated, because FOSHU approval was not directly linked to product sales (Iwatani & Yamamoto, 2019). To overcome the shrinking market for functional foods in Japan, a novel functional regulatory system was established in 2015. This new system is based on the Dietary Supplement Health and Education Act (DSHEA) system introduced in the United States in 1994. The Japanese novel system “foods with function claims” was established in 2015 based on the idea of the DSHEA. After the introduction of this system, many foods with function claims have been developed because of the more flexible health claims compared to FOSHU and the lack of a requirement for governmental approval.

17.6 Bioactive peptides and the Maillard reaction 17.6.1 The Maillard reaction Chemical reactions occur in foods during manufacture, storage, and cooking and they change color, aroma, taste, and nutritional value of foods. The Maillard reaction is an important chemical reaction in such changes (Losso, 2016; Shahidi, Samaranayaka, & Pegg, 2014). This reaction is a nonenzymatic reaction between amino groups from amino acids, peptides, or proteins and carbonyl groups of carbohydrates, especially reducing sugars. Various chemicals referred to as MRPs are produced during this reaction. Since food protein-derived peptides are widely utilized in processed foods, the changes of peptides by the Maillard reaction in foods deserve attention (Arihara et al., 2017; Fu et al., 2019). It has been reported that MRPs generated from food protein-derived peptides contribute to color and flavor, in addition to bioactivities of foods. The Maillard reaction is composed of three key stages (Fu et al., 2019) and is illustrated in Fig. 17.2. The initial stage of the reaction involves interaction between the free amino and carbonyl group (amino
carbonyl reaction) and formation of a Schiff base. This is followed by the rearrangement of the Schiff base to an Amadori compound. The intermediate stage initiates from the Amadori rearrangement compounds. They are further characterized by several pathways, such as enolization, dehydration, and Strecker degradation. During the intermediate stage of this reaction, the dicarbonyl compounds are generated and serve as precursors for developing heterocyclic compounds and flavor products. The reaction between Strecker aldehydes and intermediates generates volatile compounds, which are critical for flavor of foods. In the final stage, advanced glycation end-products, such as heterocyclic compounds and melanoidins are generated from Maillard intermediate products. Melanoidins, nitrogencontaining polymeric substances that decompose with difficulty, demonstrate physiologically positive effects because of unique partial structures in the molecules (Hayase, 1996). Melanoidins have a strong scavenging activity against active oxygen species, such as hydroxyl radicals, hydrogen peroxides, and superoxides (Morales, Somoza, & Fogliano, 2012).

414 Chapter 17 Early stage Amino compounds e.g., peptides

+

Reducing sugars e.g., glucose

Formation of Schiff base Amadori rearrangement

Intermediate stage

Enolization Dehydration Strecker reaction

Final stage Dicarbonyls

Aldehydes

Advanced glycation end-products e.g., heterocyclic compounds, melanoidins

Figure 17.2 Outline of the Maillard reaction of amino compounds and reducing sugars.

17.6.2 The Maillard reaction and meat Since meat and meat products are generally cooked before eating, they are closely related with the Maillard reaction. Although raw meat has little aroma and only a blood-like taste, cooked meat has a characteristic flavor (Shahidi et al., 2014). Numerous compounds have been identified in the aroma profiles of cooked meat products and the Maillard reaction has a critical role for generating such compounds (Pegg & Shahidi, 2014). Free amino groups of amino acids or peptides in meat can react with reducing sugars by heating. MRPs generated in cooked meat include many important classes of meat flavor compounds such as pyrazines, oxazoles, thiophenes, thiazoles, and other heterocyclic compounds. Sulfur compounds are especially important for the characteristic aroma of meat. Cheng, Song, and Wang (2011) explored the MRPs of enzymatic hydrolysates (peptides) prepared from beef proteins for the relationship between flavor products and reactants in the model system. The majority of peptides participating in the Maillard reaction have a molecular weight from 1000 to 2000 Da. The Maillard reaction of peptides is also a critical phenomenon in meat products without heat treatment (Jayasena, Ahn, Nam, & Jo, 2013; Ventanas et al., 1992; Weerawatanakorn, Wu, Pan, & Ho, 2015). Hams from Iberian breed are processed by the traditional dry-curing procedure and proteolysis and autoxidation occurred during ripening/drying. As a result, peptides, nitrogen, and carbonyl compounds are accumulated. In the next step, a rapid increase of amino acid nitrogen is observed, and aldehydes decrease notably. This change seems to be due to condensations between

Bioactivity generation from proteins of animal sources 415 carbonyls and free amino acids. While hams are kept in the cellar, the overall increase of amino acid nitrogen is in contrast to the slow rate of accumulation of some individual amino acids. This is presumably due to degradation to volatile derivatives, where Maillard reactions could be involved.

17.6.3 Bioactivities of Maillard reaction products from peptides MRPs show some biological activities, such as the antioxidative, antibacterial, and ACE inhibitory (Somoza, 2005; Van Lancker, Adams, & De Kimpe, 2011). With regard to peptides in MRPs, antioxidative activities have been reported. As the early study of MRPs from peptides, Lingnert & Eriksson (1980) reported an antioxidative activity generated from dipeptides. Later, Alfawaz, Smith, and Jeon (1994) found that the antioxidative activity of MRPs generated from protein hydrolysates was influenced by heating time and by the level of protein hydrolysates and sugar concentrations. Lu, Hao, Payne, and Ho (2005) reported that the antioxidative activity of MRPs seems to be related to both the peptide chain length and the stability of the peptide bond toward heating. Some recent studies of bioactivities of MRPs generated from animal sources are summarized in Table 17.2. For example, Hong, Meng, and Lu (2015) investigated the effect of the Maillard reaction on ACE inhibitory activity in a milk casein hydrolysate-xylose system. MRPs were prepared by heating at pH 8.0, 110 C. The ACE inhibitory activity of the MRPs increased greatly within 2 hours. We used chicken skin collagen (gelatin) for generating MRPs (Arihara, Arai, Ohata, Ishikawa, & Itoh, 2013; Arihara et al., 2017). Collagen contains approximately 33% Table 17.2: Examples of bioactivities of Maillard reaction products generated from animal protein hydrolysates. Protein source Whey protein Milk casein Chicken skin gelatin Chicken bone Fish (Anchovy) meat Fish skin gelatin

Flatfish by-product Shrimp by-product

Carbohydrate

Reaction conditions

Bioactivities

References

Lactose, lactulose Xylose Xylose



pH 6.0, 90 C, 45 min

Antioxidative

pH 8.0, 110 C, 16 h pH 8.0, 90 C, 60 min

Galactose

100 C, 1.5
7.5 h

ACE inhibitory Antioxidative antihypertensive Antioxidative

Nooshkam and Madadlou (2016) Hong et al. (2015) Arihara et al. (2013)

Ribose

pH 7.0, 110 C, 30 min 70 C, 36 h

Galactose

Ribose Glucose

ACE, Angiotensin-converting enzyme.

pH 8.3, 121 C, 38 min pH 6.5, 110 C, 10 h

Improved memory Immunomodulatory anticancer

Nie, Xu, Zhao, and Meng (2017) Su et al. (2016)

Anti-inflammatory

Karnjanapratum, O’Callaghan, Benjakul, and O’Brien (2016) Choe et al. (2016)

Antioxidative

Zha et al. (2015)

416 Chapter 17

Figure 17.3 Antihypertensive activities of MRPs prepared from collagen-peptides by the heat treatment (Maillard reaction). MRPs, Maillard reaction products. Source: Modified from Arihara, K., Arai, T. Ohata, M., Ishikawa, S., & Itoh, M. (2013). Food/pet food material having health functionality and linking-improving effect with collagen as raw material. Japan patent (No. 2013-5326489).

glycine, which is a good source for the Maillard reaction with reducing sugars. Collagen was heated for conversion into gelatin and digested by commercial proteases. Such collagen-derived peptides were added with reducing sugars (e.g., xylose) and a pH adjuster (e.g., sodium carbonate). Since the Maillard reaction prefers the alkaline condition, pH adjuster was added to the reaction solution. The solution was further heated at 90 C for the Maillard reaction. The activities by using superoxide ion and DPPH radical were increased significantly by heat treatment (the Maillard reaction). Oral administration of the MRPs to mice suppressed the stress marker (e.g., hydroperoxide value of serum). Also, continuous oral administration of the MRPs to the spontaneously hypertensive rats decreased their blood pressure compared to collagen peptides without the heat treatment (Fig. 17.3). Since this collagen peptide-based product prepared by the Maillard reaction showed good sensory properties, it will be a possible functional food ingredient.

17.6.4 Bioactivities of volatile Maillard reaction products from peptides Although MRPs include various odor components that can affect the quality of foods, limited studies have been conducted on the bioactivities of the volatile components generated by the Maillard reaction (Arihara et al., 2017, 2019; Fu et al., 2019). We investigated the effects of the odor produced by the Maillard reaction on blood pressure (Ohata, Zhou, Owashi, & Arihara, 2014). Meat protein hydrolysates (peptides) were mixed with xylose in buffer and were heated at 90 C for 240 minutes at pH 5 or pH 10. Wistar rats were exposed to the vapor of these solutions for 5 minutes, and systolic blood pressure (SBP) was measured (Fig. 17.4). The pH 10 sample significantly decreased SBP after

Bioactivity generation from proteins of animal sources 417

Figure 17.4 Measurement of systolic blood pressure of Wistar rats exposed to the vapor of each sample. Source: From Arihara, K., Zhou, L., & Ohata, M. (2017). Bioactive properties of Maillard reaction products generated from food protein-derived peptides. Advances in. Food Nutrition Research, 81, 161
185.

Figure 17.5 Odor components contributing to the odor of pH 10 sample.

10
15 minutes exposure. Volatile compounds generated in the pH 10 sample would have the effect of decreasing SBP of rats. Further experiments indicated that the pH 10 sample odor caused parasympathetic nervous system dominant and induced a relaxed and sedated state (Ohata et al., 2014). As major odor components contributing to the odor of the sample, acetic acid, 2-hydroxy-3-methyl-2-cyclopenten-1-one (cyclotene), 2,5-dimethyl-4-hydroxy-3 (2H)-furanone (DMHF), and 5-methylpyrazine-2-methanol (MPM) were identified (Fig. 17.5). Since cyclotene, DMHF, and MPM were absent in the sample before heat treatment, these compounds were generated by the Maillard reaction. Later, Zhou, Ohata, and Arihara (2016) evaluated the impacts of odor generated by the Maillard reaction on human mood and brainwaves. They demonstrated that this odor decreases negative moods in panelists, including depression, tension, fatigue, and restless moods. The increased α brainwave distribution implied a relaxing effect on the mental state of panelists and DMHF was identified as an agent for changes in α brainwave distribution.

418 Chapter 17 Also, they reported that odor generated from xylose with meat protein hydrolysates reduces SBP in Wistar rats (Zhou et al., 2018). DMHF and MPM were key components responsible for controlling blood pressure. Since exposure to DMHF increased gastric vagal nerve activity and decreased renal sympathetic nerve activity, DMHF would control blood pressure through the autonomic nervous system. Furthermore, we reported that the score of certain subjective moods, especially anger
hostility, tension
anxiety, and fatigue-inertia decreased after inhalation of DMHF, suggesting a sedative effect of this odor on mood (Ohata, Zhou, Yada, Yokoyama, & Arihara, 2020). In addition, we found that inhalation of DMHF promotes appetite and changes gene expression in the rat brain (Yokoyama, Nakai, et al., 2020) and affects some physiological parameters in rats (Yokoyama, Ohata, Komiya, Nagasao, & Arihara, 2020). Along with these findings, our latest research results will be discussed elsewhere.

17.7 Conclusion Bioactive peptides have a wide variety of activities, with antihypertensive/ACE inhibitory, antioxidant, antidiabetic, antiobesity, or antimicrobial effects. They are generated from various sources including proteins of animal products and their byproducts. Although there are still many challenges to be solved, bioactive peptides are promising food ingredients for developing functional foods. On the other hand, the Maillard reaction is a critical chemical reaction in foods, because it develops color and flavor in various foods. This reaction also delivers physiological functions in foods. By combining the protein hydrolysis and the Maillard reaction, it would be possible to develop more attractive functional foods in the near future. Such efforts would open the field of vision in the food industry.

References Agyei, D., & Danquah, M. K. (2011). Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides. Biotechnology Advances, 29, 272
277. Ahhmed, A. M., & Muguruma, M. (2010). A review of meat protein hydrolysates and hypertension. Meat Science, 86, 110
118. Albenzio, M., Santillo, A., Caroprese, M., Malva, A. D., & Marino, R. (2017). Bioactive peptides in animal food products. Foods, 6, 35. Available from https://doi.org/10.3390/foods6050035. Alfawaz, M. J., Smith, S., & Jeon, I. J. (1994). Maillard reaction products as antioxidants in pre-cooked ground beef. Food Chemistry, 51, 311
318. Arihara, K. (2006). Strategies for designing novel functional meat products. Meat Science, 74, 219
229. Arihara, K. (2013). Relevance of peptides bioactivity in foods. In F. Toldra´, & L. M. L. Nollet (Eds.), Proteomics in foods (pp. 447
463). New York: Springer. Arihara, K. (2014). Functional foods. In M. Dikeman, & C. Devine (Eds.), Encyclopedia of meat sciences (2nd ed., Volume 2). Oxford: Elsevier/Academic Press.

Bioactivity generation from proteins of animal sources 419 Arihara, K., & Komiya, Y. (2017). Bioactive properties of peptides generated from meat proteins. In F. Toldra´, & L. M. L. Nollet (Eds.), Advanced technologies for meat processing (2nd ed., pp. 371
402). Boca Raton, NJ: CRC Press. Arihara, K., & Ohata, M. (2011a). Use of probiotics and prebiotics in meat products. In J. P. Kerry, & J. F. Kerry (Eds.), Processed meats: Improving safety, nutrition and quality (pp. 403
417). Cambridge: Woodhead Publishing. Arihara, K., & Ohata, M. (2011b). Functional meat products. In M. Saarela (Ed.), Functional foods: Concept to products (2nd ed., pp. 512
533). Cambridge: Woodhead Publishing. Arihara, K., & Ohata, M. (2015). Antihypertensive peptides generated in aged pork. Japan patent (No. 2015-154703). Arihara, K., & Ohata, M. (2017). Production of bioactives from meat and animal by-products. In A. E.-D. A. Bekhit (Ed.), Advances in meat processing technology (pp. 505
520). Boca Raton: CRC Press. Arihara, K., Arai, T. Ohata, M., Ishikawa, S., & Itoh, M. (2013). Food/pet food material having health functionality and linking-improving effect with collagen as raw material. Japan patent (No. 2013-5326489). Arihara, K., Ishikawa, S., & Itoh, M. (2006). Bifidobacterium growth promoting peptides derived from meat proteins. Japan patent (No. 2011-4726129). Arihara, K., Nakashima, Y., Mukai, T., Ishikawa, S., & Itoh, M. (2001). Peptide inhibitors for angiotensin I-converting enzyme from enzymatic hydrolysates of porcine skeletal muscle proteins. Meat Science, 57, 319
324. Arihara, K., Yokoyama, I., & Ohata, M. (2019). DMHF (2,5-dimethyl-4-hydroxy-3 (2H)-furanone), a volatile food component with attractive sensory properties, brings physiological functions through inhalation. Advances in Food Nutrition Research, 89, 239
258. Arihara, K., Zhou, L., & Ohata, M. (2017). Bioactive properties of Maillard reaction products generated from food protein-derived peptides. Advances in. Food Nutrition Research, 81, 161
185. Bah, C. S. F., Bekhit, E.-D. A., Carne, A., & McConnell, M. A. (2013). Slaughterhouse blood: An emerging source of bioactive compounds. Comprehensive Reviews in Food Science and Food Safety, 12, 314
331. Banerjee, P., & Shanthi, C. (2012). Isolation of novel bioactive regions from bovine achilles tendon collagen having angiotensin I-converting enzyme-inhibitory properties. Process Biochemistry, 47, 2335
2346. Bauchart, C., Remond, D., Chambon, C., Mirand, P. P., Savary-Auzeloux, I., Reynes, C., & Morzel, M. (2006). Small peptides (,5kDa) found in ready-to-eat beef meat. Meat Science, 74, 658
666. Bechaux, J., Gatellier, P., Page, J.-F. L., Drillet, Y., & Sante-Lhoutellier, V. (2019). A comprehensive review of bioactive peptides obtained from animal byproducts and their applications. Food and Function, 10, 6244
6266. Beltra´n-Barrientos, L. M., Herna´ndez-Mendoza, A., Torres-Llanez, M. J., Gonza´lez-Co´rdova, A. F., & VallejoCo´rdoba, B. (2016). Fermented milk as antihypertensive functional food. Journal of Dairy Science, 99, 4099
4110. Bernardini, R. D., Harnedy, P., Bolton, D., Kerry, J., & O’Neil, E. (2011). Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products. Food Chemistry, 124, 1296
1307. Bhandari, D., Rafiq, S., Gat, Y., Gat, P., Waghmare, R., & Kumar, V. (2020). A review on bioactive peptides: physiological functions, bioavailability and safety. International Journal of Peptide Research and Therapeutics, 26, 139
150. Bhat, Z. F., Kumar, S., & Bhat, H. F. (2015). Bioactive peptides of animal origin: a review. Journal of Food Science and Technology, 52, 5377
5392. Bowman, L. (2015). Bioactive food peptides: Production and health benefits. NJ: Foster Academics. Brandelli, A., Daroit, D. J., & Correˆa, A. P. F. (2015). Whey as a source of peptides with remarkable biological activities. Food Research International, 73, 149
161. Brantl, V., Teschemacher, H., Henschen, A., & Lottspeich, F. (1979). Novel opioid peptides derived from casein (β-Casomorphins) I. Isolation from bovine casein peptone. Hoppe-Seyler’s Zeitschrift fu¨r Physiologische Chemie, 360, 1211
1216. Bruna, J. M., Fernandez, M., Hierro, E. M., Ordonez, J. A., & de la Hoz, L. (2000). Combined use of pronase E and a fungal extract (Penicillium aurantiogriseum) to potentiate the sensory characteristics of dry fermented sausages. Meat Science, 54, 135
145.

420 Chapter 17 Chakraborty, P. D., & Bhattacharyya, D. (2005). Isolation of fibronectin type III like peptide from human placental extract used as wound healer. Journal of Chromatography B, 818, 67
73. Chang, C.-Y., Wu, K.-C., & Chiang, S.-H. (2007). Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates. Food Chemistry, 100, 1537
1543. Cheng, L.-K., Song, H.-L., & Wang, P.-X. C. (2011). Degradation of peptides derived from enzymatic hydrolysis of beef during Maillard reaction. Food Science, 32, 46
50. Chiba, H., Tani, F., & Yoshikawa, M. (1989). Opioid antagonist peptides derived from κ-casein. Journal of Dairy Research, 56, 363
366. Choe, I. H., Jeon, H. J., Eom, S. H., Han, Y. K., Kim, Y. S., & Lee, S. H. (2016). The anti-inflammatory effect of a glycosylation product derived from the high hydrostatic pressure enzymatic hydrolysate of a flatfish byproduct. Food and Function, 7, 2557
2565. Choksawangkarn, W., Phiphattananukoon, S., Jaresitthikunchai, J., & Roytrakul, S. (2018). Antioxidative peptides from fish sauce by-product: Isolation and characterization. Agriculture and Natural Resources, 52, 460
466. Cross, K. J., Huq, N. L., & Reynolds, E. C. (2005). Anticarcinogenic peptides. In Y. Mine, & F. Shahidi (Eds.), Nutraceutical proteins and peptides in health and disease (pp. 335
351). Boca Raton, FL: Blackie Academic & Professional. Daoud, R., Dubois, V., Bors-Dodita, L., Nedjar-Arroume, N., Krier, F., Chihib, N. E., . . . Guillochon, D. (2005). New antibacterial peptide derived from bovine hemoglobin. Peptides, 26, 713
719. Diaz, O., Fernandez, M., Garcia De Fernando, G. D., Hoz, L. de la, & Ordonez, J. A. (1997). Proteolysis in dry fermented sausages: The effect of selected exogenous proteases. Meat Science, 46, 115
128. Dinika, I., Verma, D. K., Balia, R., Utama, G. L., & Patel, A. R. (2020). Potential of cheese whey bioactive proteins and peptides in the development of antimicrobial edible film composite: A review of recent trends. Trends in Food Science and Technology, 103, 57
67. Dransfield, E., & Etherington, D. (1981). Enzymes in the tenderization of meat. In G. G. Birch, N. Blakebrough, & K. J. Parker (Eds.), Enzymes and food processing (pp. 177
194). London: Applied Science Publishers. Escudero, E., Aristoy, M.-C., Nishimura, H., Arihara, K., & Toldra´, F. (2012). Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish dry-cured ham. Meat Science, 91, 306
311. Escudero, E., Sentandreu, M., Arihara, K., & Toldra´, F. (2010). Angiotensin I-converting enzyme inhibitory peptides generated from in vitro gastrointestinal digestion of pork meat. Journal of Agricultural and Food Chemistry, 58, 2895
2901. Etherington, D. J. (1984). The contribution of proteolytic enzymes to postmortem changes in muscle. Journal of Animal Science, 59, 1644
1650. Fiat, A.-M., Migliore-Samour, D., Jolle`s, P., Drout, L., Collier, C., & Caen, J. (1993). Biologically active peptides from milk proteins with emphasis on two examples concerning antithrombotic and immunomodulating activities. Journal of Dairy Science, 76, 301
310. Fogaca, A. C., da Silva, P. I., Miranda, M. T. M., Bianchi, A. G., Miranda, A., Ribolla, P. E. M., & Daffre, S. (1999). Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. Journal of Biological Chemistry, 274, 25330
25334. Froidevaux, R., Krier, F., Nedjar-Arroume, N., Vercaigne-Marko, D., Kosciarz, E., Ruckebusch, C., . . . Guillochon, D. (2001). Antimicrobial activity of a pepsin-derived bovine hemoglobin fragment. FEBS Letters, 491, 159
163. Fu, Y., Young, J. F., & Therkildsen, M. (2017). Bioactive peptides in beef: Endogenous generation through postmortem aging. Meat Science, 123, 134
142. Fu, Y., Zhang, Y., Soladoye, O. P., & Aluko, R. E. (2019). Maillard reaction products derived from food protein-derived peptides: Insights into flavor and bioactivity. Critical Reviews in Food Science and Nutrition. Available from https://doi.org/10.1080/10408398.2019.1691500. Fujita, H., Yokoyama, K., & Yoshikawa, M. (2000). Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science, 65, 564
569.

Bioactivity generation from proteins of animal sources 421 Gagnaire, V., Pierre, A., Molle, D., & Leonil, J. (1996). Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles. Journal of Dairy Research, 63, 405
422. Gallego, M., Mauri, L., Aristoy, M. C., Toldra´, F., & Mora, L. (2020). Antioxidant peptides profile in dry-cured ham as affected by gastrointestinal digestion. Journal of Functional Foods, 69. Available from https://doi. org/10.1016/j.jff.2020.103956, Article 103956. Gallego, M., Mora, L., Hayes, M., Reig, M., & Toldra´, F. (2019). Peptides with potential cardioprotective effects derived from dry-cured ham products. Journal of Agricultural and Food Chemistry, 67, 1115
1126. Garcı´a-Burgos, M., Moreno-Ferna´ndez, J., Alfe´rez, M. J. M., Dı´az-Castro, J., & Lo´pez-Aliaga, I. (2020). New perspectives in fermented dairy products and their health relevance. Journal of Functional Foods, 72. Available from https://doi.org/10.1016/j.jff.2020.104059, Article 104059. Gomez-Guillen, C. M., Gimenez, B., Lopez-Caballero, M. E., & Montero, M. P. (2011). Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocolloid, 25, 1813
1827. Harnedy, P. A., & FitzGerald, R. J. (2012). Bioactive peptides from marine processing waste and shellfish: A review. Journal of Functional Foods, 4, 6
24. Hayase, F. (1996). Scavenging of active oxygen by melanoidins. In R. Ikan (Ed.), The Maillard reaction, consequences for the chemical and life sciences (pp. 89
104). UK: John Wiley & Sons. Hayes, M., & Garcı´a-Vaquero, M. (2016). Bioactive compounds from fermented food products. In K. S. Ojha, & B. K. Tiwari (Eds.), Novel food fermentation technologies (pp. 293
310). Switzerland: Springer International Publishing. Hettiarachchy, N. S. (2012). Bioactive food proteins and peptides. Boca Raton: CRC Press. Hierro, E., de la Hoz, L., & Ordonez, J. A. (1999). Contribution of the microbial and meat endogenous enzymes to the free amino acid and amine contents of dry fermented sausages. Journal of Agricultural and Food Chemistry, 47, 1156
1161. Hong, X., Meng, J., & Lu, R. R. (2015). Improvement of ACE inhibitory activity of casein hydrolysate by Maillard reaction with xylose. Journal of the Science of Food and Agriculture, 95, 66
71. Hou, Y., Liu, W., Cheng, Y., Zhou, J., Wu, L., & Yang, G. (2014). Production, optimization and characterization of immunomodulatory peptides obtained from fermented goal placenta. Food Science and Technology (Campinas), 34, 723
729. Hu, J., Xu, M., Hang, B., Wang, L., Wang, Q., Chen, J., . . . Liu, X. (2011). Isolation and characterization of an antimicrobial peptide from bovine hemoglobin α-subunit. World Journal of Microbiology and Biotechnology, 27, 767
771. Ishak, N. H., & Sarbon, N. M. (2018). A review of protein hydrolysates and bioactive peptides deriving from wastes generated by fish processing. Food Bioprocess Technology, 11, 2
16. Iwatani, S., & Yamamoto, N. (2019). Functional food products in Japan: A review. Food Science and Human Wellness, 8, 96
101. Jakubczyk, A., Kara´s, M., Rybczy´nska-Tkaczyk, K., Zieli´nska, E., & Zieli´nski, D. (2020). Current trends of bioactive peptides - New sources and therapeutic effect. Foods. Available from https://doi.org/10.3390/ foods9070846. Jayasena, D. D., Ahn, D. U., Nam, K. C., & Jo, C. (2013). Flavour chemistry of chicken meat: A review. AsianAustralasian Journal of Animal Science, 26, 732
742. Karnjanapratum, S., O’Callaghan, Y. C., Benjakul, S., & O’Brien, N. M. (2016). In vitro cellular bioactivities of Maillard reaction products from sugar-gelatin hydrolysate of unicorn leatherjacket skin system. Journal of Functional Foods, 23, 87
94. Katayama, K., Tomatsu, M., Fuchu, H., Sugiyama, M., Kawahara, S., Yamauchi, K., . . . Muguruma, M. (2003). Purification and characterization of an angiotensin I-converting enzyme inhibitory peptide derived from porcine troponin C. Animal Science Journal, 74, 53
58. Kato, T., Matsuda, T., Tahara, T., Sugimoto, M., Sato, Y., & Nakamura, R. (1994). Effects of meat conditioning and lactic fermentation on pork muscle protein degradation. Bioscience. Biotechnology and Biochemistry, 58, 408
410.

422 Chapter 17 Kim, S.-K., Kim, Y.-T., Byun, H.-G., Park, P.-J., & Ito, H. (2001). Purification and characterization of antioxidative peptides from bovine skin. Biochemistry and Molecular. Biology, 34, 219
224. Koohmaraie, M. (1994). Muscle proteinases and meat aging. Meat Science, 36, 93
104. Korhonen, H., & Pihlanto, A. (2003). Food-derived bioactive peptides: Opportunities for designing future foods. Current Pharmaceutical Design, 9, 1297
1308. Lafarga, T., & Hayes, M. (2014). Bioactive peptides from meat muscle and by-products: Generation, functionality and application as functional ingredients. Meat Science, 98, 227
239. Lappa, I. K., Papadaki, A., Kachrimanidou, A., Terpou, A., Koulougliotis, D., Eriotou, E., & Kopsahelis, N. (2019). Cheese whey processing: Integrated biorefinery concepts and emerging food applications. Foods, 8. Available from https://doi.org/10.3390/foods8080347, Article 347. Li, B., Chen, F., Xang, X., Ji, B., & Wu, Y. (2007). Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography. Food Chemistry, 102, 1135
1143. Lingnert, H., & Eriksson, C. (1980). Antioxidative Maillard reaction products. II. products from sugars and peptides or proteins hydrolysates. Journal of Food Processing and Preservation, 4, 173
181. Liu, R., Xing, L., Fu, Q., Zhou, G.-H., & Zhang, W.-G. (2016). A review of antioxidant peptides derived from meat muscle and by-products. Antioxidants, 5, 32
46. Lo´pez, C. M., Bru, E., Vignolo, G., & Fedda, S. G. (2015). Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages. Meat Science, 104, 20
29. Losso, J. N. (2016). The Maillard reaction reconsidered. Boca Raton: CRC Press. Lu, C., Hao, Z., Payne, R., & Ho, C. (2005). Effects of water content on volatile generation and peptide degradation in the Maillard reaction of glycine, diglycine, and triglycine. Journal of Agricultural and Food Chemistry, 53, 6443
6447. Maestri, E., Pavlicevic, M., Montorsi, M., & Marmiroli. (2018). Meta-analysis for correlating structure of bioactive peptides in foods of animal origin with regard to effect and stability. Comprehensive Reviews in Food Science and Food Safety, 18, 3
30. Mazorra-Manzano, M. A., Robles-Porchas, G. R., Gonza´lez-Vela´zquez, D. A., Torres-Llanez, M. J., Martı´nezPorchas, M., Garcı´a-Sifuentes, C. O., . . . Vallejo-Co´rdoba, B. (2020). Cheese whey fermentation by its native microbiota: Proteolysis and bioactive peptides release with ACE-inhibitory activity. Fermentation, 6. Available from https://doi.org/10.3390/fermentation6010019, Article 19. Mellander, O. (1950). The physiological importance of the casein phosphopeptide calcium salts II: Peroral calcium dosage in infants. Acta Societatis Medicorum Upsaliensis, 55, 247
255. Mirdhayati, I., Hermanianto, J., Wijaya, C. H., Sajuthi, D., & Arihara, K. (2015). Angiotensin converting enzyme (ACE) inhibitory and antihypertensive activities of protein hydrolysate from meat of Kacang goat (Capra aegagrus hircus). Journal of Science of Food and Agriculture, 96, 3536
3542. Mora, L., & Toldra´, F. (2014). Bioactive peptides generated from meat industry by-products. Food Research International, 65, 344
349. Mora, L., Reig, M., & Toldra´, F. (2014). Bioactive peptides generated from meat industry by-products. Food Research International, 65, 344
349. Mora, L., Reig, M., & Toldra´, F. (2019). Bioactive compounds from animal meat byproducts. In B. K. Simpson, A. N. A. Aryee, & F. Toldra´ (Eds.), Byproducts from agriculture and fisheries: Adding value for food, feed, pharma, and fuels. NJ, USA: John Wiley & Sons, 10.1002/9781119383956.ch14. Mora, L., Sentandreu, M. A., Fraser, P. D., Toldra´, F., & Bramley, P. M. (2009). Oligopeptides arising the degradation of creatine kinase in Spanish dry-cured ham. Journal of Agricultural and. Food Chemistry, 57, 8982
8988. Morales, F., Somoza, V., & Fogliano, V. (2012). Physiological relevance of dietary melanoidins. Amino Acids, 42, 1097
1109. Nagaoka, S., Futamura, Y., Miwa, K., Awano, T., Yamauchi, K., Kanamaru, Y., . . . Kuwata, T. (2001). Identification of novel hypocholesterolemic peptides derived from bovine milk β-lactoglobulin. Biochemical and Biophysical Research Communications, 281, 11
17.

Bioactivity generation from proteins of animal sources 423 Nakamura, Y., Yamamoto, N., Sakai, K., & Takano, T. (1995). Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. Journal of Dairy Science, 78, 1253
1257. Nie, X., Xu, D., Zhao, L., & Meng, X. (2017). Antioxidant activities of chicken bone peptide fractions and their Maillard reaction products: Effects of different molecular weight distributions. International Journal of Food Properties, 20(sup1), S457
S466. Nooshkam, M., & Madadlou, A. (2016). Maillard conjugation of lactulose with potentially bioactive peptides. Food Chemistry, 192, 831
836. Nwachukwu, I. D., & Aluko, R. E. (2019). Anticancer and antiproliferative properties of food-derived protein hydrolysates and peptides. Journal of Food Bioactives. Available from https://doi.org/10.31665/ JFB.2019.7194. Nyberg, F., Sanderson, K., & Glamsta, E.-L. (1997). The hemorphins: A new class of opioid peptides derived from the blood protein hemoglobin. Biopolymers, 43, 147
156. Ohata, M., Uchida, S., Zhou, L., & Arihara, K. (2016). Antioxidant activity of fermented meat sauce and isolation of an associated antioxidant peptide. Food Chemistry, 194, 1034
1039. Ohata, M., Zhou, L., Owashi, C., & Arihara, K. (2014). The effect of odor generated from protein digests and reducing sugars by the Maillard reaction on blood pressure. IMARS News Letters, 9, 21
25. Ohata, M., Zhou, L., Yada, Y., Yokoyama, I., & Arihara, K. (2020). 2,3-Dimethylpyrazine (3DP) and 2,5dimethyl-4-hydroxy-3(2H)-furanone (DMHF) generated by the Maillard reaction in foods affect autonomic nervous activity and central nerve activity in human. Bioscience, Biotechnology, and Biochemistry. Available from https://doi.org/10.1080/09168451.2020.1775066. Ordonez, J. A., Hierro, E. M., Bruna, J. M., & de la Hoz, L. (1998). Changes in the components of dry fermented sausages during ripening. Critical Reviews in Food Science and Nutrition, 39, 329
367. Owusu-Apenten, R. (2010). Bioactive peptides applications for improving nutrition and health. Baca Raton: CRC Press. Pare´s, D., Saguer, E., & Carretero, C. (2011). Blood by-products as ingredients in processed meat. In J. P. Kerry, & J. F. Kerry (Eds.), Processed meats: Improving safety, nutrition and quality (pp. 219
242). Oxford: Woodhead Publishing. Pathera, A. K., Jairath, G., Singh, P. K., & Yadav, S. (2017). Health promoting functional properties of meat and meat products. In D. Mudgil, & S. Barak (Eds.), Functional foods: Sources and health benefits (pp. 333
362). India: Scientific Publishers. Patra, A. K., & Saxena, J. (2011). Exploitation of dietary tannins to improve rumen metabolism and ruminant nutrition. Journal of the Science of Food and Agriculture, 91, 24
37. Pavlicevic, M., Maestri, E., & Marmiroli, M. (2020). Marine bioactive peptides-an overview of generation, structure and application with a focus on food sources. Marine Drugs, 18. Available from https://doi.org/ 10.3390/md18080424, Article 424. Pegg, R. B., & Shahidi, F. (2014). Flavor development. In M. Dikeman, & C. Devine (Eds.), Encyclopedia of meat sciences (second edition, volume 1). Oxford: Elsevier/Academic Press. Pihlanto, A., & Korhonen, H. (2003). Bioactive peptides and proteins. Advances in Food and Nutritional Research, 47, 175
276. Pihlanto-Leppa¨la¨, A. (2001). Bioactive peptides derived from bovine whey proteins: opioid and ACE inhibitory peptides. Trends in Food Science and Technology, 11, 347
356. Przybylski, R., Firdaous, L., Chaˆtaigne´, G., Dhulster, P., & Nedjar, N. (2016). Production of an antimicrobial peptide derived from slaughterhouse by-product and its potential application on meat as preservative. Food Chemistry, 211, 306
313. Rezaharsamto, B., & Subroto, E. (2019). A review on bioactive peptides derived from various sources of meat and meat by-products. International Journal of Scientific and Technology Research, 8, 3151
3156. Rizzello, C. G., Losito, I., Gobbetti, M., Carbonara, T., De Bari, M. D., & Zambonin, P. G. (2005). Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties. Journal of Dairy Science, 88, 2348
2360.

424 Chapter 17 Roman, S., Sanchez-Siles, L. M., & Siegrist, M. (2017). The importance of food naturalness for consumers: results of a systematic review. Trends in Food Science and Technology, 67, 44
57. Ryan, J. T., Ross, R. P., Bolton, D., Fitzgerald, G. F., & Stanton, C. (2011). Bioactive peptides from muscle sources: meat and fish (review). Nutrients, 3, 765
791. Ryder, K., Bekhit, A. E.-D., McConnell, M., & Carne, A. (2016). Towards generation of bioactive peptides from meat industry waste proteins: generation of peptides using commercial microbial proteases. Food Chemistry, 208, 42
50. Saiga, A., Okumura, T., Makihara, T., Katsuta, S., Shimizu, T., Yamada, R., & Nishimura, T. (2003). Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract. Journal of Agricultural and Food Chemistry, 51, 1741
1745. Saiga, A., Tanabe, S., & Nishimura, T. (2003). Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. Journal of Agricultural and Food Chemistry, 51, 3661
3667. Sa´nchez, A., & Va´zquez, A. (2017). Bioactive peptides: a review. Food Quality and Safety, 1, 29
46. Sarraga, C., Gil, M., Arnau, J., Monfort, J. M., & Cusso, R. (1989). Effect of curing salt and phosphate on the activity of porcine muscle proteases. Meat Science, 25, 241
249. Sayd, T., Chambon, C., & Sante´-Lhoutellier, V. (2016). Quantification of peptides released during in vitro digestion of cooked meat. Food Chemistry, 197, 311
1323. Sentandreu, M. A., Stoeva, S., Aristoy, M. C., Laib, K., Voelter, W., & Toldra´, F. (2003). Identification of small peptides generated in Spanish dry-cured ham. Journal of Food Science, 68, 64
69. Shahidi, F., Samaranayaka, A. G. P., & Pegg, R. B. (2014). Flavor development. In M. Dikeman, & C. Devine (Eds.), Encyclopedia of meat sciences (second edition, volume 1). London: Elsevier/ Academic Press. Simonetti, A., Gambacorta, E., & Perna, A. (2016). Antioxidative and antihypertensive activities of pig meat before and after cooking and in vitro gastrointestinal digestion: Comparison between Italian autochthonous pig Suino Nero Lucano and a modern crossbred pig. Food Chemistry, 212, 590
595. Somoza, V. (2005). Five years of research on health risks and benefits of Maillard reaction products: an update. Molecular Nutrition and Food Research, 49, 663
672. Su, G. W., Zhao, T. T., Zhao, Y. Q., Sun-Waterhouse, D., Qiu, C. Y., Huang, P., & Zhao, M. M. (2016). Effect of anchovy (Coilia mystus) protein hydrolysate and its Maillard reaction product on combating memoryimpairment in mice. Food Research International, 82, 112
120. Suetsuna, K., Ukeda, H., & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. Journal of Nutritional Biochemistry, 11, 128
131. Terashima, M., Baba, T., Ikemoto, N., Katayama, M., Morimoto, T., & Matsumura, S. (2010). Novel angiotensin-converting enzyme (ACE) inhibitory peptides derived from boneless chicken leg meat. Journal of Agricultural and Food Chemistry, 58, 7432
7436. Toldra´, F. (2014). Dry. In M. Dikeman, & C. Devine (Eds.), Encyclopedia of meat sciences (2nd ed., volume 1). Oxford: Elsevier/Academic Press. Toldra´, F., & Flores, M. (1998). The role of muscle proteases and lipases in flavor development during the processing of dry-cured ham. Critical Reviews in Food Science and Nutrition, 38, 331
352. Toldra´, F., Aristoy, M.-C., Mora, L., & Reig, M. (2012). Innovations in value-addition of edible meat byproducts. Meat Science, 92, 290
296. Toldra´, F., Gallego, M., Reig, M., Aristoy, M.-C., & Mora, L. (2020a). Recent progress in enzymatic release of peptides in foods of animal origin and assessment of bioactivity. Journal of Agricultural and Food Chemistry. Available from https://doi.org/10.1021/acs.jafc.9b08297. Toldra´, F., Gallego, M., Reig, M., Aristoy, M.-C., & Mora, L. (2020b). Bioactive peptides generated in the processing of dry-cured ham. Food Chemistry, 321. Available from https://doi.org/10.1016/j. foodchem.2020.126689, Article 126689. Toldra´, F., Mora, L., & Reig, M. (2016). New insights meat by-product utilization. Meat Science, 120, 54
59. Toldra´, F., Reig, M., Aristoy, M.-C., & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395
404.

Bioactivity generation from proteins of animal sources 425 Udenigwe, C. C., & Howard, A. (2013). Meat proteome as source of functional biopeptides. Food Research International, 54, 1021
1032. Van Lancker, F., Adams, A., & De Kimpe, N. (2011). Chemical modifications of peptides and their impact on food properties. Chemical Reviews, 111, 7876
7903. Vargas-Bello-Pe´rez, E., Ma´rquez-Herna´ndez, R. I., & Herna´ndez-Castellano, L. E. (2019). Bioactive peptides from milk: Animal determinants and their implications in human health. Journal of Dairy Research, 86, 136
144. Ventanas, J., Cordoba, J. J., Antequera, T., Garcia, C., Lo´pez-bote, C., & Asensio, M. A. (1992). Hydrolysis and Maillard reactions during ripening of Iberian ham. Journal of Food Science, 57, 813
815. Weerawatanakorn, M., Wu, J.-C., Pan, M.-H., & Ho, C.-T. (2015). Reactivity and stability of selected flavor compounds. Journal of Food and Drug Analysis, 23, 176
190. Wu, J., Jahandideh, F., Yu, W., & Majumder, K. (2015). Bioactive peptides from meat proteins as functional food components. In G. Girillo, U. Gianfranco Spizzirri, & F. Iemma (Eds.), Functional polymers in food science (pp. 181
208). Beverly: Scrivener Publishing. Xiao, C., Toldra´, F., Zhou, F., Gallego, M., Zhao, M., & Mora, L. (2020). Effect of cooking and in vitro digestion on the peptide profile of chicken breast muscle and antioxidant and alcohol dehydrogenase stabilization activity. Food Research International, 136. Available from https://doi.org/10.1016/j. foodres.2020.109459, Article 109459. Xing, L., Liu, R., Cao, S., Zhang, W., & Zhou, G. (2019). Meat protein based bioactive peptides and their potential functional activity: A review. International Journal of Food Science and Technology, 54, 1956
1966. Yaghoubzadeh, Z., Ghadikolaii, F. P., Kaboosi, H., Safari, R., & Fattahi, E. (2020). Antioxidant activity and anticancer effect of bioactive peptides from rainbow trout (Oncorhynchus mykiss) skin hydrolysate. International Journal of Peptide Research and Therapeutics, 26, 625
632. Yokoyama, I., Nakai, Y., Suzuki, Y., Ohata, M., Komiya, Y., Nagasao, J., & Arihara, K. (2020). DMHF (2,5-dimethyl-4-hydroxy-4-hydroxy-3(2H)-furanone), a volatile food component generated by the Maillard reaction, promotes appetite and changes gene expression in the rat brain through inhalation. Journal of Food Science, 85, 1338
1343. Yokoyama, I., Ohata, M., Komiya, Y., Nagasao, J., & Arihara, K. (2020). Inhalation of odors containing DMHF generated by the Maillard reaction affects physiological parameters in rat. Scientific Reports, 10. Available from https://doi.org/10.1038/s41598-020-70843-z, Article 13931. Yoshikawa, M. (1996). Selection of proteases for production of biologically active peptides from food proteins. Japanese Journal of Dairy Food Science, 45, A51
A60. Young, D., & Mine, Y. (2009). Functional bioactive proteins and peptides in nutrigenomics. In Y. Mine, K. Miyashita, & F. Shahidi (Eds.), Nutrigenomics and proteomics in health and disease (pp. 129
144). Ames: Wiley-Blackwell. Yu, Y., Hu, J. N., Miyaguchi, Y., Bai, X. F., Du, Y. G., & Lin, B. C. (2006). Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin. Peptides, 27, 2950
2956. Zha, F. C., Wei, B. B., Chen, S. J., Dong, S. Y., Zeng, M. Y., & Liu, Z. Y. (2015). The Maillard reaction of a shrimp by-product protein hydrolysate: Chemical changes and inhibiting effects of reactive oxygen species in human HepG2 cells. Food and Function, 6, 1919
1927. Zhao, Q., Garreau, I., Sannier, F., & Piot, J. M. (1997). Opioid peptides derived from hemoglobin: Hemorphins. Biopolymers, 43, 75
98. Zhou, L., Ohata, M., & Arihara, K. (2016). Effects of odor generated from the glycine/glucose Maillard reaction on human mood and brainwaves. Food and Function, 7, 2574
2581. Zhou, L., Ohata, M., Owashi, C., Nagai, K., Yokoyama, I., & Arihara, K. (2018). Odors generated from the Maillard reaction affect autonomic system. Journal of the Science of Food and Agriculture, 98, 923
927. Zotta, T., Soloeri, L., Iacumin, L., Picozzi, C., & Gullo, M. (2020). Valorization of cheese whey using microbial fermentation. Applied Microbiology and Biotechnology, 104, 2749
2764.

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CHAPTER 18

Sustainable, alternative sources of bioactive peptides J.E. Aguilar-Toala´, F.G. Hall, U. Urbizo-Reyes and A.M. Liceaga Protein Chemistry and Bioactive Peptides Laboratory, Department of Food Science, Purdue University, West Lafayette, IN, United States

18.1 Introduction The rise on the worlds’ population is estimated to reach 9 billion by 2050 (FAO, 2014), indicating that world food demand will need to increase by at least 50%, with animal-based foods increasing to 70%. To meet the rise in food demand, farmers must produce B60% more crop calories (7400 trillion calories), increasing land use by 593 million hectares (Searchinger, Walte, Hanson, & Ranganathan, 2019). These data confirm the need to develop an alternative agriculture system that considers the rising population and looks beyond conventional food sources. Consumption of alternative protein sources would alleviate environmental stress caused by conventional agriculture, yet it is a matter of finding these alternatives and pleasing market trends to produce long-term changes. Rising consumer concerns on the safety and secondary effects of synthetic drugs to treat noncommunicable diseases has promoted interest toward using natural, biological active constituents that offer similar health benefits but with limited or no side effects (Mine, Li-Chan, & Jiang, 2010). In addition, over the years, there has been a shift in consumer preference for high-protein foods and consumption of sustainable protein sources such as fungi, insects, marine macroalgae, and agricultural by-products (Fig. 18.1). As consumption of these alternative protein sources continues to rise, research efforts have begun to focus on the discovery of novel bioactive peptides derived from these emerging proteins.

18.2 Fungi Fungi are categorized into four classes: (1) Chytridiomycota (chytrids), (2) Zygomycota (bread molds), (3) Ascomycota (yeasts and sac fungi), and (4) Basidiomycota (club fungi). Within these groups, mushrooms and microfungi (associated with marine microorganisms) Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00004-2 © 2021 Elsevier Inc. All rights reserved.

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428 Chapter 18

Figure 18.1 Alternative, emerging protein sources of biological active peptides.

are more commonly used as a source of bioactive peptides, whereas yeasts, molds, and other fungal microorganisms are used to liberate active peptides from various food sources. Many fungi are known for their health-promoting properties and have been widely used in traditional medicine for centuries. Their exact therapeutic properties, applications, and mechanisms of their biological activity have gained public interest in the past decade. The significance of fungi as unconventional sources of bioactive compounds progressed after the discovery of penicillin’s (Penicillium notatum) antibacterial activity, by Alexander Fleming in 1928. Since then, studies have revealed the crucial role of fungi in anticancer therapy, immune system stimulation, as well as their antimicrobial activity, and their potential for regulating and preventing hyperglycemia and hypercholesterolemia. These properties, when combined with the absence of toxicity, make these compounds ideal for functional food or nutraceutical development as consumers demand more health-conscious foods and naturally sourced medicines. A number of bioactive constituents have been isolated from or produced by fungi organisms including polysaccharides, proteins/peptides, and molecular complexes (Giavasis, 2014). Fungal polysaccharides, in particular, were evaluated in clinical trials which resulted in the treatment of various diseases including cancer (Wasser, 2017). Although polysaccharides have received the most attention, bioactive proteins constitute another important part for their functional components with relevant biological activities for human health.

18.2.1 Major fungi protein and mechanisms of extraction Fungi produce a large number of well-studied therapeutic proteins and peptides, primarily lectins, fungal immunomodulatory proteins, ribosome-inactivating proteins, antimicrobial

Sustainable, alternative sources of bioactive peptides 429 proteins, ribonucleases, ubiquitin-like peptides, proteases, laccases, and hundreds of other biologically relevant peptides (Ng, 2004; Xu, Yan, Chen, & Zhang, 2011). Proteins/peptides are generally extracted by solvent extraction, isolation, purification, or a combination of all, depending on the type of fungi (Attarat & Phermthai, 2015; Chen, Wang, & Wu, 2009). In some cases, peptides are isolated from the fungal biomass culture media using solvents, most commonly ethyl acetate, and then evaporated under vacuum at 40 C to a semi-solid residue. Extracts are usually subjected to a series of chromatographic fractionation using traditional stationary phases, such as silica gel and Sephadex, to fully purify and isolate individual peptides in their single forms. Sheu, Chien, Chien, Chen, and Chin (2004) detailed purification of an immunomodulatory protein from the fruiting body edible Jew’s Ear mushroom (Auricularia polytricha) using acid extraction, ammonium sulfate fractionation, then anion-exchange chromatography. The purified protein was found to increase splenocytes proliferation and gamma-interferon secretion, as well as to enhance nitric oxide production and tumor necrosis factor-alpha (TNF-α), suggesting a new hemoagglutinative protein, amyloid precursor protein (APP), as an immune stimulant that can strengthen the immune response. Other researchers purified immunomodulatory peptides from various fungal species with a similar approach (Kong et al., 2013; Li et al., 2017; Li, Chang, He, Chen, & Zhou, 2019; Li, Shi, Ding, Nie, & Tang, 2015; Ng, 2004). There is a great need for different technologies to overcome the time, cost, and yieldlimiting factors of traditional extraction and isolation techniques. Hence, more novel extraction methods are employed to increase protein yield by aiding cell wall degradation. For example, enzyme-assisted extraction using commercial enzymes such as chitinase or glucanase is used to break the bond between polymers in the fungal cell walls (Li, Wen, Zhang, Aa, & Liu, 2011). To improve the efficiency of enzymatic extraction, preprocessing such as conventional pulsed electric field, ultrasound, and microwave-assisted extractions demonstrates to improve efficiency to recover valuable protein components (Zhao, Huo, Qian, Ren, & Lu, 2017).

18.2.2 Bioactive properties of peptides derived from fungi To date, fungi species have been reported to render biological activity consisting mostly of antibacterial, antiproliferative, immunomodulatory, anti-inflammatory, and anticancer properties. Cordymin, for example, a low molecular weight peptide (10.9 kDa), has been purified from the mushrooms Cordyceps sinensis and Cordyceps militaris. Quan et al. reported anti-inflammatory and analgesic properties of cordymin from C. sinensis (Qian, Pan, & Guo, 2012). Cordymin treatment decreased the levels of TNF-α, interleukin-1β, and total antioxidant status. Further, this peptide was shown to dose-dependently inhibit the acetic acid-induced abdominal constrictions in mice and strong inhibitory activity (IC50 5 0.1 μM) against neurolysin (Qian et al., 2012). Wang et al. (2012) investigated the effects

430 Chapter 18 of cordymin to prevent focal cerebral ischemic/reperfusion injury. Oral treatment of cordymin significantly boosted the defense mechanism against cerebral ischemia by increasing antioxidant activity related to lesion pathogenesis. They also found that cordymin induced upregulation of C3 protein level, interleukin-1β, and TNF-α. Cordymin also demonstrated antifungal activity against HIV-1 reverse transcriptase with an IC50 of 55 μM and antiproliferative activity toward breast cancer cells (MCF-7) (Wong et al., 2011). Some fungal species were also confirmed to possess antihypertensive properties. Peptide fractions from yeast derived from spent brewer’s grains decreased systolic, diastolic, and mean blood pressure of spontaneously hypertensive rats and showed the highest antioxidant effect (Amorim et al., 2019). Amorim and their team saw that low molecular weight peptides formed during autolysis and hydrolysis of spent brewer’s yeast were efficiently absorbed in an active form to decrease the blood pressure of animals (Amorim et al., 2019). The researchers observed that fractions below 3 kDa containing triand tetra-peptides with hydrophobic amino acid residues—SPQW, PWW, and RYW— caused the most noticeable effect. Similarly, a peptide (WALKGYK) isolated from Tricholoma matsutake mushrooms exerted antihypertensive activity in spontaneously hypertensive rats at a dosage of 25 mg/kg compared to 400 mg/kg of the crude water extract. Likewise, a number of other fungi-derived peptides showed clear antihypertensive and antioxidant effects (Claude et al., 2017; Ibadallah, Abdullah, & Shuib, 2015; Khan & Tania, 2012; Lau, Abdullah, & Shuib, 2013; Lau, Abdullah, Shuib, & Aminudin, 2014; Sun, He, & Xie, 2004; Wu & Wang, 2009). There are about 131 reported peptides from fungal marine microorganisms that also contribute to the majority of fungi-derived bioactive peptides (Youssef, Ashour, Singab, & Wink, 2019). Table 18.1 summarizes some of the reports available in the literature. A majority of novel isolated fungal peptides have not yet been confirmed in vivo or preclinical trials. There are only a few commercialized peptides from fungi readily available for therapeutic treatment such as peptides from ergot alkaloid (Claviceps purpurea) containing products and penicillin (Penicillium chrysogenum). For example, cyclosporine, a cyclic nonribosomal peptide, is an immunosuppressant drug produced by a species of soil fungus (Tolypocladium inflatum). Its primary use is to suppress patients’ immune systems before transplant surgery (Fisher & Yang, 2002). Additionally, commercial fungal proteases such as corolases and flavorzyme are well-known and widely utilized in research and industry for protein extraction and generation of bioactive peptides (Ngai, Zhao, & Ng, 2005). However, protein
polysaccharide complexes have been used as chemoimmunotherapy agents for over 30 years. Polysaccharide-K and polysaccharopeptide are both protein-bound polysaccharides derived from the CM-101 and COV-1 strains of the fungus Coriolus versicolor (Cui & Chisti, 2003). Both complexes have widely documented anticancer properties in vivo and in human clinical trials (Cui & Chisti, 2003; Fisher & Yang, 2002).

Sustainable, alternative sources of bioactive peptides 431 Table 18.1: Bioactive properties from peptides derived from fungi species. Fungi Enokitake mushroom (Flammulina velutipes)

Chesnut mushroom (Agrocybe cylindracea)

Lingzhi mushroom (Ganoderma microsporum)

Medicinal mushroom (Pholiota adiposa)

Psychrotolerant fungus (Penicillium algidum)

Mushroom (Russula paludosa) Red algae fungus (Ceratodictyon spongiosum)

Medicinal mushroom (Cordyceps sinensis)

Form

Extraction/production

Peptide Glycan

Peptide Agrocybin

Ion exchange and affinity chromatography of water extract

Bioactivity

References

Antioxidant, anticancer activity, antiaging property; immunomodulatory, antiviral action Antifungal

Kong et al. (2013)

Ngai et al. (2005)

Immunomodulatory: Lin et al. suppress tumor (2010) invasion and metastasis. Antihypertensive Antiproliferative: Lectin Ion exchange and Zhang, Sun, hepatoma Hep G2 affinity Wang, and Ng and breast cancer chromatography of (2009) MCF-7 cells. HIV-1 water extract transcriptase inhibition. Antifungal Antimicrobial, Nitropeptide, C18 flash Dalsgaard, psychrophilin D chromatography, LH- antiviral, anticancer, Larsen, and and antiplasmodial Christophersen 20 Sephadex and preparative HPLC (2005) Peptide HIV-1 transcriptase KREHGQHCEF inhibition Dictyonamide Separated from algae, Antiproliferation: Komatsu, Peptide grown in PYG broth, CDK 4 inhibitor Shigemori, and solvent extraction, and Kobayashi chromatography (2001) separation (gel and C18) Peptide Cordymin Water extract; SPAnti-inflammatory Qian et al. Sepharose. and antinociceptive (2012); Wang et al. (2012); Wong et al. (2011)

Nevertheless, the bioactive potential of fungi is embodied through inhibition of disease-specific processes, targeted activation of therapeutic compounds, or immune involvement. Immunomodulatory actions seem to be common among peptides from various fungi species. From the available data, it is very probable that other, less studied, fungi species contain peptides with similar effects. Thus potential sources of bioactive peptides from fungi are seemingly endless. Future studies should continue to investigate the exact mechanisms of fungiderived peptides and their role in therapy to support their progression to in vivo testing.

432 Chapter 18

18.3 Edible insects There are approximately 2000 edible insect species (e.g., beetles, ants, grasshoppers, crickets, termites, and flies) consumed by more than 2 billion people worldwide. In fact, entomophagy, the practice of eating insects, remains prevalent in over 100 countries around the globe and holds valuable aspects of cultural identity and spiritual attributes (Ramos-Elorduy, 2009). The nutritional composition of insects consists of protein, including all essential amino acids (20%
76% of dry matter), lipids (2%
50% of dry matter), chitin (2.7
49.8 mg/kg of fresh matter), and minerals (calcium, phosphorous, potassium, and magnesium), depending on the species, life cycle stage and feed/diet composition (Klunder, Wolkers-Rooijackers, Korpela, & Nout, 2012; Sosa & Fogliano, 2017; Womeni et al., 2012). In terms of protein quality, true fecal-nitrogen levels in rat models report values between 86% and 89% for insects, which are higher than those of many plant-based proteins, but lower than those reported for other animal proteins (egg 95%, beef 98%, and casein 99%) (Finke, 2004). This is because some protein remains bound to the insects’ exoskeleton and may not be readily available when consumed. Recently, edible insects have gained attention as emerging protein sources to help alleviate the food demand in a growing world population. Interest in entomophagy has also surged in western cultures due to consumers’ willingness to eat more sustainable and environment-friendly proteins, driving insectfocused product development in these markets (Liceaga, 2019). For example, protein bars, baked goods, tortillas, pasta, and snack products formulated with insect protein are widely reported in the literature (Azzollini, Derossi, Fogliano, Lakemond, & Severini, 2018; Bußler, Rumpold, Jander, Rawel, & Schlu¨ter, 2016; Calzada-Luna, San Martin-Gonzalez, Mauer, & Liceaga, 2020; Caparros Megido et al., 2014; Gmuer, Nuessli Guth, Hartmann, & Siegrist, 2016; Gonza´lez, Garzo´n, & Rosell, 2018; Hall, Jones, O’Haire, & Liceaga, 2017; House, 2016; Leiber et al., 2017; Megido et al., 2016; Osimani et al., 2018). As insects begin to be used more as protein sources for human food and animal feed, there is great interest in exploring additional health benefits of this novel protein source (Liceaga, 2019). Research has demonstrated that extracts derived from edible insects (e.g., crickets, mealworms, larvae, and beetles) possess various disease-preventing properties including the ability to delay oxidants, regulate glucose, manage pathways involved in hypertension, reduce inflammation, and inhibit proliferation in cancerous cells.

18.3.1 Extraction of bioactive peptides from insects Insect-derived biopeptides have typically been obtained by solvent extraction, isolation, purification, or proteolysis. However, enzymatic proteolysis appears to be the primary method used to liberate biopeptides from native insect proteins, since some commercial

Sustainable, alternative sources of bioactive peptides 433 enzymes (e.g., alcalase) facilitate the separation of the insoluble chitin fraction (Dai, Ma, Luo, & Yin, 2013; Hall, Johnson, & Liceaga, 2018; Nongonierma & FitzGerald, 2017; Nongonierma, Lamoureux, & FitzGerald, 2018). The literature shows that the active peptide fractions become more readily available after proteolysis, as commonly seen with other protein-derived bioactive peptides (Korhonen & Pihlanto, 2003, 2006). Enzymatic proteolysis also seems to be the most effective approach at liberating the protein in the insect exoskeleton that is complexed with the chitin polymerized matrix (Calzada-Luna et al., 2020). As with other protein-rich resources, to improve the efficiency of enzymatic extraction, preprocessing such as oil-separation, ultrasound, and microwave-assisted extractions has been suggested as potential methods to recover bioactive components (Zhao et al., 2017). These protein extracts can also be subjected to a series of purification steps such as chromatographic fractionation, to fully purify and isolate individual peptides in their single forms.

18.3.2 Bioactivity of peptides derived from insects Research on insect-derived bioactive peptides is relatively new. Some of the early reports on insect bioactives are from Vercruysse, Smagghe, Beckers, and Van Camp (2009) who reported that enzymatic hydrolysis was necessary to release bioactive peptides with angiotensin-converting enzyme (ACE) inhibitory activity from cotton leafworm (Spodoptera littoralis). Gastrointestinal digestion of the S. littoralis peptides, using pepsin, trypsin, and chymotrypsin, also improved the IC50 values significantly (from 73 to 0.7 mg/mL). The potency of the ACE-inhibitory tri-peptides (Ala-Val-Phe) isolated from S. littoralis was later confirmed to have antihypertensive activity in vivo (Vercruysse et al., 2010). Since then, numerous studies on insect-derived bioactive peptides have emerged. Lu and Chen (2010) isolated a glycine-rich peptide (SK84) from larvae of black soldier fly (Drosophila virilis) that displayed inhibitory effects on the proliferation of several cancer cell lines (e.g., human leukemia THP-1, liver cancer HepG2, and breast cancer MCF-7 cells). SK84 also showed antimicrobial activity toward Gram-positive bacteria. Other studies have shown that protein hydrolysates from cotton leafworm larvae (S. littoralis) (Vercruysse et al., 2009), silkworm (Bombyx mori) (Wu, Jia, Yan, Du, & Gui, 2015), and modified peptides from silkworm pupa have the ability to inhibit ACE in vitro (Tao et al., 2017). Similarly, in vitro antioxidant capabilities of peptides obtained from insect protein hydrolysates have been reported. For example, cotton leafworm larvae (S. littoralis) (Vercruysse et al., 2009), cricket (Amphiacusta annulipes, Gryllodes sigillatus), super worm (Zophobas morio), and locust (Locusta migratoria) (Zielin´ ska, Karas´, & Jakubczyk, 2017) protein hydrolysates all demonstrated antioxidant activity comparable to that of other food protein hydrolysates such as fish, wheat germ, and flaxseed protein (Chalamaiah, Dinesh Kumar, Hemalatha, & Jyothirmayi, 2012; Hall et al., 2018; Karamac, Kosinska-Cagnazzo, &

434 Chapter 18 Kulczyk, 2016; Zhu, Zhou, & Qian, 2006). Zhang, Chen, et al. (2016), Zhang, Wang, et al. (2016) evaluated the potential of antidiabetic activity of silkworm (B. mori) peptides using a quantitative structure
activity relationship approach to predict α-glucoside inhibitory peptides. The authors identified two potent peptides Ser-Gln-Ser-Pro-Ala (IC50 20 μm) and Gln-Pro-Gly-Arg (IC50 65.8 μm); the former having similar values as the positive control used in the study (Zhang, Chen, et al., 2016; Zhang, Wang, et al., 2016). Table 18.2 lists a summary of representative literature reported on bioactive peptides from different insect species. Nongonierma and FitzGerald (2017), who provided a

Table 18.2: Bioactive properties from different edible insect species. Insect(s) species

Reported bioactivity

References

Bombyx mori Bombus terrestris Schistocerca gregaria Spodoptera littoralis S. littoralis

ACE inhibition

Vercruysse, Smagghe, Herregods, and Van Camp (2005)

ACE inhibition

Vercruysse, Smagghe, Matsui, and Van Camp (2008) Vercruysse et al. (2009)

S. littoralis B. mori pupae defatted dry powder B. mori larvae

B. mori Tenebrio molitor larvae flour B. mori pupae B. mori pupae B. mori pupae powder B. mori pupae powder B. mori pupae B. mori pupae

Blaptica dubia Gromphadorhina portentosa Locusta migratoria Anomonotes annulipes Zophobas morio Gryllodes sigillatus

ACE inhibition and antioxidant (DPPH scavenging and FRAP) ACE inhibition

Wang et al. (2011)

ACE inhibition and antioxidant (DPPH scavenging, Fe21chelating and Fe21reducing activities) Antioxidant (DPPH scavenging) ACE inhibition

Wu, Jia, Tan, Xu, and Gui (2011)

ACE inhibition ACE inhibition ACE inhibition ACE inhibition ACE inhibition ACE inhibition and antioxidant (DPPH scavenging, Fe21chelating and total reducing activities) Antioxidant (ABTS and DPPH scavenging, Fe21chelating, Fe21reducing an and Cu21chelating activities)

Li et al. (2014) Wang, Wang, and Zhang (2014) Jia, Wu, Yan, and Gui (2015) Wu et al. (2015) Tao et al. (2017) Zhou, Ren, Yu, Jia, and Gui (2017)

ACE inhibition and antioxidant (DPPH scavenging and FRAP), DPP-IV

Yang et al. (2013) Dai et al. (2013)

Zieli´ nska, Baraniak, and Kara´s (2017)

Hall et al. (2018); Hall and Liceaga (2020)

ACE, Angiotensin-converting enzyme; DPP-IV, dipeptidyl peptidase-IV; DPPH, 2,2-diphenyl-1-picryl-hydrazyl-hydrate; FRAP, ferric reducing antioxidant power.

Sustainable, alternative sources of bioactive peptides 435 comprehensive review study on insect bioactive peptides, reported that peptides from B. mori are some of the most extensively studied and are reported to have antioxidant, antihypertensive, antidiabetic, and hypocholesterolemic properties in vivo (Nongonierma & FitzGerald, 2017). Hall et al. (2018) showed that peptides from tropical banded cricket (G. sigillatus) had better in vitro ACE and dipeptidyl peptidase-IV (DPP-IV) inhibiting activity, compared with the native cricket protein; these bioactivities further increased after simulated gastrointestinal digestion. Increased proteolysis time resulted in the best overall bioactivity due to the presence of much smaller molecular weight peptides. In a similar study, Hall and Liceaga (2020) demonstrated that microwave-assisted enzymatic hydrolysis can be a useful method for generating bioactive peptides from insect proteins. In this study, ACE and DPP-IV inhibition was highest in the microwave-hydrolyzed protein (IC50 5 0.096 mg/mL and 0.27 mg/mL, respectively) compared to peptides derived from conventional protein hydrolysis. Although research is still in its early stage, knowledge already attained shows that edible insects can be a sustainable source of bioactive peptides for human health.

18.4 Marine macroalgae Macroalgae, also known as seaweed, are chlorophyll-containing organisms existing in three main categories green, brown, and red algae. They are composed of a group of cells arranged in colonies or as an organism (Kim, 2011). Seaweed consumption has been a common practice in East Asia; China, Korea, and Japan are considered the pioneers in industrial processing and application techniques of seaweed for food products (Paiva, Lima, Patarra, Neto, & Baptista, 2014). Seaweed has most commonly been used as a source of gelling alginates, mucoadhesive, stabilizers, and thickeners for food formulation (Chang & Wu, 2008; Hentati et al., 2020; Melanie et al., 2020; Yermak et al., 2020). Interestingly seaweeds are especially high in proteins (3%
47% dry basis) and a good source of polysaccharides, lipids, minerals, and vitamins; however, their specific composition is known to vary among species, harvesting season, and origin (Biancarosa et al., 2017; Kasimala, Mebrahtu, Magoha, Asgedom, & Kasimala, 2015). Seaweed abundance and the low environmental footprint have allowed food industries to increase their utilization as protein-dense ingredients in food formulation. Therefore seaweed’s popularity and acceptance have grown in the past few years as an alternative source of protein (Bleakley & Hayes, 2017). Within the most consumed seaweed species, we find Porphyra (nori), Laminaria (kombu), and Undaria (wakame) (McHugh, 2002). In terms of protein content, red species (Porphyra tenera) are known to have a higher protein content (47% dry basis), followed by green (Entermorpha spp., Ulva spp.) (19%
26%) and brown species (Laminaria japonica) (12%
13%) (Fleurence, 1999; Jurkovic´ , Kolb, & Colic´ , 1995; Nisizawa, Noda, Kikuchi, & Watanabe, 1987). Macroalgae are also rich in bioactive compounds including polysaccharides, proteins, lipids, and polyphenols (Holdt & Kraan, 2011).

436 Chapter 18 Recent studies identified its protein-rich matrix as a remarkable source of biologically active peptides that offer antibacterial, antidiabetic, and antihypertensive properties (Admassu, Gasmalla, Yang, & Zhao, 2018).

18.4.1 Mechanisms of extraction of bioactive peptides from marine macroalgae Macroalgae are known to have a complex, viscous, and often charged cell wall and extracellular matrix, this causes the extraction process to be laborious and very challenging. Researchers had reported that a high portion of protein in macroalgae is attached to polysaccharides in the cell cytoskeleton or bound to other macromolecules and serve as catalysts of biological reactions (Ka˛czkowski, 2003). For this reason, conventional protein extraction procedures are often inefficient and lead to low protein recovery when applied to macroalgae (Wang, Hu, Sommerfeld, & Chen, 2004). Despite this, novel approaches offer a new insight in possible processing techniques that improve the separation of protein components (e.g., proteolysis, cellulase and macerozyme hydrolysis, hydrostatic pressure, hot water extraction) (Suetsuna, Maekawa, & Chen, 2004; Suwal et al., 2019; Va´squez, Martı´nez, & Bernal, 2019). Early studies applied the utilization of a polysaccharidase mixture (β-glucanase, hemicellulase, cellulase) and showed an increase in protein extraction (Fleurence, Le Coeur, Mabeau, Maurice, & Landrein, 1995). However, the authors denoted that the implementation of this pool of enzymes was not better than other chemical approaches using sodium hydroxide (NaOH) under reductive conditions or a two-phase extraction system. For this reason, additional processes might be required to improve enzymatic techniques. Furthermore, the simultaneous utilization of carbohydrase and proteases treatments revealed an efficient way not only to increase the extraction of proteins but also to increase their overall bioactivity. For example, when Palmaria palmata was treated with polysaccharidases and proteases, the protein extraction yield increased, and their antioxidant activity was also enhanced (Wang et al., 2010). A vast number of studies focused on the use of proteases as a single-step approach to obtain bioactive peptide fractions (Cermen˜o, Stack, et al., 2019; Cian, Alaiz, Vioque, & Drago, 2013; Indumathi & Mehta, 2016; Rizzo et al., 2017; Zheng, Zhang, & San, 2020). This approach has shown a clear direction toward the generation of nutraceutical compounds from seaweed protein. Due to the efficiency of enzymes to extract, generate, and improve biologically active proteins, novel approaches have incorporated emerging technologies to support and improve the enzymatic extraction processes. A study conducted on P. palmata and Solieria chordalis implemented the use of hydrostatic pressure and simultaneous hydrolysis using polysaccharidases to augment by 2.8 times the antioxidant activity of the protein and polyphenol extract (Suwal et al., 2019). Another study evaluated the antiproliferative effects over cancer cells of hydrolysates generated from Porphyra haitanesis, by extracting proteins using ultrasonication and hydrolyzing them with trypsin (Fan, Bai, Mao, & Zhang, 2017). While new studies and technologies keep emerging, the industrial extraction of

Sustainable, alternative sources of bioactive peptides 437 bioactive peptides from macroalgae and their implementation as nutraceutical compounds is closer than ever before.

18.4.2 Bioactive properties of peptides from macroalgae proteins Even though the research in bioactive peptides from macroalgae has not been conducted vastly using in vivo models, a good number of studies have underlined their effects using cellular and in vitro analyses as denoted in Table 18.3. These studies emphasize the bioactivity offered by macroalgae proteins according to the species and amino acid composition. These bioactivities include antioxidant, antihypertensive, antibacterial, antithrombotic, and even antiproliferative effects against cancer cells. Probably the most Table 18.3: Bioactive properties from seaweed-derived peptides. Algae Sargassum maclurei Undaria pinnatifida Porphyra dioica

Bioactivity

Extraction techniques

ACE inhibition

Sequential hydrolysis (pepsin and papain) Hydrolysis (Protease S “Amano) Hydrolysis (alcalase and flavorzyme)

ACE inhibition Antioxidant, ACEand DPP-IV inhibition

Porphyra columbina

Radical scavenging activity and ACE inhibition

Porphyra columbina

Antihypertensive activity and Antioxidant capacity Antioxidant activity

Kappaphycus alvarezii Porphyra yezoensis Palmaria palmata Porphyra spp

Saccharina longicruris Porphyra haitanesis

Antithrombotic activity Renin inhibition α-amylaseinhibition Antibacterial Activity Antiproliferative activity over cancer cells

Sequential hydrolysis (fungal protease concentrates and flavorzyme) Cold water extraction and hydrolysis (trypsin, and alcalase)

Peptide or sequence identified

References

RWDISQPY

Zheng et al. (2020)

Whole protein hydrolysates ORAC (Asp-Tyr-TyrLys-Arg), ACE (ThrTyr-Ile-Ala) DPPIV (Tyr-Leu-Val-Ala) Whole protein hydrolysates

Sato et al. (2002) ˜o, Stack, Cermen et al. (2019)

Cian et al. (2013)

Whole protein hydrolysates

Cian et al. (2012)

Solvent and ultrasonication Hydrolysis (pepsin)

Whole protein hydrolysates NMEKGSSSVVSSRM

Dewi et al. (2020)

Gastrointestinal digestion (amylase, pepsin, trypsin)

IRLIIVLMPILMA

Hydrolysis (Viscozyme L) and isoelectric precipitation Hyrdrolysis (trypsin)

Gly-Gly-Ser-Lys and Glu-Leu-Ser

Indumathi and Mehta (2016) Fitzgerald, Aluko, Hossain, Rai, and Hayes (2014) Admassu et al. (2018)

10 kDa protein hydrolysate fraction VPGTPKNLDSPR

Beaulieu et al. (2015) Fan et al. (2017)

Ultrasonication followed by hydrolysis with trypsin

438 Chapter 18 studied bioactive property of macroalgae proteins is related to its antihypertensive activity. A study on Sargassum maclurei identified and synthesized a peptide (RWDISQPY) derived with an IC50 of 72.24 μM (Zheng et al., 2020). Other researchers have shown that a hydrolyzed pool of proteins derived from Porphyra columbina was especially potent as ACE inhibitor and antioxidant agents attribute this to the low molecular weight peptides and their high content in Asp, Ala, and Glu (Sato et al., 2002). Macroalgae, in general, have shown a great potential to inhibit ACE, species such as S. maclurei, Undaria pinnatifid, Porphyra dioica, and P. columbina are especially rich in peptides with high capacity of inhibition (Cermen˜o, Stack, et al., 2019; Cian et al., 2013; Cian, Martı´nez-Augustin, & Drago, 2012; Sato et al., 2002). Furthermore, other studies point at multiple bioactivities in which these peptides might serve to improve one’s health and wellness. For example, a study conducted on a hydrolyzed fraction of P. dioica showed antidiabetic, antihypertensive, and antioxidant properties (Cermen˜o, Stack, et al., 2019). Native proteins have also exhibited bioactivities, for instance, a protein (phycoerythrin) extracted from Kappaphycus alvarezii showed strong antioxidant activity when extracted using ultrasonic treatments (Dewi, Santoso, Setyaningsih, & Hardingtyas, 2020). In the case of antibacterial properties, low molecular weight peptide fractions (,10 kDa) extracted from Saccharina longicruris showed a great potential inhibiting the growth of Staphylococcus aureus. This was attributed to the presence of residual peptides from ubiquitin, which is a protein known to play a role in the innate defense of macroalgae (Beaulieu, Bondu, Doiron, Rioux, & Turgeon, 2015). Recent findings indicate the potential anticarcinogenic activity of two peptides (VPGTPKNLDSPR and MPAPSCALPRSVVPPR) extracted and synthesized from P. haitanesis that exhibited antiproliferative activities toward cancer cells MCF-7 and HepG-2. This was attributed to the capacity of these peptides to arrest the cell cycle in the G0/G1 phase and induce apoptosis (Fan et al., 2017). These studies indicate that peptides extracted from macroalgae offer numerous health benefits and might serve as nutraceuticals. However, considering that most of the research studies presented in this section are in the development state, further studies are needed to evaluate the effects of these peptides in in vivo models as well as their incorporation in functional foods and their bioavailability.

18.5 Underutilized agricultural by-products Agricultural commodities include staple crops and animals produced or raised on farms. However, in many cases these agricultural products are not consumed immediately or directly by humans, but first undergo processing to generate new products (e.g., with longer shelf life) or separate the primary product of interest from other constituents (e.g., inedible parts) (Bandara & Chalamaiah, 2019; Ramos et al., 2019). As a result of these processing steps, by-products are obtained which are discarded and in most cases represent an environmental problem or have a marginal economic value (Coelho, Pereira, Rodrigues, Teixeira, & Pintado, 2019; Lo´pez-Pedrouso et al., 2019; Peanparkdee & Iwamoto, 2019). In

Sustainable, alternative sources of bioactive peptides 439 this sense, a strategy proposed for adding value to these by-products is to explore their potential as a source of bioactive compounds (Al Khawli et al., 2020; Kumar, Yadav, Kumar, Vyas, & Dhaliwal, 2017; Ravindran & Jaiswal, 2016). These bioactive compounds are generated or extracted from these underutilized agricultural by-products, which include phytochemicals, functional lipids, and bioactive peptides (Bandara & Chalamaiah, 2019; Kumar et al., 2017; Ravindran & Jaiswal, 2016). In addition, by obtaining bioactive peptides from underutilized agricultural by-products, it is expected that their production will be more cost-effective (Radenkovs, Juhnevica-Radenkova, Go´rna´s, & Seglina, 2018). In general, bioactive peptides obtained from underutilized agricultural by-products can be differentiated mainly by their origin, which can be divided into animal and plant sources. Some examples of by-products from animal origin include muscle, collagen, and blood, as well as those derived from their products (e.g., whey derived from milk processing). Examples of by-products from plant origin include grains, vegetables, fruit, and oilseeds (e.g., palm kernel protein, olive residues, chia meal).

18.5.1 Mechanisms for extraction of bioactive peptides from underutilized agricultural by-products The mechanisms used for the extraction of bioactive peptides derived from underutilized agricultural by-products vary in the literature. For example, Haq et al. (2020) used catalystassisted subcritical water hydrolysis to generated tuna skin-collagen hydrolysates, which exhibited antioxidant and antimicrobial properties. Roblet et al. (2016) separated a peptide fraction from Atlantic salmon (Salmo salar) frame protein by using electrodialysis with ultrafiltration membrane (EDUF), which exhibited an antidiabetic effect by enhancing glucose uptake in L6 skeletal muscle cells. In similar studies, Durand, Fraboulet, Marette, and Bazinet (2019) and Ketnawa, Suwal, Huang, and Liceaga (2018), using an EDUF system, produced anionic and cationic peptide fractions from herring (Clupea harengus) and trout (Oncorhynchus mykiss) frame by-products, respectively, with ACE, DPP-IV, and antioxidant activities. The use of pretreatment methods such as microwave heating followed by conventional hydrolysis was also applied by Ketnawa and Liceaga (2017) on rainbow trout (O. mykiss) frame by-products to produce bioactive peptides with antioxidant activity and low immunoreactivity. Xu et al. (2017) generated bioactive peptides from cauliflower (Brassica oleracea) using ultrasonic-assisted extraction and hydrolysis, which exhibited ACE-inhibitory activity as well it regulated glucose metabolism. Similarly, Gonza´lezGarcı´a, Marina, and Garcı´a (2014) used ultrasonic-assisted extraction and hydrolysis on plum (Prunus domestica L.) stones to develop hydrolysates with antioxidant and ACEinhibitory activities. In other studies, Zheng, Li, Zhang, Ruan, and Zhang (2017) applied high pressure as pretreatment to palm kernel glutelin-2 followed by sequential hydrolysis to generated

440 Chapter 18 peptides that exhibited in vitro and in vivo ACE-inhibitory activity. Others, produced chia seed (Salvia hispanica) peptides using microwave-assisted sequential hydrolysis, after the protein was separated as a by-product of chia seed oil and mucilage extraction processes (Urbizo-Reyes, San Martin-Gonza´lez, Garcia-Bravo, Lo´pez Malo Vigil, & Liceaga, 2019). These chia seed peptides had DPP-IV and ACE inhibition activities, as well as chemical and cellular antioxidant activities. On the other hand, Min, Jo, and Park (2017) used hydrothermal hydrolysis systems to produce porcine skin by-products hydrolysates. Yu et al. (2020) applied ultrasound-assisted enzymatic to generated hydrolysates from brewer’s spent grain. The studies described above highlighted some of common methods used to extract bioactive peptides from diverse agricultural by-products.

18.5.2 Bioactivity of peptides derived from underutilized agricultural by-products Table 18.4 shows the scientific literature on bioactive peptides derived from animal and plant by-products, which can exhibit single or multifunctional biological properties using in vitro and in vivo studies. The bioactivities include, but are not limited to, antioxidant, antimicrobial, anti-inflammatory, and antidiabetic properties. For example, Ketnawa and Liceaga (2017) produced bioactive peptides from rainbow trout (O. mykiss) frame byproducts. Their results showed that peptides produced using microwave pretreatment followed by conventional hydrolysis increased their antioxidant activity (c.60%) and decreased immunoreactivity (55%
93%) compared with the unhydrolyzed control (0.67 μmol Trolox/mg protein by ABTS method). Based on these results, Ketnawa, Suwal, Huang, and Liceaga (2019) recovered cationic peptides from those peptides using fractionation by EDUF, which displayed ACE (IC50 5 0.0036 mg/mL) and DPP-IV (IC50 5 1.23 mg/mL) inhibitory properties. In a similar study, Nguyen, Jones, Kim, San Martin-Gonzalez, and Liceaga (2017) found that peptides obtained by microwave-assisted hydrolysis of rainbow trout (O. mykiss) fish frames improved ( . 50%) the antioxidant capacity compared with conventional hydrolysis. Slizyte et al. (2016) generated protein hydrolysates from defatted salmon backbones using different enzymes that showed diverse bioactivities. Salmon backbones hydrolysates obtained by trypsin, bromelain 1 papain, and protamex showed the highest ACE inhibitory (0.92 mg/mL), cellular glucose transporter inhibitory (39%), and antioxidant activities [c.38% 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition], respectively. Morales-Medina, Tamm, Guadix, Guadix, and Drusch (2016) produced hydrolysates of sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus) by-products with antioxidant activity between 70% and 80% of DPPH inhibition. Furthermore, Durand et al. (2019) produced an anionic peptide fraction derived from milt herring with enhanced glucose uptake (214%) and antioxidant activity (27.5%) compared with the original hydrolysate. In a related study, Roblet et al. (2016) separated a peptide

Sustainable, alternative sources of bioactive peptides 441 Table 18.4: Bioactive properties from underutilized, agricultural by-products. Type of bioactive peptide

Mechanism of extraction/ production

Bigeye tuna skin collagen

Hydrolysate

Atlantic salmon frame protein

Peptide fraction

Catalyst-assisted subcritical water hydrolysis Hydrolysis with EDUF fractionation

Rainbow trout (Oncorhynchus mykiss) muscle Cauliflower

Hydrolysate

By-product protein source

Hydrolysate

Plum (Prunus domestica L.) stones

Hydrolysate

Palm kernel glutelin-2

Hydrolysate

Chia seed

Hydrolysate

Rainbow trout (Oncorhynchus mykiss) muscle

Bioactivity or effect

Antioxidant and antimicrobial activities Antidiabetic effect (enhanced glucose uptake in L6 skeletal muscle cells) Hydrolysis Antioxidant activity and low immunoreactivity ACE-inhibitory Ultrasonic-assisted activity as well as to extraction and regulate glucose hydrolysis metabolism Antioxidant and Ultrasonic-assisted ACE-inhibitory extraction and activities hydrolysis In vitro and in vivo High-pressure ACE-inhibitory pretreatment and activity sequential hydrolysis Microwave-assisted DPP-IV and ACE sequential hydrolysis inhibition activities; chemical and cellular antioxidant activities Hydrolysis with EDUF ACE and DPP-IV fractionation inhibitory activities

Thornback rays (Raja clavata) skin collagen Bovine blood

Cationic peptides recovered from hydrolysate Hydrolysate and purified peptides Hydrolysates

Crocodile blood

Hydrolysates

Hydrolysis

Duck blood Duck plasma Pale brewers’ spent grain

Hydrolysate Hydrolysate Hydrolysate

Hydrolysis Hydrolysis Hydrolysis

Hydrolysis

Hydrolysis

ACE-inhibitory and antioxidant activities Antiproliferative activity ACE-inhibitory activity Antioxidant activity Antioxidant activity α-Glucosidase, angiotensinconverting enzyme and DPP-IV inhibitory activities

References Haq et al. (2020)

Roblet et al. (2016)

Ketnawa and Liceaga (2017) Xu et al. (2017)

Gonza´lez-Garcı´a et al. (2014) Zheng et al. (2017)

Urbizo-Reyes et al. (2019)

Ketnawa et al. (2019)

Lassoued et al. (2015)

O’Sullivan et al. (2017) Ngo-son & Katekaew (2019) Zheng et al. (2018) Yang et al. (2020) Connolly et al. (2014)

(Continued)

442 Chapter 18 Table 18.4: (Continued) Type of bioactive peptide

Mechanism of extraction/ production

Brewers’ spent grain protein-rich isolate Brewer’s spent grain

Hydrolysate

Hydrolysis

Hydrolysate

Hydrolysis

Brewer’s spent grain Palm kernel cake

Hydrolysate

Hydrolysis

Hydrolysate

Hydrolysis

Complex containing peptides (2.4 kDa) and lauric acid derivative Peptide fractions

Hydrolysis

Hydrolysis

Antioxidant activity

Hydrolysate and peptide fraction

Hydrolysis

Chemical and cellular antioxidant activity

By-product protein source

Palm kernel cake

Chia seed

Silver carp (Hypophthalmichthys molitrix) muscle

Bioactivity or effect

References

Antioxidant and anti-inflammatory activities Antioxidant and DPP-IV and ACEinhibitory activities as well as antihypertensive effect Anti-inflammatory activity In vitro and in vivo ACE-inhibitory activity Antimicrobial activity

McCarthy et al. (2013) ˜o, Connolly, Cermen et al. (2019)

Cian et al. (2020) Zarei et al. (2015)

Tan et al. (2013)

Silveira Coelho, de Araujo Aquino, Machado Latorres, and de las Mercedes Salas-Mellado (2019) Malaypally et al. (2015)

ACE, Angiotensin-I converting enzyme; DPP-IV, dipeptidylpeptidase IV; EDUF, electrodialysis with ultrafiltration membrane.

fraction from Atlantic salmon frame protein by using EDUF, which exhibited antidiabetic effect by enhanced glucose uptake in L6 skeletal muscle cells grown in culture. Other studies have used collagen from different sources as by-product protein source of bioactive peptides. In this context, Lassoued et al. (2015) generated protein hydrolysates from thornback ray (Raja clavate) skin’s gelatin using two proteases (neutrase and Bacillus subtilis A26), with ACE inhibitory (IC50 5 2.07 and 0.94 μg/μL, respectively) and antioxidant (IC50 5 21.2 and 1.98 μg/μL, respectively) activities. In addition, the most active, purified fractions contained the peptide APGAP that was the most active (IC50 5 170 mM), whereas GIPGAP derived from neutrase had an IC50 5 27.9 μM. Offengenden, Chakrabarti, and Wu (2018) generated collagen-derived peptides with antiinflammatory and antioxidant activities, as well as increased type I procollagen synthesis

Sustainable, alternative sources of bioactive peptides 443 and stimulated the cellular proliferation on human dermal fibroblasts. In a related work, Gao et al. (2019) reported bone collagen peptides from Yak (Bos grunniens) that ameliorated immunosuppression by increasing innate and adaptive immunity on cyclophosphamide-induced immunosuppression BALB/c mice. Likewise, Martini, Conte, and Tagliazucchi (2019) obtained peptide fractions from digested samples derived from pork, beef, chicken, and turkey meat by-products, showing that pork and turkey meat appeared to be the best sources of antioxidant peptides. In addition, pork by-products were found to be the best source of DPP-IV inhibitory peptides, whereas chicken meat-supplied peptides with the highest ACE-inhibitory activity. Similarly, O’Sullivan, Lafarga, Hayes, and O’Brien (2017) produced bovine blood hydrolysates with antiproliferative activity toward cancer cells in cultures including U937 lymphoma cells, MCF-7 breast cancer cells, HepG2 hepatocytes, and Caco-2 epithelial colorectal adenocarcinoma cells. However, none of the hydrolysates had significant anti-inflammatory activity. Zheng et al. (2018) generated hydrolysates from poultry blood with antioxidant activity including DPPH inhibition, reducing power, metal chelating ability, and inhibiting lipid peroxidation. Similarly, Yang et al. (2020) generated duck plasma protein hydrolysates with antioxidant activity. The authors identified seven peptides (LDGP, TGVGTK, EVGK, RCLQ, LHDVK, KLGA, and AGGVPAG) with antioxidant activities. Of these, the peptide RCLQ exhibited the highest antioxidant activity. In terms of plant-derived by-products, Connolly, Piggott, and FitzGerald (2014) generated pale brewers’ spent grain protein hydrolysates with α-glucosidase, ACE, and DPP-IV inhibitory activities. Hydrolysates produced with Corolase-PP resulted in the highest DPP-IV inhibition (75%), whereas those obtained by Prolyve-1000 displayed the highest ACE inhibition (89.25%). In a related study, McCarthy et al. (2013) used brewers’ spent grain protein-rich isolate to generate hydrolysates that exhibited antioxidant and antiinflammatory effects on concanavalin A stimulated human Jurkat T cells. Cermen˜o et al. (2019) evaluated the in vitro bioactive properties and in vivo effect of brewer’s spent grain hydrolysates. They found that the peptides exhibited in vitro antioxidant, DPP-IV, and ACE-inhibitory activities as well as in vivo antihypertensive effect in a murine model using spontaneously hypertensive rats. Their results showed that the peptide IPY had the highest antioxidant activity, IPLQP had the highest ACE-inhibitory activity, whereas IPVP displayed the highest DPP-IV inhibitory activity. Cian et al. (2020) found that brewers’ spent grain exhibited anti-inflammatory properties by regulating the immune response, involving TLR2 and TLR4 and the activation of nuclear factor kappa B and mitogenactivated protein kinases on spleen mononuclear cells ex vivo culture. Other peptides from plant by-products have been generated from hydrolysates of cauliflower by-products, exhibiting ACE-inhibitory activity (138.55 μg/mL) (Xu et al., 2017). In addition, the peptides promoted glucose consumption and enhanced the glycogen

444 Chapter 18 content in HepG2 cells. In related work, an ACE-inhibitory dipeptide (VW) was isolated and identified from cauliflower hydrolysates that exhibited ACE-inhibitory activity with an IC50 value of 31.30 μM. Similarly, protein hydrolysates from rice (Oryza sativa L.) byproducts exhibited antioxidant activity as well as anti-inflammatory activity on raw 264.7 macrophage cells (Saisavoey, Sangtanoo, Reamtong, & Karnchanatat, 2016). On the other hand, peptides with antioxidant and ACE-inhibitory capacities have been obtained from a plum (P. domestica L.) processing by-products (i.e., stones) (Gonza´lez-Garcı´a, Puchalska, Marina, & Garcı´a, 2015), where the highest antioxidant capacity was observed in the alcalase hydrolysate, while peptides yielding the highest ACE inhibition were concentrated in the ,3 kDa fraction obtained from the thermolysin hydrolysate. In a similar study, Gonza´lez-Garcı´a et al. (2014) also produced bioactive peptides from plum (P. domestica L.) stones. They extracted proteins using high-intensity focused ultrasound, followed by enzymatic hydrolysis with the peptides showing antioxidant and ACE-inhibitory activities. Zarei et al. (2015) investigated the in vitro and in vivo ACE-inhibitory activity of bioactive peptides from palm kernel cake protein obtained as a by-product from the oilseed production. The hydrolysate produced with papain exhibited the highest ACE-inhibitory activity (70.9%) compared with other enzymes (bromelain, flavorzyme, pepsin, alcalase, trypsin, chymotrypsin) with ,65% inhibition. In addition, the in vivo ACE-inhibitory activity of papain generated protein hydrolysate at various doses (150, 75, and 37.5 mg/kg body weight) also showed that a dose as low as 75 mg/kg could significantly inhibit hypertension in a normotensive rats model. Zheng et al. (2017) also evaluated the in vitro and in vivo ACE-inhibitory activity of bioactive peptides derived from oil palm kernel glutelin-2 hydrolysates. The authors found that the sequential digestion resulted in peptides with high ACE-inhibitory activity (80.24%). Tan, Ayob, and Wan Yaacob (2013) purified a complex containing peptides (B2.4 kDa) and lauric acid from a palm kernel cake, with antimicrobial activity against Bacillus species. Most of the studies have focused only on evaluating the in vitro bioactivities and there are only a few literature reports that describe findings related with the in vivo effect of bioactive peptides derived from underutilized agricultural by-products. These studies demonstrate that agricultural by-products can be a valuable source of bioactive peptides, and this can be an attractive approach to add value to these otherwise underutilized resources.

18.6 Conclusion Emerging protein sources, such as insects, fungi, agricultural by-products, and macroalgae, are becoming more widely available as we reevaluate the way in which we grow food and the types of food we grow/consume. These alternative proteins are not only sought for their nutritional value, scientists have also identified these protein-rich resources as a remarkable

Sustainable, alternative sources of bioactive peptides 445 source of biologically active peptides, which can offer antibacterial, antidiabetic, antihypertensive, antioxidant properties, among others. The methods used for extracting, isolating, and/or purifying these biopeptides, as well as pretreatment methods that can enhance their bioavailability (e.g., ultrasonication and microwave treatments) should continue to be explored. Finally, considering that most of the research available is at the developing or in vitro state, continuing research is needed to evaluate the effects of these bioactive peptides using in vivo models, clinical trials, as well as their bioavailability after incorporation in functional foods and nutraceuticals.

References Admassu, H., Gasmalla, M. A., Yang, R., & Zhao, W. (2018). Identification of bioactive peptides with α-amylase inhibitory potential from enzymatic protein hydrolysates of red seaweed (Porphyra spp). Journal of Agricultural and Food Chemistry, 66(19), 4872
4882. Al Khawli, F., Marti-Quijal, F. J., Ferrer, E., Ruiz, M.-J., Berrada, H., Gavahian, M., . . . de la Fuente, B. (2020). Aquaculture and its by-products as a source of nutrients and bioactive compounds. In Advances in food and nutrition research. Academic Press. Amorim, M., Marques, C., Pereira, J., Guarda˜o, L., Martins, M., Oso´rio, H., . . . Pintado, M. (2019). Antihypertensive effect of spent brewer yeast peptide. Process Biochemistry, 76, 213
218. Attarat, J., & Phermthai, T. (2015). Bioactive compounds in three edible Lentinus mushrooms. Walailak Journal of Science and Technology (WJST), 12(6), 491
504. Azzollini, D., Derossi, A., Fogliano, V., Lakemond, C., & Severini, C. (2018). Effects of formulation and process conditions on microstructure, texture and digestibility of extruded insect-riched snacks. Innovative Food Science & Emerging Technologies, 45, 344
353. Bandara, N., & Chalamaiah, M. (2019). Bioactives from agricultural processing by-products. In L. Melton, F. Shahidi, & P. Varelis (Eds.), Encyclopedia of food chemistry (pp. 472
480). Oxford: Academic Press. Beaulieu, L., Bondu, S., Doiron, K., Rioux, L.-E., & Turgeon, S. L. (2015). Characterization of antibacterial activity from protein hydrolysates of the macroalga Saccharina longicruris and identification of peptides implied in bioactivity. Journal of Functional Foods, 17, 685
697. Biancarosa, I., Espe, M., Bruckner, C., Heesch, S., Liland, N., Waagbø, R., . . . Lock, E. (2017). Amino acid composition, protein content, and nitrogen-to-protein conversion factors of 21 seaweed species from Norwegian waters. Journal of Applied Phycology, 29(2), 1001
1009. Bleakley, S., & Hayes, M. (2017). Algal proteins: Extraction, application, and challenges concerning production. Foods, 6(5), 33. Bußler, S., Rumpold, B. A., Jander, E., Rawel, H. M., & Schlu¨ter, O. K. (2016). Recovery and technofunctionality of flours and proteins from two edible insect species: Meal worm (Tenebrio molitor) and black soldier fly (Hermetia illucens) larvae. Heliyon, 2(12), e00218. Calzada-Luna, G., San Martin-Gonzalez, F., Mauer, L. J., & Liceaga, A. M. (2020). Cricket (Acheta domesticus) protein hydrolysates’ impact on the physicochemical, structural and sensory properties of tortillas and tortilla chips. Journal of Insects as Food and Feed. Available from https://doi.org/10.3920/JIFF2020.0010. Caparros Megido, R., Sablon, L., Geuens, M., Brostaux, Y., Alabi, T., Blecker, C., . . . Francis, F. (2014). Edible insects acceptance by Belgian consumers: Promising attitude for entomophagy development. Journal of Sensory Studies, 29(1), 14
20. Cermen˜o, M., Connolly, A., O’Keeffe, M. B., Flynn, C., Alashi, A. M., Aluko, R. E., & FitzGerald, R. J. (2019). Identification of bioactive peptides from brewers’ spent grain and contribution of Leu/Ile to bioactive potency. Journal of Functional Foods, 60103455. Available from https://doi.org/10.1016/j. jff.2019.103455.

446 Chapter 18 Cermen˜o, M., Stack, J., Tobin, P. R., O’Keeffe, M. B., Harnedy, P. A., Stengel, D. B., & FitzGerald, R. J. (2019). Peptide identification from a Porphyra dioica protein hydrolysate with antioxidant, angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities. Food & Function, 10(6), 3421
3429. Chang, H., & Wu, L. C. (2008). Texture and quality properties of Chinese fresh egg noodles formulated with green seaweed (Monostroma nitidum) powder. Journal of Food Science, 73(8), S398
S404. Chalamaiah, M., Dinesh Kumar, B., Hemalatha, R., & Jyothirmayi, T. (2012). Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review. Food chemistry, 135(4), 3020
3038. Chen, J.-N., Wang, Y.-T., & Wu, J. S.-B. (2009). A glycoprotein extracted from golden oyster mushroom Pleurotus citrinopileatus exhibiting growth inhibitory effect against U937 leukemia cells. Journal of Agricultural and Food Chemistry, 57(15), 6706
6711. Cian, R. E., Alaiz, M., Vioque, J., & Drago, S. R. (2013). Enzyme proteolysis enhanced extraction of ACE inhibitory and antioxidant compounds (peptides and polyphenols) from Porphyra columbina residual cake. Journal of Applied Phycology, 25(4), 1197
1206. Cian, R. E., Herna´ndez-Chirlaque, C., Ga´mez-Belmonte, R., Drago, S. R., Sa´nchez de Medina, F., & Martı´nezAugustin, O. (2020). Molecular action mechanism of anti-inflammatory hydrolysates obtained from brewers’ spent grain. Journal of the Science of Food and Agriculture, n/a(n/a). Available from https://doi. org/10.1002/jsfa.10313. Cian, R. E., Martı´nez-Augustin, O., & Drago, S. R. (2012). Bioactive properties of peptides obtained by enzymatic hydrolysis from protein byproducts of Porphyra columbina. Food Research International, 49 (1), 364
372. Claude, M., Bouchaud, G. g, Lupi, R., Castan, L., Tranquet, O., Denery-Papini, S., . . . Brossard, C. (2017). How proteins aggregate can reduce allergenicity: Comparison of ovalbumins heated under opposite electrostatic conditions. Journal of Agricultural and Food Chemistry, 65(18), 3693
3701. Coelho, M., Pereira, R., Rodrigues, A. S., Teixeira, J. A., & Pintado, M. E. (2019). Extraction of tomato byproducts’ bioactive compounds using ohmic technology. Food and Bioproducts Processing, 117, 329
339. Available from https://doi.org/10.1016/j.fbp.2019.08.005. Connolly, A., Piggott, C. O., & FitzGerald, R. J. (2014). In vitro α-glucosidase, angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory properties of brewers’ spent grain protein hydrolysates. Food Research International, 56, 100
107. Available from https://doi.org/10.1016/j.foodres.2013.12.021. Cui, J., & Chisti, Y. (2003). Polysaccharopeptides of Coriolus versicolor: Physiological activity, uses, and production. Biotechnology Advances, 21(2), 109
122. Dai, C., Ma, H., Luo, L., & Yin, X. (2013). Angiotensin I-converting enzyme (ACE) inhibitory peptide derived from Tenebrio molitor (L.) larva protein hydrolysate. European Food Research and Technology, 236(4), 681
689. Dalsgaard, P. W., Larsen, T. O., & Christophersen, C. (2005). Bioactive cyclic peptides from the psychrotolerant fungus Penicillium algidum. The Journal of Antibiotics, 58(2), 141
144. Dewi, N., Santoso, J., Setyaningsih, I., & Hardingtyas, S. (2020). Extraction of phycoerythrin from Kappaphycus alvarezii seaweed using ultrasonication. Paper presented at the IOP Conference Series: Earth and Environmental Science. Durand, R., Fraboulet, E., Marette, A., & Bazinet, L. (2019). Simultaneous double cationic and anionic molecule separation from herring milt hydrolysate and impact on resulting fraction bioactivities. Separation and Purification Technology, 210, 431
441. Available from https://doi.org/10.1016/j.seppur.2018.08.017. Fan, X., Bai, L., Mao, X., & Zhang, X. (2017). Novel peptides with anti-proliferation activity from the Porphyra haitanesis hydrolysate. Process Biochemistry, 60, 98
107. FAO. (2014). State of food insecurity in the world 2013: The multiple dimensions of food security. FAO. Finke, M. D. (2004). Nutrient content of insects. Springer. Fisher, M., & Yang, L.-X. (2002). Anticancer effects and mechanisms of polysaccharide-K (PSK): Implications of cancer immunotherapy. Anticancer Research, 22(3), 1737
1754.

Sustainable, alternative sources of bioactive peptides 447 Fitzgerald, C., Aluko, R. E., Hossain, M., Rai, D. K., & Hayes, M. (2014). Potential of a renin inhibitory peptide from the red seaweed Palmaria palmata as a functional food ingredient following confirmation and characterization of a hypotensive effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 62(33), 8352
8356. Fleurence, J. (1999). Seaweed proteins: Biochemical, nutritional aspects and potential uses. Trends in Food Science & Technology, 10(1), 25
28. Fleurence, J., Le Coeur, C., Mabeau, S., Maurice, M., & Landrein, A. (1995). Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata. Journal of Applied Phycology, 7(6), 577
582. Gao, S., Hong, H., Zhang, C., Wang, K., Zhang, B., Han, Q.-a, . . . Luo, Y. (2019). Immunomodulatory effects of collagen hydrolysates from yak (Bos grunniens) bone on cyclophosphamide-induced immunosuppression in BALB/c mice. Journal of Functional Foods, 60103420. Available from https://doi.org/10.1016/j. jff.2019.103420. Giavasis, I. (2014). Bioactive fungal polysaccharides as potential functional ingredients in food and nutraceuticals. Current Opinion in Biotechnology, 26, 162
173. Gmuer, A., Nuessli Guth, J., Hartmann, C., & Siegrist, M. (2016). Effects of the degree of processing of insect ingredients in snacks on expected emotional experiences and willingness to eat. Food Quality and Preference, 54, 117
127. Available from https://doi.org/10.1016/j.foodqual.2016.07.003. Gonza´lez, C. M., Garzo´n, R., & Rosell, C. M. (2018). Insects as ingredients for bakery goods. A comparison study of H. illucens, A. domestica and T. molitor flours. Innovative Food Science & Emerging Technologies. Gonza´lez-Garcı´a, E., Marina, M. L., & Garcı´a, M. C. (2014). Plum (Prunus domestica L.) by-product as a new and cheap source of bioactive peptides: Extraction method and peptides characterization. Journal of Functional Foods, 11, 428
437. Available from https://doi.org/10.1016/j.jff.2014.10.020. Gonza´lez-Garcı´a, E., Puchalska, P., Marina, M. L., & Garcı´a, M. C. (2015). Fractionation and identification of antioxidant and angiotensin-converting enzyme-inhibitory peptides obtained from plum (Prunus domestica L.) stones. Journal of Functional Foods, 19, 376
384. Available from https://doi.org/10.1016/j.jff.2015.08.033. Hall, F., Johnson, P. E., & Liceaga, A. (2018). Effect of enzymatic hydrolysis on bioactive properties and allergenicity of cricket (Gryllodes sigillatus) protein. Food Chemistry, 262, 39
47. Hall, F., & Liceaga, A. (2020). Effect of microwave-assisted enzymatic hydrolysis of cricket (Gryllodes sigillatus) protein on ACE and DPP-IV inhibition and tropomyosin-IgG binding. Journal of Functional Foods, 64, 103634. Hall, F. G., Jones, O. G., O’Haire, M. E., & Liceaga, A. M. (2017). Functional properties of tropical banded cricket (Gryllodes sigillatus) protein hydrolysates. Food Chemistry, 224, 414
422. Available from https:// doi.org/10.1016/j.foodchem.2016.11.138. Haq, M., Ho, T. C., Ahmed, R., Getachew, A. T., Cho, Y.-J., Park, J.-S., & Chun, B.-S. (2020). Biofunctional properties of bacterial collagenolytic protease-extracted collagen hydrolysates obtained using catalystsassisted subcritical water hydrolysis. Journal of Industrial and Engineering Chemistry, 81, 332
339. Available from https://doi.org/10.1016/j.jiec.2019.09.023. Hentati, F., Pierre, G., Ursu, A. V., Vial, C., Delattre, C., Abdelkafi, S., & Michaud, P. (2020). Rheological investigations of water-soluble polysaccharides from the Tunisian brown seaweed Cystoseira compressa. Food Hydrocolloids, 103, 105631. Holdt, S. L., & Kraan, S. (2011). Bioactive compounds in seaweed: Functional food applications and legislation. Journal of Applied Phycology, 23(3), 543
597. House, J. (2016). Consumer acceptance of insect-based foods in the Netherlands: Academic and commercial implications. Appetite, 107, 47
58. Ibadallah, B. X., Abdullah, N., & Shuib, A. S. (2015). Identification of angiotensin-converting enzyme inhibitory proteins from mycelium of Pleurotus pulmonarius (oyster mushroom). Planta Medica, 81(02), 123
129.

448 Chapter 18 Indumathi, P., & Mehta, A. (2016). A novel anticoagulant peptide from the Nori hydrolysate. Journal of Functional Foods, 20, 606
617. Jia, J., Wu, Q., Yan, H., & Gui, Z. (2015). Purification and molecular docking study of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide from alcalase hydrolysate of ultrasonic-pretreated silkworm pupa (Bombyx mori) protein. Process Biochemistry, 50(5), 876
883. Jurkovi´c, N., Kolb, N., & Coli´c, I. (1995). Nutritive value of marine algae Laminaria japonica and Undaria pinnatifida. Die Nahrung, 39(1), 63
66. Ka˛czkowski, J. (2003). Structure, function and metabolism of plant cell wall. Acta Physiologiae Plantarum, 25 (3), 287
305. Karamac, M., Kosinska-Cagnazzo, A., & Kulczyk, A. (2016). Use of different proteases to obtain flaxseed protein hydrolysates with antioxidant activity. International Journal of Molecular Sciences, 17. Available from https://doi.org/10.3390/ijms17071027. Kasimala, M. B., Mebrahtu, L., Magoha, P. P., Asgedom, G., & Kasimala, M. B. (2015). A review on biochemical composition and nutritional aspects of seaweeds. Caribbean Journal of Science and Technology, 3, 789
797. Ketnawa, S., & Liceaga, A. M. (2017). Effect of microwave treatments on antioxidant activity and antigenicity of fish frame protein hydrolysates. Food and Bioprocess Technology, 10(3), 582
591. Available from https://doi.org/10.1007/s11947-016-1841-8. Ketnawa, S., Suwal, S., Huang, J. Y., & Liceaga, A. M. (2018). Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates. International Journal of Food Science & Technology. Ketnawa, S., Suwal, S., Huang, J.-Y., & Liceaga, A. M. (2019). Selective separation and characterisation of dual ACE and DPP-IV inhibitory peptides from rainbow trout (Oncorhynchus mykiss) protein hydrolysates. International Journal of Food Science & Technology, 54(4), 1062
1073. Available from https://doi.org/ 10.1111/ijfs.13939. Khan, M. A., & Tania, M. (2012). Nutritional and medicinal importance of Pleurotus mushrooms: An overview. Food Reviews International, 28(3), 313
329. Kim, S.-K. (2011). Handbook of marine macroalgae: Biotechnology and applied phycology. John Wiley & Sons. Klunder, H., Wolkers-Rooijackers, J., Korpela, J., & Nout, M. (2012). Microbiological aspects of processing and storage of edible insects. Food Control, 26(2), 628
631. Komatsu, K., Shigemori, H., & Kobayashi, J. i (2001). Dictyonamides A and B, new peptides from marinederived fungus. The Journal of Organic Chemistry, 66(18), 6189
6192. Kong, X., Zhang, J., Han, X., Zhang, P., Dai, X., Liu, J., . . . Liu, S. (2013). High-yield production in Escherichia coli of fungal immunomodulatory protein isolated from Flammulina velutipes and its bioactivity assay in vivo. International Journal of Molecular Sciences, 14(2), 2230
2241. Korhonen, H., & Pihlanto, A. (2003). Food-derived bioactive peptides-opportunities for designing future foods. Current Pharmaceutical Design, 9(16), 1297
1308. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16(9), 945
960. Kumar, K., Yadav, A. N., Kumar, V., Vyas, P., & Dhaliwal, H. S. (2017). Food waste: A potential bioresource for extraction of nutraceuticals and bioactive compounds. Bioresources and Bioprocessing, 4(1), 18. Lassoued, I., Mora, L., Barkia, A., Aristoy, M. C., Nasri, M., & Toldra´, F. (2015). Bioactive peptides identified in thornback ray skin’s gelatin hydrolysates by proteases from Bacillus subtilis and Bacillus amyloliquefaciens. Journal of Proteomics, 128, 8
17. Available from https://doi.org/10.1016/j. jprot.2015.06.016. Lau, C. C., Abdullah, N., & Shuib, A. S. (2013). Novel angiotensin I-converting enzyme inhibitory peptides derived from an edible mushroom, Pleurotus cystidiosus OK Miller identified by LC-MS/MS. BMC Complementary and Alternative Medicine, 13(1), 313.

Sustainable, alternative sources of bioactive peptides 449 Lau, C. C., Abdullah, N., Shuib, A. S., & Aminudin, N. (2014). Novel angiotensin I-converting enzyme inhibitory peptides derived from edible mushroom Agaricus bisporus (JE Lange) Imbach identified by LC
MS/MS. Food Chemistry, 148, 396
401. Leiber, F., Gelencse´r, T., Stamer, A., Amsler, Z., Wohlfahrt, J., Fru¨h, B., & Maurer, V. (2017). Insect and legume-based protein sources to replace soybean cake in an organic broiler diet: Effects on growth performance and physical meat quality. Renewable Agriculture and Food Systems, 32(1), 21
27. Li, F., Wen, H., Zhang, Y., Aa, M., & Liu, X. (2011). Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor. Science China Life Sciences, 54(4), 379
385. Li, Q. Z., Chang, Y. Z., He, Z. M., Chen, L., & Zhou, X. W. (2019). Immunomodulatory activity of Ganoderma lucidum immunomodulatory protein via PI3K/Akt and MAPK signaling pathways in RAW264. 7 cells. Journal of Cellular Physiology, 234(12), 23337
23348. Li, S., Jiang, Z., Sun, L., Liu, X., Huang, Y., Wang, F., & Xin, F. (2017). Characterization of a new fungal immunomodulatory protein, FIP-dsq2 from Dichomitus squalens. Journal of Biotechnology, 246, 45
51. Li, S. Y., Shi, L. J., Ding, Y., Nie, Y., & Tang, X. M. (2015). Identification and functional characterization of a novel fungal immunomodulatory protein from Postia placenta. Food and Chemical Toxicology, 78, 64
70. Li, X., Li, Y., Huang, X., Zheng, J., Zhang, F., & Kan, J. (2014). Identification and characterization of a novel angiotensin I-converting enzyme inhibitory peptide (ACEIP) from silkworm pupa. Food Science and Biotechnology, 23(4), 1017
1023. Liceaga, A. M. (2019). Approaches for utilizing insect protein for human consumption: Effect of enzymatic hydrolysis on protein quality and functionality. Annals of the Entomological Society of America, 112(6), 529
532. Lin, C.-H., Sheu, G.-T., Lin, Y.-W., Yeh, C.-S., Huang, Y.-H., Lai, Y.-C., . . . Ko, J.-L. (2010). A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells. Process Biochemistry, 45(9), 1537
1542. Lo´pez-Pedrouso, M., Lorenzo, J. M., Cantalapiedra, J., Zapata, C., Franco, J. M., & Franco, D. (2019). Aquaculture and by-products: Challenges and opportunities in the use of alternative protein sources and bioactive compounds. In Advances in Food and Nutrition Research. Academic Press. Lu, J., & Chen, Z.-w (2010). Isolation, characterization and anti-cancer activity of SK84, a novel glycine-rich antimicrobial peptide from Drosophila virilis. Peptides, 31(1), 44
50. Malaypally, S. P., Liceaga, A. M., Kim, K.-H., Ferruzzi, M., Martin, F. S., & Goforth, R. R. (2015). Influence of molecular weight on intracellular antioxidant activity of invasive silver carp (Hypophthalmichthys molitrix) protein hydrolysates. Journal of Functional Foods, 18, 1158
1166. Available from https://doi. org/10.1016/j.jff.2014.06.011. Martini, S., Conte, A., & Tagliazucchi, D. (2019). Comparative peptidomic profile and bioactivities of cooked beef, pork, chicken and turkey meat after in vitro gastro-intestinal digestion. Journal of Proteomics, 208, 103500. Available from https://doi.org/10.1016/j.jprot.2019.103500. McCarthy, A. L., O’Callaghan, Y. C., Connolly, A., Piggott, C. O., FitzGerald, R. J., & O’Brien, N. M. (2013). In vitro antioxidant and anti-inflammatory effects of brewers’ spent grain protein rich isolate and its associated hydrolysates. Food Research International, 50(1), 205
212. Available from https://doi.org/ 10.1016/j.foodres.2012.10.022. McHugh, D. J. (2002). Prospects for seaweed production in developing countries. Megido, R. C., Gierts, C., Blecker, C., Brostaux, Y., Haubruge, E´., Alabi, T., & Francis, F. (2016). Consumer acceptance of insect-based alternative meat products in Western countries. Food Quality and Preference, 52, 237
243. Melanie, H., Taarji, N., Zhao, Y., Khalid, N., Neves, M. A., Kobayashi, I., . . . Nakajima, M. (2020). Formulation and characterisation of O/W emulsions stabilised with modified seaweed polysaccharides. International Journal of Food Science & Technology, 55(1), 211
221. Min, S.-G., Jo, Y.-J., & Park, S. H. (2017). Potential application of static hydrothermal processing to produce the protein hydrolysates from porcine skin by-products. LWT - Food Science and Technology, 83, 18
25. Available from https://doi.org/10.1016/j.lwt.2017.04.073.

450 Chapter 18 Mine, Y., Li-Chan, E., & Jiang, B. (2010). Bioactive proteins and peptides as functional foods and nutraceuticals. Oxford, UK: Wiley-Blackwell. Morales-Medina, R., Tamm, F., Guadix, A. M., Guadix, E. M., & Drusch, S. (2016). Functional and antioxidant properties of hydrolysates of sardine (S. pilchardus) and horse mackerel (T. mediterraneus) for the microencapsulation of fish oil by spray-drying. Food Chemistry, 194, 1208
1216. Available from https:// doi.org/10.1016/j.foodchem.2015.08.122. Ng, T. (2004). Peptides and proteins from fungi. Peptides, 25(6), 1055
1073. Ngai, P. H., Zhao, Z., & Ng, T. (2005). Agrocybin, an antifungal peptide from the edible mushroom Agrocybe cylindracea. Peptides, 26(2), 191
196. Ngo-son, A., & Katekaew, S. (2019). Purification and characterization of angiotensin converting enzymeinhibitory derived from crocodile blood hydrolysates. Food Science and Technology, 39, 818
823. Retrieved from. , http://www.scielo.br/scielo.php?script 5 sci_arttext&pid 5 S010120612019000400818&nrm 5 iso . . Nguyen, E., Jones, O., Kim, Y. H. B., San Martin-Gonzalez, F., & Liceaga, A. M. (2017). Impact of microwave-assisted enzymatic hydrolysis on functional and antioxidant properties of rainbow trout Oncorhynchus mykiss by-products. Fisheries Science, 83(2), 317
331. Available from https://doi.org/ 10.1007/s12562-017-1067-3. Nisizawa, K., Noda, H., Kikuchi, R., & Watanabe, T. (1987). The main seaweed foods in Japan. Hydrobiologia, 151(1), 5
29. Nongonierma, A. B., & FitzGerald, R. J. (2017). Unlocking the biological potential of proteins from edible insects through enzymatic hydrolysis: A review. Innovative Food Science & Emerging Technologies. Nongonierma, A. B., Lamoureux, C., & FitzGerald, R. J. (2018). Generation of dipeptidyl peptidase IV (DPPIV) inhibitory peptides during the enzymatic hydrolysis of tropical banded cricket (Gryllodes sigillatus) proteins. Food & function, 9(1), 407
416. Offengenden, M., Chakrabarti, S., & Wu, J. (2018). Chicken collagen hydrolysates differentially mediate antiinflammatory activity and type I collagen synthesis on human dermal fibroblasts. Food Science and Human Wellness, 7(2), 138
147. Available from https://doi.org/10.1016/j.fshw.2018.02.002. Osimani, A., Milanovi´c, V., Cardinali, F., Roncolini, A., Garofalo, C., Clementi, F., . . . Raffaelli, N. (2018). Bread enriched with cricket powder (Acheta domesticus): A technological, microbiological and nutritional evaluation. Innovative Food Science & Emerging Technologies. O’Sullivan, S. M., Lafarga, T., Hayes, M., & O’Brien, N. M. (2017). Anti-proliferative activity of bovine blood hydrolysates towards cancer cells in culture. International Journal of Food Science & Technology, 52(4), 1049
1056. Available from https://doi.org/10.1111/ijfs.13371. Paiva, L., Lima, E., Patarra, R. F., Neto, A. I., & Baptista, J. (2014). Edible Azorean macroalgae as source of rich nutrients with impact on human health. Food Chemistry, 164, 128
135. Peanparkdee, M., & Iwamoto, S. (2019). Bioactive compounds from by-products of rice cultivation and rice processing: Extraction and application in the food and pharmaceutical industries. Trends in Food Science & Technology, 86, 109
117. Available from https://doi.org/10.1016/j.tifs.2019.02.041. Qian, G.-m, Pan, G.-F., & Guo, J.-Y. (2012). Anti-inflammatory and antinociceptive effects of cordymin, a peptide purified from the medicinal mushroom Cordyceps sinensis. Natural Product Research, 26(24), 2358
2362. Radenkovs, V., Juhnevica-Radenkova, K., Go´rna´s, P., & Seglina, D. (2018). Non-waste technology through the enzymatic hydrolysis of agro-industrial by-products. Trends in Food Science & Technology, 77, 64
76. Available from https://doi.org/10.1016/j.tifs.2018.05.013. Ramos, M., Burgos, N., Barnard, A., Evans, G., Preece, J., Graz, M., . . . Jime´nez, A. (2019). Agaricus bisporus and its by-products as a source of valuable extracts and bioactive compounds. Food Chemistry, 292, 176
187. Available from https://doi.org/10.1016/j.foodchem.2019.04.035. Ramos-Elorduy, J. (2009). Anthropo-entomophagy: Cultures, evolution and sustainability. Entomological Research, 39(5), 271
288.

Sustainable, alternative sources of bioactive peptides 451 Ravindran, R., & Jaiswal, A. K. (2016). Exploitation of food industry waste for high-value products. Trends in Biotechnology, 34(1), 58
69. Available from https://doi.org/10.1016/j.tibtech.2015.10.008. Rizzo, C., Genovese, G., Morabito, M., Faggio, C., Pagano, M., Spano`, A., . . . Cigala, R. M. (2017). Potential antibacterial activity of marine macroalgae against pathogens relevant for aquaculture and human health. Journal of Pure and Applied Microbiology, 11, 1695
1706. Roblet, C., Akhtar, M. J., Mikhaylin, S., Pilon, G., Gill, T., Marette, A., & Bazinet, L. (2016). Enhancement of glucose uptake in muscular cell by peptide fractions separated by electrodialysis with filtration membrane from salmon frame protein hydrolysate. Journal of Functional Foods, 22, 337
346. Available from https:// doi.org/10.1016/j.jff.2016.01.003. Saisavoey, T., Sangtanoo, P., Reamtong, O., & Karnchanatat, A. (2016). Antioxidant and anti-inflammatory effects of defatted rice bran (Oryza sativa L.) protein hydrolysates on raw 264.7 macrophage cells. Journal of Food Biochemistry, 40(6), 731
740. Available from https://doi.org/10.1111/jfbc.12266. Sato, M., Hosokawa, T., Yamaguchi, T., Nakano, T., Muramoto, K., Kahara, T., . . . Nakano, T. (2002). Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 50(21), 6245
6252. Searchinger, T., Walte, R., Hanson, C., & Ranganathan, J. (2019). World resources report: Creating a sustainable food future. A menu of solutions to feed nearly 10 billion people by 2050. World Resources Institute., 564, July 17. Sheu, F., Chien, P.-J., Chien, A.-L., Chen, Y.-F., & Chin, K.-L. (2004). Isolation and characterization of an immunomodulatory protein (APP) from the Jew’s Ear mushroom Auricularia polytricha. Food Chemistry, 87(4), 593
600. Silveira Coelho, M., de Araujo Aquino, S., Machado Latorres, J., & de las Mercedes Salas-Mellado, M. (2019). In vitro and in vivo antioxidant capacity of chia protein hydrolysates and peptides. Food Hydrocolloids, 91, 19
25. Available from https://doi.org/10.1016/j.foodhyd.2019.01.018. Slizyte, R., Rommi, K., Mozuraityte, R., Eck, P., Five, K., & Rustad, T. (2016). Bioactivities of fish protein hydrolysates from defatted salmon backbones. Biotechnology Reports, 11, 99
109. Available from https:// doi.org/10.1016/j.btre.2016.08.003. Sosa, D. A. T., & Fogliano, V. (2017). Potential of insect-derived ingredients for food applications. In Insect physiology and ecology. InTech. Suetsuna, K., Maekawa, K., & Chen, J.-R. (2004). Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. The Journal of Nutritional Biochemistry, 15(5), 267
272. Sun, J., He, H., & Xie, B. J. (2004). Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. Journal of Agricultural and Food Chemistry, 52(21), 6646
6652. Suwal, S., Perreault, V., Marciniak, A., Tamigneaux, E´., Deslandes, E´., Bazinet, L., . . . Doyen, A. (2019). Effects of high hydrostatic pressure and polysaccharidases on the extraction of antioxidant compounds from red macroalgae, Palmaria palmata and Solieria chordalis. Journal of Food Engineering, 252, 53
59. Tan, Y. N., Ayob, M. K., & Wan Yaacob, W. A. (2013). Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake. Food Chemistry, 136(1), 279
284. Available from https://doi.org/10.1016/j.foodchem.2012.08.012. Tao, M., Wang, C., Liao, D., Liu, H., Zhao, Z., & Zhao, Z. (2017). Purification, modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate. Process Biochemistry, 54, 172
179. Available from https://doi.org/10.1016/j. procbio.2016.12.022. Urbizo-Reyes, U., San Martin-Gonza´lez, M. F., Garcia-Bravo, J., Lo´pez Malo Vigil, A., & Liceaga, A. M. (2019). Physicochemical characteristics of chia seed (Salvia hispanica) protein hydrolysates produced using ultrasonication followed by microwave-assisted hydrolysis. Food Hydrocolloids, 97, 105187. Available from https://doi.org/10.1016/j.foodhyd.2019.105187. Va´squez, V., Martı´nez, R., & Bernal, C. (2019). Enzyme-assisted extraction of proteins from the seaweeds Macrocystis pyrifera and Chondracanthus chamissoi: Characterization of the extracts and their bioactive potential. Journal of Applied Phycology, 31(3), 1999
2010.

452 Chapter 18 Vercruysse, L., Smagghe, G., Beckers, T., & Van Camp, J. (2009). Antioxidative and ACE inhibitory activities in enzymatic hydrolysates of the cotton leafworm, Spodoptera littoralis. Food Chemistry, 114(1), 38
43. Available from https://doi.org/10.1016/j.foodchem.2008.09.011. Vercruysse, L., Smagghe, G., Herregods, G., & Van Camp, J. (2005). ACE inhibitory activity in enzymatic hydrolysates of insect protein. Journal of Agricultural and Food Chemistry, 53(13), 5207
5211. Vercruysse, L., Smagghe, G., Matsui, T., & Van Camp, J. (2008). Purification and identification of an angiotensin I converting enzyme (ACE) inhibitory peptide from the gastrointestinal hydrolysate of the cotton leafworm, Spodoptera littoralis. Process Biochemistry, 43(8), 900
904. Vercruysse, L., Van Camp, J., Morel, N., Rouge´, P., Herregods, G., & Smagghe, G. (2010). Ala-Val-Phe and Val-Phe: ACE inhibitory peptides derived from insect protein with antihypertensive activity in spontaneously hypertensive rats. Peptides, 31(3), 482
488. Wang, J., Liu, Y.-M., Cao, W., Yao, K.-W., Liu, Z.-Q., & Guo, J.-Y. (2012). Anti-inflammation and antioxidant effect of Cordymin, a peptide purified from the medicinal mushroom Cordyceps sinensis, in middle cerebral artery occlusion-induced focal cerebral ischemia in rats. Metabolic Brain Disease, 27(2), 159
165. Wang, S. B., Hu, Q., Sommerfeld, M., & Chen, F. (2004). Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae). Proteomics, 4(3), 692
708. ´ ., Thorkelsson, G., & O ´ lafsdo´ttir, Wang, T., Jo´nsdo´ttir, R., Kristinsson, H. G., Hreggvidsson, G. O., Jo´nsson, J. O G. (2010). Enzyme-enhanced extraction of antioxidant ingredients from red algae Palmaria palmata. LWTFood Science and Technology, 43(9), 1387
1393. Wang, W., Wang, N., & Zhang, Y. (2014). Antihypertensive properties on spontaneously hypertensive rats of peptide hydrolysates from silkworm pupae protein. Food and Nutrition Sciences, 5(13), 1202. Wang, W., Wang, N., Zhou, Y., Zhang, Y., Xu, L., Xu, J., . . . He, G. (2011). Isolation of a novel peptide from silkworm pupae protein components and interaction characteristics to angiotensin I-converting enzyme. European Food Research and Technology, 232(1), 29
38. Wasser, S. P. (2017). Medicinal mushrooms in human clinical studies. Part I. Anticancer, oncoimmunological, and immunomodulatory activities: A review. International Journal of Medicinal Mushrooms, 19, 4. Womeni, H. M., Tiencheu, B., Linder, M., Nabayo, E. M. C., Tenyang, N., Mbiapo, P., . . . Panmentier, M. (2012). Nutritional value and effect of cooking, drying and storage process on some functional properties of Rhynchophorus phoenicis. International Journal of Life Science and Pharma Research, 2(3), 203
219. Wong, J. H., Ng, T. B., Wang, H., Sze, S. C. W., Zhang, K. Y., Li, Q., & Lu, X. (2011). Cordymin, an antifungal peptide from the medicinal fungus Cordyceps militaris. Phytomedicine, 18(5), 387
392. Wu, Q., Jia, J., Yan, H., Du, J., & Gui, Z. (2015). A novel angiotensin-I converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: Biochemical characterization and molecular docking study. Peptides, 68, 17
24. Available from https://doi.org/10.1016/ j.peptides.2014.07.026. Wu, Q.-Y., Jia, J.-Q., Tan, G.-X., Xu, J.-L., & Gui, Z.-Z. (2011). Physicochemical properties of silkworm larvae protein isolate and gastrointestinal hydrolysate bioactivities. African Journal of Biotechnology, 10(32), 6145
6153. Wu, Y., & Wang, D. (2009). A new class of natural glycopeptides with sugar moiety-dependent antioxidant activities derived from Ganoderma lucidum fruiting bodies. Journal of Proteome Research, 8(2), 436
442. Xu, X., Yan, H., Chen, J., & Zhang, X. (2011). Bioactive proteins from mushrooms. Biotechnology Advances, 29(6), 667
674. Xu, Y., Li, Y., Bao, T., Zheng, X., Chen, W., & Wang, J. (2017). A recyclable protein resource derived from cauliflower by-products: Potential biological activities of protein hydrolysates. Food Chemistry, 221, 114
122. Available from https://doi.org/10.1016/j.foodchem.2016.10.053. Yang, J., Huang, J., Dong, X., Zhang, Y., Zhou, X., Huang, M., & Zhou, G. (2020). Purification and identification of antioxidant peptides from duck plasma proteins. Food Chemistry, 319, 126534. Available from https://doi.org/10.1016/j.foodchem.2020.126534. Yang, R., Zhao, X., Kuang, Z., Ye, M., Luo, G., Xiao, G., . . . Xiong, Z. (2013). Optimization of antioxidant peptide production in the hydrolysis of silkworm (Bombyx mori L.) pupa protein using response surface methodology. Journal of Food, Agriculture and Environment, 11, 952
956.

Sustainable, alternative sources of bioactive peptides 453 Yermak, I. M., Davydova, V. N., Kravchenko, A. O., Chistyulin, D. A., Pimenova, E. A., & Glazunov, V. P. (2020). Mucoadhesive properties of sulphated polysaccharides carrageenans from red seaweed families Gigartinaceae and Tichocarpaceae. International Journal of Biological Macromolecules, 142, 634
642. Youssef, F. S., Ashour, M. L., Singab, A. N. B., & Wink, M. (2019). A comprehensive review of bioactive peptides from marine fungi and their biological significance. Marine Drugs, 17(10), 559. Yu, D., Sun, Y., Wang, W., O’Keefe, S. F., Neilson, A. P., Feng, H., . . . Huang, H. (2020). Recovery of protein hydrolysates from brewer’s spent grain using enzyme and ultrasonication. International Journal of Food Science & Technology, 55(1), 357
368. Available from https://doi.org/10.1111/ijfs.14314. Zarei, M., Forghani, B., Ebrahimpour, A., Abdul-Hamid, A., Anwar, F., & Saari, N. (2015). In vitro and in vivo antihypertensive activity of palm kernel cake protein hydrolysates: Sequencing and characterization of potent bioactive peptides. Industrial Crops and Products, 76, 112
120. Available from https://doi.org/ 10.1016/j.indcrop.2015.06.040. Zhang, G., Sun, J., Wang, H., & Ng, T. (2009). A novel lectin with antiproliferative activity from the medicinal mushroom Pholiota adiposa. Acta Biochimica Polonica, 56(3). Zhang, Y., Chen, R., Chen, X., Zeng, Z., Ma, H., & Chen, S. (2016). Dipeptidyl peptidase IV-inhibitory peptides derived from silver carp (Hypophthalmichthys molitrix Val.) proteins. Journal of Agricultural and Food Chemistry, 64, 831
839. Available from https://doi.org/10.1021/acs.jafc.5b05429. Zhang, Y., Wang, N., Wang, W., Wang, J., Zhu, Z., & Li, X. (2016). Molecular mechanisms of novel peptides from silkworm pupae that inhibit α-glucosidase. Peptides, 76, 45
50. Available from https://doi.org/ 10.1016/j.peptides.2015.12.004. Zhao, R.-J., Huo, C.-Y., Qian, Y., Ren, D.-F., & Lu, J. (2017). Ultra-high-pressure processing improves proteolysis and release of bioactive peptides with activation activities on alcohol metabolic enzymes in vitro from mushroom foot protein. Food Chemistry, 231, 25
32. Zheng, Y., Li, Y., Zhang, Y., Ruan, X., & Zhang, R. (2017). Purification, characterization, synthesis, in vitro ACE inhibition and in vivo antihypertensive activity of bioactive peptides derived from oil palm kernel glutelin-2 hydrolysates. Journal of Functional Foods, 28, 48
58. Available from https://doi.org/10.1016/j. jff.2016.11.021. Zheng, Y., Zhang, Y., & San, S. (2020). Efficacy of a novel ACE-inhibitory peptide from Sargassum Maclurei in hypertension and reduction of intracellular endothelin-1. Nutrients, 12(3), 653. Zheng, Z., Wei, X., Shang, T., Huang, Y., Hu, C., & Zhang, R. (2018). Bioconversion of duck blood cell: process optimization of hydrolytic conditions and peptide hydrolysate characterization. BMC Biotechnology, 18(1), 6767. Available from https://doi.org/10.1186/s12896-018-0475-5. Zhou, Z.-F., Ren, Z.-X., Yu, H.-Y., Jia, J.-Q., & Gui, Z.-Z. (2017). Effects of different modification techniques on molecular structure and bioactivity of Bombyx mori pupa protein. Journal of Asia-Pacific Entomology, 20(1), 35
41. Zhu, K., Zhou, H., & Qian, H. (2006). Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase. Process Biochemistry, 41(6), 1296
1302. Zieli´nska, E., Baraniak, B., & Kara´s, M. (2017). Antioxidant and anti-inflammatory activities of hydrolysates and peptide fractions obtained by enzymatic hydrolysis of selected heat-treated edible insects. Nutrients, 9 (9), 970. Zieli´nska, E., Kara´s, M., & Jakubczyk, A. (2017). Antioxidant activity of predigested protein obtained from a range of farmed edible insects. International Journal of Food Science and Technology, 52, 306
312. Available from https://doi.org/10.1111/ijfs.13282.

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CHAPTER 19

Application in nutrition: mineral binding Sarah El Hajj1,2, Tatiana Sepulveda-Rincon1,3, Ce´dric Paris1,4, Tristan Giraud5, Gizella Csire5,6, Loic Stefan5, Katalin Selmeczi6, Jean-Michel Girardet7, Ste´phane Desobry4, Said Bouhallab8, Laurence Muhr1, Caroline Gaucher2 and Laetitia Canabady-Rochelle1 1

Universite´ de Lorraine, CNRS, LRGP, Nancy, France, 2Universite´ de Lorraine, CITHEFOR, Nancy, France, 3Faculty of Pharmaceutical and Food Sciences, University of Antioquia, Medellin, Colombia, 4 Universite´ de Lorraine, LIBio, Nancy, France, 5Universite´ de Lorraine, CNRS, LCPM, Nancy, France, 6 Universite´ de Lorraine, CNRS, L2CM, Nancy, France, 7Universite´ de Lorraine, INRAE, IAM, Nancy, France, 8STLO, INRAE, Institut Agro, Rennes, France

19.1 Introduction Minerals are essentials for human health since they are involved in the inherent structure of the organism (bone and teeth constitution), in its metabolism (enzyme, hormone composition), or its functions (cardiac rhythm, muscle contraction, neuronal conductivity, and acido-basic equilibrium). Constituting about 4% of the body weight, their presence varies in the organism according to the type of mineral. They are either classified as main minerals (needs higher than 100 mg/day; including calcium, magnesium, potassium, phosphorus, sulfur, sodium, and chlorine) or as trace elements (less than 15 g in total in an adult human body, including iron, zinc, copper, fluorine, iodine, chrome and selenium). An equilibrated and diversified nutrition brings the required daily intake in minerals and compensates their natural loss in urine, sweat, or cell peeling. Nevertheless, due to our industrial way of life, the level of minerals in food decreases due to either the intensive agriculture that depletes the soil in mineral or to the industrial processes (prewashing treatment, ionization, and pasteurization). Besides, some external factors increase our needs, notably the stress of daily life, smoking, pollution, sport, age, or drug consumption. In this chapter, we focus our attention principally on calcium and magnesium as examples of main minerals, and iron and copper as trace elements (Fig. 19.1). First, we report on the importance of minerals for nutrition. Second, the potential applications of mineral-binding peptides (MBPs), their natural source of obtention, and their way of production are discussed. In the third part, the peptide
metal ion interactions, the various ways for MBP screening, and separation processes commonly used—based on these specific Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00016-9 © 2021 Elsevier Inc. All rights reserved.

455

456 Chapter 19

Screening/separation

Proteolysis Proteins

Mixture of peptides

Mineral-binding peptides

Bioactive peptides

Nutrition application

Peptide chemical synthesis Amino acids

Figure 19.1 Synoptic scheme of this chapter.

peptide
metal interactions—are reviewed. The last part of this chapter is dedicated to the evident effects of MBPs on nutrition.

19.2 Importance of minerals for nutrition 19.2.1 Main mineral involved in nutrition and their needs in human As essential micro-nutrients, minerals needs are satisfied with diet. Their repartition and their role in the human body depend on the nature of the mineral, and on physiological parameters of each individual (i.e., age, sex, specific period of life such as pregnancy, lactation, or illness). For instance, calcium accounts for about 1%
2% of the body weight (1200 g for an adult). Mainly related to the skeleton edification (99% in bone and teeth), calcium is also involved in other functions such as intracellular metabolism, muscles contraction, cardiac functions, cellular exchange, membrane permeability, hormone release, and transmission of the nerve impulses (Peng, Hou, Zhang, & Li, 2017). Magnesium is one of the most important minerals in human, with half of its total mass located in bones as well. An adult body contains approximately 25 g of magnesium (DiNicolantonio, Liu, & O’Keefe, 2018). It regulates the metabolism of carbohydrates (via glucose degradation) and lipids present in the muscular, cardiac, and nerves tissues. Magnesium participates also to the protein synthesis, to hormones activity (i.e., insulin) and is involved in the functioning of more than 300 enzymes. In humans, iron is present in the body at about 3
4 g (B50 mg/kg body weight) and is mainly included in the hemoglobin of red blood cells, the remaining being incorporated in the myoglobin present in skeletal muscle cells or stored (ferritine) in liver, spleen, or bone

Application in nutrition: mineral binding 457 marrow. Iron is mainly involved in the oxygen transport and in antioxidant enzymes activity (e.g., peroxidase and catalase). Finally, copper plays a vital role as a cofactor for various enzymes, although as a trace element (1.4
2.1 mg/kg body weight). It is involved in antiinflammation, neural communication, and acts as antibacterial. Furthermore, copper is involved in the metabolism of iron, taking part in the heme synthesis through its insertion in the cytochrome oxidase and also being able to inhibit iron absorption by binding to transferrin receptors (Chan & Rennert, 1980).

19.2.2 Safety considerations and standards/regulation The recommended daily allowance for the various minerals investigated is stated hereafter according to the FAO specifications (Table 19.1). Calcium needs are comprised between 400 and 500 mg/day and up to 1 g/day for adults, with recommendations varying between countries (Gue´guen & Pointillart, 2000). Calcium needs are higher during growth (400
700 mg/day for a child) or specific periods such as pregnancy or lactation. Milk and dairy products are the main natural sources of highly bioavailable calcium. Calcium deficiency involves rickets in children and osteoporosis in the elderly, a disease that affects more than 75 million people in the world (Gue´guen, Pointillart, 2000). Calcium nutrition, bioavailability, and fortification were nicely reviewed by Vavrusova and Skibsted (2014). Concerning magnesium, the recommended daily intake of 6 mg/kg/day is raised to 25 mg during growth, 40 mg during pregnancy, and 30 mg during lactation. Magnesium is found in cocoa, various nuts, seafood, cereals, or dried fruits. Normal magnesium concentration protects against various diseases and its deficiency induces anxiety, headache, a general fatigue in addition to a low morale (Vormann, 2003). Iron needs are about 10 mg/day for an adult, with a higher recommended daily intake for children (increase of the blood volume during growth), childbearing-aged women due to menses, during pregnancy and lactation. Anemia is related to iron deficiency while hemochromatosis is due to an excess of iron in the body. In this latter genetic illness, responsible for an excessive iron intestinal absorption, iron excess settles down in the liver, the heart, and the skin with chronic fatigue as the main symptom. Meat (animal liver, especially), fish, seafood, eggs, beans, legumes, and green vegetables (spinach, broccoli, etc.) are the main sources of iron-containing food. Adult daily recommended intake for copper is B2
5 mg/day, while 0.8
1.9 mg/day is recommended for children (Lo¨nnerdal, 1996). These recommendations are easily satisfied by copper-containing food (i.e., animal liver and kidney, shellfish, chocolate, legumes,

Table 19.1: Safety considerations and standard regulations.

Mineral Calcium

Repartition within the human body 2% of the body weight, 99% in skeleton

Role Skeleton, metabolism (muscles activity, nerves impulse, enzymes, hormone activity)

Magnesium In muscles and soft tissues (30%
40%), 1% in extracellular fluid, the reminder in skeleton (50%
60%) Iron 3
4 g (hemoglobin in red globules, myoglobins, ferritine)

Involved in metabolism (protein, RNA, DNA), cofactor of many enzymes

Zinc

All body tissues and fluid. 60% in the skeletal muscle. 2
3 g (adult)

Copper

1.4
2.1 mg/kg

In the skeleton, enzymes involved in metabolism (carbohydrate, lipid, proteins, and nucleic acid), maintenance of cell and organ integrity, immune system Constituting several enzymes, iron metabolism, antiinflammatory and antibacterial process, neural communication

O2 transport, enzymes activity (peroxidases, catalases, cytochromes)

Specific needs

MineralIllness in case of deficiency or containing food excess

400
550 mg/day (children), 700 mg/day (adult), 1200 mg/day (lactation) 280
370 mg/day for an adult (France)

Growth, pregnancy, lactation, ageing

Deficiency (osteoporosis), toxicity in case of high calcium when calcium given as carbohydrate form

Dairy products, some vegetables and legumes

FAO (2002); Gue´guen & Pointillart (2000)

Supplementary needs: lactation (50
55 mg/ day)

Rare dietary deficiency, hypermagnesemia (nausea, hypotension, diarrhea, relatively innocuous)

In Hong et al. (2015)

0.7
0.9 mg/day for infant (,1 year), 1.46 mg/ day in adult women, 1.06 mg/ day in adult men 2.1 mg/day (adult women), 3.2 mg/day (adult men)

Ferriprive anemia or Women of hemochromatosis childbearing age, pregnancy, lactation, children

Green vegetables, legume seeds, cocoa, nuts, seafood, cereals, dried fruits Meat (liver), fish, poultry, eggs, legume, green vegetables Meat (liver), fish, eggs

FAO

Liver, shellfish, chocolate, legume

www. musculaction. com

Recommended daily allowances

1.5
2 mg/day (adult)

Children, adolescence, pregnant women

Zinc deficiency: growth retardation, delay sexual and bone maturation, skin lesion, diarrhea, alopecia, defects of immune system. Zinc toxicity (nausea, vomiting, diarrhea, fever) in case of excess Anemia, Menkes syndrome (copper malabsorption) or neurodegenerative pathologies (excess)

Reference

FAO

Application in nutrition: mineral binding 459 cereals, and nuts), which are reviewed in Lo¨nnerdal (1996). Although copper intake in infants and adults is often lower than the recommendations, the status of this element is still adequate for the majority of the people. Hence, copper deficiency is rare but responsible for anemia or Menkes syndrome rather related to copper malabsorption. Inversely, an excess of copper (also known as Wilson’s disease) is toxic; it would act as a pro-oxidant and is involved in neurodegenerative diseases (i.e., Parkinson and Alzheimer disease) as reported in the literature (Kaden, Bush, Danzeisen, Bayer, & Multhaup, 2011; Rivera-Mancı´a et al., 2010). Similarly, to other sources of trace elements, iron and copper in the diet depend on their concentration in the soil (Lo¨nnerdal, 1996).

19.2.3 Bioavailability and metabolism of minerals The metabolism of minerals from food depends first on their bioavailability. In nutrition, this is defined as the part of nutrients present in food, effectively assimilated by the organism and is dependent on various parameters, either physiological (age, sex, etc.) or related to supplementation. Indeed, the nature of salt, the quantity used for supplementation, and the presence or absence of other components in the formulation can increase or decrease the mineral bioavailability. For instance, MBPs and nondigestible carbohydrates are reported to improve mineral absorption (Greger, 1999). On the contrary, some components (i.e., phytate, cellulose, and fat) present in food can negatively affect the mineral absorption, in particular calcium (Reinhold, Lahimgarzadeh, Nasr, & Hedayati, 1973; Slavin & Marlett, 1980; in Chen et al., 2014), magnesium (Pallauf, Pietsch, & Rimbach, 1998), and iron, which can be inhibited by the presence of some phytates, oxalate, and tannins. In a general way, free minerals cannot cross the intestinal barrier; they need either to be solubilized or complexed with other components to cross the intestinal barrier to become bioavailable. Calcium is hardly absorbed from foods due to the precipitation of its insoluble salts formed in a basic environment (Jin, Fu, & Ma, 2011; in Peng et al., 2017). Calcium needs to be dissolved as Ca21 in an acidic environment or complexed to organic molecules such as peptides to be absorbed by the small intestine (Wasserman, 2004; in Vavrusova & Skibsted, 2014). Besides, vitamin D also aids to increase the calcium intestinal absorption since it is a fat-soluble secosteroid (Holick, 2004). Calcium absorption decreases from 55 to 60 years old in women or 60 to 65 years old in men (Bullamore, Wilkinson, Gallagher, Nordin, & Marshall, 1970). In the elderly, calcium malabsorption is related to the reduced production of gastric acid, inducing the entrance of calcium in the intestines under the precipitated form (Straub, 2007; Vavrusova & Skibsted, 2014). Magnesium is absorbed all over the intestine, especially on the distal part of the small intestine (jejunum and ileum), via a passive intercellular process mainly (concomitant with an electrochemical gradient and solvent drag) and an active transport for a small part solely

460 Chapter 19 (Coudray et al., 2005). Considering this passive process of absorption, the quantity of Mg absorbed depends on the quantity of the Mg concentration in the intestine. The bioavailability of Mg salt used for supplementation also depends on the type of salt (Coudray et al., 2005). Dietary iron is provided either under heme or nonheme form. The first one, present in food of animal origin, is mainly absorbed at 15%
35% in the duodenum (Pereira & Vicente, 2013; in Sun et al., 2017). In comparison, less than 10% of the nonheme form of iron is absorbed (in Guo et al., 2013). The poorer absorption of this latter form of iron is related to its low solubility at pH near neutrality and its complexation to phytic acid, polyphenols, or fibers. Indeed, as inhibitors of iron absorption, these food components decrease their bioavailability (Hurrell & Egli, 2010; mentioned in Sun et al., 2017). Inversely, some other dietary compounds such as peptides or amino acids can form soluble complexes with iron, which improve its absorption (Bougle´ & Bouhallab, 2006). Compared to the widely used inorganic iron salts (i.e., ferrous sulfate, ferrous gluconate, and ferrous fumarate), iron entrapped into biomolecules/organic molecules has a better bioavailability and lower side effects, as reviewed by Li, Jiang, and Huang (2017) on protein hydrolyzates, which promote iron absorption. Indeed, the MBPs present in hydrolyzate complex iron, increase its stability and facilitate its direct absorption in the small intestine (Wang, Huang, & Jiang, 2013; mentioned in Kim et al., 2014), which finally improve the bioavailability of iron. As stated later in this chapter, MBPs obtained from various proteins constitute potential resources to overcome the mineral deficiency. Copper is absorbed mainly from the stomach but also from the duodenum to some extent. The absorption of this trace element is greatly affected by age. As reviewed by Lo¨nnerdal (1996), the bioavailability of copper is also affected by the source of proteins (animal vs vegetal) and the presence of specific amino acids (e.g., histidine has a high affinity for copper). Other compounds negatively affect the copper bioavailability, such as phytate and fibers, ascorbic acid, or other minerals with similar properties than copper (e.g., zinc, iron). Finally, copper bioavailability can be disturbed upon illness, particularly in the case of Alzheimer’s disease (Kaden et al., 2011).

19.3 Evidence of health effects of mineral-binding peptide The presence of several minerals is required in food diets for life. Mineral absorption occurs across the gastrointestinal mucosa via passive and active transport mechanisms. Sometimes minerals can interact with other substances in the diet during their gastrointestinal tract crossing, which may consequently enhance or impair mineral permeability in the small intestine (Kiela & Ghishan, 2016). Mineral-chelating peptides derived from food proteins display stability properties, enabling mineral transport across the gastrointestinal tract and improving the mineral bioavailability (Chen et al., 2017).

Application in nutrition: mineral binding 461 The effect of mineral-chelating peptides on mineral bioavailability—especially calcium and iron—has been widely evaluated in vitro on cell models and in vivo in animal and human studies (Guo et al., 2014). Calcium is one of the major required minerals in human physiology. Its absorption is mediated by passive paracellular diffusive pathway via the epithelial tight junctions claudins Cldn-2, Cldn-12, and Cldn-15 (Kiela & Ghishan, 2016). Calcium-rich foods (milk, cheese, and other dairy products) contain key proteins such as caseins, hydrolyzed upon tryptic digestion in casein phosphopeptides (CPPs) (Cross, Huq, & Reynolds, 2007). Extensively reported in mineral absorption studies, CPPs promoted calcium permeability through Caco-2 cells intestinal barrier model (Cosentino et al., 2010). Ferraretto, Signorile, Gravaghi, Fiorilli, and Tettamanti (2001) suggested that CPPs enhance calcium absorption by acting either on calcium channels at plasma membranes or as calcium-carrier proteins by endocytosis (Ferraretto et al., 2001). To favor calcium absorption, CPPs directly interact with transient receptor potential V6, a selective calcium channel (Perego et al., 2013). Sato, Noguchi, and Naito (1985) evidenced that the increase of calcium absorption at the rat small intestine level (ligated loops) was related to the inhibition of calcium precipitation due to CPPs calcium chelation properties. Indeed, increased calcium absorption by rats small intestine level was also observed when calcium solution and phytate deficient deaminated soybean globulins (another calcium chelator) were administrated together (Kumagai et al., 2004). Chabance et al. (1998) recovered casein-derived peptides from the duodenum of six healthy individuals after milk ingestion, in addition to other peptides related to casein found in the subjects’ plasma. In addition, researchers detected in human volunteers with ileostomy, the presence of CPPs in ileostomy fluid after 10 hours of milk ingestion, evidencing that these peptides can survive to the intestinal tract environment up to the ileum (Meisel et al., 2003). Moreover, fish-bone peptides from fish meals prevent also calcium deficiency by increasing calcium absorption and bone mineral density in ovariectomized rats (Jung, Lee, & Kim, 2006). Iron absorption occurs at the level of duodenal brush border, where cytochrome b reductase 1 reduces the ferric form of iron (Fe31) to its ferrous form (Fe21), the latter crossing through the brush membrane via the divalent metal transporter 1 (DMT1). Only heme iron is not absorbed via DMT1 but via a heme transporter (HCP1) (Kiela & Ghishan, 2016). Certain studies demonstrated the positive effect of iron bound to peptides for iron-deficiency treatment. Kapsokefalou and Miller (1995) explained that the solubility of iron in the small intestine was related to its binding to peptides and not to the free amino acids or fatty acids. Indeed, iron bound to CPPs was shown to be soluble and stable in the digestive tract of iron-deficient rats with increased bioavailability compared to the free iron forms (Aıˆt-Oukhatar et al., 2002; Kibangou et al., 2008; Pe´re`s et al., 1999). Kibangou et al. (2008) followed the protective effect of CPPs against enterocyte peroxidation, induced by the increase of iron absorption. Layrisse, Martı´nez-Torres, Leets, Taylor, and Ramı´rez (1984) conducted a study on 113 subjects to

462 Chapter 19 determine the effect of cysteine, histidine, reduced glutathione, and beef consumption on iron bioavailability. They showed that free cysteine had the same effect as beef consumption concerning the enhancement of food iron bioavailability and that this amino-acid residue maintained its activity when present in the reduced form of glutathione peptide. Although mineral absorption is physiologically a well-regulated phenomenon, preclinical and clinical trials on mineral-chelating peptides are needed to prove their health benefits in malabsorption syndromes. To date, various bioactive peptides have been identified in the literature from various natural resources. Nevertheless, only a few bioactive peptides are present on the market for human use due to the lack of preclinical and clinical studies. As reviewed by Chalamaiah, Ulug, Hong, and Wu (2019), all over the world, different countries have developed various regulatory frameworks to protect the consumers notably against the risk related to the bioactive peptide consumption, misleading and false claims. The regulation is indeed different according to the region (i.e., United States, Canada, Japan, China, or European countries).

19.4 Mineral-binding peptides: potential applications, sources, production, and commercialization 19.4.1 Application of mineral-binding peptides in nutrition 19.4.1.1 In case of mineral deficiency Some MBPs obtained from protein hydrolysis were reported for their positive effect on mineral absorption. Indeed, such peptides could be used as potential functional ingredients to prevent mineral deficiency. Calcium-chelating peptides were obtained from various hydrolyzed resources such as shrimp (Huang, Ren, & Jiang, 2011; Le Vo, Pham, Le, & Nguyen, 2018), whey proteins (Huang et al., 2015; Zhao et al., 2014), soy proteins (Lv et al., 2009), tilapia protein hydrolyzate (Charoenphun, Cheirsilp, Sirinupong, & Youravong, 2013), and pacific cod bone (Peng et al., 2017). Calcium-chelating peptide (GPAGPHGPPG) derived from skin Alaska pollock improved calcium absorption up to 113% in Caco-2 intestinal cells (Chen et al., 2017). There are few reports on the effects of magnesium-chelating peptides on regulating magnesium absorption. The main dietary protein source studied is milk caseins which yield bioactive multiphosphorylated peptides (Cao et al., 2017; Cuomo, Ceglie, & Lopez, 2011; Zidane et al., 2012). The latter remain stable chelates during in vitro simulated gastrointestinal digestion (Hong et al., 2015). Whey proteins are also source of bioactive peptides that carry various minerals like magnesium and enhance their bioavailability (Vegarud, Langsrud, & Svenning, 2000). Several natural protein resources are potentially rich in iron-chelating peptides, once hydrolyzed, notably those from sugar cane (De La Hoz et al., 2014), barley (Eckert et al., 2016), whey

Application in nutrition: mineral binding 463 (Caetano-Silva, Bertoldo-Pacheco, Paes-Leme, & Netto, 2015; Caetano-Silva et al., 2017), Alaska pollock skin (Guo et al., 2013, 2015), hairtail (Huang et al., 2015), αs-casein (Jaiswal, Bajaj, Mann, & Lata, 2015), spirulina (Kim et al., 2014), porcine blood plasma (Lee & Song, 2009), or anchovy muscle (Wu, Liu, Zhao, & Zeng, 2012). Li et al. (2017) nicely reviewed the potential of protein hydrolyzates for improving nonheme iron absorption. Indeed, protein hydrolyzates can promote iron absorption either by keeping the iron soluble, by reducing the Fe (III) to Fe(II), or by promoting the iron transport through the intestinal cell membrane. Lin, Deng, Huang, Li, and Song (2016) investigated the effect of ferrous-chelating hairtail peptides on iron deficiency and demonstrated that such functional peptides could improve the irondeficiency status in anemic patients. In an extensive study, our group showed that iron deficiency in rats was repleted with iron complexed with CPP from casein (Aıˆt-Oukhatar et al., 1999). Similarly, the binding of copper ion to small peptides or amino acids (i.e., histidine, methionine, cysteine) favors the absorption of copper through an amino-acid transporter. Copper-chelating peptides were produced by the hydrolysis of sunflower proteins, with various enzymes used either pure (Megı´as et al., 2008) or in mixture (Megı´as et al., 2007). Phaseolin hydrolyzate was also reported to be rich in Cu21-chelating peptides (Carrasco-Castilla et al., 2012). 19.4.1.2 In case of oxidation phenomena Transition metals, such as iron and copper, are powerful promoters for the production of reactive oxygen species (ROSs) since they are able to donate and accept electrons via intracellular reactions and help in creating free radicals. Metals can act as catalysts accelerating the formation of hydroxyl radicals via the Haber
Weiss reaction (Haber & Weiss, 1934), or can be a direct reactant for their production such as Fe(II) in Fenton reaction (Fenton, 1894). Copper is also able to catalyze such reactions (Gutteridge & Wilkins, 1982). Aging and neurodegenerative diseases (i.e., Alzheimer) are mainly caused by the dysregulation of metal-ion homeostasis, which induces oxidative stress in the brain (Adlard & Bush, 2006). Since the free forms of metals are powerful pro-oxidants, a network of transport and storage systems is expected to control their absorption, distribution, and excretion. Besides, once stored or bound to biomolecules, bioactive metals are much less toxic and cannot induce as much oxidative stress than when present in their free form (Halliwell, 1994). In addition, the exogenous metal-chelators reduce the oxidative stress resulting from environmental toxins and modern life stresses. Nevertheless, related to Alzheimer’s disease, a recent study evidenced that copper can still catalyze the formation of ROS in the brain when complexed to the amyloid-β peptide (Cheignon et al., 2017). The antioxidant metal-chelating properties of peptides are related to characteristic amino acids and their proper positioning within the peptide sequence. Indeed, amino acids can form coordination bonds with metals through their α-amino group, carboxyl group, and the side chain of certain amino acids. The carboxylate function (COO2) as well as nitrogen

464 Chapter 19 atoms (NH2) can coordinate metals to form metal carboxylate salt, amine complex, and five- or six-membered chelating rings (Hancock, 1992; Wong, Albright, & Wang, 1991). Among all 20 amino acids, histidine, cysteine, and tryptophan are well-known for their metal-chelating affinity via their respective functional groups (i.e., imidazole, thiol, and indole) (Nieba et al., 1997). Thus the metal-chelating capacity of bioactive peptides strongly depends on various parameters (see Section 19.5.1).

19.4.2 Sources of mineral-binding peptides 19.4.2.1 Mineral-binding peptide in natural resources MBPs in living organisms, such as microorganisms and plants, play various biological functions, namely, defensive and absorption functions. Rauser (1995) reviewed in his work the metal-binding peptides present in plants. Generally known as metallothioneins (MTs), these conserved sequences of peptides are rich in cysteine and comprise three classes: MT-I, MT-II, and MT-III, the latter one constituting the so-called phytochelatins. As oligomers of glutathione, phytochelatins are found in plant cell’s cytosol and serve as chelators useful for metal tolerance and detoxification (Rascio & Navari-Izzo, 2011). Moreover, minerals uptake by the plants’ roots is an important phenomenon for their growth; therefore MBPs with mineral ion homeostasis roles can be extracted from the roots of plants (Nakayama et al., 2017). Besides, microbial communities can be found wherever minerals are present and their various mechanisms of interaction with such elements have always served themselves as energy providers (Dunbar, 2017). In fact, minerals were key players in the evolution of microbes over the years (Hazen & Ferry, 2010). In a recent study, the term “mineral microbiome” was identified for the first time as the reservoir of microbial communities, in and near mineral sources. This rich source of genetic information could be used either to create synthetic and/or modified microbiomes or to modify and/or extract MBPs (Gilbert, Jansson, & Knight, 2014). Microorganisms were even engineered to take advantage of the MBPs they produce. By constructing clones in Escherichia coli, Kjærgaard, Schembri, and Klemm (2001) discovered a unique peptide sequence (HARAERHHQ) able to bind Zn21 independently from the protein scaffold. As another example, it was shown that the parasitic glutathione S-transferase, Schistosoma japonicum (SjGST) can bind Ni21 with high affinity when its Glu26 residue is muted to His residue, and thus can be used in recombinant E. coli to purify recombinant proteins (Han et al., 2010). Due to their numerous advantages related to their metal-chelation properties, the metal-binding peptides naturally produced in plants and microorganisms were a source of bio-inspiration for their biological and chemical production. Therefore many studies have developed certain methodologies to produce pure MBPs such as proteolysis of proteins and chemical synthesis of peptides.

Application in nutrition: mineral binding 465 19.4.2.2 Production of mineral-binding peptide 19.4.2.2.1 Proteolysis

Over the past few decades, protein hydrolyzates have been widely considered in human nutrition applications. Indeed, the short-chain peptides obtained upon proteolysis contain characteristic amino-acid residues, some of them being highly advantageous for targeting specific physiological or nutritional requirements (Clemente, 2000). The hydrolysis of proteins changes their functionality, resulting in the loss of their native structures, and the production of low molecular weight peptides, with enhanced interactions with their surrounding environment. MBPs obtained by proteolysis can improve minerals absorption (by avoiding precipitation) and bioavailability via peptide transporter in vivo. For the in vitro hydrolysis carried out in a reactor, the protein source and the used proteolytic enzymes specificity are important parameters to consider in addition to other physicochemical parameters to control the MBPs production (Fig. 19.2). Digestive enzymes panel, with many and various proteinases activity and their combinations—including pure (trypsin, pepsin, chymotrypsin) and crude (Alcalase, Protamex, and Flavourzyme) enzymes—have been listed in the literature with their hydrolyzing optimum conditions (Korhonen & Pihlanto, 2006).

pH, T, ionic strength

Type of reactor or process

Protein substrate

Protease

Nature of proteins Conformation Purity Concentration Solubility

Kind of protease Catalytic mechanism Pure or in mixture, Simple or sequential step Concentration Proteolytic activity

Time of hydrolysis

Hydrolysis

Nature, size, and amino acids compositon of peptides Hydrolyzate with functional-, nutritional-, and sensorial-specific properties

Bioactive peptides

Figure 19.2 Proteolysis parameters to act on to produce various MBP sequences and the most common mode of action of the commercially used enzymes (Chabanon, 2005). MBP, Mineral-binding peptide. Source: Figure adapted from Chabanon, G. (2005). Hydrolyses enzymatiques d’isolats prote´iques issus de tourteaux de colza: Cine´tique, mode´lisation, caracte´risation et fonctionnalite´s des peptides. In: The`se de l’Institut National Polytechnique de Lorraine (248 pages).

466 Chapter 19 As an example, many MBPs were produced from the proteolysis of milk proteins, especially casein and whey proteins, and have been widely reported in the literature. Calcium and iron-binding motifs were discovered in these proteins after enzymatic hydrolysis (Vegarud et al., 2000). For instance, the tryptic digestion of αS1-, αS2-, and β-caseins produce specific sequences of CPPs featured with variable calcium-binding properties depending on their content in phosphoryl groups (Bouhallab & Bougle´, 2004; Hartmann & Meisel, 2007). More generally, MBPs were discovered in animals’ protein hydrolyzates. Calcium-binding peptides were notably derived from the hydrolysis of tilapia protein and shrimp processing by-products (Charoenphun et al., 2013; Chen et al., 2014; Cheung, Cheung, Tan, & Li-Chan, 2012). From the digestion of other fish-related proteins, scientists evidenced iron- and zinc-chelating peptides and identified the contributing aminoacid sequences (Chen et al., 2017; Sun et al., 2017; Wu, Li, Hou, Zhang, & Zhao, 2017). Similarly, MBPs were obtained from food proteins derived from plant sources. Due to their wide nutritional values, soybean protein hydrolyzates were extensively examined for their bioactivity, and many studies reported the extraction and screening of calcium-, copper-, and iron-chelating peptides after various enzymatic soy proteolysis (Canabady-Rochelle et al., 2018; Bao, Lv, Yang, Ren, & Guo, 2008; Bao, Song, Zhang, Chen, & Guo, 2007; Lv et al., 2009). Other protein hydrolyzates from plants such as chickpea, rapeseed, sunflower, and bean proteins also contain bioactive MBPs (Carrasco-Castilla et al., 2012; Megı´as et al., 2007; Torres-Fuentes, Alaiz, & Vioque, 2011; Xie et al., 2015). 19.4.2.2.2 Chemical peptide synthesis

For MBPs production, chemical peptide synthesis is an alternative to proteolysis. Peptides are chains of amino acids (Aa) linearly linked together via amide bonds. The synthetic formation of an amide function requires the condensation of a carboxylic acid and an amine group. However, this coupling does not occur spontaneously at ambient temperature (Valeur & Bradley, 2009) and needs to be activated. Despite the existence of thermal activation techniques (Pedersen, Tofteng, Malik, & Jensen, 2012), in most cases, chemical activation is performed by turning the acid group into a more reactive specie like an acyl halide, an azide, an active ester, or an anhydride (Pattabiraman & Bode, 2011). The main challenge is then to choose an efficient activator [e.g., dicyclohexyl carbodiimide (DCC), carbonyl diimidazole (CDI), and onium salts] compatible with the experimental conditions and which prevents from side reactions (e.g., epimerization, guanidinization, and formation of Ncarboxyanhydride) (El-Faham & Albericio, 2011). The original whole protocol for synthetizing peptides was developed by Fisher in the 1900s (Fisher, 1903, 1907) in solution phase. This method, still used for short peptides synthesis, is very laborious for long sequences mainly due to many isolation and purification steps between each coupling (Verlander, 2007). In the 1960s, Merrifield proposed the so-called solid-phase peptide synthesis (SPPS) (Merrifield, 1963); this new procedure enables to perform all coupling

Application in nutrition: mineral binding 467 reactions successively in a single reactor. SPPS is based on the covalent attachment of the first amino acid on a solid phase followed by the stepwise addition of each amino acid of the sequence (Fig. 19.3), the peptide sequence being usually synthesized from C-term to the N-term, whereas other strategies are possible [e.g., reversed SPPS (Jaradat, 2017)]. For SPPS, peptides are fixed on insoluble solid particles, generally polymer-based resin beads, which offer good contact of the grafted compound with the dissolved reagents (Moss, 2005); hence, the reaction media can easily be washed, getting rid of reagent excess and byproducts, renewing completely the medium before the next coupling step. To avoid side reactions, lateral chains of the amino acids must be protected until the end of the synthesis, whereas N-terminal-amino protecting groups are removed at the beginning of each coupling cycle. Several systems of orthogonal protective groups have been elaborated, ´ lvarez, & Albericio, like the popular Boc/Cbz and Fmoc/tBu couples (Isidro-Llobet, A 2009). Once the whole sequence synthetized, the peptide is cleaved from the solid support and the side-chain protecting groups are discarded—sometimes both steps at once (Moss, 2005), and purification are performed through precipitation, crystallization, and/or chromatography techniques. SPPS is therefore a simple and fast method offering high efficiency and yields (Palomo, 2014), widely used in both academia and industry. Thus plenty of antioxidant peptides with an original design or with a sequence previously determined from antioxidant hydrolyzates have been reported in the literature (Liu, Yang, Zhao, & Yang, 2020; Wu, Sun, Ding, Zhu, & Lin, 2019; Zheng, Li, Zhang, & Zhao, 2016), including from our group (Csire, Canabady-Rochelle, Averlant-Petit, Selmeczi, & Stefan, 2020). However, peptide synthesis still suffers from deficiencies regarding “difficult peptide sequences” prone to side reactions or aggregation, requiring other chemical synthesis strategies (Bondalapati, Jbara, & Brik, 2016; Coin, Beyermann, & Bienert, 2007; Paradı´s-Bas, Tulla-Puche, & Albericio, 2016). Recently, new methodologies to form peptide bonds have

Figure 19.3 Principle of solid peptide phase synthesis commonly used for peptide synthesis.

468 Chapter 19 been established, in a greener and healthier way using green solvents and reagents (Varnava & Sarojini, 2019) or no solvent at all (Friˇscˇ i´c, Mottillo, & Titi, 2020), or seeking for atom economy through original amide formation pathways (Pattabiraman & Bode, 2011).

19.5 Selective extraction of mineral-binding peptides from complex hydrolyzates 19.5.1 Peptides
metal ion interactions Peptides are very efficient and versatile ligands for almost all groups of metal ions (Rodzik, Pomastowski, Sagandykova, & Buszewski, 2020). Both the thermodynamic stability and the coordination geometry of metal
peptide complexes are greatly influenced by the amino-acid composition and their sequence. Naturally occurring amino acids (Aa) are usually classified by their structure, side-chain nature, electronic, hydrophilic, or steric properties according to the specific research needs (Wu, 2010). In coordination chemistry, two classes are distinguished: Aa with noncoordinating side chains and those with side chains carrying functional groups, endowed with metal coordinating properties (strong donor atom, Lewis base character). The latter includes the imidazole group of histidine (His), the extra carboxylate function of aspartate (Asp) and glutamate (Glu), the phenolic-OH of tyrosine (Tyr), alcoholic-OH of serine (Ser) and threonine (Thr), the extra amino nitrogen of lysine (Lys) and arginine (Arg) and the sulfur atom from cysteine (Cys), and methionine (Met) side chains. His is able to strongly coordinate metal cations by the imidazole nitrogen (So´va´go´, Va´rnagy, Lihi, & Grena´cs, 2016). Besides, the thioether S-donors in Met or in S-methyl-cysteine (MeCys) have a rather low basicity compared to the thiolate-type S-donor atom in Cys, which is one of the most frequent metal-binding sites (like His) of proteinous molecules (Farkas & So´va´go´, 2017). Despite being a strong donor, the γ-carboxylic group of Glu forms only a low stability seven-membered chelate ring, as the ε-amino function of Lys is too far to form a stable chelate with other N/O-donor atoms of the chain (So´va´go´, Ka´llay, & Va´rnagy, 2012). In addition to the Aa residue composition of a peptide, their arrangement within the primary sequence also affects the peptide
metal ion interaction. Indeed, due to the large number and many different arrangements of donor atoms both in the backbone and in the side chains, small modifications in the amino-acid sequences can cause significant changes in the formation processes of peptide
metal complex. On the other side, peptides with noncoordinating side chains can use terminal-amino and amide nitrogens and/or carbonyl and terminal-carboxyl oxygens as donor atoms. At acidic pH, the metal ion is anchored to the N-terminal α-amino nitrogen, and with increasing pH, the Cu21 and Ni21 metal ion can deprotonate the skeletal amide nitrogen atoms with formation of metal
anionic nitrogen bonds. This metal ion
promoted amide deprotonation

Application in nutrition: mineral binding 469 ˝ and coordination process has a pK value around 4
8 (So´va´go´ & Osz, 2006), much lower than the pK value of the amide N-donor atom deprotonation (pKB15) (Sigel & Martin, 1982). Other factors affect the binding of a metal to peptide including the properties of metal ion, such as the oxidation state, the ionic radius, and the Lewis acidity. The hard/soft acid distinction introduced by Pearson (1963) is often used to explain the metal-ion behavior in the presence of peptide. According to this so-called Pearson theory, Fe(III), Ca(II), and Mg (II) have hard acidic character, Cu(II), Ni(II), and Fe(II) are classed as borderline, while Cu (I) is presented as soft Lewis acidic metal ion. The more stable complexes are formed between Lewis acid and Lewis base having the same character, namely hard acid with hard base, and soft acid with soft base. For example, Cu(I) has a particular affinity to bind sulfur-containing ligands. The deprotonated phenol of Tyr, β-, and γ-carboxylic group of Asp and Glu, as oxygen donor atom, are good ligands for Fe(III), alkali, and alkaline earth metal ions (Shimazaki, Takani, & Yamauchi, 2009). Transition metals like nickel(II), copper(II), and iron(II) interact preferentially with N-donor atoms from His or N-terminal-amino groups (Kozłowski, Kowalik-Jankowska, & Je˙zowska-Bojczuk, 2005; So´va´go´ et al., 2016). Finally, the effect of pH on such interactions should also be mentioned because the protonation state of amino acids in peptides depends on this former parameter. As stated earlier, metal ion
promoted deprotonation and coordination process can also occur for all O/N/S-donor atom
containing groups with pKs lower than their natural pK values in the absence of metal ion. However, at acidic pH, the dissociation of the complexes takes place by protonation of the same donor atoms, this process being a reversible equilibrium depending on pH. In addition, the ionic strength, the type of buffers used, as well as the effect of temperature on the binding constants are important factors.

19.5.2 Mineral-binding peptide screening techniques 19.5.2.1 Spectroscopic techniques 19.5.2.1.1 Principle of spectroscopic techniques

Spectroscopic techniques (Fig. 19.4), commonly applied for examining the interaction, structure, and conformational changes of peptides in metal
peptide complexes, go from simple instrumental analytical techniques to combined application of thermodynamic (potentiometric), spectroscopic, and theoretical (density functional theory) tools. Electronic absorption [UV
Vis
NIR (near infrared region)] and circular dichroism (CD) spectra correspond to the electronic transitions after absorption of isotropic or circularly polarized light, respectively (Jiskoot & Crommelin, 2005; Polavarapu, 2017). Each band is

470 Chapter 19 Absorpon (UV– Vis) and circular dichroism

Fouriertransform infrared and Raman

Fluorescence

Spectroscopic study of metal ion coordinaon sphere Nuclear magnec resonance

X-ray absorpon

Electron paramagnec resonance

Figure 19.4 Various complementary spectroscopic technics used for the investigation of peptide
metal ion interactions.

characterized by its position (λmax or ν max), intensity (εmax for absorption and Δεmax for CD), and shape (negative or positive Cotton effect in CD). Upon metal-ion binding, the electronic distribution of chromophores (amide group and side chains in Phe, Tyr, His, and Trp) is altered in the UV region, the intensity of which depends on the binding strength. More, the presence of transition metal ions involves two less intense transitions in the visible and NIRs: the charge-transfer transition (metal-to-ligand or ligand-to-metal), and the transition d
d of the metal ion. The isosbestic points, seen in the spectra upon cations addition, indicate the presence of two species in equilibrium and enable the binding constant determination. For peptides conformational analysis, changes caused by metal-ion binding, if any, are monitored by CD spectra variation. Vibrational spectroscopies, that is, Fourier-transform infrared (Barth, 2007) and Raman (Buckley & Ryder, 2017), are sensitive tools to analyze the effect of metal ions interactions on the peptide conformations. Raman, based on an inelastic scattering process of light, results in polarizability change during the molecular motion, while IR spectroscopy is associated with changes in the dipole moment during molecular vibrations. Thus both spectroscopies are complementary: transitions allowed in Raman may be forbidden in IR or vice versa (e.g., O
H stretching modes of water and alcohols). The effect of the metal-ion

Application in nutrition: mineral binding 471 coordination on the specific peptide groups is revealed by comparing the band frequencies of the complex with those of the peptide alone (shift in wavenumber, Δν). Nuclear magnetic resonance (NMR) is a powerful spectroscopy for determining the structural and dynamic properties of metal
peptide interactions in solution (De Ricco, Potocki, Kozlowski, & Valensin, 2014). Any change in the electronic environment surrounding the nuclei due to interactions with metal ions leads to selective variations in its NMR parameters (chemical shifts and nuclear relaxation rates). For example, metal ion can induce an electric field via its charge only [i.e., Ca(II), Mg(II), Cu(I), tetra-coordinated Ni (II), low spin Fe(II); classical diamagnetic NMR] or by the presence of unpaired electrons [i.e., Cu(II), hexa-coordinated Ni(II), high spin Fe(II), and Fe(III) ions; paramagnetic NMR]. The latter group can also cause line broadening, the effect of which can be selective and used to locate the metal-binding sites. Electron paramagnetic resonance (EPR) or electron spin resonance is a versatile tool for probing structural information on systems with unpaired electrons such as organic radicals or biological systems with paramagnetic metal centers (Sahu, McCarrick, & Lorigan, 2013). The EPR active metal ions have an odd spin, for example, Cu(II) (S 5 1/2), high spin Fe (III) (S 5 5/2), and the not common Ni(I) and Ni(III) (S 5 1/2) ions. The EPR “silent” metals are the diamagnetic metals with spin equal to zero [Ca(II), Mg(II), Cu(I)] or those with even spin [Ni(II) (S 5 1) and Fe(II) (S 5 2) high spin, signals difficult to detect]. The values of g-factor and hyperfine coupling constant (A) determined by simulation of the experimental spectra, and the line shape are necessary to characterize the type of metal ion, its spin state, the type and number of donor atoms and the geometry around the metal ion (Abragam & Bleaney, 1970). X-ray absorption spectroscopy is an element-specific spectroscopy, sensitive to the local chemical and structural order of the absorber element. X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) are two complementary methods. While XANES provides information on the metal oxidation state and the geometry of coordination sphere, EXAFS gives structural information on the metal-binding site [i.e., coordinating atoms (O/N/S), coordination number, and bond lengths; Ortega, Carmona, Llorens, & Solari, 2012]. Another important technique, fluorescence spectroscopy (FS), allows the binding affinity determination by measuring in a static manner the fluorescence quenching of the peptide upon metal ion addition (Tabak, Sartor, & Cavatorta, 1989). For FS, peptide must contain aromatic amino acids (Trp, Tyr, Phe), inherently fluorescent when excited with UV light (Ghisaidoobe & Chung, 2014). The metal-sensitive property of these AAs near the chelation site highly depends on metal-binding strength. While no change is observed in fluorescence emission intensity for weak complexes, strong metal
peptide complexes cause dramatic changes in the electronic environment quenching strongly the fluorescence. FS,

472 Chapter 19 suitable for moderate/strong affinities, includes some details to take into account during measurements (Van de Weert & Stella, 2011). 19.5.2.1.2 Use of spectroscopic techniques to understand metal
peptide interactions

The previously detailed analytical techniques, particularly suitable to characterize metal
peptide complexes (Faller et al., 2012; Rolinger, Ru¨dt, & Hubbuch, 2020), are more advantageous when combined given their complementarity. They are chosen according to the metal ion and its spin state, which determines the type of bonding and the donor atoms involved. The stoichiometry and geometry of the complex depending mainly on the metalligand ratio and the applied pH, the effect of these latter parameters must be investigated upon spectroscopic measurements. The chemistry of metal
peptide complexes has long been studied spectroscopically and thermodynamically by coupling spectroscopic techniques to potentiometry in bioinorganic chemistry field (Farkas & So´va´go´, 2012, 2017). The potentiometric measurement allows to describe the solution equilibria (stability constants, log K or log β) with a species distribution model containing complexes with different metal (M) and ligand (L: peptide, Aa) stoichiometry at various protonation degrees (MpLqHr) according to pM 1 qL 1 rH 5 MpLqHr equilibrium (Irving & Williams, 1953). Thus information on the MpLqHr species present in solution supplemented by spectral parameters observed as a function of the pH allows to deduce the inner coordination sphere of the metal
peptide complexes. Complemented by theoretical calculations and the solidphase structure, more and more information can be obtained on the interaction of peptides with metal ions (Lamsho¨ft & Ivanova, 2011). 19.5.2.2 Isothermal titration calorimetry 19.5.2.2.1 Principle of isothermal titration calorimetry

Biomolecular interactions, and especially mineral
peptide interactions, can be investigated by isothermal titration calorimetry (ITC) (Ladbury & Chowdhry, 1996). This technology presents the advantage of allowing characterization of weak interactions between two partner biomolecules (defined as ligands). Theory of the ITC approach of metal chelation by proteins was developed by Nielsen, Fuglsang, and Westh (2003). ITC is a thermodynamic approach where the binding equilibrium is directly determined by measuring the heat involved in the association of a ligand with its binding partner. When two molecules interact, heat is either absorbed or released. The measurement of this heat allows the accurate determination of the Gibbs free energy ΔG 5 ΔH 2 TΔS of the reaction, where ΔH is the enthalpy change and ΔS the entropy change of the thermodynamic system. According to the enthalpy change, the reaction is characterized either of endothermic (ΔH . 0) or exothermic (ΔH , 0). This determination provides general information on the nature of the linkages, that is, either electrostatic bonds enthalpically driven such as van der Waals interactions or hydrogen bonds, or hydrophobic

Application in nutrition: mineral binding 473 bonds entropically driven, or both. In addition, the molar ratio of mineral bound with peptides (stoichiometry, N) and the binding constants (KA) can be measured (in Vanhooren et al., 2002). More, the temperature dependence of the ΔH parameter, measured by performing the titration at various temperatures, describes the heat capacity (ΔCp). Finally, the stronger the binding, the less positive the ΔCp is. In a typical ITC experiment, a syringe containing a “ligand” solution is titrated into a cell containing a solution of the “macromolecule” kept at constant temperature as described hereafter (Fig. 19.5). When ligand is injected into the cell, the two materials interact. The heat (and also ΔH), released or absorbed, is related to the amount of binding occurring over the time (Fig. 19.5, top panel, right). As the macromolecule in the cell becomes saturated with ligand, the heat signal diminishes until the background heat of dilution is observed only. A binding curve and its specific binding parameters (N, KA, ΔH, and ΔS) are then obtained after integration from a plot of the heats of binding obtained from each injection against the ratio of ligand (Fig. 19.5, down panel, right). The binding titration curve and the appropriate fit of experimental data determine whether there is one or more classes of mineral-binding sites, independent or not, involving or not cooperativity phenomenon. A modern ITC instrument operates on the heat compensation principle. Upon titration, the amount of uncomplexed

Time (min)

Lead screw

Pcal/s s

Sensor

Sensor Injector Plunger

Syringe Inner shield Reference cell

Outer O t shield Sample cell

kcal/mol of injectant k

Stirring

Molar ratio

Figure 19.5 Principle of isothermal titration calorimetry. Example of the VP-ITC microcalorimeter from Microcal (North-Hampton, MA, United States).

474 Chapter 19 peptide available progressively decreases after each injection and the magnitude of the peak becomes progressively smaller. Once the saturation reached, subsequent injections produce similar peaks corresponding to dilution or mechanical effects. 19.5.2.2.2 Use of ITC for MBP screening

Several studies reported the use of ITC for the thermodynamic characterization of the interaction between metal and peptides, mainly pure synthetic peptides as short fragment of proteins. For instance, Grossoehme, Akilesh, Guerinot, and Wilcox (2006) investigated metal-binding thermodynamics of the histidine-rich sequence from the metal-transport protein IRT1 of Arabidopsis thaliana, an iron transporter overexpressed in iron-deficient conditions. The sequence investigated showed high and low binding to Fe31 and Fe21, respectively. Comba et al. (2013) studied the copper binding with synthetic derivatives of naturally occurring pseudo-octapeptides by ITC combined with square wave voltammetry (SWV) and they observed that the complex stabilities of these derivatives to Cu21 are in agreement with stabilities corresponding to natural ligands. In all of these studies, pure synthetic peptides were considered as model for a better understanding of their biological function in vivo (Grossoehme et al., 2006; Rich et al., 2012; Sacco, Skowronsky, Gade, Kenney, & Spuches, 2012; Sheftic, Snell, Jha, & Alexandrescu, 2012). Regarding natural peptides released by digestion, Zidane et al. (2012) reported the chelation of calcium, magnesium, zinc, and copper by the caseinophosphopeptide β-CN(1
25)4P investigated by ITC under experimental conditions mimicking the physiological conditions of ileum. At pH 8.0, the peptide binds two ions of calcium, magnesium, or zinc, but no copper ion. In another application, ITC is used for screening the presence of metal-binding peptides in hydrolyzates. In their work, Canabady-Rochelle, Sanchez, Mellema, and Banon (2009) and Canabady-Rochelle, Sanchez, Mellema, and Banon (2010) investigated the calciumpeptide interactions in the soy protein hydrolyzate and in other proteinous systems (soy or milk proteins). Whatever the system, the former authors evidenced a calcium-peptide interaction endothermic in nature and fitted by a one set of site model. As compared to nonhydrolyzed protein, soy protein hydrolyzate bound less calcium (2 mg/g of proteins), probably due to the conformational change and destructuration of the protein. The coupling with electrophoretic measurement or pH-cycle enabled a better understanding of calcium-peptide interactions. More recently, ITC experiments were completed by tandem mass spectrometry (MS/MS) to study the interaction between iron(II) and an heptapeptide (SNVVPLY) from barley protein hydrolyzate under physiological conditions (Eckert et al., 2016). The peptide complexed iron(II) spontaneously, with a binding constant reaching 107 M21 suggesting its potential application as dietary supplement to improve iron absorption. Similarly, Liao et al. (2019) evidenced a novel calcium-binding peptide (VLPVPQK) from casein hydrolyzate, which could potentially be developed as nutraceutical additive.

Application in nutrition: mineral binding 475 19.5.2.3 Surface plasmon resonance 19.5.2.3.1 Principle of surface plasmon resonance

Developed recently, surface plasmon resonance (SPR) is at the forefront of evolving technologies in terms of medical, nutritional, and environmental applications (Mir & Shinohara 2013; Rich & Myszka 2011; Shankaran, Gobi, & Miura, 2007). Wood (1902) was the first to observe surface plasmons; however, SPR for biosensing goals was illustrated by Liedberg in 1983 (Liedberg, Nylander, & Lunstro¨m, 1983). The Biacore technology was the first to evolve in the commercial markets based on the SPR principle and has been developed for its application since 1990. Since then, researchers have screened various interactions by SPR technology, among them drug
protein, antigen
antibody, inhibitor
enzyme, and small molecule
nucleic acid (Liu & Wilson, 2010; Navratilova et al., 2007; Sandblad, Arnell, Samuelsson, & Fornstedt, 2009; Tam et al., 2017). In SPR, the so-called ligand is immobilized onto the surface of the sensor chip and when there is an affinity, captures the target molecule or “analyte,” this latter being directly injected in solution and flows over the sensor chip. In case of ligandanalyte binding the refractive index is modified at the chip surface, proportionally to the change in mass concentration (Fig. 19.6). The analyte capture produces measurable signals represented by the sensorgram and expressed in resonance unit. Among various advantages, the SPR technology is simple, in real-time and rapid, and does not require analyte prelabeling. Applied with low amounts of the interacting partners—pure or in a mixture, the obtained sensorgram can determine the rate of complex formation and dissociation (kon, and koff, respectively). Finally, the sensor chip can be reused several times due to the regeneration treatment (Achilleos, Tailhardat, Courtellemont, Varlet, & Dupont, 2009; Maalouli et al., 2011; Treiber, Thompsett, Pipkorn, Brown, & Multhaup, 2007).

(A)

(B)

Optical detector unit Prism I II Reflected Polarized light light

Light source

Sensor chip with gold film

Intensity

I

II Flow chanel

I

II

Angle

Resonance g signal

Time

Figure 19.6 Principle of the surface plasmon resonance and the signal measured (A) and the Ni21 immobilised via NTA onto the SPR chip (B).

476 Chapter 19 19.5.2.3.2 Use of SPR for MBP screening

No longer after its introduction to the biochemical society, SPR technology has gained the scientists curiosity to detect interactions between metal ions and proteins/peptides. It offers the clearest advantages in speed and sample quantity over other alternative techniques such as immobilized metal-ion affinity chromatography (IMAC) (Bernaudat & Bu¨low, 2005). In fact, SPR signal is detected when the peptide/protein is surrounding the ion in its favorable geometry. Some available tetradentate nitrilotriacetic acid (NTA) sensor chips were commercialized by Biacore and allowed the development of a sensitive and selective screening of mineral
peptide complexes. In literature, nickel and copper ions immobilized on NTA chip were the most common methodology to screen MBPs (Canabady-Rochelle et al., 2018; Knecht, Ricklin, Eberle, & Ernst, 2009; Maalouli et al., 2011). Inversely, in other studies, peptides were immobilized on the sensor chips surface and minerals were injected as the analytes (Balliu & Baltzer, 2017; Chen et al., 2015). With convenient and well-functional platforms, the latter studies determined the kinetics of interactions between peptides/proteins and minerals such as copper, magnesium, calcium, and zinc can be determined. 19.5.2.4 Electrically switchable nanolever technology 19.5.2.4.1 Principle of the switchSENSE technology

The innovative switchSENSE technology utilizes a novel electro-switchable biosurface to characterize interactions between molecules in real-time. This technology is unlike existing methodologies; in that, it combines high sensitivity kinetics with structural information on size, shape, and conformation providing a new depth and understanding of the interaction. Studies are performed on a re-usable biochip, generated using familiar coupling, and hybridization methods. Within this biochip, DNA levers are embedded onto a series of gold electrodes. These nanolevers serve either as target for molecular interactions themselves or hold other interaction partners. To characterize interactions, the DRX instrument is used to bring about deliberate movement of these nanolevers by altering the voltage across the gold surface. When interactions occur, these movements (expressed as dynamic response, DR) are affected and, in turn, used in the calculation of kinetic and biophysical information, such as the kinetics constants of association (kon) and dissociation (koff), the affinity constant (KD) and the hydrodynamic diameter (DH) in the case where the ligand is a protein immobilized on the DNA nanolevers (Knezevic et al., 2012; Langer et al., 2013, 2015; Rant, 2012; Rant et al., 2007; Schiedel, Daub, Itzen, & Jung, 2020). 19.5.2.4.2 Application of switchSENSE for mineral-binding peptide screening

For proteins or peptides adsorption onto an immobilized metal chromatographic column, for instance, nickel ions are widely used due to their affinity with the exposed side chains of histidine and cysteine residues (Hainfeld, Liu, Halsey, Freimuth, & Powell, 1999).

Application in nutrition: mineral binding 477 (A) Hybridization

(B) Interaction

Probe

Chelation Probe

25

v

v

0

(C) Binding kinetics

100

0 0

Chip surface

200

MCP DR (dru)

(Ni - NTA)

Fluorescence (kcps)

50

Chip surface

250 500 Time (s)

MCP

(Ni2+-NTA)3

Fluorophore

– +

Peptide

(Ni2+–NTA)3

Nanolever on the chip gold surface Dissociation

Fnorm

Association

Time (s)

Time (s)

Figure 19.7 Principle of switchSENSE adapted for peptide
metal interaction study. (A) Single strands of DNA having 48 base pairs are bound on the chip gold surface and hybridized with a complementary DNA activated by trisNTA. The hybridization is followed by fluorescence measurement of a Cy5 probe fixed at the extremity of the nanolever (kcps: kilocounts per second). A multivalent ion like Ni21 is then immobilized onto the trisNTA group. (B) Binding of MCPs increases friction forces and leads to decrease the dynamic response DR (dru: dynamic response unit) of the nanolevers. (C) The kinetic (kon, koff) and affinity constants (KD 5 koff/kon) of the interaction between Ni21 and MCP can be determined by real-time measurements of the quenching of the normalized fluorescence Fnorm. MCP, Metal-chelating peptide.

With six coordination sites, Ni21 can strongly bind to a complexing agent, for example, the tetradentate nitrilotriacetic acid (NTA), while some sites remain available to interact with the target peptide (Hochuli, Do¨beli, & Schacher, 1987). With the switchSENSE technology, the ssDNA bound on the chip gold surface is hybridized with a cDNAtrisNTA, and a multivalent ion like Ni21 is loaded onto trisNTA; an excess of Ni21 is used to load each NTA moiety with one Ni21 ion (Fig. 19.7A). Binding of nickelchelating peptides increases friction forces and leads to a decrease in the DR of the nanolevers (Fig. 19.7B). The kinetic (kon, koff) and affinity constants (KD 5 koff/kon) of the interaction between Ni21 and MCP can be determined by real-time measurements of the

478 Chapter 19 quenching of the fluorescence (Fig. 19.7C). Hence, direct evidence of MCPs using switchSENSE would enable the faster detection of metal-chelating peptides before launching time-consuming separation, which should be engaged solely in the case of positive screening. For the first time, our team just developed a new screening method to highlight MCPs in dietary hydrolyzates with the switchSENSE technology using immobilized nickel (results currently under publication). 19.5.2.5 Electrospray ionization-mass spectrometry 19.5.2.5.1 Principle of electrospray ionization-mass spectrometry

Electrospray ionization-mass spectrometry (ESI-MS) is a well-known technique for the structural analysis of small and large biomolecules of variable polarity (Ho et al., 2003). For several years, ESI-MS has shown a great potential for the qualitative analysis of metal
organic ligand complexes (Keith-Roach, 2010). ESI is a soft atmospheric pressure ionization allowing the ions transfer from the liquid phase to the gas phase, before their filtering according to their mass-to-charge ratio (m/z) in a mass spectrometer (MS). The near-total absence of fragmentation in the electrospray source allows preferential observation of the pseudo-molecular ion, corresponding to a simple case where the analyte is in its mono-protonated (ESI1) or mono-deprotonated (ESI2) form. In addition, noncovalent bonds can be preserved, which is of major interest for coordination complexes study. In practice, a liquid sample circulates at a low flow rate in a capillary tube (made of stainless steel or quartz) maintained at a high electrical potential (positive or negative) relative to the entry into the MS (2.5
6 kV) (Ho et al., 2003). Under the influence of this electric field and an inert nebulizing gas (N2) applied coaxially to the capillary, fine charged droplets are formed with the same polarity as the applied potential. The solvent is then evaporated by application of a drying gas (N2) and/or gentle heating of the capillary tube (100 C
300 C) leading to the gradual decrease in droplets size. When the droplets are small enough, the electric field on the surface reaches a critical intensity allowing the direct expulsion of ions in the gas phase (Schalley & Armentrout, 2003). With ESI-MS, the MS signature is directly provided from samples in solution, and therefore the species of interest are analyzed in situ (Di Marco & Bombi, 2006). Thanks to its high sensitivity, this method can be applied to biological samples containing very low concentrations of complexes (1026 M). ESI-MS is an effective tool for determining the number of coordination species formed in solution as well as their stoichiometry, since these parameters are directly obtained from the mass spectrum (Di Marco & Bombi, 2006). In fact, the values of m/z ratios at low or high resolution as well as the isotopic patterns

Application in nutrition: mineral binding 479

Figure 19.8 Mass spectrum of a solution containing 2.5 mM Cd(II) and 10 mM glutathione-G. The inset shows the comparison of the enlarged experimental isotopic pattern (solid line) and the theoretical pattern (histogram) for selected cadmium complexes. Source: Reprinted from Keith-Roach, M. J. (2010). A review of recent trends in electrospray ionisation
mass spectrometry for the analysis of metal
organic ligand complexes. Analytica Chimica Acta, 678 (2), 140
148. https://doi.org/10.1016/j. aca.2010.08.023 with permission from Elsevier.

enable to identify the complex structure with reliability (Fig. 19.8). In addition, the complexes stoichiometry can be studied with robustness as a function of the metal:organic ligand ratio and of the pH (Keith-Roach, 2010). 19.5.2.5.2 Use of ESI-MS for MBP screening

ESI-MS has notably been used to characterize a few metal
peptide complexes. The study of the interaction between the small synthetic peptide (αMeAla-Ha AGHa, and GGGHa) and nickel(II) or copper(II) was carried out and validated the formation of different complexes in solution (Gizzi, Henry, Rubini, Giroux, & Wenger, 2005; Jancso´ et al., 2011; Selmeczi et al., 2010). Another work—based on histidine-containing synthetic oligopeptides—successfully used the ESI-MS technique to study the impact of conformational changes on the binding affinity with copper(II) and nickel(II) (Murariu, Dragan, & Drochioiu, 2010). A recent study also described the identification of several iron(II)-peptide complexes from purified peptides, thanks to a ESI-MS differential analysis conducted in the absence and in the presence of iron(II) (Wu et al., 2017). In the case of complex samples, coupled LC-ESI-MS technique allows the matrix effects suppression and is therefore able to provide relevant information on stable noncovalent

480 Chapter 19 LCMS Control (iron-free)

2

Pepde

MS spectrum control

Pepde peak

3 1

Peptide mixture + iron (test) Peptide mixture (control)

2

LCMS injection: test + control

3 Differential analysis of MS spectra 4 [Iron–peptide] complex identification 5

Chelating peptide identification

LCMS test (iron)

Pepde peak

Complex

MS spectrum test

Chelang pepde

5

4 Characterisc ∆M shi

Figure 19.9 Principle of MBP screening in complex mixture of peptides such as hydrolyzates using mass spectra differential analysis. MBP, Mineral-binding peptide.

species (Keith-Roach, 2010). As an example, hydrophilic interaction liquid chromatography (HILIC) and ESI-MS, the so-called HILIC-ESI-MS, was proven effective for the targeted analysis of iron(II)
and iron(III)
phytosiderophore complexes (Xuan et al., 2006). Recently, a promising preliminary study carried out on a pool of small synthetic peptides showed that a LC-ESI-MS-based methodology (Fig. 19.9) could be used for an efficient nontargeted screening of peptides with high iron(II)-chelating abilities (Paris, Selmeczi, Chaimbault, Desobry, & Canabady-Rochelle, 2020).

19.5.3 Immobilized metal-ion affinity chromatography separation 19.5.3.1 Principle of immobilized metal-ion affinity chromatography The IMAC principle is briefly constituted of four main steps (Fig. 19.10): (1) metal-ion immobilization onto the matrix, (2) Sample injection and target protein/peptide retention, (3) washing step to eliminate the unbound components, and (4) finally, elution of the target biomolecule. IMAC is constituted of several parts (Amiri, Mehrnia, Sobhanifard, Pourasgharian Roudsari, & Hoseini, 2017). First, the matrix (i.e., agarose, sepharose, cellulose, silica, and dextran) must be highly hydrophilic and characterized by low nonspecific adsorption, in addition to a high porosity for a high amount of ligand immobilization, or fairly large pore size and narrow pore size distribution (Gutierrez, Martı´n del Valle, & Galan, 2007). Via a spacer arm (usually short alkyl chain), a chelating

Application in nutrition: mineral binding 481

Formation of coordination bond

Washing

Caption

Elution

Complexing agent

Coordination bond Unspecific proteinous compounds

Surface of matrix

Washing

Imidazole

Imidazole competiting agent Target molecules (e.g., metal-binding peptide)

Metal-binding peptides

Figure 19.10 Principle of IMAC. IMAC, Immobilized metal-ion affinity chromatography.

agent is covalently bound onto the matrix for further Mn1 immobilization, this latter is generally tridentate such as iminodiacetic acid (IDA) or tretradentate such as NTA. The higher its denticity, the stronger the immobilization of metal ions and, hence, a lower metal-ion leakage. Yet, the fewer coordination bonds remain available for protein or peptides adsorption (Gaberc-Porekar & Menart, 2001). Once the Mn1 loaded onto the column, the complexing agent and the metal ions form complexes, letting some free coordination sites on the immobilized metal ions to further coordinate peptides or proteins (Gutierrez et al., 2007). Besides the specific coordination interactions of some amino-acid residues exposed on the biomolecule surface with the immobilized metal ions, some other aspecific interactions may occur notably, like electrostatic and hydrophobic interactions (Gutierrez et al., 2007). The nature of the surrounding chemical environment (i.e., buffer salts used and their concentration, pH, and ionic strength) can reduce these aspecific interactions. In this aim, relatively high-ionic strength buffers (often NaCl, 0.1
1.0 M) could be used during adsorption or washing, while the buffer itself should not coordinate to the immobilized metal ions (Gaberc-Porekar & Menart, 2001). Various ways of elution are considered in IMAC like acting on pH (Gonza´lez-Ortega & Guzma´n, 2015), on higher salt

482 Chapter 19 concentration, using a competition elution (e.g., imidazole, ligand exchange method) (Bresolin, Bresolin, & Pessoa, 2015), or a complexing agent elution [e.g., ethylenediaminetetraacetic acid (EDTA)] (Sun et al., 2013). Sometimes, IMAC protocols couple two ways of elution to obtain a better chromatographic resolution (Gutierrez et al., 2007). More, IMAC chromatography columns can be modeled using mathematical equations. Simulations are useful to predict liquid and solid-phase concentration profiles as a function of time, for given operating parameters. The influence of various operating parameters is thus studied numerically, reducing the number of experiments to perform for unit operation optimization. Modeling requires an understanding of the mechanisms governing the chromatographical process, that is, equilibria, hydrodynamics, and kinetics. The simulation of IMAC chromatographic separation of metal-chelating peptides was carried out in our team, from binding parameters determined in SPR (Muhr et al., 2020). Despite the high interest in this approach and considering the readership of this book, modeling of IMAC separation was not developed in this chapter. 19.5.3.2 Use of IMAC for MBP screening Introduced by Porath, Carlsson, Olsson, and Belfrage (1975), IMAC was first applied on proteins fractionation, based on their differential affinity toward immobilized metal ions due to their specific coordinating amino-acid residues exposed in surface (Gutierrez et al., 2007). In the late 1980s, recombined proteins with engineered histidine tag were efficiently purified, especially using Ni21-NTA matrix, selective for adjacent histidine groups binding (Hochuli et al., 1987). However, other matrices can be used: for instance, Fe31-IDA matrix was used to investigate the role of the phosphoseryl cluster (SerP-SerP-SerP-Glu-Glu or ΣΣΣEE) and diphosphoseryl pattern in the complexation of ferric iron and calcium by the purified caseins and the proteose peptone three component of milk and to determine the adsorption efficiency of free amino acids (Tyr, Trp, Cys, His, etc.) with the immobilized Fe31-IDA matrix (Bernos, Girardet, Humbert, & Linden, 1997). IMAC has a high selectivity, a high recovery yield of the target proteins/peptide, a high protein loading, and a complete regeneration of the solid supports (Ha et al., 2008). Today, this is one of the main methodologies used for peptide or protein purification, from laboratory to pilot/industrial scale.

19.6 Summary Minerals are essential to different biological processes in living beings. Likewise, free transition metals such as copper and iron promote the formation of ROSs. However, metalbinding peptides obtained by enzymatic hydrolysis of proteins or synthesized by SPPS avoid ROS formation catalyzed by free transition metals. Interactions between metals and MBPs are usually studied using spectroscopic techniques, ITC, and more recently by SPR

Application in nutrition: mineral binding 483 and switchSENSE. Through ESI-MS, the changes in the binding affinities can be studied. MBPs can be purified using IMAC, and its process simulation. Various cellular models are used to investigate the health effects of MBPs. Whatsoever, in the aim of human nutrition application, some scientific studies should be carried out on humans.

Acknowledgment The authors acknowledge financial support from the “Impact Biomolecules” project of the “Lorraine Universite´ d’Excellence” (in the context of the “Investissements d’avenir” program implemented by the French National Research Agency
ANR project number 15-004). The authors would like also to thank the financial support of Institut Carnot ICEEL (Project 2019, MELISSA ICEEL INTRA).

References Abragam, A., & Bleaney, B. (1970). Electron paramagnetic resonance of transition ions (p. 911) London: Oxford University Press. Achilleos, C., Tailhardat, M., Courtellemont, P., Varlet, B. L., & Dupont, D. (2009). Investigation of surface plasmon resonance biosensor for skin sensitizers studies. Toxicology In Vitro, 23(2), 308
318. Available from https://doi.org/10.1016/j.tiv.2008.11.007. Adlard, P. A., & Bush, A. I. (2006). Metals and Alzheimer’s disease. Journal of Alzheimer’s Disease, 10(2
3), 145
163. Available from https://doi.org/10.3233/JAD-2006-102-303. Aıˆt-Oukhatar, N., Bouhallab, S., Arhan, P., Maubois, J.-L., Drosdowsky, M., & Bougle´, D. (1999). Iron tissue storage and hemoglobin levels of deficient rats repleted with iron bound to the caseinophosphopeptide 1-25 of β-casein. Journal of Agricultural and Food Chemistry, 47(7), 2786
2790. Available from https://doi. org/10.1021/jf981018k. Aıˆt-Oukhatar, N., Peres, J.-M., Bouhallab, S., Neuville, D., Bureau, F., Bouvard, G., . . . Bougle, D. (2002). Bioavailability of caseinophosphopeptide-bound iron. Journal of Laboratory and Clinical Medicine, 140(4), 290
294. Available from https://doi.org/10.1067/mlc.2002.128146. Amiri, S., Mehrnia, M. R., Sobhanifard, S., Pourasgharian Roudsari, F., & Hoseini, S.-N. (2017). Evaluation of agarose-entrapped magnetic nanoparticles influence on protein adsorption isotherm and kinetics using nickel-iminodiacetic acid ligand. Separation and Purification Technology, 188, 423
430. Available from https://doi.org/10.1016/j.seppur.2017.07.030. Balliu, A., & Baltzer, L. (2017). Conjugation of a dipicolyl chelate to polypeptide conjugates increases binding affinities for human serum albumin and survival times in human serum. ChemBioChem, 18(14), 1408
1414. Available from https://doi.org/10.1002/cbic.201700049. Bao, X. L., Song, M., Zhang, J., Chen, Y., & Guo, S. T. (2007). Calcium-binding ability of soy protein hydrolysates. Chinese Chemical Letters, 18(9), 1115
1118. Available from https://doi.org/10.1016/j. cclet.2007.07.032. Bao, X.-L., Lv, Y., Yang, B.-C., Ren, C.-G., & Guo, S.-T. (2008). A study of the soluble complexes formed during calcium binding by soybean protein hydrolysates. Journal of Food Science, 73(3), C117
C121. Available from https://doi.org/10.1111/j.1750-3841.2008.00673.x. Barth, A. (2007). Infrared spectroscopy of proteins. Biochimica et Biophysica Acta (BBA)—Bioenergetics, 1767 (9), 1073
1101. Available from https://doi.org/10.1016/j.bbabio.2007.06.004. Bernaudat, F., & Bu¨low, L. (2005). Rapid evaluation of nickel binding properties of His-tagged lactate dehydrogenases using surface plasmon resonance. Journal of Chromatography A, 1066(1
2), 219
224. Available from https://doi.org/10.1016/j.chroma.2005.01.079. Bernos, E., Girardet, J.-M., Humbert, G., & Linden, G. (1997). Role of the O phosphoserine clusters in the interaction of the bovine milk αs1-, β-, κ-caseins and the PP3 component with immobilized iron(III) ions.

484 Chapter 19 Biochimica et Biophysica Acta: Protein Structure and Molecular Enzymology, 1337(1), 149
159. Available from https://doi.org/10.1016/S0167-4838(96)00159-8. Bondalapati, S., Jbara, M., & Brik, A. (2016). Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins. Nature Chemistry, 8(5), 407
418. Available from https://doi.org/10.1038/ nchem.2476. Bougle´, D., & Bouhallab, S. (2006). Mineral-binding proteins and peptides and bioavailability of trace elements. In Y. Mine, & F. Shahidi (Eds.), Nutraceutical proteins and peptides in health and disease (pp. 29
40). Taylor & Francis Group. Bouhallab, S., & Bougle´, D. (2004). Biopeptides of milk: Caseinophosphopeptides and mineral bioavailability. Reproduction Nutrition Development, EDP Sciences, 44(5), 493
498. Available from https://doi.org/ 10.1051/rnd:2004053. Bresolin, I. R. A. P., Bresolin, I. T. L., & Pessoa, A. (2015). Purification of anti-interleukin-6 monoclonal antibody using precipitation and immobilized metal-ion affinity chromatography. Adsorption Science and Technology, 33(2), 191
202. Available from https://doi.org/10.1260/0263-6174.33.2.191. Buckley, K., & Ryder, A. G. (2017). Applications of Raman spectroscopy in biopharmaceutical manufacturing: A short review. Applied Spectroscopy, 71(6), 1085
1116. Available from https://doi.org/10.1177/ 0003702817703270. Bullamore, J. R., Wilkinson, R., Gallagher, J. C., Nordin, B. E. C., & Marshall, D. H. (1970). Effect of age on calcium absorption. The Lancet, 296(7672), 535
537. Available from https://doi.org/10.1016/S0140-6736 (70)91344-9. Caetano-Silva, M. E., Alves, R. C., Lucena, G. N., Frem, R. C. G., Bertoldo-Pacheco, M. T., Lima-Pallone, J. A., & Netto, F. M. (2017). Synthesis of whey peptide-iron complexes: Influence of using different iron precursor compounds. Food Research International, 101(May), 73
81. Available from https://doi.org/ 10.1016/j.foodres.2017.08.056. Caetano-Silva, M. E., Bertoldo-Pacheco, M. T., Paes-Leme, A. F., & Netto, F. M. (2015). Iron-binding peptides from whey protein hydrolysates: Evaluation, isolation and sequencing by LC-MS/MS. Food Research International, 71, 132
139. Available from https://doi.org/10.1016/j. foodres.2015.01.008. Canabady-Rochelle, L. S., Sanchez, C., Mellema, M., & Banon, S. (2009). Study of calcium
soy protein interactions by isothermal titration calorimetry and pH cycle. Journal of Agricultural and Food Chemistry, 57, 5339
5347. Available from https://doi.org/10.1021/jf900424b. Canabady-Rochelle, L. S., Sanchez, C., Mellema, M., & Banon, S. (2010). Calcium carbonate-hydrolysed soy proteins complexation in presence of citric acid. Journal of Colloid and Interface Science, 345, 88
95. Available from https://doi.org/10.1016/j.jcis.2010.01.037. Canabady-Rochelle, L. L. S., Selmeczi, K., Collin, S., Pasc, A., Muhr, L., & Boschi-Muller, S. (2018). SPR screening of metal chelating peptides in a hydrolysate for their antioxidant properties. Food Chemistry, 239, 478
485. Available from https://doi.org/10.1016/j.foodchem.2017.06.116. Cao, Y., Miao, J., Liu, G., Luo, Z., Xia, Z., Liu, F., . . . Xiao, H. (2017). Bioactive peptides isolated from casein phosphopeptides enhance calcium and magnesium uptake in Caco-2 cell monolayers. Journal of Agricultural and Food Chemistry, 65(11), 2307
2314. Available from https://doi.org/10.1021/acs.jafc.6b05711. ´ lvarez, A. J., Jime´nez-Martı´nez, C., Jacinto-Herna´ndez, C., Alaiz, M., Giro´nCarrasco-Castilla, J., Herna´ndez-A Calle., . . . Da´vila-Ortiz, G. (2012). Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates. Food Chemistry, 135(3), 1789
1795. Available from https://doi. org/10.1016/j.foodchem.2012.06.016. Chabance, B., Marteau, P., Rambaud, J. C., Migliore-Samour, D., Boynard, M., Perrotin, P., . . . Fiat, A. M. (1998). Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. Biochimie, 80(2), 155
165. Available from https://doi.org/10.1016/S0300-9084(98)80022-9. Chabanon, G. (2005). Hydrolyses enzymatiques d’isolats prote´iques issus de tourteaux de colza: Cine´tique, mode´lisation, caracte´risation et fonctionnalite´s des peptides. In: The`se de l’Institut National Polytechnique de Lorraine (248 pages).

Application in nutrition: mineral binding 485 Chalamaiah, M., Ulug, S. K., Hong, H., & Wu, J. (2019). Regulatory requirements of bioactive peptides (protein hydrolysates) from food proteins. Journal of Functional Foods, 58, 123
129. Available from https://doi. org/10.1016/j.jff.2019.04.050. Chan, W. Y., & Rennert, O. M. (1980). The role of copper in iron metabolism. Annals of Clinical and Laboratory Science, 10(4), 338
344. Charoenphun, N., Cheirsilp, B., Sirinupong, N., & Youravong, W. (2013). Calcium-binding peptides derived from tilapia (Oreochromis niloticus) protein hydrolysate. European Food Research and Technology, 236 (1), 57
63. Available from https://doi.org/10.1007/s00217-012-1860-2. Cheignon, C., Jones, M., Atria´n-Blasco, E., Kieffer, I., Faller, P., Collin, F., & Hureau, C. (2017). Identification of key structural features of the elusive Cu
Aβ complex that generates ROS in Alzheimer’s disease. Chemical Science, 8(7), 5107
5118. Available from https://doi.org/10.1039/ C7SC00809K. Chen, D., Mu, X., Huang, H., Nie, R., Liu, Z., & Zeng, M. (2014). Isolation of a calcium-binding peptide from tilapia scale protein hydrolysate and its calcium bioavailability in rats. Journal of Functional Foods, 6(1), 575
584. Available from https://doi.org/10.1016/j.jff.2013.12.001. Chen, H., Jia, S., Zhang, J., Jang, M., Chen, X., Koh, K., & Wang, Z. (2015). Sensitive detection of copper(II) ions based on the conformational change of peptides by surface plasmon resonance spectroscopy. Analytical Methods, 7(20), 8942
8946. Available from https://doi.org/10.1039/C5AY02047F. Chen, Q., Guo, L., Du, F., Chen, T., Hou, H., & Li, B. (2017). The chelating peptide (GPAGPHGPPG) derived from Alaska pollock skin enhances calcium, zinc and iron transport in Caco-2 cells. International Journal of Food Science and Technology, 52(5), 1283
1290. Available from https://doi.org/10.1111/ ijfs.13396. Cheung, I. W. Y., Cheung, L. K. Y., Tan, N. Y., & Li-Chan, E. C. Y. (2012). The role of molecular size in antioxidant activity of peptide fractions from Pacific hake (Merluccius productus) hydrolysates. Food Chemistry, 134(3), 1297
1306. Available from https://doi.org/10.1016/j. foodchem.2012.02.215. Clemente, A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends in Food Science and Technology, 11(7), 254
262. Available from https://doi.org/10.1016/S0924-2244(01)00007-3. Coin, I., Beyermann, M., & Bienert, M. (2007). Solid-phase peptide synthesis: From standard procedures to the synthesis of difficult sequences. Nature Protocols, 2(12), 3247
3256. Available from https://doi.org/ 10.1038/nprot.2007.454. Comba, P., Dovalil, N., Haberhauer, G., Kowski, K., Mehrkens, N., & Westphal, M. C. (2013). Copper solution chemistry of cyclic pseudo-octapeptides. Zeitschrift fu¨r Anorganische und Allgemeine Chemie, 639(8
9), 1395
1400. Available from http://doi.org/10.1002/zaac.201300079. Cosentino, S., Gravaghi, C., Donetti, E., Donida, B. M., Lombardi, G., Bedoni, M., . . . Ferraretto, A. (2010). Casein phosphopeptide-induced calcium uptake in human intestinal cell lines HT-29 and Caco2 is correlated to cellular differentiation. The Journal of Nutritional Biochemistry, 21(3), 247
254. Available from https://doi.org/10.1016/j.jnutbio.2008.12.016. Coudray, C., Rambeau, M., Feillet-Coudray, C., Gueux, E., Tressol, J. C., Mazur, A., & Rayssiguier, Y. (2005). Study of magnesium bioavailability from ten organic and inorganic Mg salts in Mg-depleted rats using a stable isotope approach. Magnesium Research, 18(4), 215
223. Cross, K. J., Huq, N. L., & Reynolds, E. C. (2007). Casein phosphopeptides in oral health--chemistry and clinical applications. Current Pharmaceutical Design, 13(8), 793
800. Available from https://doi.org/ 10.2174/138161207780363086. Csire, G., Canabady-Rochelle, L., Averlant-Petit, M.-C., Selmeczi, K., & Stefan, L. (2020). Both metalchelating and free radical-scavenging synthetic pentapeptides as efficient inhibitors of reactive oxygen species generation. Metallomics, 1
10. Available from https://doi.org/10.1039/d0mt00103a. Cuomo, F., Ceglie, A., & Lopez, F. (2011). Temperature dependence of calcium and magnesium induced caseinate precipitation in H2O and D2O. Food Chemistry, 126(1), 8
14. Available from https://doi.org/ 10.1016/j.foodchem.2010.10.021.

486 Chapter 19 De La Hoz, L., Ponezi, A. N., Milani, R. F., Da Silva, V. S. N., De Souza, A. S., & Bertoldo-Pacheco, M. T. (2014). Iron-binding properties of sugar cane yeast peptides. Food Chemistry, 142, 114
120. Available from https://doi.org/10.1016/j.foodchem.2013.06.133. De Ricco, R., Potocki, S., Kozlowski, H., & Valensin, D. (2014). NMR investigations of metal interactions with unstructured soluble protein domains. Coordination Chemistry Review, 269, 1
12. Available from https:// doi.org/10.1016/j.ccr.2014.02.014. Di Marco, V. B. D., & Bombi, G. G. (2006). Electrospray mass spectrometry (ESI-MS) in the study of metal
ligand solution equilibria. Mass Spectrometry Reviews, 25(3), 347
379. Available from https://doi. org/10.1002/mas.20070. Dinicolantonio, J. J., Liu, J., & O’keefe, J. H. (2018). Magnesium for the prevention and treatment of cardiovascular disease. Open Heart, 5, e000775. Available from http://doi.org/10.1136/openhrt-2018-000775. Dunbar, W. S. (2017). Biotechnology and the mine of tomorrow. Trends in Biotechnology, 35(1), 79
89. Available from https://doi.org/10.1016/j.tibtech.2016.07.004. Eckert, E., Lu, L., Unsworth, L. D., Chen, L., Xie, J., & Xu, R. (2016). Biophysical and in vitro absorption studies of iron chelating peptide from barley proteins. Journal of Functional Foods, 25, 291
301. Available from https://doi.org/10.1016/j.jff.2016.06.011. El-Faham, A., & Albericio, F. (2011). Peptide coupling reagents, more than a letter soup. Chemical Reviews, 111(11), 6557
6602. Available from https://doi.org/10.1021/cr100048w. Faller, P., Hureau, C., Dorlet, P., Hellwig, P., Coppel, Y., Collin, F., & Alies, B. (2012). Methods and techniques to study the bioinorganic chemistry of metal
peptide complexes linked to neurodegenerative diseases. Coordination Chemistry Reviews, 256(19
20), 2381
2396. Available from https://doi.org/ 10.1016/j.ccr.2012.03.015. FAO. (2002). FAO specification human vitamin and mineral requirements. Report of a Joint FAO/WHO Expert Consultation. FAO/WHO non-series publication. Rome: Food and Agriculture Organization. (En). Farkas, E., & So´va´go´, I. (2012). Amino acids, peptides and proteins, . Amino acids, peptides and proteins (Vol. 37, pp. 66
118). The Royal Society of Chemistry. Available from https://doi.org/10.1039/9781849734677. Farkas, E., & So´va´go´, I. (2017). Metal complexes of amino acids and peptides, . Amino acids, peptides and proteins (Vol. 41, pp. 100
151). The Royal Society of Chemistry. Available from https://doi.org/10.1039/ 9781782626619-00100. Fenton, H. J. H. (1894). LXXIII.—Oxidation of tartaric acid in presence of iron. Journal of the Chemical Society, Transactions, 65, 899
910. Available from https://doi.org/10.1039/CT8946500899. Ferraretto, A., Signorile, A., Gravaghi, C., Fiorilli, A., & Tettamanti, G. (2001). Casein phosphopeptides influence calcium uptake by cultured human intestinal HT-29 tumor cells. The Journal of Nutrition, 131(6), 1655
1661. Available from https://doi.org/10.1093/jn/131.6.1655. Fisher, E. (1903). Synthese von derivaten der polypeptide. ChemPlusChem, 36(2), 2094
2106. Available from https://doi-org.bases-doc.univ-lorraine.fr/10.1002/cber.190303602127. Fisher, E. (1907). Synthese von polypeptiden. ChemPlusChem, 40(2), 1754
1767. Available from https://doiorg.bases-doc.univ-lorraine.fr/10.1002/cber.19070400273. Friˇscˇ i´c, T., Mottillo, C., & Titi, H. M. (2020). Mechanochemistry for synthesis. Angewandte Chemie International Edition, 59(3), 1018
1029. Available from https://doi.org/10.1002/anie.201906755. Gaberc-Porekar, V., & Menart, V. (2001). Perspectives of immobilized-metal affinity chromatography. Journal of Biochemical and Biophysical Methods, 49(1
3), 335
360. Available from https://doi.org/10.1016/ S0165-022X(01)00207-X. Ghisaidoobe, A., & Chung, S. (2014). Intrinsic tryptophan fluorescence in the detection and analysis of proteins: A focus on Fo¨rster resonance energy transfer techniques. International Journal of Molecular Sciences, 15 (12), 22518
22538. Available from https://doi.org/10.3390/ijms151222518. Gilbert, J. A., Jansson, J. K., & Knight, R. (2014). The earth microbiome project: Successes and aspirations. BMC Biology, 12(1), 69. Available from https://doi.org/10.1186/s12915-014-0069-1. Gizzi, P., Henry, B., Rubini, P., Giroux, S., & Wenger, E. (2005). A multi-approach study of the interaction of the Cu(II) and Ni(II) ions with alanylglycylhistamine, a mimicking pseudopeptide of the serum albumine

Application in nutrition: mineral binding 487 N-terminal residue. Journal of Inorganic Biochemistry, 99(5), 1182
1192. Available from https://doi.org/ 10.1016/j.jinorgbio.2005.02.018. Gonza´lez-Ortega, O., & Guzma´n, R. (2015). Purification of human serum immunoglogulins using immobilized metal affinity chromatography with ethylendiamine triacetic acid as chelating agent. Journal of Liquid Chromatography and Related Technologies, 38(1), 74
81. Available from https://doi.org/10.1080/ 10826076.2014.883534. Greger, J. L. (1999). Nondigestible carbohydrates and mineral bioavailability. The Journal of Nutrition, 129(7), 1434S
1435S. Available from https://doi.org/10.1093/jn/129.7.1434S. Grossoehme, N. E., Akilesh, S., Guerinot, M. L., & Wilcox, D. E. (2006). Metal-binding thermodynamics of the histidine-rich sequence from the metal-transport protein IRT1 of Arabidopsis thaliana. Inorganic Chemistry, 45(21), 8500
8508. Available from https://doi.org/10.1021/ic0606431. Gue´guen, L., & Pointillart, A. (2000). The bioavailability of dietary calcium. Journal of the American College of Nutrition, 19(Suppl. 2), 119S
136S. Available from https://doi.org/10.1080/ 07315724.2000.10718083. Guo, L., Harnedy, P. A., Li, B., Hou, H., Zhang, Z., Zhao, X., & Fitzgerald, R. J. (2014). Food protein-derived chelating peptides: Biofunctional ingredients for dietary mineral bioavailability enhancement. Trends in Food Science & Technology, 37(2), 92
105. Available from https://doi.org/10.1016/j.tifs.2014.02.007. Guo, L., Harnedy, P. A., O’keeffe, M. B., Zhang, L., Li, B., Hou, H., & Fitzgerald, R. J. (2015). Fractionation and identification of Alaska pollock skin collagen-derived mineral chelating peptides. Food Chemistry, 173, 536
542. Available from https://doi.org/10.1016/j.foodchem.2014.10.055. Guo, L., Hou, H., Li, B., Zhang, Z., Wang, S., & Zhao, X. (2013). Preparation, isolation and identification of iron-chelating peptides derived from Alaska pollock skin. Process Biochemistry, 48(5
6), 988
993. Available from https://doi.org/10.1016/j.procbio.2013.04.013. Gutierrez, R., Martı´n Del Valle, E. M., & Galan, M. A. (2007). Immobilized metal-ion affinity chromatography: Status and trends. Separation & Purification Reviews, 36, 71
111. Available from https://doi.org/10.1080/ 15422110601166007. Gutteridge, J. M. C., & Wilkins, S. (1982). Copper-dependent hydroxyl radical damage to ascorbic acid. FEBS Letters, 137(2), 327
330. Available from https://doi.org/10.1016/0014-5793(82)80377-3. Ha, E.-J., Kim, Y.-J., An, S. S. A., Kim, Y.-R., Lee, J.-O., Lee, S.-G., & Paik, H. (2008). Purification of Histagged proteins using Ni21
poly(2-acetamidoacrylic acid) hydrogel. Journal of Chromatography B, 876 (1), 8
12. Available from https://doi.org/10.1016/j.jchromb.2008.10.020. Haber, F., & Weiss, J. (1934). The catalytic decomposition of hydrogen peroxide by iron salts. Proceedings of the Royal Society of London. Series A—Mathematical and Physical Sciences, 147(861), 332
351. Available from https://doi.org/10.1098/rspa.1934.0221. Hainfeld, J. F., Liu, W., Halsey, C. M. R., Freimuth, P., & Powell, R. D. (1999). Ni-NTA-gold clusters target His-tagged proteins. Journal of Structural Biology, 127(2), 185
198. Available from https://doi.org/ 10.1006/jsbi.1999.4149. Halliwell, B. (1994). Free radicals, antioxidants, and human disease: Curiosity, cause, or consequence? The Lancet, 344(8924), 721
724. Available from https://doi.org/10.1016/S0140-6736(94)92211-X. Han, Y.-H., Seo, H.-A., Kim, G.-H., Lee, C.-K., Kang, Y. K., Ryu, K. H., & Chung, Y. J. (2010). A histidine substitution confers metal binding affinity to a Schistosoma japonicum glutathione S-transferase. Protein Expression and Purification, 73(1), 74
77. Available from https://doi.org/10.1016/j.pep.2010.03.014. Hancock, R. D. (1992). Chelate ring size and metal ion selection. The basis of selectivity for metal ions in open-chain ligands and macrocycles. Journal of Chemical Education, 69(8), 615. Available from https:// doi.org/10.1021/ed069p615. Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity: From research to food applications. Current Opinion in Biotechnology, 18(2), 163
169. Available from https://doi.org/10.1016/j. copbio.2007.01.013. Hazen, R. M., & Ferry, J. M. (2010). Mineral evolution: Mineralogy in the fourth dimension. Elements, 6(1), 9
12. Available from https://doi.org/10.2113/gselements.6.1.9.

488 Chapter 19 Ho, C. S., Lam, C. W. K., Chan, M. H. M., Cheung, R. C. K., Law, L. K., Lit, L. C. W., . . . Tai, H. L. (2003). Electrospray ionisation mass spectrometry: Principles and clinical applications. The Clinical Biochemist Reviews, 24(1), 3
12, Retrieved from. Available from https://pubmed.ncbi.nlm.nih.gov/18568044. Hochuli, E., Do¨beli, H., & Schacher, A. (1987). New metal chelate adsorbent selective for proteins and peptides containing neighboring histidine residues. Journal of Chromatography A, 411, 177
184. Available from https://doi.org/10.1016/S0021-9673(00)93969-4. Holick, M. F. (2004). Sunlight and vitamin D for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. The American Journal of Clinical Nutrition, 80(6), 1678S
1688S. Available from https://doi.org/10.1093/ajcn/80.6.1678S. Hong, H. J., Kim, Ej, Park, I. S., Ryu, J. H., Ryu, T., Kim, B. G., & Chung, K. S. (2015). Preparation and characterisation of an easily absorbable Mg-casein hydrolysate complex produced through enzymatic hydrolysis and ultrafiltration. International Journal of Food Science and Technology, 50(2), 365
371. Available from https://doi.org/10.1111/ijfs.12650. Huang, G., Ren, L., & Jiang, J. (2011). Purification of a histidine-containing peptide with calcium binding activity from shrimp processing byproducts hydrolysate. European Food Research and Technology, 232(2), 281
287. Available from https://doi.org/10.1007/s00217-010-1388-2. Huang, S. L., Zhao, L. N., Cai, X., Wang, S. Y., Huang, Y. F., Hong, J., & Rao, P. F. (2015). Purification and characterization of a glutamic acid-containing peptide with calcium-binding capacity from whey protein hydrolysate. Journal of Dairy Research, 82(1), 29
35. Available from https://doi.org/10.1017/ S0022029914000715. Hurrell, R., & Egli, I. (2010). Iron bioavailability and dietary reference values. The American Journal of Clinical Nutrition, 91(5), 1461S
1467S. Available from https://doi.org/10.3945/ajcn.2010.28674F. Irving, H., & Williams, R. J. P. (1953). The stability of transition-metal complexes. Journal of the Chemical Society, 3192
3210. Available from https://doi.org/10.1039/jr9530003192. ´ lvarez, M., & Albericio, F. (2009). Amino acid-protecting groups. Chemical Reviews, 109 Isidro-Llobet, A., AA (6), 2455
2504. Available from https://doi.org/10.1021/cr800323s. Jaiswal, A., Bajaj, R., Mann, B., & Lata, K. (2015). Iron (II)-chelating activity of buffalo αS-casein hydrolysed by corolase PP, alcalase and flavourzyme. Journal of Food Science and Technology, 52(6), 3911
3918. Available from https://doi.org/10.1007/s13197-014-1626-x. Jancso´, A., Selmeczi, K., Gizzi, P., Nagy, N. V., Gajda, T., & Henry, B. (2011). The role of terminal amino group and histidine at the fourth position in the metal ion binding of oligopeptides revisited: Copper(II) and nickel(II) complexes of glycyl-glycyl-glycyl-histamine and its N-Boc protected derivative. Journal of Inorganic Biochemistry, 105, 92
101. Available from https://doi.org/10.1016/j.jinorgbio.2010.09.004. Jaradat, D. M. M. (2017). Thirteen decades of peptide synthesis: Key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation. Amino Acids, 50(1), 39
68. Available from https://doi.org/10.1007/s00726-017-2516-0. Jin, Y.-G., Fu, W.-W., & Ma, M.-H. (2011). Preparation and structure characterization of soluble bone collagen peptide chelating calcium. African Journal of Biotechnology, 10(50), 10204
10211. Available from https:// doi.org/10.5897/AJB10.1923. Jiskoot, W., & Crommelin, D. (2005). Methods for structural analysis of protein pharmaceuticals (Vol. 3, pp. 1
26). Springer Science and Business Media. Jung, W.-K., Lee, B.-J., & Kim, S.-K. (2006). Fish-bone peptide increases calcium solubility and bioavailability in ovariectomised rats. British Journal of Nutrition, 95(1), 124
128. Available from https://doi.org/ 10.1079/BJN20051615. Kaden, D., Bush, A. I., Danzeisen, R., Bayer, T. A., & Multhaup, G. (2011). Disturbed copper bioavailability in Alzheimer’s disease. International Journal of Alzheimer’s Disease, 2011. Available from https://doi.org/ 10.4061/2011/345614. Kapsokefalou, M., & Miller, D. D. (1995). Iron speciation in intestinal contents of rats fed meals composed of meat and nonmeat sources of protein and fat. Food Chemistry, 52(1), 47
56. Available from https://doi. org/10.1016/0308-146(94)P4180-N.

Application in nutrition: mineral binding 489 Keith-Roach, M. J. (2010). A review of recent trends in electrospray ionisation
mass spectrometry for the analysis of metal
organic ligand complexes. Analytica Chimica Acta, 678(2), 140
148. Available from https://doi.org/10.1016/j.aca.2010.08.023. Kibangou, I., Bouhallab, S., Bureau, F., Allouche, S., Thouvenin, G., & Bougle´, D. (2008). Caseinophosphopeptide-bound iron: Protective effect against gut peroxidation. Annals of Nutrition and Metabolism, 52(3), 177
180. Available from https://doi.org/10.1159/000136384. Kiela, P. R., & Ghishan, F. K. (2016). Physiology of intestinal absorption and secretion. Best Practice & Research. Clinical Gastroenterology, 30(2), 145
159. Available from https://doi.org/10.1016/j. bpg.2016.02.007. Kim, N. H., Jung, S. H., Kim, J., Kim, S. H., Ahn, H. J., & Song, K. B. (2014). Purification of an iron-chelating peptide from spirulina protein hydrolysates. Journal of the Korean Society for Applied Biological Chemistry, 57(1), 91
95. Available from https://doi.org/10.1007/s13765-013-4211-5. Kjærgaard, K., Schembri, M. A., & Klemm, P. (2001). Novel Zn21-chelating peptides selected from a fimbriadisplayed random peptide library. Applied and Environmental Microbiology, 67(12), 5467
5473. Available from https://doi.org/10.1128/AEM.67.12.5467-5473.2001. Knecht, S., Ricklin, D., Eberle, A. N., & Ernst, B. (2009). Oligohis-tags: Mechanisms of binding to Ni21-NTA surfaces. Journal of Molecular Recognition, 22(4), 270
279. Available from https://doi.org/10.1002/ jmr.941. Knezevic, J., Langer, A., Hampel, P. A., Kaiser, W., Strasser, R., & Rant, U. (2012). Quantitation of affinity, avidity, and binding kinetics of protein analytes with a dynamically switchable biosurface. Journal of the American Chemical Society, 134(37), 15225
15228. Available from https://doi.org/10.1021/ja3061276. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16(9), 945
960. Available from https://doi.org/10.1016/j.idairyj.2005.10.012. Kozłowski, H., Kowalik-Jankowska, T., & Je˙zowska-Bojczuk, M. (2005). Chemical and biological aspects of Cu21 interactions with peptides and aminoglycosides. Coordination Chemistry Reviews, 249(21
22), 2323
2334. Available from https://doi.org/10.1016/j.ccr.2005.04.027. Kumagai, H., Koizumi, A., Suda, A., Sato, N., Sakurai, H., & Kumagai, H. (2004). Enhanced calcium absorption in the small intestine by a phytate-removed deamidated soybean globulin preparation. Bioscience, Biotechnology, and Biochemistry, 68(7), 1598
1600. Available from https://doi.org/10.1271/bbb.68.1598. Ladbury, J. E., & Chowdhry, B. Z. (1996). Sensing the heat: The application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chemistry and Biology, 3(10), 791
801. Available from https://doi.org/10.1016/S1074-5521(96)90063-0. Lamsho¨ft, M., & Ivanova, B. (2011). Protonation and coordination ability of small peptides
Theoretical and experimental approaches for elucidation. Journal of Coordination Chemistry, 64(14), 2419
2442. Available from https://doi.org/10.1080/00958972.2011.598926. Langer, A., Hampel, P. A., Kaiser, W., Knezevic, J., Welte, T., Villa, V., . . . Rant, U. (2013). Protein analysis by time-resolved measurements with an electro-switchable DNA chip. Nature Communications, 4, 2099. Available from https://doi.org/10.1038/ncomms3099. Langer, A., Schra¨ml, M., Strasser, R., Daub, H., Myers, T., Heindl, D., & Rant, U. (2015). Polymerase/DNA interactions and enzymatic activity: Multi-parameter analysis with electro-switchable biosurfaces. Scientific Reports, 5, 1
15. Available from https://doi.org/10.1038/srep12066. Layrisse, M., Martı´nez-Torres, C., Leets, I., Taylor, P., & Ramı´rez, J. (1984). Effect of histidine, cysteine, glutathione or beef on iron absorption in humans. The Journal of Nutrition, 114(1), 217
223. Available from https://doi.org/10.1093/jn/114.1.217. Le Vo, T. D. L., Pham, K. T., Le, L. T., & Nguyen, T. T. H. (2018). Identification of a new calcium-binding peptide from enzymatic proteolysate of Acetes japonicus. Journal of Food Processing and Preservation, 42 (12), 2
7. Available from https://doi.org/10.1111/jfpp.13837. Lee, S. H., & Song, K. B. (2009). Purification of an iron-binding nona-peptide from hydrolysates of porcine blood plasma protein. Process Biochemistry, 44(3), 378
381. Available from https://doi.org/10.1016/j. procbio.2008.12.001.

490 Chapter 19 Li, Y., Jiang, H., & Huang, G. (2017). Protein hydrolysates as promoters of non-haem iron absorption. Nutrients, 9(6), 1
18. Available from https://doi.org/10.3390/nu9060609. Liao, W., Yang, Z., Liu, X., Duan, S., Xiao, S., Liu, S., & Miao, J. (2019). The purification, identification and bioactivity study of a novel calcium-binding peptide from casein hydrolysate. Food & Function, 10, 7724
7732. Available from https://doi.org/10.1039/c9fo01383k. Liedberg, B., Nylander, C., & Lunstro¨m, I. (1983). Surface plasmon resonance for gas detection and biosensing. Sensors and Actuators, 4, 299
304. Available from https://doi.org/10.1016/0250-6874(83)85036-7. Lin, H. M., Deng, S. G., Huang, S. B., Li, Y. J., & Song, R. (2016). The effect of ferrous-chelating hairtail peptides on iron deficiency and intestinal flora in rats. Journal of the Science of Food and Agriculture, 96 (8), 2839
2844. Available from https://doi.org/10.1002/jsfa.7452. Liu, Q., Yang, M., Zhao, B., & Yang, F. (2020). Isolation of antioxidant peptides from yak casein hydrolysate. RSC Advances, 34. Available from https://doi.org/10.1039/d0ra02644a. Liu, Y., & Wilson, W. D. (2010). Quantitative analysis of small molecule
nucleic acid interactions with a biosensor surface and surface plasmon resonance detection. In R. F. Keith (Ed.), Drug-DNA interaction protocols (613, pp. 1
23). Totowa, NJ: Humana Press. Available from https://doi.org/10.1007/978-160327-418-0_1. Lo¨nnerdal, B. (1996). Bioavailability of copper. The American Journal of Clinical Nutrition, 63(5), 821S
829S. Available from https://doi.org/10.1093/ajcn/63.5.821. Lv, Y., Liu, Q., Bao, X., Tang, W., Yang, B., & Guo, S. (2009). Identification and characteristics of ironchelating peptides from soybean protein hydrolysates using IMAC-Fe31. Journal of Agricultural and Food Chemistry, 57(11), 4593
4597. Available from https://doi.org/10.1021/jf9000204. Maalouli, N., Gouget-Laemmel, A. C., Pinchemel, B., Bouazaoui, M., Chazalviel, J.-N., Ozanam, F., . . . Szunerits, S. (2011). Development of a metal-chelated plasmonic interface for the linking of His-peptides with a droplet-based surface plasmon resonance read-off scheme. Langmuir, 27(9), 5498
5505. Available from https://doi.org/10.1021/la2005437. Megı´as, C., Pedroche, J., Yust, M. M., Giro´n-Calle, J., Alaiz, M., Milla´n, F., & Vioque, J. (2007). Affinity purification of copper-chelating peptides from sunflower protein hydrolysates. Journal of Agricultural and Food Chemistry, 55(16), 6509
6514. Available from https://doi.org/10.1021/jf0712705. Megı´as, C., Pedroche, J., Yust, M. M., Giro´n-Calle, J., Alaiz, M., Milla´n, F., & Vioque, J. (2008). Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin. LWT—Food Science and Technology, 41(10), 1973
1977. Available from https://doi.org/10.1016/j.lwt.2007.11.010. Meisel, H., Meisel, H., Fairweather-Tait, S., Fitzgerald, R. J., Hartmann, R., Lane, C. N., . . . Wal, J. M. (2003). Detection of caseinophosphopeptides in the distal ileostomy fluid of human subjects. British Journal of Nutrition, 89(3), 351
358. Available from https://doi.org/10.1079/BJN2002803. Merrifield, R. B. (1963). Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society, 85(14), 2149
2154. Available from https://doi.org/10.1021/ja00897a025. Mir, T. A., & Shinohara, H. (2013). Two-dimensional surface plasmon resonance imager: An approach to study neuronal differentiation. Analytical Biochemistry, 443(1), 46
51. Available from https://doi.org/10.1016/j.ab.2013.08.008. Moss, J. A. (2005). Guide for resin and linker selection in solid-phase peptide synthesis. Current Protocols in Protein Science, 40(1), 18.7.1
18.7.19. Available from https://doi.org/10.1002/0471140864.ps1807s40. Muhr, L., Pontvianne, S., Selmeczi, K., Paris, C., Boschi-Muller, S., & Canabady-Rochelle, L. (2020). Chromatographic separation simulation of metal-chelating peptides from surface plasmon resonance binding parameters. Journal of Separation Science, 43(11), 2031
2041. Available from https://doi.org/ 10.1002/jssc.201900882. Murariu, M., Dragan, E. S., & Drochioiu, G. (2010). Model peptide-based system used for the investigation of metal ions binding to histidine-containing polypeptides. Biopolymers, 93(6), 497
508. Available from https://doi.org/10.1002/bip.21385. Nakayama, T., Shinohara, H., Tanaka, M., Baba, K., Ogawa-Ohnishi, M., & Matsubayashi, Y. (2017). A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots. Science, 355(6322), 284
286. Available from https://doi.org/10.1126/science.aai9057.

Application in nutrition: mineral binding 491 Navratilova, I., Papalia, G. A., Rich, R. L., Bedinger, D., Brophy, S., Condon, B., . . . Myszka, D. G. (2007). Thermodynamic benchmark study using Biacore technology. Analytical Biochemistry, 364(1), 67
77. Available from https://doi.org/10.1016/j.ab.2007.01.031. Nieba, L., Nieba-Axmann, S. E., Persson, A., Ha¨ma¨la¨inen, M., Edebratt, F., Hansson, A., . . . Plu¨ckthun, A. (1997). Biacore analysis of histidine-tagged proteins using a chelating NTA sensor chip. Analytical Biochemistry, 252(2), 217
228. Available from https://doi.org/10.1006/abio.1997.2326. Nielsen, A. D., Fuglsang, C. C., & Westh, P. (2003). Isothermal titration calorimetric procedure to determine protein
metal ion binding parameters in the presence of excess metal ion or chelator. Analytical Biochemistry, 314(2), 227
234. Available from https://doi.org/10.1016/S0003-2697(02) 00655-3. Ortega, R., Carmona, A., Llorens, I., & Solari, P. L. (2012). X-ray absorption spectroscopy of biological samples. A tutorial. Journal of Analytical Atomic Spectrometry, 27(12), 2054. Available from https://doi. org/10.1039/c2ja30224a. Pallauf, J., Pietsch, M., & Rimbach, G. (1998). Dietary phytate reduces magnesium bioavailability in growing rats. Nutrition Research, 18(6), 1029
1037. Available from https://doi.org/10.1016/S0271-5317(98)00085-2. Palomo, J. M. (2014). Solid-phase peptide synthesis: An overview focused on the preparation of biologically relevant peptides. RSC Advances, 4(62), 32658
32672. Available from https://doi.org/10.1039/ C4RA02458C. Paradı´s-Bas, M., Tulla-Puche, J., & Albericio, F. (2016). The road to the synthesis of “difficult peptides. Chemical Society Reviews, 45(3), 631
654. Available from https://doi.org/10.1039/C5CS00680E. Paris C., Selmeczi K., Chaimbault P., Desobry S., & Canabady-Rochelle, L. Optimization of a LCMS Method for screening of iron-chelating peptides by differential mass spectrometry analysis. Pittcon Conference, Chicago, Mc Cormick place, 1
5 March 2020. Oral communication. Pattabiraman, V. R., & Bode, J. W. (2011). Rethinking amide bond synthesis. Nature, 480(7378), 471
479. Available from https://doi.org/10.1038/nature10702. Pearson, R. G. (1963). Hard and soft acids and bases. Journal of the American Chemical Society, 85(22), 3533
3539. Available from https://doi.org/10.1021/ja00905a001. Pedersen, S. L., Tofteng, A. P., Malik, L., & Jensen, K. J. (2012). Microwave heating in solid-phase peptide synthesis. Chemical Society Reviews, 41(5), 1826
1844. Available from https://doi.org/10.1039/ C1CS15214A. Peng, Z., Hou, H., Zhang, K., & Li, B. (2017). Effect of calcium-binding peptide from Pacific cod (Gadus macrocephalus) bone on calcium bioavailability in rats. Food Chemistry, 221, 373
378. Available from https://doi.org/10.1016/j.foodchem.2016.10.078. Perego, S., Zabeo, A., Marasco, E., Giussani, P., Fiorilli, A., Tettamanti, G., & Ferraretto, A. (2013). Casein phosphopeptides modulate calcium uptake and apoptosis in Caco2 cells through their interaction with the TRPV6 calcium channel. Journal of Functional Foods, 5(2), 847
857. Available from https://doi.org/ 10.1016/j.jff.2013.01.032. Pereira, P. Md. C. C., & Vicente, A. Fd. R. B. (2013). Meat nutritional composition and nutritive role in the human diet. Meat Science, 93(3), 586
592. Available from https://doi.org/10.1016/j.meatsci.2012.09.018. Pe´re`s, J.-M., Bouhallab, S., Bureau, F., Neuville, D., Maubois, J.-L., Devroede, G., . . . Bougle´, D. (1999). Mechanisms of absorption of caseinophosphopeptide bound iron. The Journal of Nutritional Biochemistry, 10(4), 215
222. Available from https://doi.org/10.1016/S0955-2863(98)00101-6. Polavarapu, P. L. (2017). Chiroptical spectroscopy. Boca Raton, FL: CRC Press 448 pages. Available from https://doi.org/10.1201/9781315374888. Porath, J., Carlsson, J., Olsson, I., & Belfrage, G. (1975). Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 258(5536), 598
599. Available from https://doi.org/10.1038/258598a0. Rant, U. (2012). Sensing with electro-switchable biosurfaces. Bioanalytical Reviews, 4(2
4), 97
114. Available from https://doi.org/10.1007/s12566-012-0030-0. Rant, U., Arinaga, K., Scherer, S., Pringsheim, E., Fujita, S., Yokoyama, N., . . . Abstreiter, G. (2007). Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets. Proceedings of the

492 Chapter 19 National Academy of Sciences of the United States of America, 104(44), 17364
17369. Available from https://doi.org/10.1073/pnas.0703974104. Rascio, N., & Navari-Izzo, F. (2011). Heavy metal hyperaccumulating plants: How and why do they do it? And what makes them so interesting? Plant Science, 180(2), 169
181. Available from https://doi.org/10.1016/j. plantsci.2010.08.016. Rauser, W. E. (1995). Phytochelatins and related peptides (structure, biosynthesis, and function). Plant Physiology, 109(4), 1141
1149. Available from https://doi.org/10.1104/pp.109.4.1141. Reinhold, J., Lahimgarzadeh, A., Nasr, K., & Hedayati, H. (1973). Effects of purified phytate and phytate-rich bread upon metabolism of zinc, calcium, phosphorus, and nitrogen in man. The Lancet, 301(7798), 283
288. Available from https://doi.org/10.1016/S0140-6736(73)91538-9. Rich, A. M., Bombarda, E., Schenk, A. D., Lee, P. E., Cox, E. H., Spuches, A. M., . . . Wilcox, D. E. (2012). Thermodynamics of Zn21 binding to Cys2His2 and Cys2HisCys zinc fingers and a Cys4 transcription factor site. Journal of the American Society, 134, 10405
10418. Available from https://doi.org/10.1021/ ja211417g. Rich, R. L., & Myszka, D. G. (2011). Survey of the 2009 commercial optical biosensor literature. Journal of Molecular Recognition, 24(6), 892
914. Available from https://doi.org/10.1002/jmr.1138. Rivera-Mancı´a, S., Pe´rez-Neri, I., Rı´os, C., Trista´n-Lo´pez, L., Rivera-Espinosa, L., & Montes, S. (2010). The transition metals copper and iron in neurodegenerative diseases. Chemico-Biological Interactions, 186(2), 184
199. Available from https://doi.org/10.1016/j.cbi.2010.04.010. Rodzik, A., Pomastowski, P., Sagandykova, G. N., & Buszewski, B. (2020). Interactions of whey proteins with metal ions. International Journal of Molecular Sciences, 21(6), 2156. Available from https://doi.org/ 10.3390/ijms21062156. Rolinger, L., Ru¨dt, M., & Hubbuch, J. (2020). A critical review of recent trends, and a future perspective of optical spectroscopy as PAT in biopharmaceutical downstream processing. Analytical and Bioanalytical Chemistry, 412(9), 2047
2064. Available from https://doi.org/10.1007/s00216-020-02407-z. Sacco, C., Skowronsky, R. A., Gade, S., Kenney, J. M., & Spuches, A. M. (2012). Calorimetric investigation of copper(II) binding to Aβ peptides: Thermodynamics of coordination plasticity. Journal of Biological Inorganic Chemistry, 17, 531
541. Available from https://doi.org/10.1007/s00775-012-0874-3. Sahu, I. D., Mccarrick, R. M., & Lorigan, G. A. (2013). Use of electron paramagnetic resonance to solve biochemical problems. Biochemistry, 52(35), 5967
5984. Available from https://doi.org/10.1021/bi400834a. Sandblad, P., Arnell, R., Samuelsson, J., & Fornstedt, T. (2009). Approach for reliable evaluation of drug proteins interactions using surface plasmon resonance technology. Analytical Chemistry, 81(9), 3551
3559. Available from https://doi.org/10.1021/ac900299p. Sato, R., Noguchi, T., & Naito, H. (1985). The formation and iron-binding property of phosphopeptides in the small intestinal contents of rats fed egg yolk diet. Nutrition Reports International, 31(1), 245
252. Schalley, C. A., & Armentrout, P. B. (2003). In C. A. Schalley (Ed.), Modern mass spectrometry (Vol. 225). Berlin: Springer. Schiedel, M., Daub, H., Itzen, A., & Jung, M. (2020). Validation of the slow off-kinetics of sirtuin-rearranging ligands (SirReals) by means of label-free electrically switchable nanolever technology. ChemBioChem, 21 (8), 1161
1166. Available from https://doi.org/10.1002/cbic.201900527. Selmeczi, K., Gizzi, P., Champmartin, D., Rubini, P., Aubert, E., Dahaoui, S., & Henry, B. (2010). Nickel(II)dipeptidoamine-based tetrameric complex: Structural study in solution and in solid state. Inorganic Chemistry, 49(18), 8222
8229. Available from https://doi.org/10.1021/ic101167f. Shankaran, D., Gobi, K., & Miura, N. (2007). Recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest. Sensors and Actuators B: Chemical, 121(1), 158
177. Available from https://doi.org/10.1016/j. snb.2006.09.014. Sheftic, S. R., Snell, J. M., Jha, S., & Alexandrescu, A. T. (2012). Inhibition of semen-derived enhancer of virus infection (SEVI) fibrillogenesis by zinc and copper. European Biophysical Journal, 41, 695
704. Available from https://doi.org/10.1007/s00249-012-0846-0.

Application in nutrition: mineral binding 493 Shimazaki, Y., Takani, M., & Yamauchi, O. (2009). Metal complexes of amino acids and amino acid side chain groups. Structures and properties. Dalton Transactions (38), 7854. Available from https://doi.org/10.1039/b905871k. Sigel, H., & Martin, R. B. (1982). Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands. Chemical Reviews, 82(4), 385
426. Available from https:// doi.org/10.1021/cr00050a003. Slavin, J. L., & Marlett, J. A. (1980). Influence of refined cellulose on human bowel function and calcium and magnesium balance. The American Journal of Clinical Nutrition, 33(9), 1932
1939. Available from https://doi.org/10.1093/ajcn/33.9.1932. ˝ So´va´go´, I., & Osz, K. (2006). Metal ion selectivity of oligopeptides. Dalton Transactions, 32, 3841
3854. Available from https://doi.org/10.1039/B607515K. So´va´go´, I., Ka´llay, C., & Va´rnagy, K. (2012). Peptides as complexing agents: Factors influencing the structure and thermodynamic stability of peptide complexes. Coordination Chemistry Reviews, 256(19
20), 2225
2233. Available from https://doi.org/10.1016/j.ccr.2012.02.026. ´ . (2016). Coordinating properties of peptides containing histidyl So´va´go´, I., Va´rnagy, K., Lihi, N., & Grena´cs, A residues. Coordination Chemistry Reviews, 327
328, 43
54. Available from https://doi.org/10.1016/j. ccr.2016.04.015. Straub, D. A. (2007). Calcium supplementation in clinical practice: A review of forms, doses, and indications. Nutrition in Clinical Practice, 22(3), 286
296. Available from https://doi.org/10.1177/ 0115426507022003286. Sun, N., Cui, P., Jin, Z., Wu, H., Wang, Y., & Lin, S. (2017). Contributions of molecular size, charge distribution, and specific amino acids to the iron-binding capacity of sea cucumber (Stichopus japonicus) ovum hydrolysates. Food Chemistry, 230, 627
636. Available from https://doi.org/10.1016/j. foodchem.2017.03.077. Sun, X., Yu, G., Xu, Q., Li, N., Xiao, C., Yin, X., . . . He, Q.-Y. (2013). Putative cobalt- and nickel-binding proteins and motifs in Streptococcus pneumoniae. Metallomics, 5(7), 928. Available from https://doi.org/ 10.1039/c3mt00126a. Tabak, M., Sartor, G., & Cavatorta, P. (1989). On the interactions of metal ions with tryptophan and glyciltryptophan: A fluorescence study. Journal of Luminescence, 43(6), 355
361. Available from https:// doi.org/10.1016/0022-2313(89)90038-0. Tam, Y. J., Zeenathul, N. A., Rezaei, M. A., Mustafa, N. H., Azmi, M. L. M., Bahaman, A. R., . . . Rasedee, A. (2017). Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA. Biotechnology and Applied Biochemistry, 64(5), 735
744. Available from https://doi.org/10.1002/bab.1528. Torres-Fuentes, C., Alaiz, M., & Vioque, J. (2011). Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates. Food Chemistry, 129(2), 485
490. Available from https://doi. org/10.1016/j.foodchem.2011.04.103. Treiber, C., Thompsett, A. R., Pipkorn, R., Brown, D. R., & Multhaup, G. (2007). Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein. JBIC—Journal of Biological Inorganic Chemistry, 12(5), 711
720. Available from https://doi.org/10.1007/s00775-007-0220-3. Valeur, E., & Bradley, M. (2009). Amide bond formation: Beyond the myth of coupling reagents. Chemical Society Reviews, 38(2), 606
631. Available from https://doi.org/10.1039/B701677H. Van De Weert, M., & Stella, L. (2011). Fluorescence quenching and ligand binding: A critical discussion of a popular methodology. Journal of Molecular Structure, 998(1
3), 144
150. Available from https://doi.org/ 10.1016/j.molstruc.2011.05.023. Vanhooren, A., Vanhee, K., Noyelle, K., Majer, Z., Joniau, M., & Hanssens, I. (2002). Structural basis for difference in heat capacity increments for Ca21 binding to two α-lactalbumins. Biophysical Journal, 82(1), 407
417. Available from https://doi.org/10.1016/S0006-3495(02)75405-2. Varnava, K. G., & Sarojini, V. (2019). Making solid-phase peptide synthesis greener: A review of the literature. Chemistry
An Asian Journal, 14(8), 1088
1097. Available from https://doi.org/10.1002/ asia.201801807.

494 Chapter 19 Vavrusova, M., & Skibsted, L. H. (2014). Calcium nutrition, bioavailability and fortification. LWT—Food Science and Technology, 59(2), 1198
1204. Available from https://doi.org/10.1016/j.lwt.2014.04.034. Vegarud, G. E., Langsrud, T., & Svenning, C. (2000). Mineral-binding milk proteins and peptides; Occurrence, biochemical and technological characteristics. British Journal of Nutrition, 84(S1), 91
98. Available from https://doi.org/10.1017/S0007114500002300. Verlander, M. (2007). Industrial applications of solid-phase peptide synthesis
A status report. International Journal of Peptide Research and Therapeutics, 13(1
2), 75
82. Available from https://doi.org/10.1007/ s10989-006-9075-7. Vormann, J. (2003). Magnesium: Nutrition and metabolism. Molecular Aspects of Medicine, 24(1
3), 27
37. Available from https://doi.org/10.1016/S0098-2997(02)00089-4. Wang, P.-F., Huang, G.-R., & Jiang, J.-X. (2013). Optimization of hydrolysis conditions for the production of iron-binding peptides from mackerel processing byproducts. Advance Journal of Food Science and Technology, 5(7), 921
925. Available from https://doi.org/10.19026/ajfst.5.3183. Wasserman, R. H. (2004). Vitamin D and the dual processes of intestinal calcium absorption. The Journal of Nutrition, 134(11), 3137
3139. Available from https://doi.org/10.1093/jn/134.11.3137. Wong, J. W., Albright, R. L., & Wang, N.-H. L. (1991). Immobilized metal ion affinity chromatography (IMAC): Chemistry and bioseparation applications. Separation and Purification Methods, 20(1), 49
106. Available from https://doi.org/10.1080/03602549108021408. Wood, R. W. (1902). On a remarkable case of uneven distribution of light in a diffraction grating spectrum. Proceedings of the Physical Society of London, 18(1), 269
275. Available from https://doi.org/10.1088/ 1478-7814/18/1/325. Wu, D., Sun, N., Ding, J., Zhu, B., & Lin, S. (2019). Evaluation and structure
activity relationship analysis of antioxidant shrimp peptides. Food & Function, 10, 5605
5615. Available from https://doi.org/10.1039/ C9FO01280J. Wu, G. (2010). Amino acids
Biochemistry and nutrition (1st ed.). Boca Raton, FL: CRC Press. Available from https://doi.org/10.1201/b14661. Wu, H., Liu, Z., Zhao, Y., & Zeng, M. (2012). Enzymatic preparation and characterization of iron-chelating peptides from anchovy (Engraulis japonicus) muscle protein. Food Research International, 48(2), 435
441. Available from https://doi.org/10.1016/j.foodres.2012.04.013. Wu, W., Li, B., Hou, H., Zhang, H., & Zhao, X. (2017). Identification of iron-chelating peptides from pacific cod skin gelatin and the possible binding mode. Journal of Functional Foods, 35, 418
427. Available from https://doi.org/10.1016/j.jff.2017.06.013. Xie, N., Huang, J., Li, B., Cheng, J., Wang, Z., Yin, J., & Yan, X. (2015). Affinity purification and characterization of zinc chelating peptides from rapeseed protein hydrolysates: Possible contribution of characteristic amino acid residues. Food Chemistry, 173, 210
217. Available from https://doi.org/10.1016/ j.foodchem.2014.10.030. Xuan, Y., Scheuermann, E. B., Meda, A. R., Hayen, H., Von Wire´n, N., & Weber, G. (2006). Separation and identification of phytosiderophores and their metal complexes in plants by zwitterionic hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry. Journal of Chromatography A, 1136(1), 73
81. Available from https://doi.org/10.1016/j.chroma.2006.09.060. Zhao, L., Huang, Q., Huang, S., Lin, J., Wang, S., Huang, Y., . . . Rao, P. (2014). Novel peptide with a specific calcium-binding capacity from whey protein hydrolysate and the possible chelating mode. Journal of Agricultural and Food Chemistry, 62(42), 10274
10282. Available from https://doi.org/10.1021/jf502412f. Zheng, Y., Li, Y., Zhang, Y., & Zhao, S. (2016). Purification, characterization and synthesis of antioxidant peptides from enzymatic hydrolysates of coconut (Cocos nucifera L.) cake protein isolates. RSC Advances, 6(59), 54346
54356. Available from https://doi.org/10.1039/C6RA07086H. Zidane, F., Mate´os, A., Cakir-Kiefer, C., Miclo, L., Rahuel-Clermont, S., Girardet, J.-M., & Corbier, C. (2012). Binding of divalent metal ions to 1-25 β-caseinophosphopeptide: An isothermal titration calorimetry study. Food Chemistry, 132(1), 391
398. Available from https://doi.org/10.1016/j.foodchem.2011.11.006.

CHAPTER 20

Applications in nutrition: clinical nutrition Wen-Ying Liu, Liang Chen, Ying Wei, Guo-Ming Li, Yan Liu, Yu-Chen Wang, Yu-Qing Wang, Xiu-Yuan Qin, Xin-Yue Cui, Rui-Zeng Gu and Jun Lu Beijing Engineering Research Center of Protein and Functional Peptides, China National Research Institute of Food and Fermentation Industries Co., Ltd., Beijing, P.R. China

20.1 Introduction Peptides, which are defined as compounds containing two or more amino acid residues connected via peptide bonds, play essential physiological roles in the human body. To date, many peptides have been identified in living organisms, where they serve as crucial participants in numerous complex physiological processes. For example, peptides are involved in the regulation of various systems, organs, and cells as well as hormone release, nerve transmission, cell growth, and reproduction. Mellander discovered the first food-derived bioactive peptide, casein phosphorylated peptide, which has the ability to enhance vitamin D-independent bone calcification in infants (Mellander, 1951). In recent years, biologically active peptides have attracted widespread attention owing to their various physiological effects, such as antioxidant, antihypertensive, antibacterial, and immunomodulatory activities, and an increasing number of peptides have been applied in clinical nutritional support and clinical nutrition therapy.

20.1.1 Overview of clinical nutritional support and clinical nutrition therapy Clinical nutrition support and clinical nutrition therapy began to be implemented in the 1960s and have saved the lives of countless patients with severe intestinal failure, malnutrition, trauma, burns, infections, and other serious conditions. The significance of micronutrients and gut function on metabolism and the immune system and the importance of components such as amino acids and fats in the development of specific diseases have been recognized, and nutrition support therapy has emerged as an essential means of clinical treatment (Iwasaki & Ohyanagi, 2004). In a set of guidelines published in 2009, the American Society for Parenteral and Enteral Nutrition (ASPEN) proposed the concept of “nutrition support therapy”, further emphasizing the importance of nutrition support and nutrition therapy (American Society for Parenteral & Enteral Nutrition A.S.P. Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00019-4 © 2021 Elsevier Inc. All rights reserved.

495

496 Chapter 20 E.N. Board of Directors, 2009). At present, patients may receive nutrition support therapy in hospitals, nursing homes, rehabilitation facilities, and their homes. Clinical nutrition treatment includes parenteral nutrition (PN) and enteral nutrition (EN). PN provides patients with a variety of nutrients to meet their physiological and disease treatment needs, including carbohydrates, fats, amino acids or proteins, both fat- and water-soluble vitamins, trace elements, electrolytes, and water. PN is mainly employed in patients who require nutrition support but suffer from gastrointestinal dysfunction or poor tolerance to EN. However, in contrast to PN, EN can stimulate the secretion of hormones, digestive enzymes, and immunoglobulins in the gastrointestinal tract, maintain intestinal immune function, and promote normal growth of intestinal bacteria to preserve the intestinal biological barrier (Wereszczynska-Siemiatkowska, Swidnicka-Siergiejko, & Siemiatkowski, 2013). With the continuous development of clinical practice and medical research, the disadvantages of high-energy-density nutrition support and excessive dependence on PN have been recognized, and the preferred approach for nutrition support has gradually shifted toward a concept of “EN as the main, PN as the auxiliary; when necessary, a combination of the two”. For example, the ASPEN guidelines of nutrition support for critically ill patients stipulate that EN should be administered within 24
48 hours of admission to hospital, and the target amount should be reached within 48
72 hours. If the target dose is not achieved by this time, EN and PN should be administered in combination. This approach is supported by abundant theoretical and clinical evidence. In addition, the European Society for Clinical Nutrition and Metabolism (ESPEN), the American College of Gastroenterology (ACG), the Chinese Society for Parenteral and Enteral Nutrition (CSPEN), and other clinical nutrition organizations from various countries have issued additional guidelines regarding enteral and parenteral nutrition support and therapy. However, some problems remain, including the coexistence of insufficient and excessive nutrition support, unreasonable application structure of PN, and irregular PN infusion. Therefore, to provide safe nutrition support to patients, it is necessary to establish a multidisciplinary nutrition support team including doctors, nurses, nutritionists, and pharmacists, in which the nutritionists are important members (Shin, Chun, & Ryu, 2018). Studies into the physical condition of patients have revealed that patients receiving nutrition support from nutritionists exhibit improved nutritional status, shorter hospital stays, and fewer complications (Brugler & Bernstein, 1998; Delegge & True Kelley, 2013). In 2014, ASPEN issued a professional standard for nutrition support pharmacists, which affirmed the role of nutrition support pharmacists (Tucker, Ybarra, & Bingham, 2015). According to ESPEN, prior to diagnosing malnutrition, healthcare professionals should first use reasonable nutritional screening tools such as Nutritional Risk Screening

Applications in nutrition: clinical nutrition 497 2002 (NRS 2002) and Subjective Global Assessment (SGA) for patients with nutritional risk. The application of both SGA and NRS 2002 may have a complementary effect on improving nutritional status classification and predicting poor clinical outcomes (Raslan, Gonzalez, & Torrinhas, 2011). With the improvement of medical technology and the shortening of the average hospital stay, home enteral nutrition (HEN) is a continuation of nutrition support that permits many patients to receive long-term and effective nutrition support treatment outside of the hospital setting (Peladic, Gagliardi, & Fagnani, 2017). However, the implementation and management of HEN is a clinical approach that requires the cooperation and supervision of a multidisciplinary team. In April 2019, ESPEN issued clinical guidelines regarding HEN for the first time, in which detailed recommendations were given for members of the medical team. The significance of clinical nutrition support and therapy cannot be overstated and this significance has been increasingly recognized in recent years. The growing number of clinical nutritionists is expected to lead to improved clinical services and the gradual standardization of nutrition support and therapy.

20.1.2 Application of biologically active peptides in clinical nutritional support and therapy In recent years, the relationships between health and many natural active compounds have been elucidated. Among the various classes of active compounds, proteins and peptides are of considerable interest. Considering that the specificity of peptide activity is dependent on the amino acid sequence and structure, it is easy to appreciate the enormously diverse range of activities that peptides may exhibit. Biologically active peptides from food-derived proteins typically contain 2
20 amino acids. The low molecular weight, high bioavailability, and flexible molecular behavior of these small biologically active peptides allow them to easily interact with a large variety of proteins/receptors both in vitro and in the human body. Biologically active peptides exhibit numerous health benefits and play various roles in mitigating disease, including reducing blood pressure (angiotensin-Iconverting enzyme (ACE) inhibitors), inhibiting blood coagulation, and mediating antiinflammatory, antimicrobial, and antitumor activities. Several studies have demonstrated that biologically active peptides can be found in bovine (Fu, Young, & Rasmussen, 2016), soybean (Fan et al., 2009; Rho, Lee, & Chung, 2009), porcine (O’Keeffe, Norris, & Alashi, 2017), egg white (Rizzetti, Martı´n, & Corrales, 2017), milk (Tidona, Criscione, & Guastella, 2009), and marine byproducts (Rushikesh, Pravin, & Seetharama, 2017). Approximately 150 types of peptides are currently being investigated in preclinical and clinical studies (Lau & Dunn, 2017).

498 Chapter 20 Food-derived bioactive peptides exhibiting anticancer activity have attracted increasing attention in clinical nutritional support owing to the neurotoxic, gonadotoxic, nephrotoxic, and cardiotoxic side effects of most synthetic anticancer agents. The main mechanisms of action of these peptides include the induction of apoptosis, inhibition of gene proliferation, tumor angiogenesis, or cell migration, disorganization of tubulin structure, and antioxidant activity (Atul, Abhishek, & Priya, 2015; Schweizer, 2009). In the search for food-derived bioactive peptides with anticancer activity, particular interest has been directed toward the antineoplastic effects of lunasin, the antiproliferative activity of the pentapeptide EQRPR purified from rice bran, and peptides of animal origin derived from milk, eggs, and marine sources (Hernandez-Ledesma, Hsieh, & C, 2013; Kannan, Hettiarachchy, & Lay, 2010). Peptides derived from milk, whey, fish, and plants have demonstrated mild but significant antihypertensive effects in humans based on the inhibition of ACE, the renin
angiotensin system, or an increase in endothelial NO levels (Bhat, 2015). Two casein tripeptides, VPP and IPP, have been reported to significantly reduce hypertension in humans (Fekete, Givens, & Lovegrove, 2015). The bioactive peptides with the most clinical evidence for ameliorating cholesterolemia are those derived from soy, lupine, and milk proteins (Butteiger, Hibberd, & Mcgraw, 2016; Lammi, Zanoni, & Scigliuolo, 2014). Antimicrobial activity has been identified for peptide fragments of casein, β-lactoglobulin, and α-lactalbumin (Piotto, Sessa, & Concilio, 2012), as well as peptides isolated from fish and fish products (SIFIQRFTT from mackerel and GLSRLFTALK from anchovies) (Ennaas, Hammami, Beaulieu, & Fliss, 2015; Tang, Zhang, Wang, Qian, & Qi, 2015). Antioxidant peptides have been identified in marine organisms such as oysters, shrimp, squid, and blue mussels (Harada et al., 2010). Some bioactive peptides also display analgesic activity owing to their affinity for opiate receptors. For example, α- and β-lactorphin are opioid peptides derived from α-lactalbumin and β-lactoglobulin, respectively, that are released during the in vitro proteolysis of bovine whey proteins and exhibit pharmacological activity (Pihlanto-Leppa¨la¨, 2000). Research into food containing antidiabetic peptides is also increasing. Fermented soybeans contain low-molecular-weight peptides, some of which have been found to induce insulin-stimulated glucose uptake in 3T3-L1 cells (Kwon et al., 2011) and antagonize peroxisome proliferator-activated receptorγ activity. Furthermore, several peptides isolated from black bean protein hydrolysates, namely, AKSPLF, ATNPLF, FEELN, and LSVSVL, were reported to effectively inhibit glucose transporter 2 and sodium-dependent glucose transporter 1 and thereby reduce blood glucose levels (Mojica, de Mejia, Granados-Silvestre, & Menjivar, 2017).

20.2 Application of biologically active peptides in disease treatment To date, a variety of biologically active peptides have been isolated from animals, plants, and microorganisms via the enzymatic hydrolysis of proteins in vitro, with a

Applications in nutrition: clinical nutrition 499 range of physiological functions that may be promising for the treatment of cardiovascular diseases, tumors, liver injury, and diabetes in clinical patients. In addition, some biologically active peptides also exhibit other nutritional and physiological effects on the body, such as immunomodulatory, anti-osteoporosis, antiobesity, opioid agonist, radioprotective, antifatigue, anxiolytic, and anti-inflammatory activities. Some biologically active peptides that are relevant to clinical nutrition are listed in Table 20.1.

Table 20.1: Biologically active peptides relevant to clinical nutrition. Peptide sequence

Origin

Function

Reference

EQRPR

Rice bran

Kannan et al. (2010)

VPP, IPP

Casein

Antiproliferative activity Antihypertensive activity Antimicrobial activity Antimicrobial activity Hypoglycemic activity Antihypertensive activity Antithrombotic activity Serum cholesterollowering effect Hypotriglyceridemic activity Anticancer activity Facilitates ethanol metabolism Antihepatitis activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity Anti-obesity activity Anxiolytic activity

Wang et al. (2008)

SIFIQRFTT GLSRLFTALK AKSPLF, ATNPLF, FEELN, LSVSVL VVYPWTQRF

Mackerel Anchovy Black bean

MAIPPKKNQDK, KNQDK

Bovine κ-casein

Oyster

LPYPR

Soy

VVYP, VYP, VTL

Soy

AFNIHNRNLL QLLPF

Shellfish Corn

pyroEL IPAVF

Wheat gluten Whey

VAGTWY

β-Lactoglobulin

LPQNIPPL

Gouda-type cheese β-Lactoglobulin

LKPTPEGDL, LKPTPEGDLE, LKPTPEGDLEIL VA, VL, WL, WI

Whey

LPQNIPPL

Casein

DIVDKIEI VYLPR

Tuna Ovalbumin

Fekete et al. (2015) Ennaas et al. (2015) Tang et al. (2015) Mojica et al. (2017)

Jolle`s et al. (1986) Takenaka et al. (2000) Kagawa et al. (1996) Kim et al. (2012) ma et al. (2012) Sato et al. (2013) Silvana et al. (2013) Uchida et al. (2011) Uenishi et al. (2011) Lacroix and Li-chan (2014), Lacroix et al. (2016) Le maux et al. (2015) Uenishi et al. (2011) Kim et al. (2015) Oda et al. (2012)

500 Chapter 20

20.2.1 Application of biologically active peptides in the clinical treatment of cardiovascular diseases According to the definition of the World Health Organization (WHO), cardiovascular diseases (CVDs) are a group of heart and blood vessel disorders that include coronary heart disease. CVDs have become the main cause of death globally and are associated with several risk factors, such as high blood pressure, serum glucose level, and obesity (Hajar, 2016). Food-derived bioactive peptides are one source of health-enhancing components that may improve cardiovascular health after dietary digestion. Numerous studies have examined the potential of food-derived bioactive peptides in the prevention or treatment of chronic disease. Depending on the amino acid sequence, these peptides may exhibit a diverse range of activities (Erdmann, Cheung, & Schro¨der, 2008). ACE mediates the conversion of angiotensin I to the vasoconstrictor angiotensin II and inactivates the vasodilator bradykinin. Some food-derived peptides have been identified as ACE inhibitors, such as those derived from marine fish, plant, or dairy proteins (FitzGerald, Murray, & Walsh, 2004). ACE inhibitory peptides are typically oligopeptides containing two to nine amino acids (Meisel, 1997). These oligopeptides have been demonstrated to be absorbed more rapidly. It is worth noting that binding to ACE appears to be strongly influenced by C-terminal sequences containing proline, lysine, or arginine residues, which are resistant to degradation by digestive enzymes (Korhonen & Pihlanto, 2003). In a recent study, Wang et al. reported that an oyster protein pepsin hydrolysate exhibited antihypertensive activity upon oral administration in spontaneously hypertensive rats at a dose of 20 mg/kg (Wang, Hu, & Cui, 2008). A purified peptide with the sequence VVYPWTQRF was isolated, and the IC50 value of the ACE inhibitory activity was determined to be 66 μmol/L. Whey protein hydrolysate derived from curdled and strained milk also contains a variety of bioactive sequences (Pan, Cao, & Guo, 2012). Nine different Lactobacillus species were used for whey fermentation prior to testing the ACE inhibitory activity. The results revealed that the ACE inhibition rate of the resulting peptides ranged from 93.3% to 100%. Furthermore, the fraction isolated from Lactobacillus helveticus displayed in vitro IC50 values of 5.3 and 7.8 mg/mL, respectively. Thrombus formation is mainly attributable to abnormalities in coagulation, with the risk factors including platelet hyperreactivity, high levels of hemostatic proteins, defective fibrinolysis, and blood hyperviscosity (Grundy, 1999). Food-derived antithrombotic peptides have been obtained from the hydrolysis of bovine, ovine, or human κ-casein (Chabance, Jolle`s, & Izquierdo, 1995). Jolle`s et al. reported that the undecapeptide MAIPPKKNQDK (residues 106
116) derived from the soluble C-terminal fragment of bovine κ-casein inhibits ADP-induced platelet aggregation as well as binding of the human fibrinogenin ϒ-chain receptor in a concentration-dependent manner (Jolle`s, Le´vy-Toledano, & Fiat, 1986). Moreover, the pentapeptide KNQDK (residues 112
116) and caseinoglycopeptide

Applications in nutrition: clinical nutrition 501 (residues 106
169) can also affect platelet function. The mechanism pathway of antithrombotic peptide is intact into the bloodstream. Human and bovine κ-caseinoglycomacropeptides have been detected at physiologically active concentrations in the plasma of newborn babies following the ingestion of breast or cow milk (Jolle`s et al., 1986). In addition, no detectable toxic effects have been reported. Hence, caseinoglycomacropeptides could potentially be used to treat or prevent thrombosis. Hyperlipidemia, which includes hypercholesterolemia and hypertriglyceridemia, is associated with various diseases. Improving the serum lipid distribution via dietary modification can have a positive effect on such conditions. To date, peptides derived from soy, whey, and fish have been reported to alter the plasma profile from atherogenic to cardioprotective (Hori, Wang, & Chan, 2001; Zhang & Beynen, 1993). The exact mechanism underlying the hypocholesterolemic activity of peptides has not been entirely elucidated. However, it has been reported that dietary proteins containing low methionine: glycine and lysine:arginine ratios (e.g., soy and fish proteins) favor a hypocholesterolemic effect. Oral administration of a peptide isolated from soy glycinin, LPYPR, was found to reduce serum cholesterol in mice. The structure of this peptide is similar to that of enterostatin (VPDPR), which also exhibits hypocholesterolemic and enorectic effects (Takenaka, UTsUMI, & Yoshikawa, 2000). Hypotriglyceridemic activity has also been associated with decreased intestinal fat absorption and enhanced lipolysis of triglycerides, especially in the liver. Iritani et al. reported that the oral administration of soy protein to rats can significantly reduce the triglyceride concentration in the plasma and liver (Iritani, Nagashima, & Fukuda, 1986). This may be associated with significant reductions in the activities of hepatic lipogenic enzymes, indicating that soy protein reduces liver triglycerides or fats in part by inhibiting hepatic fatty acid synthesis. The peptides VVYP, VYP, and VTL have also been demonstrated to exhibit hypotriglyceridemic activity in rats (Kagawa, Matsutaka, & Fukuhama, 1996). Owing to the limited production of these bioactive peptides, they have not yet been used to treat CVDs. However, they could be investigated as possible supplemental treatments for patients suffering from CVDs, with a low risk of adverse side effects such as dry cough and allergic symptoms.

20.2.2 Application of biologically active peptides in the clinical treatment of cancer Cancer is a major global public health issue and there has been a significant increase in the number of patients over the past few years. It is also known as malignant tumor by the control of cell growth proliferation mechanism of arrhythmia caused by disease. Cancer cells exhibit higher division and growth rates than normal cells and may metastasize to other organs. Cancer is a major cause of death and the incidence and mortality continue to increase. A variety of risk factors for cancer have been identified, such as immune

502 Chapter 20 disorders, genetic factors, microorganisms, free radicals and toxins, radiation, and environmental pollution. Biologically active peptides play a variety of roles in various stages of digestion and absorption, and numerous studies have indicated that peptides are applicable to the diagnosis or treatment of cancer. As described in a review article by Okarvi (Okarvi, 2004), radiolabeled peptides permit the delivery of radioactivity to target receptors for the imaging of certain types of cancer (e.g., breast cancer, prostate cancer). Another successful clinical application has been the long-term use of nonradioactive somatostatin analogs to relieve the symptoms of hormone-secreting neuroendocrine tumors (Kwekkeboom, Krenning, & de Jong, 2000). Numerous bioactive peptides with anticancer activity have been investigated. Several studies have reported the extraction of peptides with potential anticancer functions from various marine animals and botanical plants. The bioactivation pathways of peptides for cancer treatment mainly depend on the membranolytic mode (Suarez-Jimenez, BurgosHernandez, & Ezquerra-Brauer, 2012). Briefly, the target sequence sites on the cancerous cells are recognized by the peptides, resulting in inhibition of growth or proliferation. One report demonstrated that oyster hydrolysates can inhibit tumor growth by exerting strong immunostimulatory effects in S108-bearing mice, indicating a potential role in tumor therapy (Schweizer, 2009). Anticancer peptides have also been identified in and purified from pepsin hydrolysates of the shellfish Mytilus coruscus, with the sequence AFNIHNRNLL effectively inducing cell death in prostate, breast, and lung cancer cells (Kim, Joung, & Kim, 2012). Dolastatin 10, a novel peptide isolated from a marine shell-less mollusk found in the Indian Ocean, was investigated as an anticancer agent in phase I and phase II clinical trials (Turner, Jackson, & Pettit, 1998). At a concentration of 1 nM (IC50 5 0.5 nM), dolastatin 10 completely inhibited the growth of D-145 human prostate cancer cells. The mechanism of action of this peptide was found to involve tubulin depolymerization but not the induction of apoptosis. With respect to botanical peptides, studies have demonstrated that peptides derived from plants can inhibit the proliferation of various tumor cell lines. For example, dianthin E derived from Dianthus superbus was found to significantly inhibit the proliferation of HepG2 human liver cancer cells with an IC50 of 2.37 μg/mL (Hsieh, Chang, & Wu, 2005). RA-VII isolated from Rubia akane Nakai is currently undergoing phase I clinical trials as an anticancer drug in Japan, with reported IC50 values against KB cells and P388 cells of approximately 1.2 and 3.5 μg/mL, respectively (Hitotsuyanagi, Ishikawa, & Hasuda, 2004). Tepkeeva et al. studied the antitumor effects of peptide extracts from eight kinds of medicinal plants in human breast cancer (Tepkeeva, Moiseeva, & Chaadaeva, 2008). The results revealed that the administration of a mixture of Chelidonium majus L., Inula helenium L., Equisetum arvense L., and Inonotus obliquus over four weeks exhibited

Applications in nutrition: clinical nutrition 503 maximum activity for inhibiting the growth and appearance of palpable breast cancer in mice. Wu et al. reported that a soy peptide inhibited the growth of gastric cancer cells; however, the series of sequence was not qualified (Fei, Shengnan, & liang, 2011). The mechanisms underlying the anticancer effects of these peptides are complex but include interfering with microtubule polymerization and depolymerization, inducing cell apoptosis, inhibiting angiogenesis, overcoming multidrug resistance, and even interrupting signal transduction. However, owing to the complex structure and diversity of plant compounds, research has not progressed as rapidly as that for peptides from other sources. Peptides from other animals have also been reported to exhibit antitumor activity. Bee products have long been used in traditional medicine. Several studies have indicated that peptides found in bee products can induce apoptosis in human cell lines derived from renal, lung, liver, prostate, bladder, and lymphoid cancers (Premratanachai & Chanchao, 2014). A peptide isolated from goat spleens or livers, referred to as bioactive peptide-3 (ACPB-3), was reported to exhibit anticancer activity against human gastric cancer cell line BGC-823 and gastric cancer stem cells both in vitro and in vivo (Su, Xu, Shen, & Tuo, 2010). Furthermore, milk and dairy products contain numerous components that exhibit a wide variety of physiological and functional activities. A number of studies have reported the anticancer effects of milk-protein-derived peptides on various cancer cells. For example, Meisel and Fitzgerald reported that casein phosphopeptides can inhibit cancer cell growth and stimulate the activity of immunocompetent cells (Meisel & FitzGerald, 2003). Cancer not only impacts public health but also places a substantial burden on economic development. Many traditional anticancer drugs cannot adequately differentiate between cancerous and normal cells, leading to systemic toxicity and adverse side effects. Therefore, biologically active peptides from natural sources are currently being considered as new drug candidates for cancer treatment.

20.2.3 Application of biologically active peptides in the clinical treatment of liver injury Hepatic injury is a prevalent ailment worldwide and can be divided into alcoholic and nonalcoholic liver diseases. Oxidative stress is considered to be the major factor underlying the pathogenesis of both types of liver disease, with ethanol consumption, a high-fat diet, obesity, and diabetes among the main causes (Leung & Nieto, 2013). Owing to their easy absorption, peptides are good candidates for advanced clinical nutrition treatments and supplements (Kreider, Iosia, & Cooke, 2011). Compared with synthetic compounds, foodderived peptides are natural products that can be digested by the human body with less potential for toxicity. Recent studies have demonstrated that various food-derived peptides can play a hepatoprotective role both in vitro and in vivo.

504 Chapter 20 Kang et al. examined the protective effects of peptides from the marine microalgae Navicula incerta against ethanol toxicity in HepG2/CYP2E1 cells (Kang, Qian, & Ryu, 2012). The results revealed a dose-dependent decrease in γ-glutamyl transpeptidase activity when the ethanol-treated cells were exposed to increasing peptide concentrations. In vivo studies have also demonstrated that peptides derived from cod skin collagen, corn, the mushroom Ganoderma lucidum, and wheat gluten exert potent hepatoprotective activity in various rat and mice models of liver injury induced by carbon tetrachloride, acetaminophen, thioacetamide, bacillus Calmette
Gue´rin, lipopolysaccharides, ethanol, or D-galactosamine (Han, Xie, & Gao, 2015; Hui, Jie, & Hui, 2009; Li, Guo, & Hu, 2007; Lv, Nie, & Zhang, 2013; Sato, Yukari, & Shin, 2013; Shi, Sun, & He, 2008; Yu, Lv, & He, 2012; Zhang, Zhang, & Li, 2012). Food-derived peptides exert their hepatoprotective effects by decreasing the serum activities of aspartate transaminase (AST) and alanine transaminase (ALT) and the levels of malondialdehyde (MDA), nitric oxide (NO), and triacylglycerol (TG) in the liver, while increasing the serum levels of albumin and antioxidants and the levels of superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione (GSH) in the liver. Ma et al. reported that the corn-derived pentapeptide QLLPF facilitates ethanol metabolism (ma, Zhang, Yu, He, & Zhang, 2012). The results revealed that administration of this pentapeptide (10 mg/kg) led to increased ethanol elimination in vivo compared with the mixed peptides (MW ,5 kDa, 200 mg/kg). A subsequent study revealed that Leu-Leu and Pro-Phe were the key structural units of the pentapeptide responsible for the improved ethanol metabolism (ma et al., 2016). Sato et al. also identified pyroglutamyl leucine (pyroGlu-Leu) as a hepatoprotective peptide in wheat gluten hydrolysate (Sato et al., 2013). The ingestion of small doses (20 mg/kg body weight) of pyroGlu-Leu was found to exert a strong hepatoprotective effect in a rat model of D-galactosamine-induced acute hepatitis and significantly decreased the serum concentrations of AST and ALT to almost normal levels. However, the efficacy of pyroGlu-Leu and foods containing this dipeptide has yet to be evaluated in human trials. Furthermore, the potential applications of biologically active peptides in clinical nutrition therapy have been studied. Ma et al. evaluated the effects of corn peptides on facilitating ethanol metabolism in a crossover experiment (Magoichi, Fumi, & Michiko, 1997). In this study, ten healthy male volunteers ingested five grams of corn peptides, wheat peptides, pea peptides, alanine, or leucine 30 minutes prior to ethanol intake at a dose of 0.5 g/kg, and their blood ethanol and plasma amino acid concentrations were measured after 2 hours. The results revealed that the corn peptide group displayed the lowest blood ethanol level during the observation period, as well as significantly higher plasma alanine and leucine levels compared with the other groups. These findings indicate that corn peptides may reduce the blood ethanol level after ethanol intake owing to the marked elevation of plasma alanine and leucine levels. To further assess

Applications in nutrition: clinical nutrition 505 the hepatoprotective effect of corn peptides, Wu et al. conducted a nine-week, randomized, double-blind, placebo-controlled study involving a total of 161 male participants, of which 146 completed the study (Wu, Pan, & Zhang, 2014). The participants were randomly assigned to receive corn peptides (n 5 53), whey protein (n 5 54), or corn starch placebo (n 5 54) at the same dose of two grams twice daily. The results revealed that corn peptide supplementation (4 g/d) for nine weeks significantly lowered the serum levels or activities of total cholesterol (CHO), AST, ALT, MDA, TG, and tumor necrosis factor-α (TNF-α) and significantly increased the serum activities of SOD and GPX, which indicates that corn peptides may exert their hepatoprotective effect by modulating lipid metabolism and oxidative stress, whereas neither whey protein nor corn starch exhibited these effects. The hepatoprotective effects of bioactive peptides may be explained by their capacity to prevent oxidative liver injury mediated by reactive oxygen species and scavenge free radicals via decreasing the CHO, AST, ALT, MDA, NO, TG, TNF-α, and transforming growth factor beta 1 (TGF-β1) activities to normal levels while restoring the contents of SOD, GPX, and GSH (Fei et al., 2011; Hitotsuyanagi et al., 2004; Su et al., 2010; Tepkeeva et al., 2008; Yu et al., 2012). In addition, bioactive peptides may suppress hepatocyte apoptosis by regulating the expression of Bcl-2, Bax, and cytochrome c in the intrinsic pathway and that of Fas, FasL, and NF-κB in the extrinsic pathway, as well as preventing the activation of caspase 3 (Zhili, Tao, & Wen, 2015).

20.2.4 Application of biologically active peptides in the clinical treatment of diabetes mellitus Type 2 diabetes mellitus is a significant global public health concern that is characterized by increased insulin resistance, decreased insulin secretion following glucose-induced stimulation, and high glucagon levels, ultimately resulting in high blood glucose concentrations. The two most important incretin hormones are glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1), which increase in concentration rapidly following food intake and are quickly degraded by the metabolic enzyme dipeptidyl peptidase 4 (DPP-IV) in the human body (Demuth, McIntosh, & Pederson, 2005). Antidiabetic treatment strategies can be divided into two types, namely, the use of DPP-IV inhibitors to prevent GLP-1 and GIP degradation and the enhancement or protection of incretin activities. DPP-IV inhibitors are naturally found in dietary proteins in the form of peptide fragments. For example, the pentapeptide IPAVF was separated from whey protein hydrolysate by Silveira et al. Silvana, Martı´nez-Maqueda, and Recio (2013). This peptide exhibits notable DPP-IV inhibitory activity (IC50 5 44.7 μM) and homology with β-lactoglobulin f (78
82). This peptide may be a beneficial component of foods to protect against type 2

506 Chapter 20 diabetes. The hexapeptide VAGTWY isolated from trypsin-treated β-lactoglobulin also exhibited a concentration-dependent inhibitory effect with an IC50 value of 174 μM (Uchida, Ohshiba, & Mogami, 2011). This structure corresponds to β-lactoglobulin f (15
20). The octapeptide LPQNIPPL with DPP-IV inhibitory activity (IC50 5 46 μM) was isolated by Uenishi et al. from Gouda-type cheese (Uenishi, Kabuki, & Seto, 2011). In addition, three homologous peptides of different lengths from β-lactoglobulin, namely, LKPTPEGDL, LKPTPEGDLE, and LKPTPEGDLEIL, were found to be effective DPP-IV inhibitors with IC50 values of 45, 42, and 57 μM, respectively (Lacroix & Li-chan, 2014; Lacroix, Meng, & Cheung, 2016). There has also been considerable interest in identifying short peptides with potential antidiabetic activity. Le Maux et al. identified short peptide inhibitors of DPP-IV with molecular weights of less than 200 Da from whey protein hydrolysate via nanofiltration fractionation (Le maux, Nongonierma, & Murray, 2015). Four dipeptides, namely, VA, VL, WL, and WI, displayed IC50 values of less than 170 μM, with WL exhibiting the lowest value of 43.6 μM. In addition to animal-derived peptides, peptides originating from plants can also inhibit DPP-IV. For example, quinoa protein hydrolysate was reported to display an IC50 value of 0.88 mg/mL. However, further characterization is required to identify which peptide sequences within the hydrolysate are responsible for the DPP-IV inhibition. The octapeptide LPQNIPPL identified by Uenishi et al. was further investigated in vivo. Glucose tolerance tests were performed in rats by orally administering synthetic LPQNIPPL (30 mg per 100 g body weight) with a crossover experimental design. The postprandial area under the plasma glucose curve was lower for the LPQNIPPL-administered group compared with the control (Uenishi et al., 2011). Owing to their serum-glucose-lowering properties, food-derived peptides have potential as functional ingredients. However, the DPP-IV inhibitory activities of reported food-derived peptides remain approximately 10-fold lower than diprotin A, a well-known DPP-IV inhibitory peptide, and 100-fold lower than commercial drugs (Uenishi et al. Demuth & Kim 2005, 2011Mcintosh). As mentioned above, the other antidiabetic treatment strategy is to enhance and protect the incretin activities. Native GLP-1 is very rapidly degraded by DPP-IV with a half-life of approximately 2 min (Vilsbøll, Agersø, & Krarup, 2003). Several glucagon-like peptide-1 receptor (GLP-1R) agonists with longer half-lives have emerged for the treatment of type 2 diabetes. In the case of these drugs, the structure of native GLP-1 was altered to avoid degradation by DPP-IV. Exenatide, the first GLP-1R agonist to become available, was introduced to the U.S. and European markets in 2005 and 2007, respectively (Lund, Knop, & Vilsbøll, 2014). The sequence of exenatide was based on a modified version of exendin-4. This peptide has 53% amino acid sequence homology with human GLP-1 and is primarily cleared by the kidneys via glomerular filtration (Linnebjerg, Kothare, & Park, 2007). The half-life of exenatide

Applications in nutrition: clinical nutrition 507 following subcutaneous injection is approximately 2
3 hours, with detectable plasma concentrations up to 10 hours following administration (Lund et al., 2014). Exenatide is recommended for twice-daily administration with a typical dose of 5 μg. Lixisenatide, another GLP-1R agonist, was approved for the treatment of type 2 diabetes in Europe in February 2013 (Lund et al., 2014). This peptide has a similar sequence to exenatide but with a proline removed and six lysine residues added to the C terminus (Christensen, Filip-K, & Vilsbøll, 2011). The half-life of lixisenatide after subcutaneous injection is 2
3 hours. Lixisenatide is recommended for once-daily administration with a typical dose of 20 μg (Neumiller, Sonnett, & Wood, 2010). Liraglutide was approved for clinical use in Europe and the U.S. in 2009 and 2010, respectively (Lund et al., 2014). The structure of liraglutide is based on that of GLP-1, with the addition of a 16-carbon fatty acid side chain and an altered amino acid residue (Neumiller et al., 2010). The fatty acid side chain enables liraglutide to bind to albumin and circulate slowly in the plasma following subcutaneous injection. Consequently, the half-life of liraglutide is 11
15 hours and the recommended dose is 0.6 mg for the first week and 1.2 mg thereafter (Lund et al., 2014). Several other GLP-1R agonists, including albiglutide, dulaglutide, and semaglutide are also currently in clinical use (Lund et al., 2014; Shaddinger, Soffer, & Vlasakakis, 2019; Umpierrez, Blevins, & Rosenstock, 2011).

20.2.5 Application of biologically active peptides in the clinical treatment of other diseases In addition to the antithrombotic, cholesterol-lowering, anticancer, antineoplastic, antihypertensive, hepatoprotective, and glycemic regulation properties discussed in the preceding subsections, bioactive peptides may exert other nutritional and physiological effects on living organisms, such as immunomodulatory, anti-osteoporosis, anti-obesity, opioid agonist, radioprotective, antifatigue, anxiolytic, and anti-inflammatory properties. In fact, several peptides can often simultaneously play multiple crucial roles in controlling multiple symptoms (Agyei, Pan, Acquah, & Danquah, 2017). Immunomodulatory peptides have been reported that both affect the immune system and participate in cell proliferation responses (Pihlanto-Leppa¨la¨, 2002). Some casein-derived peptides were reported to stimulate non-specific immune responses, such as human or murine macrophages, including αs1-casein f(194
199) and β-casein f(63
68) and f(191
193) (Woo, Song, & Kang, 2018), whereas others act on lymphocyte proliferation, such as human T cells, including sodium caseinate hydrolysates (O’Sullivan, O’Callaghan, & O’Keeffe, 2015). The in vitro and in vivo modulation of lymphocyte proliferation has also been reported for peptides derived from β-lactoglobulin, α-lactalbumin, lactoferrin, lactoperoxidase, and mixtures of whey proteins (Gauthier, Pouliot, & Saint-Sauveur, 2006).

508 Chapter 20 Collagen peptides are frequently employed for the prevention or treatment of osteoporosis. Collagen peptides, such as sheep bone collagen peptide (Nai-Rui, Li-Zhen, & Run-Ying, 2009) and marine fish skin collagen peptide (Pan, Liu, & Dai, 2011), exert their antiosteoporosis effects by inhibiting bone resorption or promoting bone growth (Nai-Rui et al., 2009). In addition, some non-collagen peptides, such as krill peptides (Fei, Zhao, & Liu, 2017) and particular peptide fragments containing RGD or RGDS motifs, have also been reported to mitigate osteoporosis. The RGD and RGDS motifs mediate cell adhesion based on biophysical interactions between cells and type I collagen, making these peptides a natural biomaterial for regenerative therapies (Monteiro, Fernandes, & Sundararaghavan, 2011; Zhang & Huang, 2008). Anti-obesity treatment has been attracting increasing clinical attention as a major problem arising from overnutrition. From a genetic viewpoint, the octapeptide DIVDKIEI derived from tuna was reported to inhibit the expression of lipogenic and adipogenic genes and activate the Wnt/β-catenin signaling pathway (Kim, Kim, & Choi, 2015). From a proteinbinding viewpoint, the dipeptide YY or its analog could activate the Y2 receptor to regulate appetite, which could play a role in obesity (Nishizawa, Niida, & Masuda, 2017). From a lipid metabolism perspective, a skate skin collagen peptide was reported to downregulate the expression of several hepatic proteins involved in fatty acid synthesis (e.g., fatty acid synthase and acetyl-CoA carboxylase) and cholesterol synthesis (Woo et al., 2018). Several opioid agonist peptides have been reported. These peptides are usually synthesized in clinical application working on nociceptive and neuropathic, including dependence or independence of opioid receptor stimulation. To date, natural bioactive peptides extracted from milk, including milk protein hydrolysates, have been reported to act as opioid agonists. The effects of radiation on cells include those of reactive oxygen species generated upon the irradiation of water. However, in a clinical setting, radiation can also be used to kill tumors. The reactive oxygen species generated by radiation, including OH, O2, and H2O2, can damage nucleic acids, proteins, and other biological macromolecules. H-D-Arg-DmtLys-phe-NH2 was reported to reduce the apoptotic rate following acute radiation (Jia, 2012). Scorpion venom peptide II isolated from Buthus martensii Karsch was also reported to work on stem cells (Li, Wang, & Wang, 2012). Several peptides displaying antifatigue effects have also been reported, such as a highFischer-ratio corn oligopeptide that increases the glycogen contents of the liver and muscles (Zhang, Yin, & Liu, 2007), a collagen oligopeptide from Cyanea nozaki that decreases blood urea nitrogen and lactic acid content (Dan, Qi, & Chao, 2010), and similarly including soybean peptide or other edible protein. Anxiety is related to the nervous system. For anxiolytic research, mental stress and liferelated disease are usually modeled together. A peptide derived from the β subunit of soy

Applications in nutrition: clinical nutrition 509 β-conglycinin was reported to display anxiolytic activity and interfere with glucose and lipid metabolism (Ohinata, Muraki, & Oie, 2010). The tripeptide GHK was also reported to exhibit anxiolytic activity (Bobyntsev, Chernysheva, & Dolgintsev, 2015) by weakening of neurotropic effects. The pentapeptide ovolin, VYLPR, released from natural ovalbumin was also reported to act as an orally active anxiolytic (Oda, Kaneko, & Mizushige, 2012). Anti-inflammatory peptides have also attracted substantial attention (Sato, Ono, & Suzuki, 2011). Anti-inflammatory peptides have also been derived from living organisms or their products, such as bee venom (Billingham, Morley, & Hanson, 1973) and sponges (Festa, Marino, & Sepe, 2009). These peptides function by downregulating inflammatory factors. Some anti-inflammatory peptides are listed in Table 20.2. Several peptides have been proved to have anti-inflammatory activity by cell and animal experiments. According to these experimental results, they may be potential peptides for clinical application. Nutritional intervention is widely accepted as a safe and effective approach for improving or maintaining the health of sub-healthy or sick people. Bioactive peptides and chemically synthesized small-molecule therapeutics both offer distinct advantages for improving physiological functions, while recent advances concerning the role of food-derived biomolecules in health promotion have indicated that bioactive peptides may be superior for clinical nutrition. In particular, bioactive peptides also have higher target specificity, wider bioactivity spectrum, lower toxicity, and reduced mass accumulation in bodily systems. Furthermore, bioactive peptides generated from edible proteins offer a more natural alternative for disease prevention and management, an approach that is gaining increasing prominence among health-conscious consumers. Table 20.2: Some anti-inflammatory peptides. Peptide sequence

Chemical mass

Reference

KIPYIL RRPYIL ELYENKPRRPYIL HDMNKVLDL

745.9410 816.9830 1690.9400 1084.2490

DSDPR DTEAR KGHYAERVG

588.5630 590.5770 1016.1060

SKWQHQQDSCRKQLQGVNLTPCEK HIMEKIQGRGDDDDDDDDD IPP VPP

5062.0900

Wei and Thomas (1993) Wei and Thomas (1993) Wei and Thomas (1993) Miele, Cordella-Miele, Fachiano, and Mukherjee (1988) Hamburger (1975) Hamburger (1975) Brodsky, Erlanger-Rosengarten, Proscura, Shapira, and Wormser (2008) Gonzalez de Mejia and Dia (2009)

325.3940 311.3670

Chakrabarti and Wu (2015) Chakrabarti and Wu (2015)

510 Chapter 20

20.3 Application of biologically active peptides in clinical nutritional foods 20.3.1 Determination of proportions of biologically active peptides in products with specific nutritional requirements 20.3.1.1 Characteristics of clinical nitrogen supplementation products Protein metabolism in the body is usually measured clinically via the nitrogen balance status. Different levels of stress require different amounts of nitrogen; patients with no stress or mild stress require 0.15 g nitrogen/(kg  d), those with moderate stress require 0.2 g nitrogen/(kg  d), and those with severe stress require 0.3 g of nitrogen/(kg  d) (Fujisaki, Tashiro, & Mashima, 1992). At present, the main nitrogen supplementation products on the market are based on proteins, peptides, amino acids, and other components. Proteins typically exert a strong stimulatory effect on mucous membranes and should be administered to patients with adequate digestion and absorption functions. In addition, macromolecular substances can stimulate the growth of the intestinal mucosa to prevent atrophy. Peptides and amino acids exhibit excellent water solubility. Furthermore, peptides, especially short peptides, possess the characteristics of easy absorption, rich physiological functions, a lower incidence of adverse gastrointestinal reactions, and significant improvement on the nutritional level of proteins. People with gastrointestinal dysfunction, such as pancreatitis, short bowel syndrome, inflammatory bowel disease, and other conditions, can use preparations containing a peptide-type nitrogen source. 20.3.1.2 Nitrogen intake requirements for different patients In a 1985 report, the FAO/WHO/UNU recommended that an essential amino acid content of 40% (relative to total amino acids) was beneficial for obtaining a good nitrogen balance in the body (FAO/WHO/UNU, 1985); the optimal E/T (amino acid vs. total nitrogen) ratio of 2.8 is almost equivalent to human protein, which should be beneficial for protein synthesis. According to currently accepted best practices (Demira˘g, Uyar, & Gu¨lbahar, 2012; Guilhermet & Cochard, 1998), a nitrogen supplementation product should contain moderate amounts of glutamine (Gln) and arginine (Arg) and a small amount of glycine (Gly). Studies have demonstrated that glutamine is the most abundant non-essential amino acid in the human body and plays important roles in improving nitrogen balance, promoting intestinal mucosa and pancreas growth, preventing fatty liver lesions, and increasing skeletal muscle protein synthesis. As glutamine is relatively unstable and readily decomposes to products such as ammonia and pyroglutamic acid, it is generally added to nitrogen supplementation products in the form of dipeptides such as AQ and GQ; however, glutamine-containing products are not suitable for patients with kidney, liver, or central nervous system dysfunction (Demira˘g et al., 2012). Arginine plays an important role in

Applications in nutrition: clinical nutrition 511 treating postoperative trauma, burns, and sepsis as it can enhance immune function, promote wound healing, and act as a secretagogue for certain hormones (Guilhermet & Cochard, 1998). A high glycine content can lead to hyperammonemia and subsequent brain injury, especially in patients with severe liver dysfunction. Consequently, it is generally not appropriate to use nitrogen supplementation products containing large amounts of glycine and ammonium ions (Kristiansen, Rose, & Ytreb, 2016). Patients with hepatic encephalopathy should be supplemented with abundant branched-chain amino acids to promote a stable ratio of branched-chain amino acids and aromatic amino acids, for which plant-derived protein sources are more suitable than animal-derived ones (Kawaguchi, Taniguchi, & Sata, 2013). Patients with renal dysfunction must be supplemented with rich essential amino acids and reduce their consumption of non-essential amino acids. 20.3.1.3 Design requirements for clinical biologically active peptide products Clinicians and clinical dietitians must implement personalized solutions for different patients with specific nitrogen intake requirements. Therefore, the manufacturers of nitrogen supplementation products must strictly manage and evaluate the form, specifications, and purity and establish strict standards. In addition, the product composition must be specified in detail, including the total nitrogen content, peptide content, free amino acid content, nature of the identified peptides, and other parameters (Zhu, LI, & Peng, 2010). For example, the main components of the short-peptide-based enteral nutrient Peptisorb produced by the German company Milupa GmbH are hydrolyzed whey protein, carbohydrates, fats, minerals, vitamins, and taurine, which are clearly specified in the product description. Clinicians and clinical nutritionists should devise the appropriate treatment strategy on the basis of the general treatment plan combined with the nitrogen intake requirements of each patient and the composition information of the nitrogen supplementation product to develop the optimal personalized adjuvant treatment plan for each patient (Xian-Ju, Shi-Yu, & Chun-Li, 2017).

20.3.2 Source selection of biologically active peptides in products for patients with specific health needs The nutritional needs of patients are a top priority when preparing clinical nutritional foods. The form and source of the nutritional supply may need to be modified depending on the nutritional requirements and health status of the patient. In general, essential nutrients should be present in sufficient amounts for daily consumption. Nutrients should also be provided in easily digestible forms, such as peptides and fatty acids. In this subsection, the selection of peptides will be discussed. A diverse range of sources, including plants, animals, and microbes, have been investigated and developed for the manufacture of bioactive peptides. Most of these bioactive peptides

512 Chapter 20 can be used in normal food as a main ingredient without restriction. However, in clinical nutritional food, the available sources of raw materials are limited. First, safety is a primary concern when designing recipes. Raw materials that are supported by many studies and proteins with a well-defined structure and composition are preferred. For instance, soy, wheat, corn, and rice, which are widely grown and used in the food industry, are considered safer than other sources such as insects, wild animals, and traditional herbs. Second, the amino acid profile of the protein source should meet the daily requirements of patients, especially in terms of essential amino acids. To meet the daily requirements of patients, more than one source of bioactive peptides can be used in a recipe. However, the combination of protein sources should be simple and the raw materials should be traceable. Third, the flavor, stability, and product form are also important. Palatable products that are easy to chew and swallow are preferred by patients, especially those with a low appetite. Finally, the personal needs or preferences of the patients should also be considered. For example, it is desirable to know whether the bioactive peptides are derived from kosher/ halal sources and whether the food is suitable for vegetarian patients. In addition to these four principles, some studies have indicated that certain bioactive peptides are beneficial for the recovery of patients with specific health needs. Diabetics and patients with blood glucose control disorders could have impaired insulin synthesis and should avoid foods with a high glycemic index, which means that the intake of foods rich in monosaccharides, disaccharides, and starch must be controlled. In these circumstances, protein-based foods are a good choice for energy supply. In addition to a lower glycemic index, protein-based foods help to promote satiety. Numerous studies have demonstrated that bioactive peptides derived from whey protein or marine fish are beneficial for lowering blood glucose. The underlying mechanism is that bioactive peptides with a specific amino acid composition and sequence after hydrolysis act as DPP-IV inhibitors. Many bioactive peptides prepared from industrialized proteins serve as ACE inhibitors and have been applied in functional foods and clinical nutritional foods to lower blood pressure. Compared with non-peptidomimetic inhibitors, such as captopril and ilapril, food-derived bioactive peptides typically exhibit high safety and no obvious side effects. The most common sources of these bioactive peptides that may assist in lowering blood pressure are soy and casein. These two sources are widely used as raw materials in the food industry. Owing to the large market size, stable quality, low price, and easy availability of these two protein sources, they are good sources of protein for clinical nutritional foods. Furthermore, the amino acid compositions of casein and soy meet the daily needs of the human body and are complementary to some degree. This is also in line with the four principles of raw material selection outlined above. Patients with malignant tumors often experience side effects such as loss of appetite and weakened digestive function due to radiotherapy and chemotherapy. In clinical treatment,

Applications in nutrition: clinical nutrition 513 patients are generally encouraged to eat actively and less frequently if permitted by the patient’s digestive system. In this case, easily digestible and safe bioactive peptides are a good choice for patients to meet their protein needs. In some studies, marine fish skin oligopeptides were used as part or as full nitrogen source in patients undergoing chemotherapy for malignant tumors, which led to certain nutritional improvements in visceral proteins and anthropometries. For patients with liver disease, limited liver function leads to a diminished ability to metabolize aromatic amino acids and therefore an increase in the bodily content of these amino acids, while the content of branched-chain amino acids decreases. In severe cases, hepatic encephalopathy may occur. For such patients, the ratio of branched-chain amino acids to aromatic amino acids in the protein composition (Fischer value) should be noted during protein supplementation. Among large-scale industrially produced proteins, whey protein and corn protein contain relatively high contents of branched-chain amino acids. Therefore, these two proteins are often used as raw materials to prepare clinical nutritional foods via varying degrees of hydrolysis. Proteins present in some foods, such as peanut, gluten, and shellfish proteins, can act as allergens. Owing to such allergies, many patients have insufficient intake of protein or certain essential amino acids. Peptides, especially oligopeptides, can be an alternative choice for these patients as the enzymatic hydrolysis of allergenic proteins often significantly decreases their allergenicity. When selecting clinical nutritional foods for such patients, some safe and widely used heavily hydrolyzed oligopeptides can be specifically selected. In some clinical trials, soy peptides, pea peptides, and wheat peptides were used for nutrition support. For patients with chronic kidney disease, the administration of high-quality and bioavailable protein sources to support protein synthesis, prevent protein breakdown, and alleviate azotemia is the key to dietary support. Hence, highly utilizable proteins should be primarily considered. In general, the digestibility of animal-derived foods, such as egg protein, milk protein (especially whey protein), and fish protein, is higher than that of plant-derived foods. Osteoporosis due to aging, hormonal changes, and drug side effects is a health problem that plagues many patients. Osteoporosis is thought to be associated with the loss of calcium from bones, and this loss generally cannot be mitigated by medical intervention. Increasing calcium intake provides an alternative way to treat osteoporosis. Among various types of bioactive peptides, casein phosphopeptides obtained from the hydrolysis of casein have proved to be efficient in cations transmit. Complexation by these peptides ensures that the calcium is maintained in a soluble form and does not undergo precipitation as calcium phosphate. In addition to the use of casein phosphopeptides, the treatment of osteoporosis via clinical nutritional foods requires supplementation with a sufficient amount of vitamin D, while too much oxalic acid, phytic acid, and other resistance factors should be avoided.

514 Chapter 20 To summarize, although numerous food-derived bioactive peptides exhibit beneficial physiological functions, their main role in clinical applications is to provide energy and nutrition. The primary concern in the selection of bioactive peptides should always be safety, followed by function. A well-designed clinical nutritional food recipe can lead to improved patient outcomes with less medical treatment.

20.3.3 Product forms As products designed to assist patient recovery, clinical nutritional foods are still mainly in the form of mainstream food processing. Therefore, clinical nutritional foods are typically supplied as liquids or solids. As clinical nutritional foods are often used in patients with reduced immunity, product safety, including microbial safety and the absence of contaminants, is a major consideration. Microbial safety is mainly achieved via adequate sterilization during manufacture and the use of sealed packaging. The absence of contaminants must be ensured by adequate screening and inspection of packaging materials. In this context, the typical forms of clinical nutritional foods are discussed below, and some typical clinical food products are listed in Table 20.3. Powders are one of the most common dosage forms for clinical nutritional foods. In general, powders can be prepared via two methods, namely, powder mixing or wet mixing and drying. The powder is dissolved prior to use, and the packaging is typically individual depending on the dosage. Powder formulations have the advantages of good stability and easy storage and transportation, assuming that moisture absorption and high temperatures can be avoided. Enteral nutritional emulsions or suspensions are suitable for patients with complete or partial gastrointestinal function who cannot or are unwilling to eat a sufficient quantity of conventional food to meet the nutritional demands of the body. These products are typically bottled separately, strictly sterilized during manufacture, and contain a specified number of calories and balanced nutrients, which gives medical workers the ability to precisely control the nutritional intake of the patient. However, because these products usually contain both oil-phase and water-phase components, they can exhibit poorer stability than solid or beverage products containing only a single phase. Beverage products are used to supplement vitamins, minerals, and soluble fiber. These products typically contain a specified number of calories and possess good flavor and palatability. In general, one bottle is sufficient to meet the daily nutritional needs of a patient. Semi-solid products in gel or pulp form are usually packaged individually in bottles or soft packs and can provide increased satiety compared to beverages and enteral nutritional

Applications in nutrition: clinical nutrition 515 Table 20.3: Selected clinical nutritional food products. Product name

Product form

Target patients

Peptide or protein source

EleCare Nutrition Powder (Abbott)

Powder

Children under 13 who cannot tolerate milk, soy, or protein hydrolysate formulas

None, all protein nutrient was presented in amino acids

Leskon Jiaying Nutritional Fluid Food (Xian Libang Clinical Nutrition)

Enteral nutritional suspension

People unable to eat normally or who must control nutritional intake

Milk (casein and whey)

Dongzeping 400 Kcal protein functional beverage (Daisy FSMP)

Beverage

People in need of nutrition

Milk (casein and whey)

Dongzeneng nutritional powder (BCAA type, Daisy FSMP)

Powder

Ensure nutritional powder (Abbott)

Powder

People over 10 with restricted eating, digestive, absorption, or metabolic disorders

Casein protein isolate Whey protein isolate Soy protein isolate

Peptamen Junior (Nestle)

Powder

Children with gastrointestinal discomfort and feeding difficulties due to various causes

Hydrolyzed whey protein

Product packaging

Soy peptide

(Continued)

516 Chapter 20 Table 20.3: (Continued) Target patients

Peptide or protein source

Powder

Children with refractory epilepsy

Milk (casein and whey)

Ruineng Enteral Nutrition Emulsion (Huarui SINO-SWED Pharmaceutical)

Enteral nutritional emulsion

Cancer patients

Proteins from various sources

Leskon High BCAA Complete Nutrition Powder (Xian Libang Clinical Nutrition)

Powder

Patients with liver disease

Collagen peptide

Product name Ketocal ketogenic formula powder (Nutricia)

Product form

Product packaging

suspensions. These products are suitable for patients with impaired chewing ability or the need to control energy intake. In summary, clinical nutritional foods are usually packaged individually, taking safety and convenience into consideration, and with the dosage, method of use, storage conditions, and applicable population clearly indicated. These products may be supplied as solids (powders), liquids (enteral nutritional suspensions and nutritional beverages), or semi-solids (gels and pulps).

20.4 Summary and prospects In recent years, biologically active peptides and their roles in clinical nutrition and disease have attracted increasing research attention. This chapter has focused on the

Applications in nutrition: clinical nutrition 517 applications of biologically active peptides in clinical nutrition support and treatment, the treatment of CVDs, tumors, liver injury, diabetes, and other conditions, and clinical nutritional food. The continued discovery of biologically active peptides and further investigation of their structure, function, and mechanism are expected to be of great significance to both theoretical research and clinical practice. Biologically active peptides have become a very popular research topic with great development prospects in the global nutrition and food industry. To date, nutritional foods containing short peptides have been investigated for the supportive care of patients suffering from cancer, liver disease, kidney disease, postoperative complications, diabetes, and other conditions. These nutritional foods typically rely on the use of food-derived oligopeptides as the nitrogen source, exploiting the synergistic effect of biologically active peptides and phytochemicals such as polysaccharides, to improve the condition of patients. With the continued research into biologically active peptides, the application of food-derived oligopeptides in clinical therapeutic foods is expected to become increasingly common, leading to a corresponding improvement in human health. Although most research into bioactive peptides remains at the stage of basic research or preliminary clinical research, the relationship between bioactive peptides and clinical nutrition or disease is attracting increasing attention. With the continuous improvement in research methods, especially the rapid development of protein engineering and enzyme engineering technologies, research into bioactive peptides, such as those displaying immunomodulatory, anticancer, or insulin-like activities, is expected to lead to the discovery of more biologically active peptides with special functions and the ability to alleviate the suffering of clinical patients and improve clinical outcomes. In the future, gene engineering, DNA recombination, and other technologies may be used to integrate genes expressing biologically active peptides into microorganisms or animals to permit direct expression of the desired peptide, which could improve the output and reduce the cost of treatment, while also playing a decisive role in the promotion of enteral or parenteral nutrition. In addition, because peptides are very susceptible to enzymatic hydrolysis in vivo and typically possess a short half-life, one possible research direction is the development of peptide analog drugs amenable to oral administration with a longer duration of action in vivo. In the future, research into the clinical application of biologically active peptides is expected to focus on the continued discovery and identification of new biologically active peptides, in-depth analysis of their physiological function, release mechanism, receptor binding, degradation, and deactivation, structure
activity relationships, and drug development and applications. Consequently, biologically active peptides are expected to make substantial contributions to human health in numerous ways, such as in health foods, drugs for disease treatment, and treatment regimens.

518 Chapter 20

References Agyei, D., Pan, S., Acquah, C., & Danquah, M. K. (2017). Bioactivity profiling of peptides from food proteins. Soft Chemistry and Food Fermentation (pp. 49
77). Academic Press. American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.) Board of Directors. (2009). Clinical guidelines for the use of parenteral and enteral nutrition in adult and pediatric patients. Journal of Parenteral & Enteral Nutrition, 33(3), 255
259. Atul, T., Abhishek, T., Priya, A., et al. (2015). CancerPPD: A database of anticancer peptides and proteins. Nucleic Acids Research, 43, D837
D843. Bhat, Z. (2015). Bioactive peptides of animal origin: A review. Journal of Food Science and Technology, 52(9), 5377
5392. Billingham, M. E. J., Morley, J., Hanson, J. M., et al. (1973). An anti-inflammatory peptide from bee venom. Nature, 245(5421), 163
164. Bobyntsev, I. I., Chernysheva, O. I., Dolgintsev, M. E., et al. (2015). Anxiolytic effects of Gly-His-Lys peptide and its analogs. Bulletin of Experimental Biology and Medicine, 158(6), 726
728. Brodsky, B., Erlanger-Rosengarten, A., Proscura, E., Shapira, E., & Wormser, U. (2008). From topical antidote against skin irritants to a novel counter-irritating and antiinflammatory peptide. Toxicology and Applied Pharmacology, 229, 342
350. Brugler, L., & Bernstein, L. (1998). An acuity based clinical nutrition staffing model improves acute care clinical effectiveness and maintains program viability in a managed care environment. Journal of the American Dietetic Association, 98(9-supp-S), A23. Butteiger, D. N., Hibberd, A. A., Mcgraw, N. J., et al. (2016). Soy protein compared with milk protein in a western diet increases gut microbial diversity and reduces serum lipids in golden syrian hamsters. Journal of Nutrition, 146(4), 697
705. Chabance, B., Jolle`s, P., Izquierdo, C., et al. (1995). Characterization of an antithrombotic peptide from α-casein in newborn plasma after milk ingestion. British Journal of Nutrition, 73(4), 583
590. Chakrabarti, S., & Wu, J. (2015). Milk-derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) promote adipocyte differentiation and inhibit inflammation in 3T3-F442A cells. PLoS ONE, 10, Article No e0117492. Christensen, M., Filip-K, K., Vilsbøll, T., et al. (2011). Lixisenatide for type 2 diabetes mellitus. Expert opinion on investigational drugs, 20(4), 549
557. Dan, L., Qi, W., Chao, D., et al. (2010). Study on the anti-fatigue effect of collagen peptide and oligopeptide from Cyanea nozakii. Natural Product Research & Development, 22(1), 113
116. Delegge, M. H., & True Kelley, A. (2013). State of nutrition support teams. Nutrition in Clinical Practice, 28(6), 691
697. Demira˘g, K., Uyar, M., Gu¨lbahar, O., et al. (2012). The effect of parenteral glutamine supplementation on the immune system and nitrogen balance in critically ill patients receiving glutamine enriched enteral nutrition[J]. egetipdergisi.com.tr. Demuth, H. U., McIntosh, C. H., & Pederson, R. A. (2005). Type 2 diabetes—Therapy with dipeptidyl peptidase IV inhibitors[J]. BBA - Proteins and Proteomics, 1751(1), 33
44. FAO/WHO/UNU (1985). Energy and protein requirements. Report of a joint FAO/WHO/UNU expert consultation. World Health Organization technical report series, 1
206. Ennaas, N., Hammami, R., Beaulieu, L., & Fliss, I. (2015). Purification and characterization of four antibacterial peptides from protamex hydrolysate of Atlantic mackerel (Scomber scombrus) by-products. Biochemical and Biophysical Research Communications, 462(3), 195
200. Erdmann, K., Cheung, B. W. Y., & Schro¨der, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease[J]. The Journal of Nutritional Biochemistry, 19(10), 643
654. Fan, J. F., Hu, et al. (2009). Isolation and characterisation of a novel angiotensin I-converting enzyme-inhibitory peptide derived from douchi, a traditional Chinese fermented soybean food[J]. Journal of the Science of Food and Agriculture, 89(4), 603
608.

Applications in nutrition: clinical nutrition 519 Fei, W., Shengnan, Y., liang, L., et al. (2011). Preparation of soybean antihypertensive peptide and anti-cancer peptide [J]. China Oils and Fats, 36(7), 58
61. Fei, W., Zhao, Y., Liu, Y., et al. (2017). Peptides from Antarctic krill (Euphausia superba) ameliorate senile osteoporosis via activating osteogenesis related BMP2/Smads and Wnt/β-catenin pathway. Journal of Food Biochemistry, 41(4), E12381. ´ . A., Givens, D. I., & Lovegrove, J. A. (2015). Casein-derived lactotripeptides reduce systolic and Fekete, A diastolic blood pressure in a meta-analysis of randomised clinical trials. Nutrients, 7(1), 659
681. Festa, C., Marino, S. D., Sepe, V., et al. (2009). Perthamides C and D, two new potent anti-inflammatory cyclopeptides from a Solomon Lithistid sponge Theonella swinhoei[J]. Tetrahedron, 65(50), 10424
10429. FitzGerald, R. J., Murray, B. A., & Walsh, D. J. (2004). Hypotensive peptides from milk proteins[J]. The Journal of Nutrition, 134(4), 980S
988S. Fu, Y., Young, J. F., Rasmussen, M. K., et al. (2016). Angiotensin I-converting enzyme
inhibitory peptides from bovine collagen: insights into inhibitory mechanism and transepithelial transport[J]. Food Research International, 89(1), 373
381. Fujisaki, Y., Tashiro, T., Mashima, Y., et al. (1992). Adequate requirements of energy and protein in postoperative total parenteral nutrition[J]. Nihon Geka Gakkai zasshi, 93(2), 119
127. Gauthier, S. F., Pouliot, Y., & Saint-Sauveur, D. (2006). Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins[J]. International Dairy Journal, 16(11), 0
1323. Gonzalez de Mejia, E., & Dia, V. P. (2009). Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-κB pathway in the macrophage. Peptides, 30, 2388
2398. Grundy, S. M. (1999). Primary prevention of coronary heart disease: Integrating risk assessment with intervention[J]. Circulation, 100(9), 988
998. Guilhermet, R. G., & Cochard, A. (1998). Arginine: Wound healing, immune and endocrine functions[J]. Cahiers De Nutrition Et De Die´te´tique, 33(1), 19
24. Hajar, R. (2016). Framingham contribution to cardiovascular disease[J]. Heart views: the official journal of the Gulf Heart Association, 17(2), 78
81. Hamburger, R. N. (1975). Peptide inhibition of Prausnitz-Ku¨stner reaction. Science, 189, 389
390. Han, Y., Xie, J., Gao, H., et al. (2015). Hepatoprotective effect of collagen peptides from cod skin against liver oxidative damage in vitro and in vivo[J]. Cell Biochemistry and Biophysics, 71(2), 95
1089. Harada, K., Maeda, T., Hasegawa, Y., Tokunaga, T., Tamura, Y., & Koizumi, T. (2010). Antioxidant activity of fish sauces including puffer (Lagocephalus wheeleri) fish sauce measured by the oxygen radical absorbance capacity method. Molecular Medicine Reports, 3(4), 663
668. Hernandez-Ledesma, B., Hsieh, C. C., & de Lumen, B. O. (2013). Chemopreventive properties of peptide lunasin: A review[J]. Protein & Peptide Letters, 20(4), 424
432. Uenishi, H., Kabuki, T., Seto, Y., et al. (2011). Isolation and identification of casein-derived dipeptidylpeptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats[J]. International Dairy Journal, 22(1), 24
30. Hitotsuyanagi, Y., Ishikawa, H., Hasuda, T., et al. (2004). Isolation, structural elucidation, and synthesis of RAXVII, a novel bicyclic hexapeptide from Rubia cordifolia, and the effect of side chain at residue 1 upon the conformation and cytotoxic activity[J]. Tetrahedron Letters, 45(5), 935
938. Hori, G., Wang, M. F., Chan, Y. C., et al. (2001). Soy protein hydrolyzate with bound phospholipids reduces serum cholesterol levels in hypercholesterolemic adult male volunteers[J]. Bioscience, Biotechnology, and Biochemistry, 65(1), 72
78. Hsieh, P. W., Chang, F. R., Wu, C. C., et al. (2005). Longicalycinin A, a new cytotoxic cyclic peptide from Dianthus superbus var. longicalycinus (Maxim.) Will[J]. Chemical & Pharmaceutical Bulletin, 53(37), 336
338. Hui, G., Jie, S., Hui, H., et al. (2009). Antihepatotoxic effect of corn peptides against Bacillus Calmette-Guerin/ lipopolysaccharide-induced liver injury in mice[J]. Food and Chemical Toxicology, 47(10), 2431
2435. Iritani, N., Nagashima, K., Fukuda, H., et al. (1986). Effects of dietary proteins on lipogenic enzymes in rat liver[J]. The Journal of Nutrition, 116(2), 190
197.

520 Chapter 20 Iwasaki, T., & Ohyanagi, H. (2004). Advance and perspective of clinical nutrition[J]. Nippon Geka Gakkai Zasshi, 105(2), 196
199. Jia, Z. (2012). An in vitro preliminary study of the radio-protective properties of antioxidant peptide SS31[J]. Journal of Radiation Research and Radiation Processing, 30(5), 291
296. Jolle`s, P., Le´vy-Toledano, S., Fiat, A. M., et al. (1986). Analogy between fibrinogen and casein: Effect of an undecapeptide isolated from k-casein on platelet function[J]. European Journal of Biochemistry, 158(2), 379
382. Kagawa, K., Matsutaka, H., Fukuhama, C., et al. (1996). Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect[J]. Life Sciences, 58(20), 1745
1755. Kang, K.-H., Qian, Z.-J., Ryu, B., et al. (2012). Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells[J]. Food chemistry, 132(2), 677
685. Kannan, A., Hettiarachchy, N. S., Lay, J. O., et al. (2010). Human cancer cell proliferation inhibition by a pentapeptide isolated and characterized from rice bran[J]. Peptides, 31(9), 1629
1634. Kawaguchi, T., Taniguchi, E., & Sata, M. (2013). Effects of oral branched-chain amino acids on hepatic encephalopathy and outcome in patients with liver cirrhosis. Nutrition in Clinical Practice, 28(5), 580
588. Kim, E. K., Joung, H. J., Kim, Y. S., et al. (2012). Purification of a novel anticancer peptide from enzymatic hydrolysate of Mytilus coruscus[J]. Journal of Microbiology and Biotechnology, 22(10), 1381
1387. Kim, Y.-M., Kim, I.-H., Choi, J.-W., et al. (2015). The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/β-catenin signaling pathway[J]. International Journal of Molecular Medicine, 36(2), 327
334. Korhonen, H., & Pihlanto, A. (2003). Food-derived bioactive peptides-opportunities for designing future foods[J]. Current Pharmaceutical Design, 9(16), 1297
1308. Kreider, R.-B., Iosia, M., Cooke, M., et al. (2011). Bioactive properties and clinical safety of a novel milk protein peptide[J]. BioMed Central, 10(99), 1
8. Kristiansen, R. G., Rose, C. F., & Ytreb, L. M. (2016). Glycine and hyperammonemia: Potential target for the treatment of hepatic encephalopathy(Article)[J]. Metabolic Brain Disease, 31(6), 1269
1273. Kwekkeboom, D., Krenning, E. P., & de Jong, M. (2000). Peptide receptor imaging and therapy[J]. Journal of Nuclear Medicine, 41(10), 1704
1713. Kwon, D. Y., Hong, S. M., Ahn, I. S., Kim, M. J., Yang, H. J., & Park, S. (2011). Isoflavonoids and peptides from meju, long-term fermented soybeans, increase insulin sensitivity and exert insulinotropic effects in vitro. Nutrition, 27(2), 244
252. Lacroix, I.-M. E., & Li-chan, E.-C. Y. (2014). Isolation and characterization of peptides with dipeptidyl peptidase-IV inhibitory activity from pepsin-treated bovine whey proteins[J]. Peptides, 54(1), 39
48. Lacroix, I.-M. E., Meng, G., Cheung, I.-W. Y., et al. (2016). Do whey protein-derived peptides have dual dipeptidyl-peptidase IV and angiotensin I-converting enzyme inhibitory activities[J]. Journal of Functional Foods, 21(1), 87
96. Lammi, C., Zanoni, C., Scigliuolo, G. M., et al. (2014). Lupin peptides lower low-density lipoprotein (LDL) cholesterol through an up-regulation of the LDL receptor/sterol regulatory element binding protein 2 (SREBP2) pathway at HepG2 cell line[J]. Journal of Agricultural & Food Chemistry, 62(29), 7151
7159. Lau, J. L., & Dunn, M. K. (2017). Therapeutic peptides: Historical perspectives, current development trends, and future directions[J]. Bioorganic & Medicinal Chemistry, 26, 2700
2707. Le maux, S., Nongonierma, A.-B., Murray, B., et al. (2015). Identification of short peptide sequences in the nanofiltration permeate of abioactive whey protein hydrolysate[J]. Food Research International, 77(1), 534
539. Leung, T.-m, & Nieto, N. (2013). CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease[J]. Journal of Hepatology, 58(2), 8
395.

Applications in nutrition: clinical nutrition 521 Li, H.-m, Guo, P., Hu, X., et al. (2007). Preparation of corn (Zea mays) peptides and their protective effect against alcohol-induced acute hepatic injury in NH mice.[J]. Biotechnology and Applied Biochemistry, 47(3), 74
169. Li, T., Wang, C. X., Wang, Y., et al. (2012). The radioprotective effects of scorpion venom peptide Ⅱ on mice hematopoietic cells[J]. Guangdong Medical Journal, 033(22), 3360
3363. Linnebjerg, H., Kothare, P. A., Park, S., et al. (2007). Effect of renal impairment on the pharmacokinetics of exenatide[J]. British Journal of Clinical Pharmacology, 64(3), 317
327. Lund, A., Knop, F. K., & Vilsbøll, T. (2014). Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes: Differences and similarities[J]. European Journal of Internal Medicine, 25(5), 14
407. Lv, J., Nie, Z.-k, Zhang, J.-l, et al. (2013). Corn peptides protect against thioacetamide-induced hepatic fibrosis in rats. Journal of Medicinal Food, 16(10), 9
912. Ma, Z.-l, Hou, T., Shi, W., Liu, W.-W., Ibrahim, S. A., & He, H. (2016). Purification and identification of corn peptides that facilitate alcohol metabolism by semi-preparative high-performance liquid chromatography and nano liquid chromatography with electrospray ionization tandem mass spectrometry[J]. Journal of separation science, 39(1), 4234
4242. Ma, Z.-l, Zhang, W.-J., Yu, G.-C., He, H., & Zhang, Y. (2012). The primary structure identification of a corn peptide facilitating alcohol metabolism by HPLC
MS/MS[J]. Peptides, 37(1), 138
143. Magoichi, Y., Fumi, N., Michiko, I., et al. (1997). The effects of corn peptide ingestion on facilitating alcohol metabolism in healthy men. Japan Society for Bioscience, Biotechnology, and Agrochemistry, 61(9), 1474
1481. Uchida, M., Ohshiba, Y., & Mogami, O. (2011). Novel dipeptidyl peptidase-4
inhibiting peptide derived from β-lactoglobulin. The Japanese Pharmacological Society, 117(1), 63
66. Mcintosh, C. H. S., Demuth, H.-U., Kim, S.-J., et al. (2005). Applications of dipeptidyl peptidase IV inhibitors in diabetes mellitus. International Journal of Biochemistry and Cell Biology, 38(5), 860
872. Meisel, H. (1997). Biochemical properties of regulatory peptides derived from mil proteins[J]. Peptide Science, 43(2), 119
128. Meisel, H., & FitzGerald, R. J. (2003). Biofunctional peptides from milk proteins: Mineral binding and cytomodulatory effects[J]. Current Pharmaceutical Design, 9(16), 1289
1295. Mellander, O. (1951). The physiological importance of the casein phosphopeptide calcium salts. II. Peroral calcium dosage of infants[J]. Acta Societatis Medicorum Upsaliensis, 55(5
6), 247
255. Miele, L., Cordella-Miele, E., Fachiano, A., & Mukherjee, A. B. (1988). Novel anti inflammatory peptides from region of high similarity between uteroglobin and lipocortin I. Nature, 335, 726
730. ´ ., & Menjivar, M. (2017). Evaluation of the hypoglycemic Mojica, L., de Mejia, E. G., Granados-Silvestre, M. A potential of a black bean hydrolyzed protein isolate and its pure peptides using in silico, in vitro and in vivo approaches. Journal of Functional Foods, 31, 274
286. Monteiro, G. A., Fernandes, A. V., Sundararaghavan, H. G., et al. (2011). Positively and negatively modulating cell adhesion to type I collagen via peptide grafting[J]. Tissue engineering. Part A, 17(13
14), 1663
1673. Nai-Rui, H., Li-Zhen, M. A., Run-Ying, Z., et al. (2009). The preventive and therapeutic effect of sheep bone collagen peptide on osteoporosis of ovariectomized rats[J]. Acta Nutrimenta Sinica. Neumiller, J. J., Sonnett, T. E., Wood, L. D., et al. (2010). Pharmacology, efficacy and safety of liraglutide in the management of type 2 diabetes[J]. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 29(12), 1617
1626. Nishizawa, N., Niida, A., Masuda, Y., et al. (2017). Antiobesity effect of a short-length peptide YY analogue after continuous administration in mice[J]. ACS Medicinal Chemistry Letters, 8(6), 628
631. O’Keeffe, M. B., Norris, R., Alashi, M. A., et al. (2017). Peptide identification in a porcine gelatin prolyl endoproteinase hydrolysate with angiotensin converting enzyme (ACE) inhibitory and hypotensive activity[J]. Journal of Functional Foods, 34, 77
88. Oda, A., Kaneko, K., Mizushige, T., et al. (2012). Characterization of ovolin, an orally active tryptic peptide released from ovalbumin with anxiolytic-like activity[J]. Journal of Neurochemistry, 122(2), 356
362.

522 Chapter 20 Ohinata, K., Muraki, A., Oie, M., et al. (2010). Research and application of bioactive peptide derived from soy protein with anxiolytic-like activity. Soy Protein Research Japan, 32(8), 88
94. Okarvi, S. M. (2004). Peptide-based radiopharmaceuticals: Future tools for diagnostic imaging of cancers and other diseases[J]. Medicinal Research Reviews, 24(3), 357
397. O’Sullivan, S. M., O’Callaghan, Y. C., O’Keeffe, M. B., et al. (2015). Potential immunomodulatory effects of casein-derived bioactive peptides in human T cells[J]. Proceedings of The Nutrition Society, 74(OCE1), E107. Pan, D., Cao, J., Guo, H., et al. (2012). Studies on purification and the molecular mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate[J]. Food Chemistry, 130(1), 121
126. Pan, X. C., Liu, Y., Dai, T. T., et al. (2011). Observation on the effect of marine fish collagen oligopeptide on osteoporosis[J]. Journal of Preventive Medicine Information, 27(5), 393
395. Peladic, N. J., Gagliardi, C., Fagnani, D., et al. (2017). Home enteral nutrition therapy: Difficulties, satisfactions and support needs of caregivers assisting older patients[J]. Clinical Nutrition, 36(4), 1062
1067. Pihlanto-Leppa¨la¨, A. (2000). Bioactive peptides derived from bovine whey proteins: Opioid and ace-inhibitory peptides. Trends in Food Science & Technology, 11(9
10), 347
356. Pihlanto-Leppa¨la¨, A. (2002). Milk proteins
bioactive peptides[J]. Encyclopedia of Dairy Sciences, 1960
1967. Piotto, S. P., Sessa, L., Concilio, S., et al. (2012). YADAMP: yet another database of antimicrobial peptides[J]. International Journal of Antimicrobial Agents, 39(4), 346
351. Premratanachai, P., & Chanchao, C. (2014). Review of the anticancer activities of bee products[J]. Asian Pacific Journal of Tropical Biomedicine, 4(5), 337
344. Raslan, M., Gonzalez, M. C., Torrinhas, R. S. M. M., et al. (2011). Complementarity of subjective global assessment (SGA) and nutritional risk screening 2002 (NRS 2002) for predicting poor clinical outcomes in hospitalized patients[J]. Clinical Nutrition, 30(1), 49
53. Rho, S. J., Lee, J. S., Chung, Y. I., et al. (2009). Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract[J]. Process Biochemistry, 44(4), 490
493. Rizzetti, D. A., Martı´n, a´, Corrales, P., et al. (2017). Egg white-derived peptides prevent cardiovascular disorders induced by mercury in rats: Role of angiotensin-converting enzyme (ACE) and NADPH oxidase[J]. Toxicology Letters, 281, 158
174. Rushikesh, S., Pravin, P., & Seetharama, J. (2017). Peptides, peptidomimetics, and polypeptides from marine sources: A wealth of natural sources for pharmaceutical applications[J]. Marine drugs, 15(4), E124. Sato K., Ono S., Suzuki Y. (2011). Anti-inflammatory peptide. United States Patent Application, 20110183925. Sato, K., Yukari, E., Shin, O., et al. (2013). Identification of a hepatoprotective peptide in wheat gluten hydrolysate against d-galactosamine-induced acute hepatitis in rats[J]. Journal of Agricultural and Food Chemistry, 61, 6304
6310. Schweizer, F. (2009). Cationic amphiphilic peptides with cancer-selective toxicity[J]. European Journal of Pharmacology, 625(1
3), 190
194. Shaddinger, B. C., Soffer, J., Vlasakakis, G., et al. (2019). Efficacy and safety of an albiglutide liquid formulation compared with the lyophilized formulation: A 26-week randomized, double-blind, repeat-dose study in patients with type 2 diabetes mellitus[J]. Diabetes Research and Clinical Practice, 152(1), 125
134. Shi, Y., Sun, J., He, H., et al. (2008). Hepatoprotective effects of Ganoderma lucidum peptides against D-galactosamine-induced liver injury in mice. Journal of Ethnopharmacology, 117(3), 9
415. Shin, B. C., Chun, I. A., Ryu, S. Y., et al. (2018). Association between indication for therapy by nutrition support team and nutritional status[J]. Medicine, 97(52), e13932. Silvana, T. S., Martı´nez-Maqueda, D., Recio, I., et al. (2013). Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin[J]. Food Chemistry, 141(2), 1072
1077. Su, L. Y., Xu, G. H., Shen, J., Tuo., et al. (2010). Anticancer bioactive peptide suppresses human gastric cancer growth through modulation of apoptosis and the cell cycle[J]. Oncology Reports, 23(1), 3
9.

Applications in nutrition: clinical nutrition 523 Suarez-Jimenez, G. M., Burgos-Hernandez, A., & Ezquerra-Brauer, J. M. (2012). Bioactive peptides and depsipeptides with anticancer potential: Sources from marine animals[J]. Marine drugs, 10(5), 963
986. Takenaka, Y., UTsUMI, S., & Yoshikawa, M. (2000). Introduction of enterostatin (VPDPR) and a related sequence into soybean proglycinin A1aB1b subunit by site-directed mutagenesis[J]. Bioscience, Biotechnology, and Biochemistry, 64(12), 2731
2733. Tang, W., Zhang, H., Wang, L., Qian, H., & Qi, X. (2015). Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis. Food chemistry, 168, 115
123. Tepkeeva, I. I., Moiseeva, E. V., Chaadaeva, A. V., et al. (2008). Evaluation of antitumor activity of peptide extracts from medicinal plants on the model of transplanted breast cancer in CBRB-Rb (8.17) 1Iem mice[J]. Bulletin of Experimental Biology and Medicine, 145(4), 464
466. Tidona, F., Criscione, A., Guastella, A. M., et al. (2009). Bioactive peptides in dairy products[J]. Anim. Sci, 8(3), 315
340. Tucker, A., Ybarra, J., Bingham, A., et al. (2015). American society for parenteral and enteral nutrition (A.S.P. E.N.) standards of practice for nutrition support pharmacists[J]. Nutrition in Clinical Practice, 30(1), 139
146. Turner, T., Jackson, W. H., Pettit, G. R., et al. (1998). Treatment of human prostate cancer cells with dolastatin 10, a peptide isolated from a marine shell-less mollusc[J]. The Prostate, 34(3), 175
181. Umpierrez, G. E., Blevins, T., Rosenstock, J., et al. (2011). The effects of LY2189265, a long-acting glucagonlike peptide-1 analogue, in a randomized, placebo-controlled, double-blind study of overweight/obese patients with type 2 diabetes: The EGO study[J]. Diabetes, obesity & amp; metabolism, 13(5), 25
418. Vilsbøll, T., Agersø, H., Krarup, T., et al. (2003). Similar elimination rates of glucagon-like peptide-1 in obese type 2 diabetic patients and healthy subjects[J]. The Journal of Clinical Endocrinology & amp; Metabolism, 88(1), 220
224. Wang, J., Hu, J., Cui, J., et al. (2008). Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in spontaneously hypertensive rats[J]. Food chemistry, 111(2), 302
308. Wei, E. T., & Thomas, H. A. (1993). Anti-inflammatory peptide agonists. Annual Review of Pharmacology and Toxicology, 33, 91
108. Wereszczynska-Siemiatkowska, U., Swidnicka-Siergiejko, A., Siemiatkowski, A., et al. (2013). Early enteral nutrition is superior to delayed enteral nutrition for the prevention of infected necrosis and mortality in acute pancreatitis[J]. Pancreas, 42(4), 640
646. Woo, M. J., Song, Y. O., Kang, K. H., et al. (2018). Anti-obesity effects of collagen peptide derived from skate (Raja kenojei) skin through regulation of lipid metabolism[J]. Marine drugs, 16(9), 306
317. Wu, Y., Pan, X., Zhang, S., et al. (2014). Protective effect of corn peptides against alcoholic liver injury in men with chronic alcohol consumption: A randomized double-blind placebo-controlled study[J]. Lipids in Health and Disease, 13(192), 1
9. Xian-Ju, D., Shi-Yu, W., Chun-Li, L. I., et al. (2017). Application of short peptide enteral nutrition in preoperative bowel preparation for patients with gynecological laparoscopy[J]. parenteral & enteral nutrition. Yu, G. -C., Lv, J., He, H., et al. (2012). Hepatoprotective effects of corn peptides against carbon tetrachlorideinduced liver injury in mice. Journal of Food Biochemistry, 36(4), 458
464. Zhang, F., Zhang, J., & Li, Y. (2012). Corn oligopeptides protect against early alcoholic liver injury in rats. Food and Chemical Toxicology, 50(6), 2149
2154. Zhang, T., Yin, Y., Liu, J., et al. (2007). Study on function of anti-fatigue high F ratio oligo-peptide from corn gluten meal and preparation of its beverage. Food Science, 28(5), 308
312. Zhang, X., & Beynen, A. C. (1993). Lowering effect of dietary milk-whey protein v. casein on plasma and liver cholesterol concentrations in rats[J]. British Journal of Nutrition, 70(1), 139
146. Zhang, Y., & Huang, W. X. (2008). Research progress of bone matrix non-collagen protein containing RGD peptides in osteoporosis[J]. Progress in physiological sciences, 39(39), 81
83.

524 Chapter 20 Zhili, M., Tao, H., Wen, S., et al. (2015). Inhibition of hepatocyte apoptosis: An important mechanism of corn peptides attenuating liver injury induced by ethanol. International Journal of Molecular Sciences, 16(9), 22062
22080. Zhu, C.-F., Li, G.-Z., Peng, H.-B., et al. (2010). Effect of marine collagen peptides on markers of metabolic nuclear receptors in type 2 diabetic patients with/without hypertension[J]. Biomedical and Environmental Sciences, 23(2), 113
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CHAPTER 21

Applications in nutrition: sport nutrition ¨nig and C. Centner J. Kohl, S. Jerger, D Ko Department of Sport and Sport Science, University of Freiburg, Freiburg, Germany

21.1 Introduction The importance of nutrition in sports is nowadays well acknowledged and many athletes implement concepts of nutritional science in their daily diet. A balanced nutrition not only helps to maintain health and prevents injuries or illnesses despite intense daily training or competition but also improves athletic performance and delay fatigue (Ja¨ger et al., 2017; Kerksick et al., 2018). In this regard, several systems of the human body can be optimized which ultimately leads to improved muscle and connective tissue function (Stellingwerff, Bovim, & Whitfield, 2019). Therefore muscle recovery and adaptive training responses can be augmented by adequate sports nutrition. The strongest evidence in sports nutrition is found regarding carbohydrate intake in endurance-type sports and protein intake in strength-type sports (Ja¨ger et al., 2017; Kerksick et al., 2018; Thomas, Erdman, & Burke, 2016). These findings are well established in competitive sports and there are several recommendations from expert societies such as the American College of Sports Medicine and the International Society of Sports Nutrition (Ja¨ger et al., 2017; Kerksick et al., 2018). In addition, to improve the interaction between training and nutrition (Marquet, Brisswalter, et al., 2016; Marquet, Hausswirth, et al., 2016; Stellingwerff, Morton, & Burke, 2019), the use of supplements is highly common in athletes (Garthe & Maughan, 2018; Knapik et al., 2016). Many nutritional supplements are being discussed in regards to a performance-enhancing effect (Kerksick et al., 2018; Peeling, Binnie, Goods, Sim, & Burke, 2018); however, the evidence for many substances is weak. In a larger context, findings from sports nutrition research are not only important for competitive athletes but also for subjects with chronic diseases or older individuals who aim for optimizing the health-related effects of exercise. The increase of functional performance and muscle mass, and an improvement of mitochondrial function and connective tissue structures are for example highly relevant for each individual regardless of high-performance sports (Distefano & Goodpaster, 2018; Gan, Fu, Kelly, & Vega, 2018; Kulkarni et al., 2012; Moreira et al., 2017; Svensson, Heinemeier, Couppe, Kjaer, & Magnusson, 2016). For this purpose, nutritional strategies that enhance the stimulus of physical training and thus maintain or even increase physical performance are sought after. Therefore research interest Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00024-8 © 2021 Elsevier Inc. All rights reserved.

525

526 Chapter 21 in bioactive peptides in sports has increased and various numbers of pathways have been investigated so far to elucidate potential mechanisms.

21.2 Rationale The nutrition of an athlete can positively influence physical performance in different ways. Depending on the type of exercise, different nutritional approaches are necessary to satisfy different demands. A very good overview on this topic is given by Stellingwerff et al. (2019), who summarized the different requirements and potentially useful nutritional strategies depending on the running distance of an athlete. In addition, regardless of running distance, the body composition is frequently in the athletes’ focus, especially in endurance, esthetic, and weight-class sports (Sundgot-Borgen & Garthe, 2011). For many athletes, a diet that supports the increase in muscle mass and the decrease in fat mass is desirable (Aragon et al., 2017). Furthermore, recovery after intensive training units or competitions plays an important role in today’s competitive sports (Mohr et al., 2016; Ranchordas, Dawson, & Russell, 2017). An accelerated regeneration from exhausting exercise sessions induced by dietary interventions could potentially enable a better performance and is therefore especially important in sports with limited regeneration periods (McCartney, Desbrow, & Irwin, 2018). Another important aspect with potentially dual effects regarding regeneration and adaptation in sports is the existence or the degree of oxidative stress. While reactive oxygen species (ROS) are important for the induction of adaptation processes on a cellular level, excessive oxidative stress can also hinder and slow down regeneration (Margaritelis, Paschalis, Theodorou, Kyparos, & Nikolaidis, 2018; Merry & Ristow, 2016; Rothschild & Bishop, 2020). Food with antioxidant potential may therefore be beneficial in competitive sports, while excessive antioxidant supplements may not be helpful to improve training-induced adaptive processes (Margaritelis et al., 2018). Nevertheless, not only metabolic improvements or gains in muscle mass are important in sports, structural and functional adaptations in the connective tissue as well as a balanced interaction between the myotendinous complex and the skeletal system are also necessary to withstand increased stress and facilitate human movement. Little is known at present to what extent nutritive factors can positively influence training-induced adaptations in the connective tissue. So far, the benefit and influence of bioactive peptides in these areas have been underestimated, since mostly only the amino acid composition of proteins was considered. However, many aspects that make up the beneficial effects of various fields in sports nutrition could possibly be better explained by improving our knowledge on bioactive peptides with multiple signaling effects (see Fig. 21.1). According to current knowledge, metabolic and structural improvements are possible (reviewed in Chapter 3: Novel Technologies in Bioactive Peptides Production and Stability). The current data indicate that the cellular effects

Applications in nutrition: sport nutrition 527

Figure 21.1 Summary of the potential sport-specific influence of bioactive peptides according to the current state of knowledge.

of strength and endurance training with their signal cascades (e.g., mTOR) offer the possibility of interaction with specific nutrients, for example, proteins, peptides, or amino acids (e.g., leucine) thus influencing the adaptation process (Hoppeler, 2016). To date, several studies have found that both sports performance and tissue repair and recovery might be improved by the presence of specific bioactive peptides. In addition, the majority of available studies investigated the effects of hydrolyzed proteins and not individual bioactive peptides. Much of our knowledge is based on cell culture and animal studies, while studies with known amino acid sequences in vivo are missing. For a better understanding, peptide sequences need to be consistently identified and their effects investigated. To our knowledge, individual peptides have rarely been tested in humans. Instead, human studies have so far only been carried out with protein hydrolyzates, whereby the amino acids or the peptides contained therein can each have different effects. We are just at the beginning to investigate and understand the proportion of the individual amino acids or peptides in a protein hydrolyzate as well as the respective specific effects. In the following section, the current findings regarding the effects of bioactive peptides in sport are summarized. In addition, potential mechanisms of bioactive peptides are identified and possible applications in sports are discussed.

21.3 Application in sports nutrition 21.3.1 Bioactive peptides, body composition, and muscular performance It has been shown that a number of food-derived peptides could potentially influence signaling pathways with regard to body composition, strength, and endurance. An increase in muscle

528 Chapter 21 mass and a reduction in fat mass play an important role in many sports. For the induction of respective changes, anabolic signals are necessary to facilitate muscle hypertrophy (Gonzalez, Hoffman, Stout, Fukuda, & Willoughby, 2016). Especially, the mechanistic target of rapamycin (mTOR) signaling pathway is considered a decisive factor in muscle protein synthesis (Kim, 2017). While it is known that leucine and some other amino acids are potent stimulators of mTOR (Dickinson et al., 2011), the potential influence of smaller peptides is relatively unknown. Previous evidence suggests that the dipeptide hydroxyproline-glycine can activate this signaling pathway in vitro and thereby promote hypertrophy of myotubes (Kitakaze et al., 2016). The fact that the dipeptide is also bioavailable and can be accordingly detected in the blood has been demonstrated in humans after the ingestion of collagen peptides (Sugihara, Inoue, Kuwamori, & Taniguchi, 2012). These results could partly explain the increased fat-free mass produced by collagen peptides compared to a placebo in intervention studies with strength training (Jendricke, Centner, Zdzieblik, Gollhofer, & Ko¨nig, 2019; Zdzieblik, Oesser, Baumstark, Gollhofer, & Ko¨nig, 2015). It should be noted that collagen peptides are considered a low-quality protein due to the incomplete amino acid spectrum. On this basis, a low anabolic effect would be expected with regard to the biological value and low leucine content (Phillips, 2017). Nevertheless, positive effects on the fat-free mass could be observed in several training studies. Collagen peptide supplementation in combination with 12 weeks of resistance training has had a positive effect on the fat-free mass in young men (Kirmse, Oertzen-Hagemann, de Marees, Bloch, & Platen, 2019; Oertzen-Hagemann et al., 2019), elderly sarcopenic men (Zdzieblik et al., 2015), and premenopausal women (Jendricke et al., 2019) compared to placebo. Combined with blood flow restriction training, the intake of collagen hydrolyzate showed a trend toward increasing the cross-sectional area of the thigh muscles measured by magnetic resonance imaging in older men compared to placebo (Centner, Zdzieblik, Roberts, Gollhofer, & Ko¨nig, 2019). In a recent trial from Oikawa et al. (2019) the supplementation with collagen peptides during periods of intensive endurance training in endurance-trained men and women also increased myofibrillar and sarcoplasmic protein synthesis compared to the washout phase in a crossover design, although the effect of lactalbumin was greater. However, the interpretation of the results is difficult due to the lack of an adequate control condition. In contrast to these results, Oikawa et al. (2018) have found that collagen peptides have little or no effect on muscle protein synthesis or lean mass. Elderly people did not benefit from a high protein intake through supplementation with whey protein or collagen peptides during a hypocaloric diet and inactivity. Thus the leg lean mass could not be maintained by either of the two supplements. During the recovery period, only whey protein brought statistically significant on the leg lean mass (Oikawa et al., 2018). The effect on skeletal muscle protein synthesis was also greater with whey protein than with collagen peptides in a study with older women, again without comparison to a placebo (Oikawa et al., 2020).

Applications in nutrition: sport nutrition 529 The positive effect of whey protein on the fat-free mass is generally accepted (Naclerio & Larumbe-Zabala, 2016). The high availability of essential amino acids (EAAs) and especially the high content of leucine are responsible for the anabolic effects (Churchward-Venne et al., 2012; Churchward-Venne et al., 2014; Devries et al., 2018; Dickinson et al., 2011). Nevertheless, peptides in whey hydrolyzates could also play a role in the activation of anabolic signaling pathways. For example, the dipeptide leucine-valine increases the expression of mTOR in rats and could cause anabolic effects similar to leucine as a single amino acid (Moura, Lollo, Morato, Risso, & Amaya-Farfan, 2017). The authors conclude from these data that peptides in addition to the individual amino acids could be coresponsible for positive effects through supplementation with whey hydrolyzate. Due to the positive effect of whey protein on fat-free and muscle mass, the positive influence of bioactive peptides in whey hydrolyzate is difficult to evaluate. Two studies from 2017 found no advantage of whey hydrolyzate over whey protein in combination with a resistance training in young men (Lockwood et al., 2017; Mobley et al., 2017). By means of increasing muscle mass, bioactive peptides could potentially also positively influence muscular strength. That protein supplementation in general can augment the training-induced effects of resistance training on lean mass and muscle strength was shown in a recent metaanalysis by Morton et al. (2018). Bioactive peptides have been shown in some studies to improve both lean mass and muscle strength. Jendricke et al. (2019) observed an increase in hand strength through supplementation with collagen peptides in elderly woman. Zdzieblik et al. (2015) found an increase in the isokinetic quadriceps strength due to collagen peptides compared to placebo. In the study by Centner et al. (2019), no statistically significant advantage of collagen peptides following a 8-week low-load resistance training could be found in relation to the one-repetition maximum (1-RM) of the leg press, even though a positive trend was observed. While the placebo group increased by 4.8% with the same training, the collagen group improved their performance in the 1-RM by 10.2%. Also, Kirmse et al. (2019) and Oertzen-Hagemann et al. (2019) found more pronounced improvements in muscle strength in young men in the collagen group compared to the placebo group, although the differences were not always significant. In summary, there is a promising potential for bioactive peptides in relation to muscle hypertrophy and a resulting increase in strength. Whether protein supplementation brings advantages in terms of endurance performance is currently still controversially discussed. It is even less clear whether bioactive peptides could have a positive effect on endurance performance. From a mechanistic point of view, endurance performance could be improved via several pathways by bioactive peptides. The positive effect of strength training on running economy has already been observed numerous times (Alcaraz-Ibanez & Rodriguez-Perez, 2018; Balsalobre-Fernandez, Santos-Concejero, & Grivas, 2016; Blagrove, Howatson, & Hayes, 2018). Therefore it might be speculated that enhancing the effects of strength training by supplementation with bioactive peptides could

530 Chapter 21 increase the positive influence on the running economy. This relationship, however, needs to be elucidated by future studies. There is limited evidence that, for example, whey hydrolyzate improves performance during endurance testing. Especially in combination with carbohydrates, whey hydrolyzate also seems to offer acute benefits during endurance load. A possible explanation could be an increase in glucose uptake and skeletal muscle glycogen. Filling up the glycogen stores before and after intensive exercise is regarded as one of the decisive factors in all sports with high energy consumption (Hearris, Hammond, Fell, & Morton, 2018). In vitro, the dipeptide isoleucine-valine contained in whey hydrolyzate was identified as a stimulant for glucose uptake (Morifuji, Koga, Kawanaka, & Higuchi, 2009). The positive effect of isoleucine-valine on muscle glycogen content was also confirmed in rats (Moura et al., 2017). A possible explanation could be the observed increase of GLUT-4 transporters in the plasma membrane by increasing the translocation from the cytoplasm in rats (Morato et al., 2013). In mice, an activation of glycogen synthase was also demonstrated (Kanda et al., 2012). The intake of whey hydrolyzate resulted in a higher glucose uptake into the muscles compared to whey protein (Kanda et al., 2012; Morato et al., 2013). The combination of whey hydrolyzate and glucose seems to be more effective to increase skeletal muscle glycogen than whey protein and glucose or glucose alone (Morifuji, Kanda, Koga, Kawanaka, & Higuchi, 2010). Human studies have so far not been able to show this advantage from a combined administration of protein hydrolyzate from casein, whey, or wheat plus carbohydrates compared to a similar dose of carbohydrates (Cogan et al., 2018; Van Hall, Shirreffs, & Calbet, 2000; van Loon, Saris, Kruijshoop, & Wagenmakers, 2000). It should be noted that other peptides, for example, from soy, might also improve the absorption of glucose into the muscles, but human studies are lacking (Roblet et al., 2014). Few human studies have investigated whether protein hydrolyzate has a positive effect on endurance performance. One study investigated the effect of whey hydrolyzate plus carbohydrates compared to carbohydrates alone on performance during a 1 week training camp for elite orienteering runners. The runners supplemented whey hydrolyzate plus carbohydrates or carbohydrates only before and after each training session. The performance of the runners with protein ingestion during a 4 km time trial improved compared to the runners with carbohydrate supplementation only (Hansen, Bangsbo, Jensen, Bibby, & Madsen, 2015). However, other hydrolyzates such as those from casein may also appear to have a positive effect on performance during endurance load. Carbohydrate supplementation plus casein hydrolyzate resulted in improved performance at the end of the exercise compared to carbohydrate supplementation alone (Saunders, Moore, Kies, Luden, & Pratt, 2009). The reasons for this could be the beneficial changes in metabolism with an increase in fat oxidation, thus saving glycogen stores. In cyclists, this benefit on fat oxidation was observed with a combined intake of casein hydrolyzate plus carbohydrate compared to

Applications in nutrition: sport nutrition 531 carbohydrate alone, while performance was significantly improved by this combination only compared to a placebo (Oosthuyse, Carstens, & Millen, 2015). Another explanatory approach for potential improvements in endurance sports through bioactive peptides is the enhancement of endothelial function. That the endothelial function has an influence on performance was shown in studies with nitrate supplementation. Nitrate supplementation extends the time-to-exhaustion during exercise by increasing nitric oxide (McMahon, Leveritt, & Pavey, 2017). Various foods and nutrients such as Omega-3 fatty acids could increase the endurance performance through their influence on endothelial function via an improved supply of energy sources and oxygen (Zebrowska, Mizia-Stec, Mizia, Gasior, & Poprzecki, 2015). Some bioactive peptides from eggs (Liu, Oey, Bremer, Carne, & Silcock, 2018), whey (Ballard et al., 2009; Ballard et al., 2013; Martin et al., 2019; Oliveira, Volino-Souza, Cordeiro, Conte-Junior, & Alvares, 2020), and plants (Daskaya-Dikmen, Yucetepe, Karbancioglu-Guler, Daskaya, & Ozcelik, 2017) could have a beneficial effect on endothelial function by inhibiting angiotensin-converting enzyme. Whether these peptides can improve endurance performance via endothelial function has to be investigated in studies first. Nevertheless, protein hydrolyzates such as those from whey with their bioactive peptides that influence glucose uptake and endothelial function could bring benefits during intensive exercise, especially in combination with carbohydrates. Other signaling pathways for the improvement of endurance performance could be promoted by bioactive peptides, but there is a lack of studies with sports science issue.

21.3.2 Bioactive peptides and muscle damage Exercise-induced muscle damage (EIMD) is a frequently occurring phenomenon particularly resulting from unaccustomed and eccentric exercise (Clarkson & Hubal, 2002; Ebbeling & Clarkson, 1989). It is currently well known, that the lengthening of a contracting muscle (e.g., during eccentric maneuvers) leads to structural damage including Z-line streaming, sarcomere disruption, or local disorganization within the contractile machinery (Morgan & Allen, 1999; Proske & Morgan, 2001). Evidence indicates that in addition to muscular tissue, connective tissue is also affected by high-intensity exercise that may lead to excessive structure strain and facilitate disruption of the extracellular matrix (ECM) (Stauber, Clarkson, Fritz, & Evans, 1990; Armstrong, Warren, & Warren, 1991). As a consequence of EIMD, muscle soreness as well as a reduction in force-generating capacity can frequently be observed (Byrne, Twist, & Eston, 2004). Given that athletic performance can be affected up to several days after cessation of exercise (Doma et al., 2018; Sieljacks et al., 2016), highlights the importance of identifying new strategies to attenuate EIMD (Harty, Cottet, Malloy, & Kerksick, 2019). Besides pharmacological and physical therapy interventions (Dupuy, Douzi, Theurot, Bosquet, & Dugue, 2018),

532 Chapter 21 nutritional strategies, especially the intake of bioactive peptides, have recently gained more awareness in the scientific community (Harty et al., 2019). To date, several studies have been conducted to examine the influence of various peptides and hydrolyzates on muscular performance following fatiguing exercise. For this purpose, Buckley et al. (2010) performed a randomized, placebo-controlled trial and investigated the changes in muscle soreness, serum creatine kinase activity, and tumor necrosis factor alpha (TNF-α) concentrations before and after unaccustomed eccentric exercise with and without whey hydrolyzate. The findings revealed that hydrolyzed whey led to a rapid regeneration, measured by performing maximal isometric contractions, which was significantly augmented compared to the control group ingesting flavored water. This led the authors to speculate that this supplement enhanced repair processes within the damaged muscle tissue (Buckley et al., 2010). Interestingly, no changes in muscle soreness or any indirect marker of muscle damage and inflammation were observed. In 2018, a more recent study by Brown, Stevenson, and Howatson (2018) examined the effects of whey protein hydrolyzate on exercise recovery following a fatiguing repeated-sprint protocol. Markers of EIMD were assessed before and after, as well as 2, 24, 48, and 72 hours postexercise. The findings revealed that whey protein hydrolyzate reduced circulating creating kinase levels and attenuated the decline in muscle function. Furthermore, the supplementation accelerated the recovery of hamstring flexibility compared to a control group. These results are in accordance with the findings from other studies (Cooke, Rybalka, Stathis, Cribb, & Hayes, 2010; Hansen et al., 2015), although there are also some conflicting trials (Gee, Woolrich, & Smith, 2019). Using a longitudinal (12-week) study design, Lollo et al. (2014) found that the supplementation with whey hydrolyzate significantly decreased markers of muscle damage such as creatine kinase and lactate dehydrogenase compared to control. Apart from whey peptides, collagen peptides have received increasing attention. A recent randomized-controlled trial by Clifford et al. (2019) investigated the effects of a collagen peptide supplementation on maximal and explosive force production and muscle soreness. The results demonstrated that explosive force production was more rapidly restored in the group which ingested collagen peptides compared to the control group. In addition, high effect sizes for reduced muscle soreness were observed. Interestingly, such enhancements in muscular recovery were also seen following the ingestion of isolated soy protein (Shenoy, Dhawan, & Singh Sandhu, 2016). In general, however, evidence is very limited and far from conclusive whether peptides or protein hydrolyzates improve muscle recovery and decrease muscle damage. It has to be mentioned that all these studies did not directly assess the specific peptide composition in their supplements, limiting the conclusions about the effects of bioactive peptides on muscle damage. Since some studies point toward positive effects, a more thorough look on the bioactivity of these peptides is warranted.

Applications in nutrition: sport nutrition 533 21.3.2.1 Mechanisms 21.3.2.1.1 Effects on protein synthesis

It is well acknowledged that muscle protein synthesis and degradation are increased during EIMD and that an adequate nutritional stimulus is needed to provide a positive net protein balance (Pasiakos, Lieberman, & McLellan, 2014). Bioactive peptides are increasingly considered as a crucial factor for regulating muscle protein turnover. As mentioned in Section 21.3.1, previous evidence suggests that the collagen-derived dipeptide hydroxyprolyl-glycine (Hyp-Gly) can induce myogenic differentiation and increase muscle protein synthesis in vitro (Kitakaze et al., 2016). Therefore Hyp-Gly might promote skeletal anabolism (Kitakaze et al., 2016) and aid the recovery of contractile tissue, allowing higher force production after EIMD. This evidence, however, is derived from in vitro studies and needs to be confirmed by future clinical trials. 21.3.2.1.2 Antiinflammatory effect

During the first stage following exercise-induced muscle injury, an inflammatory response is induced (Fielding et al., 2000; Tidball, 1995). Depending on the type of exercise and its intensity, markers of inflammation can be elevated by up to several days (Tidball, 2005). Although an adequate level of inflammation is needed for wound healing (Lin, Kotani, & Lowry, 1998) and satellite cell activation (Hawke & Garry, 2001), excess amount of inflammatory products impair muscle regeneration (Perandini, Chimin, Lutkemeyer, & Camara, 2018) and induce lysis of muscle membranes (Tidball, 2005). Therefore it might be speculated that specific nutritional requirements are demanded to maintain an optimal level of muscular inflammation following EIMD. Much of the recent insights into the antiinflammatory potential of bioactive peptides have been derived from mammalian cell culture experiments (Guha & Majumder, 2019). Tripeptides with the sequence Val-Pro-Pro obtained from bacterial fermentation of casein have been shown to have antiinflammatory effects by inhibiting activation of the nuclear factor kappa B pathway and reducing adipokine levels (Chakrabarti & Wu, 2015). In a further study, another peptide derived from casein (Gln-Glu-Pro-Val-Leu) was reported to regulate nitric oxide release and augmenting the production of antiinflammatory cytokines including interleukin (IL) 4 and IL-10 (Jiehui et al., 2014). A recent in vivo study by Raizel et al. (2016) investigated the effects of a specific dipeptide (L-alanyl-L-glutamine) in Wistar rats. The findings indicated that this dipeptide revealed a potent cytoprotective effect and was able to reduce muscle damage and inflammation markers (e.g., creatine kinase, TNF-α) following progressive resistance exercise. However, research in humans is still scarce and it remains unknown to what extent these antiinflammatory effects can contribute.

534 Chapter 21 21.3.2.1.3 Antioxidant activity

A further mechanism that might help to reduce EIMD lays within the antioxidative effects of certain peptide forms. Interestingly, a large number of peptides have been demonstrated to hold a high antioxidative capacity. This antioxidant activity is extremely helpful for scavenging ROS and thus prevents oxidative stress. ROS are highly reactive oxygen-containing molecules characterized by their unpaired valence electron (Droge, 2002). In case of an inadequate neutralization of ROS by the antioxidative system, free radicals can lead to an oxidative modification of various macromolecules (such as DNA, proteins, or lipids) (Brieger, Schiavone, Miller, & Krause, 2012). Although physiological levels of ROS are necessary for contractile function of the skeletal muscle (Powers, Ji, Kavazis, & Jackson, 2011), excessive amounts have been shown to be involved in inflammation and also muscle damage (Aoi et al., 2004). Bioactive peptides derived from milk have been shown to possess antioxidant properties. From the principal components of milk (casein B80% and whey B20%), both have been used to create antioxidant peptides, mostly by means of enzymatic or chemical hydrolysis (Power, Jakeman, & FitzGerald, 2013). In one study, for example, casein peptides with the sequence Tyr-Phe-Tyr-Pro-Glu-Leu have been revealed to have a high superoxide anion scavenging activity (Suetsuna, Ukeda, & Ochi, 2000). After removal of amino acid residues, the highest antioxidant potential was found in the Glu-Leu dipeptide portion of the sequence. Moreover, phosphopeptides from casein have also been shown to possess antioxidant capacity (Kitts, 2005) by demonstrating both direct free radical quenching activity and also sequestering of potential iron prooxidants. For further information the interested reader is referred to the review by Power et al. (2013). Besides casein, whey peptides also have been found to possess potent antioxidant activity. For example, the enzymatic hydrolysis of α-lactobumin and β-lactoglobulin resulted in antioxidant hydrolyzates (e.g., Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) demonstrating a considerable radical scavenging activity (Hernandez-Ledesma, Davalos, Bartolome, & Amigo, 2005). To identify the most potent amino acids and peptide fragments, the authors reported that Trp, Tyr, and Met demonstrated the highest antioxidant activity (Hernandez-Ledesma et al., 2005), which was attributed to the capacity of indolic (Trp) and phenolic (Tyr) groups potentially serving as hydrogen donors. Similar to whey peptides, antioxidant potential has also been found for various other peptides (Power et al., 2013) including collagen (Barzideh, Latiff, Gan, Abedin, & Alias, 2014), marine (Wu et al., 2015), or soy (Ma et al., 2016; Park, Lee, Baek, & Lee, 2010). To what extent this antioxidant effect might alleviate the effects of EIMD is still under debate and needs further clarification. 21.3.2.2 Interim conclusion In summary, a variety of peptides exist which have been demonstrated to possess bioactive components that could be of special interest in counteracting EIMD and accelerate the

Applications in nutrition: sport nutrition 535 recovery process. Since both antiinflammatory and antioxidant properties are essential for muscular repair and exercise adaptations (Powers & Jackson, 2008; Powers, Duarte, Kavazis, & Talbert, 2010), future trials should shed light on the in vivo benefits of these peptides and investigate whether the outlined properties might also negatively interfere with the pro-inflammatory phase of muscular repair and thus blunt the repair progression. Given the existing evidence on detrimental effects of excessive amounts of antioxidants on muscular adaptations (Merry & Ristow, 2016), individually tailored intervention programs are warranted to take individual antioxidant deficiencies and redox profiles into account (Margaritelis et al., 2018).

21.3.3 Bioactive peptides and connective tissue The execution of human movement requires a proper functioning of connective tissue. Particularly during repeated powerful contractions in sports connective tissue plays a crucial role and determines athletic performance (Albracht & Arampatzis, 2013; Stafilidis & Arampatzis, 2007). It is thereby exposed to enhanced stress and strain which is induced by extensive physical activity. As a result, tendinopathy and functional joint pains are common injuries among athletes (Lopes, Hespanhol Junior, Yeung, & Costa, 2012). To optimally support athletic performance, to cope with enhanced loading, and to prevent degeneration, connective tissue needs to adapt its structure and composition permanently to the physical requirements (Kjaer, 2004). Besides exercise-induced adaptions, nutrition seems to influence connective tissue. More specifically, the effect of biologically active peptides on material and morphological adaptions of connective tissue has gained increasing attention in the scientific community (Alcock, Shaw, Tee, & Burke, 2019). Mechanical properties of connective tissue depend on the structure of the ECM and specifically the protein composition (McKee, Perlman, Morris, & Komarova, 2019). Collagen is the most abundant matrix molecule in all connective tissue and vital for the biomechanical properties (Kjaer, 2004; McKee et al., 2019). Thus research of connective tissue adaptions has focused on its synthesis in fibroblasts (Kjaer et al., 2009). 21.3.3.1 Tendon The most common collagen types present in tendons are types I and III (Franchi, Trire, Quaranta, Orsini, & Ottani, 2007). The ordered assembly of those collagen molecules in fibrils contributes to its characteristic elastic behavior and allows efficient storing and returning of energy during tensile loads (Kannus, 2000). An elevated collagen synthesis rate of tenocytes leads to a reinforcement of ECM, increasing its ability to withstand higher loads (Galloway, Lalley, & Shearn, 2013). Potential positive effects of an intact and healthy tendon are improved athletic performances such as running economy (Albracht & Arampatzis, 2013) and a decreased risk of injury (Galloway et al., 2013).

536 Chapter 21 Several studies have been conducted to examine the stimulating effect of peptides on collagen synthesis in tendon. Schunck and Oesser (2013) seeded primary fibroblasts derived from human tendons and ligaments in a culture medium. After adding collagen hydrolyzate, the authors reported a pronounced stimulatory effect on RNA expression and biosynthesis of collagen and other matrix molecules. These findings were confirmed by an in vivo animal study, which revealed that collagen peptides supplemented to rabbits over a time period of 56 days increased collagen fibril diameter and possibly improved mechanical properties of the Achilles tendon (Minaguchi et al., 2005). The authors attributed this effect to the high concentration of glycine within the supplement, since oral ingestion of glycine is known to trigger biological reactions (Yin et al., 1998, 2000). Besides single amino acids, the authors also suspected a promoting effect of the tri-peptide glycine-proline-arginine (Gly-Pro-Arg), since it shows various functions in the human body. For example, antiplatelet effects have been proven (Nonaka et al., 1997). Furthermore, in a randomized, placebo-controlled trial by Shaw, Lee-Barthel, Ross, Wang, and Baar (2017) gelatin supplementation before rope-skipping lead to increased levels of collagen synthesis markers in blood. It has to be pointed out that conclusions from these results on biologically active peptides are speculative, since gelatin per se does not contain collagen peptides. But their formation during digestive degradation processes could potentially contribute to the stimulating effect. Because of a lack of studies in vivo, there is neither sufficient evidence highlighting the effects of a supplementation with collagen peptides on tendon adaptions in humans nor enough knowledge to reliably differentiate between the stimulating effects of single amino acids and specific peptides. Given that a stimulating effect of collagen peptides on tendon fibroblasts is indicated in vitro (Schunck & Oesser, 2013) together with the promoting effect of collagen hydrolyzate on tendon adaptions in animals (Minaguchi et al., 2005) and increased collagen synthesis after gelatin intake in humans (Shaw et al., 2017), a beneficial influence of collagen peptide supplementation is conceivable. Further research is needed for practical recommendations. Supplementation with proteins containing high concentrations of EAAs (e.g., leucine) is currently a major field of myofibrillar protein synthesis research (Morton et al., 2018). Given this, its impact on tendinous adaptions has also gained interest. In an animal study, Barbosa et al. (2012) examined the effect of a leucine-rich diet on collagen content and biomechanical adaptions of the deep digital flexor tendon in malnourished rats conducting aerobic physical exercise. The results revealed a stimulating effect. The authors contributed it to the promoting influence of leucine on collagen synthesis (Barbosa et al., 2012). In humans, supplementation with high-leucine whey hydrolyzate in combination with a 12-week high-intensity resistance training led to an augmented cross-sectional area of the proximal patellar tendon (Farup et al., 2014). This indicates a beneficial impact of a supplementation with EAA rich peptides on exercise-induced adaptions in healthy tendons. It has to be mentioned that no specific peptides have been identified in these studies.

Applications in nutrition: sport nutrition 537 Tendinopathy is a disorder characterized by loss of normal tendon architecture caused by accumulated microtears (Fung et al., 2010) which lead to degenerative responses (Galloway et al., 2013). Due to the pronounced and repetitive loading of tendons in sports, tendinopathy is a severe issue among athletes (Lopes et al., 2012). Besides other therapies, peptide intake was the subject of research about tendon healing. Praet et al. (2019) investigated the promoting effect of collagen hydrolyzate supplementation on symptoms of Achilles tendinopathy combined with exercise. The results indicate a beneficial effect of collagen supplementation on tendon healing. According to the authors, this might be attributed to the high glycine uptake of 1.1 g per day. Glycine was related to improved matrix organization strength and tenocyte remodeling, by modulation of TNF-α, matrix metalloproteases, and collagen precursors (Praet et al., 2019; Vieira et al., 2015). The potential influence of specific peptides has not been examined. Another clinical trial showed a positive influence of a supplement containing i.a. arginine and collagen on extracorporeal shockwave therapy in Achilles tendinopathic patients (Notarnicola et al., 2012). These results indicate a beneficial effect of single amino acids and may be specific peptides on tendon healing therapy (Baar, 2019). Further research is needed to characterize potential mechanisms and the role of biologically active peptides in detail. 21.3.3.2 Cartilage and functional joint pain Physically active athletes show a pronounced risk of articular cartilage lesions leading to functional joint pain (McAdams, Mithoefer, Scopp, & Mandelbaum, 2010). To date, different therapeutic attempts are conducted with the aim of pain reduction and enhanced joint mobility. One attempt includes the treatment and prevention of degenerative articular processes (in collagen-containing tissues such as bone and cartilage) by stimulation of collagen synthesis with biologically active peptides (Alcock et al., 2019). Comprehensive studies were conducted examining cartilage tissue reactions to collagen peptides in vitro. Cultured chondrocytes showed stimulation of type II collagen and proteoglycans as wells as increased protease activity (Fichter et al., 2006; Jennings et al., 2001). In line with the results in vitro, several studies detected a beneficial impact of collagen peptide supplementation on pain and joint mobility of athletic subjects suffering from functional joint pains (Dressler et al., 2018; Zdzieblik, Oesser, Gollhofer, & Ko¨nig, 2017). According to the authors, a likely explanation for these results could be the stimulatory effect of collagen peptides on ECM-protein synthesis. Even though the exact mechanism remains unclear, some researchers speculate that those effects might be attributed to specific biologically active signaling peptides derived from hydrolyzed collagen such as hydroxyproline-proline-glycine (Hyp-Pro-Gly) and hydroxyproline-glycine (Hyp-Gly) (Kitakaze et al., 2016; Watanabe-Kamiyama et al., 2010; Walrand, Chiotelli, Noirt, Mwewa, & Lassel, 2008; Zdzieblik et al., 2017).

538 Chapter 21 Studies indicate that supplementation with whey hydrolyzate might have a stimulating effect on collagen synthesis (Barbosa et al., 2012) which could not only be beneficial in tendon adaptions but also in cartilage regeneration. To date, it is unclear to what extend single amino acids or bioactive peptides contribute to the effect. Thus the potential influence of whey hydrolyzate supplementation on recovery of functional joint pains could be discussed. However, because of a lack of studies, no statements about the effects of a whey hydrolyzate supplementation on articular cartilage can be drawn. 21.3.3.3 Interim conclusion In summary, there are a number of studies that examined the effects of biologically active peptides on exercise-related adaptions of connective tissue. The majority uses supplementation of hydrolyzed forms of collagen or whey proteins. Although the exact mechanisms remain unclear, potential explanations refer to collagen synthesizing effects of the amino acids leucine (whey) and glycine (collagen). Furthermore, specific Hyp-Gly and Hyp-Pro-Gly peptides are suspected to act as signal peptides promoting collagen synthesis (Kitakaze et al., 2016; Zdzieblik et al., 2017). Whereas there is substantial evidence for a soothing effect of peptide supplementation on sports-related functional joint pains (Dressler et al., 2018; Zdzieblik et al., 2017), studies examining the promoting effect on traininginduced tendon adaptions are rare. Thus reliable recommendations about the effects of protein supplementation on connective tissue adaptions in sports nutrition require more research.

21.4 Limitations Nutritional supplements in sports commonly contain proteins such as whey, casein, soy, or collagen. Studies examining the effects of proteins in sports nutrition often use supplements that contain hydrolyzed forms rather than intact whole proteins. Protein hydrolyzates are composed of a mixture of single amino acids and peptides of different lengths (Manninen, 2009). Since di- and tripeptides are known to be absorbed intact and thus faster (Grimble et al., 1987), they are the most abundant peptide forms in common sports-supplements. As a result, a classification of those studies with regard to the effects of biologically active peptides implies several limitations. First, the mixed composition of the used supplements does not allow discrimination between the biological effects of amino acids and peptides. Furthermore, hydrolyzed protein supplements always contain a series of different peptides which are built up from various combinations of amino acids. Thus a differentiation of the effects of certain peptides cannot directly be concluded from human studies, it can only be assumed on the basis of knowledge from in vitro and animal studies. For instance, both single amino acids and peptide compositions may influence an anabolic response after ingestion (Hulmi, Lockwood, & Stout, 2010). As shown in Section 21.3.1, leucine as well as the dipeptide leucine-valine is able to stimulate the mTOR pathway in vitro (Anthony, Anthony, Kimball, & Jefferson, 2001;

Applications in nutrition: sport nutrition 539 Moura et al., 2017). Thus exercise studies that show a beneficial effect of whey hydrolyzate supplementation (which contains leucine and leucine-valine) are unable to elucidate to what extend single amino acids or peptides affected the outcome. In addition, the protein source of the supplement is always reported, yet only few authors assess and report the specific peptide composition. Since different hydrolyzation of the same protein results in different peptide compositions and absorption kinetics (Manninen, 2009), even studies that refer to the same protein source are hardly comparable. According to Farup et al. (2014), this could be responsible for contradictory results within studies examining the effects of supplements from the same protein source; therefore the authors highlight the importance of a detailed description of the amino acid and peptide profile. Furthermore, mixed supplement compositions impede findings about underlying modes of action. Studies examining the signal pathways of specific peptides in vitro and in vivo are required to elucidate potential mechanisms that may contribute to the effects of peptide supplementation. Remodeling of the supplement during digestion seems to be a severe problem regarding the bioavailability of peptides after intake. It is under debate whether peptides have to be consumed purely or in forms of proteins and hydrolyzates to enter the bloodstream effectively. Studies have shown that numerous di- and tripeptides (i.e., Pro-Hyp, Pro-Hyp-Gly, Ala-Hyp) can be found in the blood after oral ingestion of a hydrolyzed collagen supplement (Ichikawa et al., 2010; Ohara, Matsumoto, Ito, Iwai, & Sato, 2007). Results from Sugihara et al. (2012) point out that the measured concentrations vary individually between subjects. These results indicate that simple conclusions from the peptide profile of the supplement to biological availability might also be misleading. As a consequence, future research should aim to display the whole process from intake to physical adaptions in the human body to enable reliable recommendations for peptide supplementation in sports. First, more cell culture studies are needed, elucidating the effects and mechanisms of specific peptides in different tissues. Second, it requires more knowledge about the bioavailability of a specific peptide from trials examining its concentration in the blood after intake by mass spectrometry. Third, longitudinal studies with combined exercise and nutritional interventions should evaluate the effects on exercise-related adaptions in vivo. Summed up, statements toward the role of biologically active peptides in training-induced adaptations require more peptide-specific research. At present, our knowledge about the effects of specific peptides in combination with exercise in humans and its underlying mechanisms is not sufficient to draw reliable conclusions.

21.5 Practical applications Athletes subject themselves to various mechanical and metabolic stimuli which aim to improve training status and exercise performance (Campbell & Wisniewski, 2017). Besides

540 Chapter 21 these predominantly exercise-induced stimuli, adequate dietary strategies have also been incorporated into training and competition routines (Cintineo, Arent, Antonio, & Arent, 2018; Drummond & Rasmussen, 2008; Moore, 2019). Various peptides and hydrolyzed proteins have been identified as possible candidates with significant roles in sports and exercise (see Fig. 21.2). As indicated in Sections 21.3.1 and 21.3.2, several bioactive peptides (such as hydroxyproline-glycine) have been shown to increase muscle protein synthesis (Kitakaze et al., 2016) and thus have an indirect positive effect on strength (Zdzieblik et al., 2015) and muscle recovery (Clifford et al., 2019). This might be of high relevance for sports, which require exerting large forces against an object or an opponent, including judo, wrestling, or weightlifting. Previous studies also point toward beneficial effects of hydrolyzed proteins on endurance performance and revealed that casein or whey hydrolyzate in combination with carbohydrate enhanced running

Figure 21.2 Summary of the potential effects of bioactive peptides on athletic performance. ACE, Angiotensinconverting enzyme; CK, creatinkinase; COL I/II/III, collagen type I/II/III; CSA, cross-sectional area; GLUT-4, Glucose transporter type 4; IL-10, interleukin 10; IL-4, interleukin 4; LDH, lactate dehydrogenase; MMP, matrix metalloproteases; mTOR, mechanistic target of rapamycin; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SOD, superoxide dismutase; TNF-α, tumor necrosis factor alpha. Source: Illustration of the human designed by kjpargeter.

Applications in nutrition: sport nutrition 541 capacity compared to carbohydrate alone (Hansen et al., 2015; Saunders et al., 2009). This indicates that also endurance-type sports might benefit from nutritional interventions with certain peptide forms. However, it needs to be mentioned that this topic is controversially discussed and the evidence is still weak (McLellan, Pasiakos, & Lieberman, 2014). Besides improving athletic performance (e.g., strength increases and optimization of body composition), recent investigations have assumed that the intake of these components might also be beneficial for minimizing the risk of injury and accelerating return-to-sport (Baar, 2017). In terms of injury prevention, a recent study by Shaw et al. (2017) revealed that an increase in circulating glycine, proline, hydroxyproline, and hydroxylysine after gelatine supplementation was positively associated with an augmented collagen synthesis following an intermittent exercise program. Furthermore, the authors reported an increase in collagen content after treating an engineered ligament with vitamin C-enriched gelatine (Shaw et al., 2017). Interestingly, McAlindon et al. (2011) demonstrated that a supplementation of collagen hydrolyzate (10 g) for 24 weeks resulted in an increased proteoglycan content in the knee cartilage. In agreement with these observations a randomized-controlled trial in athletes reported that the same amount of collagen hydrolyzate facilitated improvements in joint pain (Clark et al., 2008). These data support the use of bioactive collagen peptides and collagen hydrolyzate for connective tissue injury prevention. In this context, sports with high tendon stress including sprinting (Monte & Zamparo, 2019) or jumping elements (Bayliss et al., 2016) might benefit from the intake of bioactive peptides derived from collagen. In terms of peptide timing, it might be beneficial to have the peptides available in the blood before engaging in physical exercise (Baar, 2017). This hypothesis is based on the fact that nutrient delivery to inactive tendons might be limited compared to tendons during exercise (Baar, 2017). In addition to timing, future studies are needed to quantify the exact amounts of peptides that are needed for optimal adaptations. It should be kept in mind that little is known about potential side effects from taking bioactive peptides and the intake of a highly effective isolated peptide could likely be comparable with a drug rather than a dietary supplement (Chakrabarti, Guha, & Majumder, 2018). Therefore, with regard to sports, it might be speculated that the use of isolated bioactive peptides with performance-enhancing effects might fall into the category of doping.

21.6 Summary In summary, several studies have demonstrated that both exercise performance and recovery and tissue repair might be improved by the presence of specific bioactive peptides. Although the observed effects are promising, it is important to take into account that more studies are needed before definite conclusions can be drawn. In addition, the majority of

542 Chapter 21 available studies investigated the effects of hydrolyzed proteins but not individual bioactive peptides. Therefore further research is needed to elucidate the modes of action both in vitro and in vivo.

References Albracht, K., & Arampatzis, A. (2013). Exercise-induced changes in triceps surae tendon stiffness and muscle strength affect running economy in humans. European Journal of Applied Physiology, 113, 1605
1615. Alcaraz-Ibanez, M., & Rodriguez-Perez, M. (2018). Effects of resistance training on performance in previously trained endurance runners: A systematic review. Journal of Sports Sciences, 36, 613
629. Alcock, R. D., Shaw, G. C., Tee, N., & Burke, L. M. (2019). Plasma amino acid concentrations after the ingestion of dairy and collagen proteins, in healthy active males. Frontiers in Nutrition, 6, 163. Anthony, J. C., Anthony, T. G., Kimball, S. R., & Jefferson, L. S. (2001). Signaling pathways involved in translational control of protein synthesis in skeletal muscle by leucine. The Journal of Nutrition, 131, 856
860. Aoi, W., Naito, Y., Takanami, Y., Kawai, Y., Sakuma, K., Ichikawa, H., et al. (2004). Oxidative stress and delayed-onset muscle damage after exercise. Free Radical Biology & Medicine, 37, 480
487. Aragon, A. A., Schoenfeld, B. J., Wildman, R., Kleiner, S., VanDusseldorp, T., Taylor, L., et al. (2017). International Society of Sports Nutrition position stand: Diets and body composition. Journal of the International Society of Sports Nutrition, 14, 16. Armstrong, R. B., Warren, G. L., & Warren, J. A. (1991). Mechanisms of exercise-induced muscle fibre injury. Sports Medicine, 12, 184
207. Baar, K. (2017). Minimizing injury and maximizing return to play: Lessons from engineered ligaments. Sports Medicine (Auckland, NZ), 47, 5
11. Baar, K. (2019). Stress relaxation and targeted nutrition to treat patellar tendinopathy. International Journal of Sport Nutrition and Exercise Metabolism, 29, 453
457. Ballard, K. D., Bruno, R. S., Seip, R. L., Quann, E. E., Volk, B. M., Freidenreich, D. J., et al. (2009). Acute ingestion of a novel whey-derived peptide improves vascular endothelial responses in healthy individuals: A randomized, placebo controlled trial. Nutrition Journal, 8, 34. Ballard, K. D., Kupchak, B. R., Volk, B. M., Mah, E., Shkreta, A., Liptak, C., et al. (2013). Acute effects of ingestion of a novel whey-derived extract on vascular endothelial function in overweight, middle-aged men and women. The British Journal of Nutrition, 109, 882
893. Balsalobre-Fernandez, C., Santos-Concejero, J., & Grivas, G. V. (2016). Effects of strength training on running economy in highly trained runners: A systematic review with meta-analysis of controlled trials. Journal of Strength and Conditioning Research, 30, 2361
2368. Barbosa, A. W., Benevides, G. P., Alferes, L. M., Salomao, E. M., Gomes-Marcondes, M. C., & Gomes, L. (2012). A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery. Amino Acids, 42, 329
336. Barzideh, Z., Latiff, A. A., Gan, C.-Y., Abedin, M. Z., & Alias, A. K. (2014). ACE inhibitory and antioxidant activities of collagen hydrolysates from the ribbon jellyfish (Chrysaora sp.). Food Technology and Biotechnology, 52, 495
504. Bayliss, A. J., Weatherholt, A. M., Crandall, T. T., Farmer, D. L., McConnell, J. C., Crossley, K. M., et al. (2016). Achilles tendon material properties are greater in the jump leg of jumping athletes. Journal of Musculoskeletal & Neuronal Interactions, 16, 105
112. Blagrove, R. C., Howatson, G., & Hayes, P. R. (2018). Effects of strength training on the physiological determinants of middle- and long-distance running performance: A systematic review. Sports Medicine, 48, 1117
1149. Brieger, K., Schiavone, S., Miller, F. J., Jr., & Krause, K. H. (2012). Reactive oxygen species: From health to disease. Swiss Medical Weekly, 142, 304
310.

Applications in nutrition: sport nutrition 543 Brown, M. A., Stevenson, E. J., & Howatson, G. (2018). Whey protein hydrolysate supplementation accelerates recovery from exercise-induced muscle damage in females. Applied Physiology, Nutrition, and Metabolism 5 Physiologie appliquee, nutrition et metabolisme, 43, 324
330. Buckley, J. D., Thomson, R. L., Coates, A. M., Howe, P. R. C., DeNichilo, M. O., & Rowney, M. K. (2010). Supplementation with a whey protein hydrolysate enhances recovery of muscle force-generating capacity following eccentric exercise. Journal of Science and Medicine in Sport, 13, 178
181. Byrne, C., Twist, C., & Eston, R. (2004). Neuromuscular function after exercise-induced muscle damage. Sports Medicine, 34, 49
69. Campbell, S. C., & Wisniewski, P. J. (2017). Chapter 13—Nutritional recommendations for athletes. In A. M. Coulston, C. J. Boushey, M. G. Ferruzzi, & L. M. Delahanty (Eds.), Nutrition in the prevention and treatment of disease (4th ed., pp. 255
271). Academic Press. Centner, C., Zdzieblik, D., Roberts, L., Gollhofer, A., & Ko¨nig, D. (2019). Effects of blood flow restriction training with protein supplementation on muscle mass and strength in older men. Journal of Sports Science & Medicine, 18, 471
478. Chakrabarti, S., & Wu, J. (2015). Milk-derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) promote adipocyte differentiation and inhibit inflammation in 3T3-F442A cells. PLoS One, 10, e0117492. Chakrabarti, S., Guha, S., & Majumder, K. (2018). Food-derived bioactive peptides in human health: Challenges and opportunities. Nutrients, 10, 1738. Churchward-Venne, T. A., Breen, L., Di Donato, D. M., Hector, A. J., Mitchell, C. J., Moore, D. R., et al. (2014). Leucine supplementation of a low-protein mixed macronutrient beverage enhances myofibrillar protein synthesis in young men: A double-blind, randomized trial. The American Journal of Clinical Nutrition, 99, 276
286. Churchward-Venne, T. A., Burd, N. A., Mitchell, C. J., West, D. W. D., Philp, A., Marcotte, G. R., et al. (2012). Supplementation of a suboptimal protein dose with leucine or essential amino acids: Effects on myofibrillar protein synthesis at rest and following resistance exercise in men. The Journal of Physiology—London, 590, 2751
2765. Cintineo, H. P., Arent, M. A., Antonio, J., & Arent, S. M. (2018). Effects of protein supplementation on performance and recovery in resistance and endurance training. Frontiers in Nutrition, 5, 83. Clark, K. L., Sebastianelli, W., Flechsenhar, K. R., Aukermann, D. F., Meza, F., Millard, R. L., et al. (2008). 24-Week study on the use of collagen hydrolysate as a dietary supplement in athletes with activity-related joint pain. Current Medical Research and Opinion, 24, 1485
1496. Clarkson, P. M., & Hubal, M. J. (2002). Exercise-induced muscle damage in humans. American Journal of Physical Medicine and Rehabilitation, 81, 52
69. Clifford, T., Ventress, M., Allerton, D. M., Stansfield, S., Tang, J. C. Y., Fraser, W. D., et al. (2019). The effects of collagen peptides on muscle damage, inflammation and bone turnover following exercise: A randomized, controlled trial. Amino Acids, 51, 691
704. Cogan, K. E., Evans, M., Iuliano, E., Melvin, A., Susta, D., Neff, K., et al. (2018). Co-ingestion of protein or a protein hydrolysate with carbohydrate enhances anabolic signaling, but not glycogen resynthesis, following recovery from prolonged aerobic exercise in trained cyclists. European Journal of Applied Physiology, 118, 349
359. Cooke, M. B., Rybalka, E., Stathis, C. G., Cribb, P. J., & Hayes, A. (2010). Whey protein isolate attenuates strength decline after eccentrically-induced muscle damage in healthy individuals. Journal of the International Society of Sports Nutrition, 7, 30. Daskaya-Dikmen, C., Yucetepe, A., Karbancioglu-Guler, F., Daskaya, H., & Ozcelik, B. (2017). Angiotensin-Iconverting enzyme (ACE)-inhibitory peptides from plants. Nutrients, 9, 316. Devries, M. C., McGlory, C., Bolster, D. R., Kamil, A., Rahn, M., Harkness, L., et al. (2018). Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: A randomized, controlled trial. American Journal of Clinical Nutrition, 107, 217
226. Dickinson, J. M., Fry, C. S., Drummond, M. J., Gundermann, D. M., Walker, D. K., Glynn, E. L., et al. (2011). Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids. The Journal of Nutrition, 141, 856
862.

544 Chapter 21 Distefano, G., & Goodpaster, B. H. (2018). Effects of exercise and aging on skeletal muscle. Cold Spring Harbor Perspectives in Medicine, 8, a029785. Doma, K., Leicht, A., Sinclair, W., Schumann, M., Damas, F., Burt, D., et al. (2018). Impact of exerciseinduced muscle damage on performance test outcomes in elite female basketball players. Journal of Strength and Conditioning Research, 32, 1731
1738. Dressler, P., Gehring, D., Zdzieblik, D., Oesser, S., Gollhofer, A., & Ko¨nig, D. (2018). Improvement of functional ankle properties following supplementation with specific collagen peptides in athletes with chronic ankle instability. Journal of Sports Science & Medicine, 17, 298
304. Droge, W. (2002). Free radicals in the physiological control of cell function. Physiological Reviews, 82, 47
95. Drummond, M. J., & Rasmussen, B. B. (2008). Leucine-enriched nutrients and the regulation of mammalian target of rapamycin signalling and human skeletal muscle protein synthesis. Current Opinion in Clinical Nutrition and Metabolic Care, 11, 222
226. Dupuy, O., Douzi, W., Theurot, D., Bosquet, L., & Dugue, B. (2018). An evidence-based approach for choosing post-exercise recovery techniques to reduce markers of muscle damage, soreness, fatigue, and inflammation: A systematic review with meta-analysis. Frontiers in Physiology, 9, 403. Ebbeling, C. B., & Clarkson, P. M. (1989). Exercise-induced muscle damage and adaptation. Sports Medicine, 7, 207
234. Farup, J., Rahbek, S. K., Vendelbo, M. H., Matzon, A., Hindhede, J., Bejder, A., et al. (2014). Whey protein hydrolysate augments tendon and muscle hypertrophy independent of resistance exercise contraction mode. Scandinavian Journal of Medicine and Science in Sports, 24, 788
798. Fichter, M., Korner, U., Schomburg, J., Jennings, L., Cole, A. A., & Mollenhauer, J. (2006). Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes. Journal of Orthopaedic Research, 24, 63
70. Fielding, R. A., Violan, M. A., Svetkey, L., Abad, L. W., Manfredi, T. J., Cosmas, A., et al. (2000). Effects of prior exercise on eccentric exercise-induced neutrophilia and enzyme release. Medicine and Science in Sports and Exercise, 32, 359
364. Franchi, M., Trire, A., Quaranta, M., Orsini, E., & Ottani, V. (2007). Collagen structure of tendon relates to function. The Scientific World Journal, 7, 404
420. Fung, D. T., Wang, V. M., Andarawis-Puri, N., Basta-Pljakic, J., Li, Y., Laudier, D. M., et al. (2010). Early response to tendon fatigue damage accumulation in a novel in vivo model. Journal of Biomechanics, 43, 274
279. Galloway, M. T., Lalley, A. L., & Shearn, J. T. (2013). The role of mechanical loading in tendon development, maintenance, injury, and repair. The Journal of Bone and Joint Surgery. American Volume, 95, 1620
1628. Gan, Z., Fu, T., Kelly, D. P., & Vega, R. B. (2018). Skeletal muscle mitochondrial remodeling in exercise and diseases. Cell Research, 28, 969
980. Garthe, I., & Maughan, R. J. (2018). Athletes and supplements: Prevalence and perspectives. International Journal of Sport Nutrition and Exercise Metabolism, 28, 126
138. Gee, T. I., Woolrich, T. J., & Smith, M. F. (2019). Effectiveness of whey protein hydrolysate and milk-based formulated drinks on recovery of strength and power following acute resistance exercise. Journal of Human Kinetics, 68, 193
202. Gonzalez, A. M., Hoffman, J. R., Stout, J. R., Fukuda, D. H., & Willoughby, D. S. (2016). Intramuscular anabolic signaling and endocrine response following resistance exercise: Implications for muscle hypertrophy. Sports Medicine, 46, 671
685. Grimble, G. K., Rees, R. G., Keohane, P. P., Cartwright, T., Desreumaux, M., & Silk, D. B. (1987). Effect of peptide chain length on absorption of egg protein hydrolysates in the normal human jejunum. Gastroenterology, 92, 136
142. Guha, S., & Majumder, K. (2019). Structural-features of food-derived bioactive peptides with anti-inflammatory activity: A brief review. Journal of Food Biochemistry, 43, e12531. Hansen, M., Bangsbo, J., Jensen, J., Bibby, B. M., & Madsen, K. (2015). Effect of whey protein hydrolysate on performance and recovery of top-class orienteering runners. International Journal of Sport Nutrition and Exercise Metabolism, 25, 97
109.

Applications in nutrition: sport nutrition 545 Harty, P. S., Cottet, M. L., Malloy, J. K., & Kerksick, C. M. (2019). Nutritional and supplementation strategies to prevent and attenuate exercise-induced muscle damage: A brief review. Sports Medicine—Open, 5, 1. Hawke, T. J., & Garry, D. J. (2001). Myogenic satellite cells: Physiology to molecular biology. Journal of Applied Physiology (1985), 91, 534
551. Hearris, M. A., Hammond, K. M., Fell, J. M., & Morton, J. P. (2018). Regulation of muscle glycogen metabolism during exercise: Implications for endurance performance and training adaptations. Nutrients, 10, 298. Hernandez-Ledesma, B., Davalos, A., Bartolome, B., & Amigo, L. (2005). Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/ MS. Journal of Agricultural and Food Chemistry, 53, 588
593. Hoppeler, H. (2016). Molecular networks in skeletal muscle plasticity. The Journal of Experimental Biology, 219, 205
213. Hulmi, J. J., Lockwood, C. M., & Stout, J. R. (2010). Effect of protein/essential amino acids and resistance training on skeletal muscle hypertrophy: A case for whey protein. Nutrition & Metabolism, 7, 51. Ichikawa, S., Morifuji, M., Ohara, H., Matsumoto, H., Takeuchi, Y., & Sato, K. (2010). Hydroxyprolinecontaining dipeptides and tripeptides quantified at high concentration in human blood after oral administration of gelatin hydrolysate. International Journal of Food Sciences and Nutrition, 61, 52
60. Ja¨ger, R., Kerksick, C. M., Campbell, B. I., Cribb, P. J., Wells, S. D., Skwiat, T. M., et al. (2017). International Society of Sports Nutrition position stand: Protein and exercise. Journal of the International Society of Sports Nutrition, 14, 20. Jendricke, P., Centner, C., Zdzieblik, D., Gollhofer, A., & Ko¨nig, D. (2019). Specific collagen peptides in combination with resistance training improve body composition and regional muscle strength in premenopausal women: A randomized controlled trial. Nutrients, 11, 892. Jennings, L., Wu, L., King, K. B., Hammerle, H., Cs-Szabo, G., & Mollenhauer, J. (2001). The effects of collagen fragments on the extracellular matrix metabolism of bovine and human chondrocytes. Connective Tissue Research, 42, 71
86. Jiehui, Z., Liuliu, M., Haihong, X., Yang, G., Yingkai, J., Lun, Z., et al. (2014). Immunomodulating effects of caseinderived peptides QEPVL and QEPV on lymphocytes in vitro and in vivo. Food & Function, 5, 2061
2069. Kanda, A., Morifuji, M., Fukasawa, T., Koga, J., Kanegae, M., Kawanaka, K., et al. (2012). Dietary whey protein hydrolysates increase skeletal muscle glycogen levels via activation of glycogen synthase in mice. Journal of Agricultural and Food Chemistry, 60, 11403
11408. Kannus, P. (2000). Structure of the tendon connective tissue. Scandinavian Journal of Medicine and Science in Sports, 10, 312
320. Kerksick, C. M., Wilborn, C. D., Roberts, M. D., Smith-Ryan, A., Kleiner, S. M., Ja¨ger, R., et al. (2018). ISSN exercise & sports nutrition review update: Research & recommendations. Journal of the International Society of Sports Nutrition, 15, 38. Kim, Y. (2017). Effects of exercise training alone on depot-specific body fat stores in youth: Review of recent literature. Pediatric Exercise Science, 30, 1
25. Kirmse, M., Oertzen-Hagemann, V., de Marees, M., Bloch, W., & Platen, P. (2019). Prolonged collagen peptide supplementation and resistance exercise training affects body composition in recreationally active men. Nutrients, 11, 1154. Kitakaze, T., Sakamoto, T., Kitano, T., Inoue, N., Sugihara, F., Harada, N., et al. (2016). The collagen derived dipeptide hydroxyprolyl-glycine promotes C2C12 myoblast differentiation and myotube hypertrophy. Biochemical and Biophysical Research Communications, 478, 1292
1297. Kitts, D. D. (2005). Antioxidant properties of casein-phosphopeptides. Trends in Food Science & Technology, 16, 549
554. Kjaer, M. (2004). Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiological Reviews, 84, 649
698. Kjaer, M., Langberg, H., Heinemeier, K., Bayer, M. L., Hansen, M., Holm, L., et al. (2009). From mechanical loading to collagen synthesis, structural changes and function in human tendon. Scandinavian Journal of Medicine and Science in Sports, 19, 500
510.

546 Chapter 21 Knapik, J. J., Steelman, R. A., Hoedebecke, S. S., Austin, K. G., Farina, E. K., & Lieberman, H. R. (2016). Prevalence of dietary supplement use by athletes: Systematic review and meta-analysis. Sports Medicine, 46, 103
123. Kulkarni, S. S., Salehzadeh, F., Fritz, T., Zierath, J. R., Krook, A., & Osler, M. E. (2012). Mitochondrial regulators of fatty acid metabolism reflect metabolic dysfunction in type 2 diabetes mellitus. Metabolism: Clinical and Experimental, 61, 175
185. Lin, E., Kotani, J. G., & Lowry, S. F. (1998). Nutritional modulation of immunity and the inflammatory response. Nutrition, 14, 545
550. Liu, Y. F., Oey, I., Bremer, P., Carne, A., & Silcock, P. (2018). Bioactive peptides derived from egg proteins: A review. Critical Reviews in Food Science and Nutrition, 58, 2508
2530. Lockwood, C. M., Roberts, M. D., Dalbo, V. J., Smith-Ryan, A. E., Kendall, K. L., Moon, J. R., et al. (2017). Effects of hydrolyzed whey versus other whey protein supplements on the physiological response to 8 weeks of resistance exercise in college-aged males. Journal of the American College of Nutrition, 36, 16
27. Lollo, P. C. B., Amaya-Farfan, J., Faria, I. C., Salgado, J. V. V., Chacon-Mikahil, M. P. T., Cruz, A. G., et al. (2014). Hydrolysed whey protein reduces muscle damage markers in Brazilian elite soccer players compared with whey protein and maltodextrin. A twelve-week in-championship intervention. International Dairy Journal, 34, 19
24. Lopes, A. D., Hespanhol Junior, L. C., Yeung, S. S., & Costa, L. O. (2012). What are the main running-related musculoskeletal injuries? A systematic review. Sports Medicine, 42, 891
905. Ma, H., Liu, R., Zhao, Z., Zhang, Z., Cao, Y., Ma, Y., et al. (2016). A novel peptide from soybean protein isolate significantly enhances resistance of the organism under oxidative stress. PLoS One, 11, e0159938. Manninen, A. H. (2009). Protein hydrolysates in sports nutrition. Nutrition & Metabolism, 6, 38. Margaritelis, N. V., Paschalis, V., Theodorou, A. A., Kyparos, A., & Nikolaidis, M. G. (2018). Antioxidants in personalized nutrition and exercise. Advances in Nutrition (Bethesda, MD), 9, 813
823. Marquet, L. A., Brisswalter, J., Louis, J., Tiollier, E., Burke, L. M., Hawley, J. A., et al. (2016). Enhanced endurance performance by periodization of carbohydrate intake: “Sleep low” strategy. Medicine and Science in Sports and Exercise, 48, 663
672. Marquet, L. A., Hausswirth, C., Molle, O., Hawley, J. A., Burke, L. M., Tiollier, E., et al. (2016). Periodization of carbohydrate intake: Short-term effect on performance. Nutrients, 8. Martin, M., Hagemann, D., Nguyen, T. T., Schwarz, L., Khedr, S., Moskopp, M. L., et al. (2019). Plasma concentrations and ACE-inhibitory effects of tryptophan-containing peptides from whey protein hydrolysate in healthy volunteers. European Journal of Nutrition, 59, 1135
1147. McAdams, T. R., Mithoefer, K., Scopp, J. M., & Mandelbaum, B. R. (2010). Articular cartilage injury in athletes. Cartilage, 1, 165
179. McAlindon, T. E., Nuite, M., Krishnan, N., Ruthazer, R., Price, L. L., Burstein, D., et al. (2011). Change in knee osteoarthritis cartilage detected by delayed gadolinium enhanced magnetic resonance imaging following treatment with collagen hydrolysate: A pilot randomized controlled trial. Osteoarthritis and Cartilage, 19, 399
405. McCartney, D., Desbrow, B., & Irwin, C. (2018). Post-exercise ingestion of carbohydrate, protein and water: A systematic review and meta-analysis for effects on subsequent athletic performance. Sports Medicine, 48, 379
408. McKee, T. J., Perlman, G., Morris, M., & Komarova, S. V. (2019). Extracellular matrix composition of connective tissues: A systematic review and meta-analysis. Scientific Reports, 9, 10542. McLellan, T. M., Pasiakos, S. M., & Lieberman, H. R. (2014). Effects of protein in combination with carbohydrate supplements on acute or repeat endurance exercise performance: A systematic review. Sports Medicine, 44, 535
550. McMahon, N. F., Leveritt, M. D., & Pavey, T. G. (2017). The effect of dietary nitrate supplementation on endurance exercise performance in healthy adults: A systematic review and meta-analysis. Sports Medicine, 47, 735
756.

Applications in nutrition: sport nutrition 547 Merry, T. L., & Ristow, M. (2016). Do antioxidant supplements interfere with skeletal muscle adaptation to exercise training? The Journal of Physiology, 594, 5135
5147. Minaguchi, J., Koyama, Y., Meguri, N., Hosaka, Y., Ueda, H., Kusubata, M., et al. (2005). Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in Achilles tendon. Journal of Nutritional Science and Vitaminology, 51, 169
174. Mobley, C. B., Haun, C. T., Roberson, P. A., Mumford, P. W., Romero, M. A., Kephart, W. C., et al. (2017). Effects of whey, soy or leucine supplementation with 12 weeks of resistance training on strength, body composition, and skeletal muscle and adipose tissue histological attributes in college-aged males. Nutrients, 9, 972. Mohr, M., Draganidis, D., Chatzinikolaou, A., Barbero-Alvarez, J. C., Castagna, C., Douroudos, I., et al. (2016). Muscle damage, inflammatory, immune and performance responses to three football games in 1 week in competitive male players. European Journal of Applied Physiology, 116, 179
193. Monte, A., & Zamparo, P. (2019). Correlations between muscle-tendon parameters and acceleration ability in 20 m sprints. PLoS One, 14, e0213347. Moore, D. R. (2019). Maximizing post-exercise anabolism: The case for relative protein intakes. Frontiers in Nutrition, 6. Morato, P. N., Lollo, P. C., Moura, C. S., Batista, T. M., Camargo, R. L., Carneiro, E. M., et al. (2013). Whey protein hydrolysate increases translocation of GLUT-4 to the plasma membrane independent of insulin in Wistar rats. PLoS One, 8, e71134. Moreira, O. C., Estebanez, B., Martinez-Florez, S., de Paz, J. A., Cuevas, M. J., & Gonzalez-Gallego, J. (2017). Mitochondrial function and mitophagy in the elderly: Effects of exercise. Oxidative Medicine and Cellular Longevity, 2017, 13. Morgan, D. L., & Allen, D. G. (1999). Early events in stretch-induced muscle damage. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology, 87, 2007
2015. Morifuji, M., Kanda, A., Koga, J., Kawanaka, K., & Higuchi, M. (2010). Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats. Amino Acids, 38, 1109
1115. Morifuji, M., Koga, J., Kawanaka, K., & Higuchi, M. (2009). Branched-chain amino acid-containing dipeptides, identified from whey protein hydrolysates, stimulate glucose uptake rate in L6 myotubes and isolated skeletal muscles. Journal of Nutritional Science and Vitaminology, 55, 81
86. Morton, R. W., Murphy, K. T., McKellar, S. R., Schoenfeld, B. J., Henselmans, M., Helms, E., et al. (2018). A systematic review, meta-analysis and meta-regression of the effect of protein supplementation on resistance training-induced gains in muscle mass and strength in healthy adults. British Journal of Sports Medicine, 52, 376
384. Moura, C. S., Lollo, P. C., Morato, P. N., Risso, E. M., & Amaya-Farfan, J. (2017). Bioactivity of food peptides: Biological response of rats to bovine milk whey peptides following acute exercise. Food & Nutrition Research, 611290740. Naclerio, F., & Larumbe-Zabala, E. (2016). Effects of whey protein alone or as part of a multi-ingredient formulation on strength, fat-free mass, or lean body mass in resistance-trained individuals: A meta-analysis. Sports Medicine, 46, 125
137. Nonaka, I., Katsuda, S. I., Ohmori, T., Shigehisa, T., Nakagami, T., & Maruyama, S. (1997). In vitro and in vivo anti-platelet effects of enzymatic hydrolysates of collagen and collagen-related peptides. Bioscience, Biotechnology, and Biochemistry, 61, 772
775. Notarnicola, A., Pesce, V., Vicenti, G., Tafuri, S., Forcignano, M., & Moretti, B. (2012). SWAAT study: Extracorporeal shock wave therapy and arginine supplementation and other nutraceuticals for insertional Achilles tendinopathy. Advances in Therapy, 29, 799
814. Oertzen-Hagemann, V., Kirmse, M., Eggers, B., Pfeiffer, K., Marcus, K., de Marees, M., et al. (2019). Effects of 12 weeks of hypertrophy resistance exercise training combined with collagen peptide supplementation on the skeletal muscle proteome in recreationally active men. Nutrients, 11, 1072. Ohara, H., Matsumoto, H., Ito, K., Iwai, K., & Sato, K. (2007). Comparison of quantity and structures of hydroxyproline-containing peptides in human blood after oral ingestion of gelatin hydrolysates from different sources. Journal of Agricultural and Food Chemistry, 55, 1532
1535.

548 Chapter 21 Oikawa, S. Y., Kamal, M. J., Webb, E. K., McGlory, C., Baker, S. K., & Phillips, S. M. (2020). Whey protein but not collagen peptides stimulate acute and longer-term muscle protein synthesis with and without resistance exercise in healthy older women: A randomized controlled trial. The American Journal of Clinical Nutrition, 111, 708
718. Oikawa, S. Y., MacInnis, M. J., Tripp, T. R., McGlory, C., Baker, S. K., & Phillips, S. M. (2019). Lactalbumin, not collagen, augments muscle protein synthesis with aerobic exercise. Medicine and Science in Sports and Exercise, 52, 1394
1403. Oikawa, S. Y., McGlory, C., D’Souza, L. K., Morgan, A. K., Saddler, N. I., Baker, S. K., et al. (2018). A randomized controlled trial of the impact of protein supplementation on leg lean mass and integrated muscle protein synthesis during inactivity and energy restriction in older persons. The American Journal of Clinical Nutrition, 108, 1060
1068. Oliveira, G. V., Volino-Souza, M., Cordeiro, E. M., Conte-Junior, C. A., & Alvares, T. S. (2020). Effects of fish protein hydrolysate ingestion on endothelial function compared to whey protein hydrolysate in humans. International Journal of Food Sciences and Nutrition, 71, 242
248. Oosthuyse, T., Carstens, M., & Millen, A. M. (2015). Whey or casein hydrolysate with carbohydrate for metabolism and performance in cycling. International Journal of Sports Medicine, 36, 636
646. Park, S. Y., Lee, J.-S., Baek, H.-H., & Lee, H. G. (2010). Purification and characterization of antioxidant peptides from soy protein hydrolysate. Journal of Food Biochemistry, 34, 120
132. Pasiakos, S. M., Lieberman, H. R., & McLellan, T. M. (2014). Effects of protein supplements on muscle damage, soreness and recovery of muscle function and physical performance: A systematic review. Sports Medicine (Auckland, NZ), 44, 655
670. Peeling, P., Binnie, M. J., Goods, P. S. R., Sim, M., & Burke, L. M. (2018). Evidence-based supplements for the enhancement of athletic performance. International Journal of Sport Nutrition and Exercise Metabolism, 28, 178
187. Perandini, L. A., Chimin, P., Lutkemeyer, D. D. S., & Camara, N. O. S. (2018). Chronic inflammation in skeletal muscle impairs satellite cells function during regeneration: Can physical exercise restore the satellite cell niche? The FEBS Journal, 285, 1973
1984. Phillips, S. M. (2017). Current concepts and unresolved questions in dietary protein requirements and supplements in adults. Frontiers in Nutrition, 4, 13. Power, O., Jakeman, P., & FitzGerald, R. J. (2013). Antioxidative peptides: Enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides. Amino Acids, 44, 797
820. Powers, S. K., Duarte, J., Kavazis, A. N., & Talbert, E. E. (2010). Reactive oxygen species are signalling molecules for skeletal muscle adaptation. Experimental Physiology, 95, 1
9. Powers, S. K., & Jackson, M. J. (2008). Exercise-induced oxidative stress: Cellular mechanisms and impact on muscle force production. Physiological Reviews, 88, 1243
1276. Powers, S. K., Ji, L. L., Kavazis, A. N., & Jackson, M. J. (2011). Reactive oxygen species: Impact on skeletal muscle. Comprehensive Physiology, 1, 941
969. Praet, S. F. E., Purdam, C. R., Welvaert, M., Vlahovich, N., Lovell, G., Burke, L. M., et al. (2019). Oral supplementation of specific collagen peptides combined with calf-strengthening exercises enhances function and reduces pain in Achilles tendinopathy patients. Nutrients, 11, 76. Proske, U., & Morgan, D. L. (2001). Muscle damage from eccentric exercise: Mechanism, mechanical signs, adaptation and clinical applications. The Journal of Physiology, 537, 333
345. Raizel, R., Leite, J. S., Hypolito, T. M., Coqueiro, A. Y., Newsholme, P., Cruzat, V. F., et al. (2016). Determination of the anti-inflammatory and cytoprotective effects of L-glutamine and L-alanine, or dipeptide, supplementation in rats submitted to resistance exercise. The British Journal of Nutrition, 116, 470
479. Ranchordas, M. K., Dawson, J. T., & Russell, M. (2017). Practical nutritional recovery strategies for elite soccer players when limited time separates repeated matches. Journal of the International Society of Sports Nutrition, 14, 35.

Applications in nutrition: sport nutrition 549 Roblet, C., Doyen, A., Amiot, J., Pilon, G., Marette, A., & Bazinet, L. (2014). Enhancement of glucose uptake in muscular cell by soybean charged peptides isolated by electrodialysis with ultrafiltration membranes (EDUF): Activation of the AMPK pathway. Food chemistry, 147, 124
130. Rothschild, J. A., & Bishop, D. J. (2020). Effects of dietary supplements on adaptations to endurance training. Sports Medicine, 50, 25
53. Saunders, M. J., Moore, R. W., Kies, A. K., Luden, N. D., & Pratt, C. A. (2009). Carbohydrate and protein hydrolysate coingestions improvement of late-exercise time-trial performance. International Journal of Sport Nutrition and Exercise Metabolism, 19, 136
149. Schunck, M., & Oesser, S. (2013). Specific collagen peptides benefit the biosynthesis of matrix molecules of tendons and ligaments. Journal of the International Society of Sports Nutrition, 10, 23. Shaw, G., Lee-Barthel, A., Ross, M. L., Wang, B., & Baar, K. (2017). Vitamin C-enriched gelatin supplementation before intermittent activity augments collagen synthesis. The American Journal of Clinical Nutrition, 105, 136
143. Shenoy, S., Dhawan, M., & Singh Sandhu, J. (2016). Four weeks of supplementation with isolated soy protein attenuates exercise-induced muscle damage and enhances muscle recovery in well trained athletes: A randomized trial. Asian Journal of Sports Medicine, 7, e33528. Sieljacks, P., Matzon, A., Wernbom, M., Ringgaard, S., Vissing, K., & Overgaard, K. (2016). Muscle damage and repeated bout effect following blood flow restricted exercise. European Journal of Applied Physiology, 116, 513
525. Stafilidis, S., & Arampatzis, A. (2007). Muscle-tendon unit mechanical and morphological properties and sprint performance. Journal of Sports Sciences, 25, 1035
1046. Stauber, W. T., Clarkson, P. M., Fritz, V. K., & Evans, W. J. (1990). Extracellular matrix disruption and pain after eccentric muscle action. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology, 69, 868
874. Stellingwerff, T., Bovim, I. M., & Whitfield, J. (2019). Contemporary nutrition interventions to optimize performance in middle-distance runners. International Journal of Sport Nutrition and Exercise Metabolism, 29, 106
116. Stellingwerff, T., Morton, J. P., & Burke, L. M. (2019). A framework for periodized nutrition for athletics. International Journal of Sport Nutrition and Exercise Metabolism, 29, 141
151. Suetsuna, K., Ukeda, H., & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. The Journal of Nutritional Biochemistry, 11, 128
131. Sugihara, F., Inoue, N., Kuwamori, M., & Taniguchi, M. (2012). Quantification of hydroxyprolyl-glycine (HypGly) in human blood after ingestion of collagen hydrolysate. Journal of Bioscience and Bioengineering, 113, 202
203. Sundgot-Borgen, J., & Garthe, I. (2011). Elite athletes in aesthetic and Olympic weight-class sports and the challenge of body weight and body composition. Journal of Sports Sciences, 29, 101
114. Svensson, R. B., Heinemeier, K. M., Couppe, C., Kjaer, M., & Magnusson, S. P. (2016). Effect of aging and exercise on the tendon. Journal of Applied Physiology (1985), 121, 1237
1246. Thomas, D. T., Erdman, K. A., & Burke, L. M. (2016). Position of the Academy of Nutrition and Dietetics, Dietitians of Canada, and the American College of Sports Medicine: Nutrition and athletic performance. Journal of the Academy of Nutrition and Dietetics, 116, 501
528. Tidball, J. G. (1995). Inflammatory cell response to acute muscle injury. Medicine and Science in Sports and Exercise, 27, 1022
1032. Tidball, J. G. (2005). Inflammatory processes in muscle injury and repair. American Journal of PhysiologyRegulatory, Integrative and Comparative Physiology, 288, 345
353. Van Hall, G., Shirreffs, S. M., & Calbet, J. A. L. (2000). Muscle glycogen resynthesis during recovery from cycle exercise: No effect of additional protein ingestion. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology, 88, 1631
1636. van Loon, L. J., Saris, W. H., Kruijshoop, M., & Wagenmakers, A. J. (2000). Maximizing postexercise muscle glycogen synthesis: Carbohydrate supplementation and the application of amino acid or protein hydrolysate mixtures. The American Journal of Clinical Nutrition, 72, 106
111.

550 Chapter 21 Vieira, C. P., De Oliveira, L. P., Da Re Guerra, F., Dos Santos De Almeida, M., Marcondes, M. C., & Pimentel, E. R. (2015). Glycine improves biochemical and biomechanical properties following inflammation of the Achilles tendon. Anatomical Record (Hoboken, NJ: 2007), 298, 538
545. Walrand, S., Chiotelli, E., Noirt, F., Mwewa, S., & Lassel, T. (2008). Consumption of a functional fermented milk containing collagen hydrolysate improves the concentration of collagen-specific amino acids in plasma. Journal of Agricultural and Food Chemistry, 56, 7790
7795. Watanabe-Kamiyama, M., Shimizu, M., Kamiyama, S., Taguchi, Y., Sone, H., Morimatsu, F., et al. (2010). Absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats. Journal of Agricultural and Food Chemistry, 58, 835
841. Wu, R., Wu, C., Liu, D., Yang, X., Huang, J., Zhang, J., et al. (2015). Overview of antioxidant peptides derived from marine resources: The sources, characteristic, purification, and evaluation methods. Applied Biochemistry and Biotechnology, 176, 1815
1833. Yin, M., Ikejima, K., Arteel, G. E., Seabra, V., Bradford, B. U., Kono, H., et al. (1998). Glycine accelerates recovery from alcohol-induced liver injury. The Journal of Pharmacology and Experimental Therapeutics, 286, 1014
1019. Yin, M., Rusyn, I., Schoonhoven, R., Graves, L. M., Rusyn, E. V., Li, X., et al. (2000). Inhibition of chronic rejection of aortic allografts by dietary glycine. Transplantation, 69, 773
780. Zdzieblik, D., Oesser, S., Baumstark, M. W., Gollhofer, A., & Ko¨nig, D. (2015). Collagen peptide supplementation in combination with resistance training improves body composition and increases muscle strength in elderly sarcopenic men: A randomised controlled trial. The British Journal of Nutrition, 114, 1237
1245. Zdzieblik, D., Oesser, S., Gollhofer, A., & Ko¨nig, D. (2017). Improvement of activity-related knee joint discomfort following supplementation of specific collagen peptides. Applied physiology, Nutrition, and Metabolism 5 Physiologie appliquee, nutrition et metabolisme, 42, 588
595. Zebrowska, A., Mizia-Stec, K., Mizia, M., Gasior, Z., & Poprzecki, S. (2015). Omega-3 fatty acids supplementation improves endothelial function and maximal oxygen uptake in endurance-trained athletes. European Journal of Sport Science, 15, 305
314.

CHAPTER 22

Application in nutrition: cholesterollowering activity Carmen Lammi1, Carlotta Bollati1, Gilda Aiello2 and Anna Arnoldi1 1

Department of Pharmaceutical Sciences, University of Milan, Milan, Italy, 2Department of Human Science and Quality of Life Promotion, Telematic University San Raffaele, Rome, Italy

22.1 Introduction Cardiovascular disease (CVD) is a major cause of deaths in industrialized countries and a constantly growing cause of morbidity and mortality worldwide (De Backer et al., 2003). Hypercholesterolemia, which is considered as the presence of high levels of cholesterol in the blood, is one of the main risk factors for CVD progression that may be prevented by lifestyle changes, including diet (Stampfer, Hu, Manson, Rimm, & Willett, 2000). In this context, plant proteins are considered useful regulators of serum cholesterol concentrations (Sirtori, Galli, Anderson, Sirtori, & Arnoldi, 2009), since the dietary intake of soy protein and soy-based food products is linked with a reduction in hypercholesterolemia (Ramdath, Padhi, Sarfaraz, Renwick, & Duncan, 2017). Specifically, the substantially lower CVD mortality and morbidity in Asian countries than in Western countries has been explained with considerably higher intakes of soy protein in these countries; indeed, prospective observational studies in the Japanese population (Nagata, Takatsuka, Kurisu, & Shimizu, 1998) and in Chinese women (Zhang et al., 2003) indicate a reduction of total and low-density lipoprotein cholesterol (LDL-C) as well as of ischemic and cerebrovascular events with a regular daily intake of soy foods. Other studies have indicated that the consumption of different grain legumes may be beneficial for high cholesterol prevention (Arnoldi, Zanoni, Lammi, & Boschin, 2015). The observation that the kind of dietary protein may substantially influence the blood cholesterol level and the consideration that during digestion food proteins are hydrolyzed in the gastrointestinal tract have suggested that the observed outcome might be attributed to the release of hypocholesterolemic peptides during protein cleavage. Indeed, over the years, different researchers have identified numerous hypocholesterolemic hydrolysates from different food proteins, out of which a number of bioactive peptides have been sorted out

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00017-0 © 2021 Elsevier Inc. All rights reserved.

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552 Chapter 22 and characterized (Chakrabarti, Guha, & Majumder, 2018; Lammi, Aiello, Boschin, & Arnoldi, 2019).

22.2 Rationale: peptides activity and characterization This chapter will discuss the main hypocholesterolemic peptides identified from plant and animal food sources. Cholesterol homeostasis is maintained by a complex mechanism of sterol absorption, anabolism, catabolism, and excretion (Nagaoka, 2018). The very diversified structures of the hypocholesterolemic peptides so far identified explain why they exert their activity through different mechanisms of action that will be extensively described in this chapter. Indeed, the literature describes peptides from different food matrices that are able to decrease cholesterol by one of the following mechanisms: (1) binding of the bile acids, (2) in vitro inhibition of the micellar solubility of cholesterol, (3) in vivo impairment of cholesterol absorption, (4) inhibition of the activity of 3-hydroxy-3methylglutaryl CoA reductase (HMGCoAR) and PCSK9, and (5) modulation of the expression of proteins involved in cholesterol metabolism. Although clinical studies are more in favor of a relevant role of plant proteins in the prevention of hypercholesterolemia, bioactive peptides have been isolated also from some animal sources. Therefore this chapter will consider peptides either from plant or animal sources.

22.3 Peptides from plant proteins 22.3.1 Soybean peptides Soybean is the most diffuse source of plant proteins for human nutrition. In the presence of high and mild hypercholesterolemia, numerous clinical studies have associated soy food consumption with a reduction of total and LDL-C levels (Harland and Haffner, 2008; Jenkins et al., 2010; Sirtori, Eberini, & Arnoldi, 2007). Specifically, a systemic metaanalysis of available randomized controlled studies, mainly in subjects with moderate hypocholesterolemia, has confirmed that the consumption of 25 g per day of soybean protein leads to a reduction of mean total cholesterol (TC) by 0.22 mmol/L [95% confidence interval (CI): 20.142 to 20.291, P , .0001], mean LDL-cholesterol by 0.23 mmol/L (95% CI: 20.160 to 20.306, P , .0001), and mean blood triglycerides by 0.08 mmol/L (95% CI: 20.004 to 20.158, P 5 .04), concluding that a modest amount of soybean protein into the diet of mild hypercholesterolemic patients results in a small but significant 6% reduction of LDL-C (Harland and Haffner, 2008). These results have substantiated the Food and Drug Administration’s approval of the health claim that a regular daily intake of soybean protein (25 g) is useful to reduce blood cholesterol level and

Application in nutrition: cholesterol-lowering activity 553 therefore the CVD risk (FDA, 1999). In this context, it is not surprising that numerous investigations have tried to sort out bioactive peptides in the soy protein sequences. Recently, a total protein extract from soybean has been hydrolyzed with either pepsin or trypsin and, after analysis by mass spectrometry, the biological activity of the hydrolysates has been evaluated either in vitro with biochemical assays or in situ using suitable cell models. Both hydrolysates inhibit the HMGCoAR activity increasing the low-density lipoprotein receptor (LDLR) level on HepG2 cell membranes, with a consequently improved capacity of the HepG2 cells to uptake LDL from the extracellular environment (Lammi, Arnoldi, & Aiello, 2019). These results are in line with previous studies in which some hypocholesterolemic peptides have been identified in the sequences of glycinin and β-conglycinin, two major soybean globulins (Lammi, Zanoni, Arnoldi, & Vistoli, 2015; Lammi, Zanoni, & Arnoldi, 2015). Three peptides (LPYP, IAVPTGVA, and IAVPGEVA) have been isolated and characterized after digesting glycinin with trypsin or pepsin, which are able to inhibit the activity of HMGCoAR (Pak, Koo, Kasymova, & Kwon, 2005). In vitro experiments performed with the catalytic domain of HMGCoAR showed that these peptides act as competitive inhibitors. Further experiments in HepG2 cells have demonstrated that the inhibition leads to an increase of the LDLR protein levels by the activation of the SREBP2 pathway and an enhanced LDL-uptake in the same cells (Lammi, Zanoni, & Arnoldi, 2015). Moreover, these peptides are able to increase the phosphorylation level of HMGCoAR on Ser 872 (the inactive form of HMGCoAR) via the activation of the AMPK pathway. Being the intestinal absorption a critical issue for the bioavailability of these peptides, a detailed study has been carried out to assess the absorbability and stability of these peptides in the presence of differentiated human intestinal Caco-2 cells (Aiello et al., 2018). These experiments have shown that the transport of peptide IAVPGEVA is more efficient than that of IAVPTGVA and that LPYP is very poorly transported. In addition, IAVPGEVA and IAVPTGVA are also degraded by active peptidases, which are expressed on the brush border of mature Caco-2 cells. Degradation, however, does not mean an automatic loss of activity. In fact, an in silico study has permitted us to predict that the longest fragments (AVPTGVA and AVPGEVA) maintain the ability to inhibit the HMGCoAR since they retain all the key contacts of the parent peptides, whereas the shortest metabolites (IAVPT and IAVP) show less stable complexes, in which they are able to maintain the ion-pairs elicited by their carboxyl terminus, but lose the key contacts involving their amino group (Aiello et al., 2018). By using the same model of the intestinal barrier, the absorption of peptides deriving from the hydrolysis of β-conglycinin has been assessed (Amigo-Benavent et al., 2014). Among absorbed peptides, two interesting fragments of β-conglycinin (YVVNPDNDEN and YVVNPDNNEN) have been identified (Lammi, Zanoni, Arnoldi, & Vistoli, 2015).

554 Chapter 22 Experiments in HepG2 cells aimed at investigating their effects on cholesterol metabolism showed that they are able to upregulate the LDLR protein levels and behave as competitive inhibitors of HMGCoAR activity with a statin-like mechanism (Lammi, Zanoni, Arnoldi, & Vistoli, 2015). Interestingly, the former is a fragment of peptide LRVPAGTTFYVVNPDNDENLRMIA, previously shown to increase the LDL-uptake and degradation in hepatocytes (Lovati et al., 2000). In another study, peptide FVVNATSN has been identified as able to increase the LDL-receptor mRNA in HepG2 cells. However, the researchers did not provide any corroborative evidences from in vivo experiments (Cho, Juillerat, & Lee, 2008). LPYPR and WGAPSL are two peptides deriving from soybean that displace cholesterol from mixed micelles in vitro, that is, behave similarly to plant sterol and stanols. An in vivo study has shown that both peptides increase plasma cholesterol and LDL in mice fed with a hypercholesterolemic diet (Zhang et al., 2013). VAWWMY is a glycinin-derived peptide, named soy statin, which acts as an inhibitor of cholesterol absorption in vivo. More in details, it has been demonstrated that this peptide binds bile acids and through this mechanism of action, it reduces the cholesterol level in vivo (Nagaoka, Nakamura, Shibata, & Kanamaru, 2010). Soybean is a rich source of lunasin, a unique 43-amino acid polypeptide sequence encoded within the soybean Gm2S-1 gene (Odani, Koide, & Ono, 1987), with a concentration ranging from 4.4 to 70.5 mg lunasin/g of protein depending on the genotypes (Herna´ndezLedesma, Hsieh, & de Lumen, 2009). Recent studies have demonstrated that this protein is present also in cereals and other seeds, such as wheat, barley, oat, rye, quinoa, and amaranth (Jeong et al., 2009). Although lunasin has been investigated mostly for its anticancer activity (Galvez, Chen, Macasieb, & de Lumen, 2001), evidences suggest that this polypeptide is also antioxidant, hypocholesterolemic, and anti-inflammatory. The transcriptional activation of HMGCoAR via specific acetylation of histone H3 by P300/CBP-associated factor (PCAF) is an essential step in hepatic cholesterol biosynthesis. In relation to this, the capacity of lunasin of reducing serum LDL-C levels is based on different mechanisms. Lunasin selectively reduces the acetylation of the histone H3 tail at K14 position by PCAF, thus lowering the HMGCoAR gene expression and making HMGCoAR unavailable for cholesterol biosynthesis, and also increases the expression of the LDLR gene, which raises the amount of LDLR to clear LDL-C from the bloodstream. In the presence of lunasin, the levels of SP1 proteins, the coactivators of SREBP, increase twice more than without lunasin (Galvez, 2012). Furthermore, a study revealed that a casein diet supplemented with a lunasin-enriched soy extract lowered the LDL-C levels more than a simple casein diet in pigs carrying mutated LDLR gene (Galvez, 2012). Finally, lunasin downregulates the proprotein convertase subtilisin/kexintype 9 (PCSK9) via the downregulation of the hepatocyte nuclear factor-1α (HNF-1α) (Gu et al., 2017).

Application in nutrition: cholesterol-lowering activity 555 Interestingly, the final effects of lunasin are very similar to those of some soybean peptides (IAVPGEVA, IAVPTGVA, and LPYP), although the modes of action are quite different. In particular, lunasin inhibits the expression of HMGCoAR, which leads to an increased LDLR expression at transcriptional level (Galvez, 2012), whereas the other peptides produce a direct inhibition of HMGCoAR activity leading to an increase of the LDLR protein level and finally to an improved ability of HepG2 cells to clear extracellular LDL-C (Lammi, Zanoni, Arnoldi, & Vistoli, 2015). A study has provided evidence that lunasin is absorbed in the intestine, since it has been found intact in the plasma of volunteers after soybean consumption (Dia, Torres, De Lumen, Erdman, & De Mejia, 2009). The high bioavailability has been explained with the simultaneous presence of protease inhibitors, which allow 30% of lunasin to reach the target tissues.

22.3.2 Lupin peptides Lupin is grain legume whose food applications have expanded during the last two decades owing to the protein content ranging from 35%
40% and the lack of commercially available genetically modified varieties. After a pioneer investigation in the hyperlipidemic rat model, which has shown that its protein may have hypocholesterolemic effects (Sirtori et al., 2004), human studies have confirmed the interest of this protein as cholesterollowering ingredient (Sirtori et al., 2012). In particular, a 4-week-long double-blind randomized clinical trial has evaluated the potential hypocholesterolemic effects of lupin proteins, versus casein as a control protein, in mild hypercholesterolemic patients (Sirtori et al., 2012). The consumption of dietary bars containing lupin proteins (30 g/day) results in a significant reduction of TC (211.6 mg/dL 5 24.2%), whereas no significant changes are observed in the subjects consuming the control protein casein. These observations have stimulated investigation initially on lupin protein hydrolysates and then on single peptides. A study on human hepatic HepG2 cells has provided the first evidences of the hypocholesterolemic activity of lupin peptides and a detailed elucidation of the molecular mechanism of action. Both tryptic and peptic hydrolysates from lupin protein are able to interfere with the HMGCoAR activity, upregulating the LDLR due to the activation of SREBP-2, through the modulation of the Akt/GSK3β pathway (Lammi, Zanoni, Scigliuolo, D’Amato, & Arnoldi, 2014). In addition, from a functional point of view, both peptic and tryptic hydrolysates increase the LDLR protein levels inducing an increased LDL-uptake by HepG2 cells. Absorption experiments performed using differentiated human intestinal Caco-2 cells demonstrated that only 8 peptic and 11 tryptic peptides among all those present in the hydrolysates are transported by the mature intestinal cells (Lammi, Aiello, Vistoli, Zanoni, et al., 2016). Among absorbed peptides, the research activity was mainly focused on the decapeptide P5 (LILPKHSDAD) and the nonapeptide P7 (LTFPGSAED), both deriving from the peptic

556 Chapter 22 hydrolysis of β-conglutin. Interestingly, an in silico approach has suggested that both peptides might be able to bind the HMGCoAR catalytic site inhibiting the enzyme activity. Indeed, biochemical and cellular studies have confirmed that these peptides are able to modulate in a positive manner cholesterol metabolism leading to an increase of the LDLR protein levels (Zanoni, Aiello, Arnoldi, & Lammi, 2017b). Briefly, both peptides inhibit in vitro the HMGCoAR activity leading to an increase of LDLR protein levels due to the activation of the SREBP-2 transcription factors. Moreover, through the activation of the AMPK pathway, both peptides lead to an increase of the phosphorylation level of HMGCoAR that becomes inactive. Another useful observation has been provided by a recent clinical study that has shown that the consumption of 30 g/day lupin protein for 3 months leads to 12.7% reduction of plasma PCSK9 level (Lammi, Zanoni, Calabresi, & Arnoldi, 2016). The molecular mechanism of this modulation has been investigated using HepG2 cells, demonstrating that either the peptic or the tryptic lupin hydrolysates decrease the mature PCSK9 protein levels and the secretion in the extracellular environment. An investigation on absorbable peptides indicated that P5 is mainly responsible for this mechanism of action, since it reduces the mature PCSK9 protein level and its secretion through its ability to decrease its transcription factor HNF-1α (Zanoni et al., 2017b). In addition, P5 is also able to inhibit the protein
protein interaction (PPI) between PCSK9 and LDLR with an IC50 equal to 1.6 μM. A bioinformatics tool has permitted us to build an in silico docking model of the interaction of P5 with the LDLR binding site of PCSK9, thus allowing us to explain how this peptide impairs the PPI between these two crucially important proteins (Lammi, Zanoni, Aiello, Arnoldi, & Grazioso, 2016). Interestingly, P5 is the first hypocholesterolemic peptide characterized by this dual inhibitory activity. T9 (GQEQSHQDEGVIVR) is another absorbed peptide, deriving from the tryptic hydrolysis of lupin β-conglutin, which is able to exert a direct inhibition of the PCSK9/ LDLR PPI, even though with a lower potency (Lammi, Zanoni, Aiello, et al., 2016). However, only T9 inhibits the PPI between the LDLR and the dangerous gain of function PCSK9 mutant named PCSK9D374Y (Grazioso, Bollati, Sgrignani, Arnoldi, & Lammi, 2018), which is responsible for a severe form of hypercholesterolemia. Furthermore, this peptide is also able to reduce the mature PCSK9D374Y protein level in HepG2 cells, showing unique features among food-derived peptides endowed with cholesterol-lowering activity (Lammi, Bollati, Lecca, Abbracchio, & Arnoldi, 2019).

22.3.3 Hempseed peptides Hempseed (Cannabis sativa L.) contains about 25%
30% proteins and it is currently gaining a great interest in human nutrition (Aiello et al., 2016). A recent article has reported the preparation of different hydrolysates from hempseed protein produced using pepsin, trypsin,

Application in nutrition: cholesterol-lowering activity 557 pancreatin, or a mixture of these enzymes (Aiello, Lammi, Boschin, Zanoni, & Arnoldi, 2017). The extensive protein cleavage exerted by pancreatin alone or in combination with pepsin leads to the generation of hempseed hydrolysates with a scarce capacity of modulating in vitro the HMGCoAR activity, whereas pepsin and trypsin generate more active hydrolysates. The molecular mechanism through which the peptic hydrolysate exerts a cholesterol-lowering activity has been elucidated in depth using HepG2 cells. Upon HMGCoAR inhibition, hempseed peptides induce an augment of the LDLR protein levels that has the consequence of an increased ability of HepG2 cells to absorb the LDL from the extracellular environment (Zanoni, Aiello, Arnoldi, & Lammi, 2017a). Similarly to statin, the hempseed hydrolysate increases the mature PCSK9 protein levels versus the untreated samples: this is the main divergence of action between hempseed and lupin peptides. Literature does not report any specific hempseed hypocholesterolemic peptides yet.

22.4 Hypocholesterolemic peptide from other seeds: amaranth, cowpea, and rice Cowpea is an annual grain legume from the genus Vigna. After simulation of human digestion, peptides generated from raw and cooked cowpea proteins modulate in vitro lipid metabolism. Cowpea peptides inhibit the HMGCoAR activity and reduce cholesterol micellar solubilization in vitro (Marques, Fontanari, Pimenta, Soares-Freitas, & Areas, 2015). Rice is one of the main world crops, whose pericarp removing produces huge amounts of bran, which contains 10%
16% of highly nutritional protein that may be separated and valorized in different ways. In particular, it has been shown that rice bran peptides, obtained by trypsin hydrolysis, inhibit in vitro the micellar solubility of cholesterol (Zhang and Yokoyama, 2012). Furthermore, Wang and coworkers showed that rice bran peptides decrease serum TC levels accompanied with increasing excretion of fecal steroids in vivo, confirming that rice bran peptides bind to bile acids and inhibit the micellar solubility of cholesterol in vitro (Wang, Shimada, Kato, Kusada, & Nagaoka, 2015). Amaranth is a pseudocereal cultivated especially in South America. A recent investigation has provided evidences that peptides derived from amaranth protein hydrolysis exert a hypocholesterolemic effect. In particular, in vitro experiments using the catalytic domain of HMGCoAR demonstrated that the peptides GGV, IVG, and VGVL are able to inhibit the HMGCoAR activity (Soares, Mendonca, de Castro, Menezes, & Areas, 2015). These findings suggest that these peptides act as HMGCoAR competitive inhibitors, similarly to some peptides from soybean (IAVPGEVA, IAVPTGVA, LPYP, YVVNPDNDEN, and YVVNPDNNEN) and lupin (P5 and P7). However, more detailed studies should be performed to better characterize the hypocholesterolemic mechanism of action at cellular level and to assess the potential bioavailability of these peptides.

558 Chapter 22

22.5 Peptides from animal sources 22.5.1 Milk peptides Numerous cellular and animal studies suggest that milk proteins, either casein or whey proteins, are very interesting sources of bioactive peptides, which are known to exhibit many physiological effects (Meisel, 2004). Nagaoka et al. identified, for the first time IIAEK, a pentapeptide named lactostatin, as the active component responsible at least in part for the hypocholesterolemic action of a tryptic hydrolysate of cow milk β-lactoglobulin (Nagaoka, 2019; Nagaoka et al., 2001). This peptide shows a greater activity in the modulation of lipid metabolism. In particular, using a Caco-2 cell screening method, the β-lactoglobulin-derived peptides IIAEK, GLDIQK, ALPMH, and VYVEELKPTPEGDLEILLQK have been demonstrated to inhibit the cholesterol absorption in vitro (Nagaoka et al., 2001). Moreover, in rats fed by oral administration, the hypocholesterolemic activity IIAEK was better than that of β-sitosterol. The cholesterollowering mechanism of lactostatin has been investigated also in vitro in HepG2 cells, in which extracellular signal-regulated kinase pathway is involved as well as the calciumchannel-related mitogen-activated protein kinase signaling pathway of cholesterol degradation (Morikawa, Kondo, Kanamaru, & Nagaoka, 2007). More in details, lactostatin induces the calcium channel in cholesterol 7a-hydroxylase (CYP7A1) transactivation in HepG2 cells (Morikawa, Ishikawa, Kanamaru, Hori, & Nagaoka, 2007). As CYP7A1 is the rate-limiting enzyme for cholesterol degradation in the process of bile acid synthesis, the regulation of CYP7A1 gene expression and activity is a useful approach to modulate hypercholesterolemia and atherosclerosis. In fact, it has been established that the overexpression of CYP7A1 ameliorates hypercholesterolemia and atherosclerosis in animal models (Spady, Cuthbert, Willard, & Meidell, 1995, 1998). Upon binding to its receptor and transactivation of CYP7A1, IIAEK and its fragments (IAEK, AEK, and EK) activate CYP7A1 gene expression (Morikawa et al., 2007). Another in vitro approach effort to identify the hypocholesterolemic peptide has evaluated the increase of cholesterol CYP7A1 mRNA in HepG2 cells in vitro. In this system, a mixture of milk casein peptides increased CYP7A1 mRNA without corroborative evidence from animal experiments in vivo (Nass et al., 2008). Peptide HIRL, obtained after hydrolysis of β-lactoglobulin, shows many health-promoting activities, including antinociceptive, anti-stress, and memory-enhancing activities (Ohinata et al., 2007; Yamauchi, Ohinata, & Yoshikawa, 2003; Yamauchi, Wada, Yamada, Yoshikawa, & Wada, 2006). In addition, HIRL is able to reduce serum LDL and VLDL via neurotensin receptor 2 (NT2), similarly to neurotensin in animal studies (Yamauchi et al., 2003). As mentioned before, milk-derived peptides may exert cholesterol-lowering action modulating the lipid metabolism by the impairment of micellar cholesterol solubility. In this

Application in nutrition: cholesterol-lowering activity 559 context, the peptides LQPE, VLPVPQ, and VAPFPE, obtained by the casein protein hydrolysate, have been recently identified (Jiang et al., 2020). One of the mechanisms through which these peptides inhibit the intestinal cholesterol absorption in Caco-2 cells is linked to the reduction of Niemann-Pick C1-like 1 (NPC1L1) protein levels; this transporter mediates the passage of cholesterol from the intestinal lumen to the enterocytes and is thus crucial for cholesterol absorption (Altmann et al., 2004). It has also been demonstrated that VLPVPQ downregulates the acetyl-CoA-acetyltransferase 2 (ACAT2) gene expression, obtaining the inhibition of the micellar cholesterol solubility (Castro-Torres et al., 2014; Jiang et al., 2020). Additionally, both LQPE and VLPVPQ increase the transcription of ABCA1, which is responsible for the biogenesis of high-density lipoprotein (HDL), indeed by promoting HDL production, these peptides could decrease the intracellular cholesterol levels (Jiang et al., 2020).

22.5.2 Meat peptides Dietary beef proteins show cholesterol-lowering activity in rats fed with a rich-cholesterol diet (Jacques, Deshaies, & Savoie, 1986) and, in human studies, where the hypocholesterolemic effects of a soy protein diet were reported to be similar to those of a beef protein-based diet (Holmes, Rubel, & Hood, 1980). A cattle heart protein hydrolysate and a cattle heart protein hydrolysate ultrafiltrate exert strong hypocholesterolemic activity in animal experiments and suppress the cholesterol absorption in enterocytes. An in vivo study has shown that the ultrafiltrate hydrolysate has a greater ability than the non-ultrafiltrated sample to decrease the cholesterol levels in both serum and liver of rats, suggesting that the ultrafiltration process increases the concentration of bioactive species (Nakade et al., 2009). In a recent study, the sequence of the active dipeptide FP (Phe-Pro) has been identified in the ultrafiltrate hydrolysate and its hypocholesterolemic properties have been assessed by in vivo studies and in vitro assays. The oral administration of FP in rats fed a hyperlipidemic diet results in significant reductions in serum total and non-HDL-cholesterol concentrations. The atherogenic index (TC/HDL-cholesterol) is also reduced, suggesting that FP may be responsible for the cholesterol-lowering effect of heart protein hydrolysate ultrafiltrate (Banno et al., 2019). The proton-coupled oligopeptide transporter 1 (PepT1) is a highly preserved transporter present in various mammalian species and it has an important role in the protein assimilation process (Hu et al., 2008). It has been shown that peptide FP is absorbed by Caco-2 cells through PepT1 and it downregulates ABCA1 expression, thus mediating a novel cholesterol-lowering pathway via a nonmembrane receptor at intestinal level. In PepT1KO mice, the FP-induced hypocholesterolemic activity disappears, confirming that PepT1 is a crucial target for the improvement of cholesterol metabolism (Banno et al., 2019). In the same study, FP significantly increases the fecal steroid and cholesterol

560 Chapter 22 excretion in vitro. This result is in agreement with the capacity of peptide FP to modulate cholesterol micellar solubility and cholesterol absorption ability in Caco-2 cells (Banno et al., 2019).

22.5.3 Fish peptides Fish consumption is known to provide health benefits in both animal and humans studies. In animal experiments, proteins from different fish species display hypocholesterolemic effects when compared with casein. The cholesterol-lowering mechanisms include the increased hepatic LDLR expression, the enhancement of fecal cholesterol and bile acid excretions and CYP7A1 expression levels (Hosomi et al., 2012; Shukla et al., 2006; Zhang and Beynen, 1993). In a very recent study, 10 peptides with antioxidant activities have been purified and identified from protein hydrolysate of miiuy croaker (Miichthys miiuy) muscle and, among them, VIAPW and IRWWW peptides show inhibitory activity on lipid accumulation in hepatocytes (He, Pan, Chi, Sun, & Wang, 2019). Cellular experiments on hepatic HepG2 cells, in which lipid accumulation is induced with an oleic acid treatment, show that the two pentapeptides are able to modulate the AMPK signaling pathway (Wang, Xi, He, Chi, & Wang, 2020). The literature reports that AMPK has an enzymatic activity capable of AMP-activated phosphorylation and inhibition of acetyl-CoA carboxylase (ACC) and HMGCoA reductase, two rate-limiting enzymes in fatty acid (FA) and cholesterol metabolism, whose expression may be upregulated by the LDLR-sterol regulatory element-binding protein 1c and 2 (SREBP-1, SREBP-2) (Carling, Zammit, & Hardie, 1987; Yeh, Lee, & Kim, 1980). By downregulating the expression of SREBP-1c, SREBP-2, FA synthetase (FAS), ACC, and HMGCoAR genes levels, VIAPW and IRWWW can rapidly decrease the cholesterol biosynthesis. In addition, both peptides significantly reduce the TC accumulation as a function of the dose with a final decrease of the lipid accumulation in hepatic cells (Wang et al., 2020).

22.5.4 Egg peptides Although there is a general tendency to moderate the consumption of eggs to avoid an excessive dietary cholesterol intake, several articles report the potential beneficial effects of this food in CVD prevention (Alexander, Miller, Vargas, Weed, & Cohen, 2016; Fernandez, 2006; Nakamura et al., 2006; Qureshi et al., 2007; Rong et al., 2013). In particular, there are numerous evidences supporting a positive role of egg proteins in cholesterol homeostasis. The main components of egg white protein are ovalbumin (54%), ovotransferrin (13%), ovomucoid (11%), ovomucin (3%
4%), and lysozyme (3.5%) (Nakamura, Takayama, Nakamura, & Umemura, 1980). Ovomucin shows cholesterol

Application in nutrition: cholesterol-lowering activity 561 modulating properties both in vivo and in vitro studies (Nagaoka, Masaoka, Zhang, Hasegawa, & Watanabe, 2002). Rats fed with egg ovomucin have lower serum and liver TC levels, and this protein inhibits cholesterol absorption in Caco-2 cells. The cholesterolcontaining micelles interact with intestinal epithelial cells inhibiting the absorption of cholesterol and, additionally, the reabsorption of bile acids in the ileum is also promoted, with a mechanism that may explain the cholesterol-lowering effect of ovomucin (Nagaoka et al., 2002). The pepsin hydrolysates of the water-soluble protein fraction of egg white, containing ovalbumin and ovotransferrin, promote the inhibition of the cholesterol micellar solubility. In rats fed egg white protein, reduced cholesterol levels are observed in the serum, liver, and intestinal mucosa as well as increased fecal excretions of sterols and bile acids (Matsuoka et al., 2008). In a human study involving young women with moderate hypercholesterolemia, the decrease of total serum cholesterol and the increase in HDL were more evident in the groups treated with egg white protein than in those fed tofu or cheese. One of the last in vivo studies reports the ability of peptide VSEE, a duck egg white-derived peptide, to decrease the content of serum TC, triglyceride, LDL-C, and increase the HDL-cholesterol levels after 8-week treatments in ovariectomized rats. The intestinal absorption of VSEE has been analyzed by Caco-2/HT-29 cocultured cell monolayer and pharmacokinetic experiments that have shown that VSEE is integrally absorbed by the intestine to enter into the circulation (Guo et al., 2019).

22.5.5 Royal jelly peptides Royal jelly is secreted by the hypopharyngeal and mandibular glands of the worker honeybees and contains high amounts of essential amino acids. Recent studies suggest that the consumption of a royal jelly hydrolysate ameliorates hypercholesterolemia in an experimental animal model and human subjects (Guo et al., 2007; Vittek, 1995). Specifically, the micellar solubility of cholesterol is significantly decreased in vitro in the presence of major royal jelly protein 1(MRJP1) compared with casein. Liver bile acids levels are significantly increased, and cholesterol 7a-hydroxylase (CYP7A1) mRNA and protein tend to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels are significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. Therefore the cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids, induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion, and also enhances the hepatic cholesterol catabolism. In fact, the increase of SREBP-1 and LDLR protein levels is also detected in HepG2 cells treated with MRJP1 peptides (Kashima et al., 2014).

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22.6 Structure
activity relationship of hypocholesterolemic peptides The preceding paragraphs have shown that many food peptides have been sorted out that exert many biological effects. This suggests that these peptides may represent good candidates for the development of new therapeutic agents as well as functional foods and/or supplements. For attaining fruitful applications, it would be important to identify which function as inhibitors of known target enzymes and which are involved in cellular pathways related with specific diseases. An even superficial review of the available literature clearly indicates that some activities have been studied much more extensively than others. In particular, the literature provides clear evidences on the ability of many food peptides to act as inhibitors of angiotensinconverting enzyme (ACE) or dipeptidyl peptidase-IV (DPP-IV) enzyme. In both cases, the huge amounts of available data permit to successfully speculate the structure
function relationship. On the contrary, only scarce and incomplete information is available in the case of hypocholesterolemic peptides, a fact that underlines the importance of filling the structure
function relationship knowledge gap. Basically, behind the hypocholesterolemic activity of peptides, different mechanisms of action may occur. In particular, to function as a competitive inhibitor of HMGCoAR, a peptide should mimic the hydroxymethylglutaryl moiety. To achieve this goal, the conformation and the side chain groups play a more important role than the total hydrophobicity. Moreover, the correlation of the inhibitory activity with the peptide length is still unclear. Based on these considerations, it has been assessed that VPTG and VPGE fragments acquire bioactive “turn” conformations. The Pro residue in each soybean and lupin peptide mimics the nicotinamide moiety of NADPH, which is the enzyme cofactor (Pak et al., 2005; Pak, Koo, Kwon, & Yun, 2012). Moreover, it has been established that a Leu, Ile, and/or Tyr residue at the N-terminus and a Glu residue at the C-terminus play important roles for the peptide inhibitory property (Pak et al., 2005; Pak et al., 2012). Indeed, all these peptides satisfy these features. However, only peptide LTFPGSAED comprises two negatively charged side chains at the C-terminal tail that improve its ability to interact with the receptor site and make it the best HMGCoAR inhibitor. Recently, it has been figured out that during an absorption study of LTFPGSDAD, Caco-2 cells produce a metabolite, LTFPG, which is less active than the parent peptide as HMGCoAR inhibitor (Lammi et al., 2020). This result clearly confirms the importance of the negatively charged side chains at the C-terminal tail for achieving an effective enzyme inhibition. As regards the physical interaction of peptides with bile acids and micelles, the presence of hydrophobic cores in peptides is important for cholesterol and bile acid binding. Amphipathicity of peptides is also thought to enhance their ability to interact with free and micellar bile acids, which can decrease the emulsification, solubility, and total amount of

Application in nutrition: cholesterol-lowering activity 563 dietary cholesterol absorbed in the small intestine. In fact, the sequence EK is important for the hypocholesterolemic function of IIAEK, as dipeptides having a C-terminal lysine are important to identify a hypocholesterolemic peptide (Morikawa et al., 2007). In addition, the screening of dipeptides having a C-terminal lysine evaluated with CYP7A1 mRNA level revealed that the DK, EK, and WK dipeptides can significantly increase the CYP7A1 mRNA level in HepG2 cells (Morikawa et al., 2007).

22.7 Summary The literature provides interesting insights regarding the hypocholesterolemic activity of food-derived peptides. It is clear that their potential cholesterol-lowering effect might be attributed to more than one mechanism of action that involves not only the regulation of key targets in the cholesterol metabolism but also physicochemical interactions, which lead to a reduction of cholesterol absorption and increase of its extraction. However, compared to some other food-derived peptides displaying other biological activities (ACE and DPP-IV inhibitors), hypocholesterolemic peptides are much less studied. Doubtlessly, their potential use in nutritional application is desirable, however, only few of them are assessed in vivo. Therefore more efforts need to be pursued for singling out good candidates for the development of functional foods or dietary supplements. In addition, increasing investigations will be also useful to establish a good correlation between the structure and the hypocholesterolemic function as well as the assessment of absorption, distribution, metabolism, excretion, and toxicity of the peptides and their derivatives.

References Aiello, G., Fasoli, E., Boschin, G., Lammi, C., Zanoni, C., Citterio, A., & Arnoldi, A. (2016). Proteomic characterization of hempseed (Cannabis sativa L.). Journal of Proteomics. Aiello, G., Ferruzza, S., Ranaldi, G., Sambuy, Y., Arnoldi, A., Vistoli, G., & Lammi, C. (2018). Behavior of three hypocholesterolemic peptides from soy protein in an intestinal model based on differentiated Caco-2 cell. Journal of Functional Foods, 45, 363
370. Aiello, G., Lammi, C., Boschin, G., Zanoni, C., & Arnoldi, A. (2017). Exploration of potentially bioactive peptides generated from the enzymatic hydrolysis of hempseed proteins. Journal of Agricultural and Food Chemistry, 65, 10174
10184. Alexander, D. D., Miller, P. E., Vargas, A. J., Weed, D. L., & Cohen, S. S. (2016). Meta-analysis of egg consumption and risk of coronary heart disease and stroke. Journal of the American College of Nutrition, 35, 704
716. Altmann, S. W., Davis, H. R., Zhu, L. J., Yao, X. R., Hoos, L. M., Tetzloff, G., . . . Graziano, M. P. (2004). Niemann-Pick C1 like 1 protein is critical for intestinal cholesterol absorption. Science (New York, N.Y.), 303, 1201
1204. Amigo-Benavent, M., Clemente, A., Caira, S., Stiuso, P., Ferranti, P., & del Castillo, M. D. (2014). Use of phytochemomics to evaluate the bioavailability and bioactivity of antioxidant peptides of soybean β-conglycinin. Electrophoresis, 35, 1582
1589. Arnoldi, A., Zanoni, C., Lammi, C., & Boschin, G. (2015). The role of grain legumes in the prevention of hypercholesterolemia and hypertension. Critical Reviews in Plant Sciences, 34, 144
168.

564 Chapter 22 Banno, A., Wang, J., Okada, K., Mori, R., Mijiti, M., & Nagaoka, S. (2019). Identification of a novel cholesterol-lowering dipeptide, phenylalanine-proline (FP), and its down-regulation of intestinal ABCA1 in hypercholesterolemic rats and Caco-2 cells. Scientific Reports, 9, 19416. Carling, D., Zammit, V. A., & Hardie, D. G. (1987). A common bicyclic protein-kinase cascade inactivates the regulatory enzymes of fatty-acid and cholesterol-biosynthesis. FEBS Letters, 223, 217
222. Castro-Torres, I. G., De la O-Arciniega, M., Bravo-Duarte, G. A., Gallegos-Estudillo, J., Domı´nguez-Ortı´z, M., & Martı´nez-Va´zquez, M. (2014). Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease? European Journal of Pharmacology, 728, 77
81. Chakrabarti, S., Guha, S., & Majumder, K. (2018). Food-derived bioactive peptides in human health: Challenges and opportunities. Nutrients, 10. Cho, S., Juillerat, M., & Lee, C. (2008). Identification of LDL-receptor transcription stimulating peptides from soybean hydrolysate in human hepatocytes. Journal of Agricultural and Food Chemistry, 56, 4372
4376. De Backer, G., Ambrosioni, E., Borch-Johnsen, K., Brotons, C., Cifkova, R., Dallongeville, J., . . . Wood, D. (2003). European guidelines on cardiovascular disease and prevention in clinical practice. Atherosclerosis, 171, 145
155. Dia, V. P., Torres, S., De Lumen, B. O., Erdman, J. W., & De Mejia, E. G. (2009). Presence of lunasin in plasma of men after soy protein consumption. Journal of Agricultural and Food Chemistry, 57, 1260
1266. FDA. (1999). Food labeling health claims: Soybean protein and coronary heart disease. Final rule, . Federal Register (64, pp. 57699
57733). Fernandez, M. L. (2006). Dietary cholesterol provided by eggs and plasma lipoproteins in healthy populations. Current Opinion in Clinical Nutrition and Metabolic Care, 9, 8
12. Galvez, A. F., Chen, N., Macasieb, J., & de Lumen, B. O. (2001). Chemopreventive property of a soybean peptide (lunasin) that binds to deacetylated histones and inhibits acetylation. Cancer Research, 61, 7473
7478. Galvez, A. F. (2012). Identification of lunasin as the active component in soy protein responsible for reducing LDL cholesterol and risk of cardiovascular disease. Circulation, 126. Grazioso, G., Bollati, C., Sgrignani, J., Arnoldi, A., & Lammi, C. (2018). The first food-derived peptide inhibitor of the protein-protein interaction between Gain-of-Function PCSK9D374Yand the LDL receptor. Journal of Agricultural and Food Chemistry. Available from https://doi.org/10.1021/acs.jafc.8b03233. Gu, L., Wang, Y., Xu, Y., Tian, Q., Lei, G., Zhao, C., . . . Tan, S. (2017). Lunasin functionally enhances LDL uptake via inhibiting PCSK9 and enhancing LDLR expression. Oncotarget, 8, 80826
80840. Guo, D., Liu, W., Zhang, X., Zhao, M., Zhu, B., Hou, T., & He, H. (2019). Duck egg white-derived peptide VSEE (Val-Ser-Glu-Glu) regulates bone and lipid metabolisms by Wnt/β-catenin signaling pathway and intestinal microbiota. Molecular Nutrition & Food Research, 63, e1900525. Guo, H., Saiga, A., Sato, M., Miyazawa, I., Shibata, M., Takahata, Y., & Morimatsu, F. (2007). Royal jelly supplementation improves lipoprotein metabolism in humans. Journal of Nutritional Science and Vitaminology, 53, 345
348. Harland, J., & Haffner, T. (2008). Systematic review, meta-analysis and regression of randomised controlled trials reporting an association between an intake of circa 25g soya protein per day and blood cholesterol. Atherosclerosis, 200, 13
27. He, Y., Pan, X., Chi, C. F., Sun, K. L., & Wang, B. (2019). Ten new pentapeptides from protein hydrolysate of miiuy croaker (Miichthys miiuy) muscle: Preparation, identification, and antioxidant activity evaluation. LWT-Food Science and Technology, 105, 1
8. Herna´ndez-Ledesma, B., Hsieh, C. C., & de Lumen, B. O. (2009). Lunasin, a novel seed peptide for cancer prevention. Peptides, 30, 426
430. Holmes, W. L., Rubel, G. B., & Hood, S. S. (1980). Comparison of the effect of dietary meat versus dietary soybean protein on plasma-lipids of hyperlipidemic individuals. Atherosclerosis, 36, 379
387. Hosomi, R., Fukunaga, K., Arai, H., Kanda, S., Nishiyama, T., & Yoshida, M. (2012). Fish protein hydrolysates affect cholesterol metabolism in rats fed non-cholesterol and high-cholesterol diets. Journal of Medicinal Food, 15, 299
306.

Application in nutrition: cholesterol-lowering activity 565 Hu, Y., Smith, D. E., Ma, K., Jappar, D., Thomas, W., & Hillgren, K. M. (2008). Targeted disruption of peptide transporter Pept1 gene in mice significantly reduces dipeptide absorption in intestine. Molecular Pharmaceutics, 5, 1122
1130. Jacques, H., Deshaies, Y., & Savoie, L. (1986). Relationship between dietary proteins, their in vitro digestion products, and serum cholesterol in rats. Atherosclerosis, 61, 89
98. Jenkins, D. J., Mirrahimi, A., Srichaikul, K., Berryman, C. E., Wang, L., Carleton, A., . . . Kris-Etherton, P. M. (2010). Soy protein reduces serum cholesterol by both intrinsic and food displacement mechanisms. The Journal of Nutrition, 140, 2302S
2311S. Jeong, H. J., Lee, J. R., Jeong, J. B., Park, J. H., Cheong, Y.-K., & de Lumen, B. O. (2009). The cancer preventive seed peptide lunasin from rye is bioavailable and bioactive. Nutrition and Cancer International Journal, 61, 680
686. Jiang, X., Pan, D., Zhang, T., Liu, C., Zhang, J., Su, M., . . . Guo, Y. (2020). Novel milk casein-derived peptides decrease cholesterol micellar solubility and cholesterol intestinal absorption in Caco-2 cells. Journal of Dairy Science, 103, 3924
3936. Kashima, Y., Kanematsu, S., Asai, S., Kusada, M., Watanabe, S., Kawashima, T., . . . Nagaoka, S. (2014). Identification of a novel hypocholesterolemic protein, major royal jelly protein 1, derived from royal jelly. PLoS One, 9, e105073. Lammi, C., Aiello, G., Boschin, G., & Arnoldi, A. (2019). Multifunctional peptides for the prevention of cardiovascular disease: A new concept in the area of bioactive food-derived peptides. Journal of Functional Foods, 55, 135
145. Lammi, C., Aiello, G., Dellafiora, L., Bollati, C., Boschin, G., Ranaldi, G., . . . Arnoldi, A. (2020). Assessment of the multifunctional behavior of lupin peptide P7 and its metabolite using an integrated strategy. Journal of Agricultural and Food Chemistry. Lammi, C., Aiello, G., Vistoli, G., Zanoni, C., Arnoldi, A., Sambuy, Y., . . . Ranaldi, G. (2016). A multidisciplinary investigation on the bioavailability and activity of peptides from lupin protein. Journal of Functional Foods, 24, 297
306. Lammi, C., Arnoldi, A., & Aiello, G. (2019). Soybean peptides exert multifunctional bioactivity modulating 3hydroxy-3-methylglutaryl-CoA reductase and dipeptidyl peptidase-IV targets in vitro. Journal of Agricultural and Food Chemistry, 67, 4824
4830. Lammi, C., Bollati, C., Lecca, D., Abbracchio, M. P., & Arnoldi, A. (2019). Lupin peptide T9 (GQEQSHQDEGVIVR) modulates the mutant PCSK9. Nutrients, 11. Lammi, C., Zanoni, C., Aiello, G., Arnoldi, A., & Grazioso, G. (2016). Lupin peptides modulate the proteinprotein interaction of PCSK9 with the low density lipoprotein receptor in HepG2 cells. Scientific Reports, 6. Lammi, C., Zanoni, C., & Arnoldi, A. (2015). IAVPGEVA, IAVPTGVA, and LPYP, three peptides from soy glycinin, modulate cholesterol metabolism in HepG2 cells through the activation of the LDLR-SREBP2 pathway. Journal of Functional Foods, 14, 469
478. Lammi, C., Zanoni, C., Arnoldi, A., & Vistoli, G. (2015). Two peptides from soy β-conglycinin induce a hypocholesterolemic effect in HepG2 Cells by a statin-like mechanism: Comparative in vitro and in silico modeling studies. Journal of Agricultural and Food Chemistry, 63, 7945
7951. Lammi, C., Zanoni, C., Calabresi, L., & Arnoldi, A. (2016). Lupin protein exerts cholesterol-lowering effects targeting PCSK9: From clinical evidences to elucidation of the in vitro molecular mechanism using HepG2 cells. Journal of Functional Foods, 23, 230
240. Lammi, C., Zanoni, C., Scigliuolo, G. M., D’Amato, A., & Arnoldi, A. (2014). Lupin peptides lower lowdensity lipoprotein (LDL) cholesterol through an up-regulation of the LDL receptor/sterol regulatory element binding protein 2 (SREBP2) pathway at HepG2 cell line. Journal of Agricultural and Food Chemistry, 62, 7151
7159. Lovati, M. R., Manzoni, C., Gianazza, E., Arnoldi, A., Kurowska, E., Carroll, K. K., & Sirtori, C. R. (2000). Soy protein peptides regulate cholesterol homeostasis in Hep G2 cells. The Journal of Nutrition, 130, 2543
2549.

566 Chapter 22 Marques, M. R., Fontanari, G. G., Pimenta, D. C., Soares-Freitas, R. M., & Areas, J. A. G. (2015). Proteolytic hydrolysis of cowpea proteins is able to release peptides with hypocholesterolemic activity. Food Research International, 77, 43
48. Matsuoka, R., Kimura, M., Muto, A., Masuda, Y., Sato, M., & Imaizumi, K. (2008). Mechanism for the cholesterol-lowering action of egg white protein in rats. Bioscience, Biotechnology, and Biochemistry, 72, 1506
1512. Meisel, H. (2004). Multifunctional peptides encrypted in milk proteins. Biofactors (Oxford, England), 21, 55
61. Morikawa, K., Ishikawa, K., Kanamaru, Y., Hori, G., & Nagaoka, S. (2007). Effects of dipeptides having a C-terminal lysine on the cholesterol 7alpha-hydroxylase mRNA level in HepG2 cells. Bioscience, Biotechnology, and Biochemistry, 71, 821
825. Morikawa, K., Kondo, I., Kanamaru, Y., & Nagaoka, S. (2007). A novel regulatory pathway for cholesterol degradation via lactostatin. Biochemical and Biophysical Research Communications, 352, 697
702. Nagaoka, S. (2019). Structure-function properties of hypolipidemic peptides. Journal of Food Biochemistry, 43, e12539. Nagaoka, S. (2018). Mystery of cholesterol-lowering peptides, lactostatin and soystatin. Journal of Agricultural and Food Chemistry, 66, 3993
3994. Nagaoka, S., Futamura, Y., Miwa, K., Awano, T., Yamauchi, K., Kanamaru, Y., . . . Kuwata, T. (2001). Identification of novel hypocholesterolemic peptides derived from bovine milk beta-lactoglobulin. Biochemical and Biophysical Research Communications, 281, 11
17. Nagaoka, S., Masaoka, M., Zhang, Q., Hasegawa, M., & Watanabe, K. (2002). Egg ovomucin attenuates hypercholesterolemia in rats and inhibits cholesterol absorption in Caco-2 cells. Lipids, 37, 267
272. Nagaoka, S., Nakamura, A., Shibata, H., & Kanamaru, Y. (2010). Soystatin (VAWWMY), a novel bile acidbinding peptide, decreased micellar solubility and inhibited cholesterol absorption in rats. Bioscience, Biotechnology, and Biochemistry, 74, 1738
1741. Nagata, C., Takatsuka, N., Kurisu, Y., & Shimizu, H. (1998). Decreased serum total cholesterol concentration is associated with high intake of soy products in Japanese men and women. The Journal of Nutrition, 128, 209
213. Nakade, K., Kaneko, H., Oka, T., Ahhmed, A. M., Muguruma, M., Numata, M., & Nagaoka, S. (2009). A cattle heart protein hydrolysate ameliorates hypercholesterolemia accompanied by suppression of the cholesterol absorption in rats and Caco-2 cells. Bioscience, Biotechnology, and Biochemistry, 73, 607
612. Nakamura, R., Takayama, M., Nakamura, K., & Umemura, O. (1980). Constituent proteins of globulin fraction obtained from egg-white. Agricultural and Biological Chemistry, 44, 2357
2362. Nakamura, Y., Iso, H., Kita, Y., Ueshima, H., Okada, K., Konishi, M., . . . Tsugane, S. (2006). Egg consumption, serum total cholesterol concentrations and coronary heart disease incidence: Japan Public Health Center-based prospective study. The British Journal of Nutrition, 96, 921
928. Nass, N., Schoeps, R., Ulbrich-Hofmann, R., Simm, A., Hohndorf, L., Schmelzer, C., . . . Eder, K. (2008). Screening for nutritive peptides that modify cholesterol 7 alpha-hydroxylase expression. Journal of Agricultural and Food Chemistry, 56, 4987
4994. Odani, S., Koide, T., & Ono, T. (1987). Amino-acid-sequence of a soybean (Glycine max) seed polypeptide having a poly(L-aspartic acid) structure. The Journal of Biological Chemistry, 262, 10502
10505. Ohinata, K., Sonoda, S., Inoue, N., Yamauchi, R., Wada, K., & Yoshikawa, M. (2007). beta-Lactotensin, a neurotensin agonist peptide derived from bovine beta-lactoglobulin, enhances memory consolidation in mice. Peptides, 28, 1470
1474. Pak, V. V., Koo, M., Kwon, D. Y., & Yun, L. (2012). Design of a highly potent inhibitory peptide acting as a competitive inhibitor of HMG-CoA reductase. Amino Acids, 43, 2015
2025. Pak, V. V., Koo, M. S., Kasymova, T. D., & Kwon, D. Y. (2005). Isolation and identification of peptides from soy 11S-globulin with hypocholesterolemic activity. Chemistry of Natural Compounds, 41, 710
714. Qureshi, A. I., Suri, F. K., Ahmed, S., Nasar, A., Divani, A. A., & Kirmani, J. F. (2007). Regular egg consumption does not increase the risk of stroke and cardiovascular diseases. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research, 13, CR1-8.

Application in nutrition: cholesterol-lowering activity 567 Ramdath, D. D., Padhi, E. M., Sarfaraz, S., Renwick, S., & Duncan, A. M. (2017). Beyond the cholesterollowering effect of soy protein: A review of the effects of dietary soy and its constituents on risk factors for cardiovascular disease. Nutrients, 9. Rong, Y., Chen, L., Zhu, T., Song, Y., Yu, M., Shan, Z., . . . Liu, L. (2013). Egg consumption and risk of coronary heart disease and stroke: Dose-response meta-analysis of prospective cohort studies. British Medical Journal, 346, e8539. Shukla, A., Bettzieche, A., Hirche, F., Brandsch, C., Stangl, G. I., & Eder, K. (2006). Dietary fish protein alters blood lipid concentrations and hepatic genes involved in cholesterol homeostasis in the rat model. The British Journal of Nutrition, 96, 674
682. Sirtori, C. R., Eberini, I., & Arnoldi, A. (2007). Hypocholesterolaemic effects of soya proteins: Results of recent studies are predictable from the Anderson meta-analysis data. British Journal of Nutrition, 97, 816
822. Sirtori, C. R., Galli, C., Anderson, J. W., Sirtori, E., & Arnoldi, A. (2009). Functional foods for dyslipidaemia and cardiovascular risk prevention. Nutrition Research Reviews, 22, 244
261. Sirtori, C. R., Lovati, M. R., Manzoni, C., Castiglioni, S., Duranti, M., Magni, C., . . . Arnoldi, A. (2004). Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells. The Journal of Nutrition, 134, 18
23. Sirtori, C. R., Triolo, M., Bosisio, R., Bondioli, A., Calabresi, L., Vergori, V., . . . Arnoldi, A. (2012). Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals. British Journal of Nutrition, 107, 1176
1183. Soares, R. A. M., Mendonca, S., de Castro, L. I. A., Menezes, A., & Areas, J. A. G. (2015). Major peptides from amaranth (Amaranthus cruentus) protein inhibit HMG-CoA reductase activity. International Journal of Molecular Sciences, 16, 4150
4160. Spady, D. K., Cuthbert, J. A., Willard, M. N., & Meidell, R. S. (1995). Adenovirus-mediated transfer of a gene encoding cholesterol 7 alpha-hydroxylase into hamsters increases hepatic enzyme activity and reduces plasma total and low density lipoprotein cholesterol. The Journal of Clinical Investigation, 96, 700
709. Spady, D. K., Cuthbert, J. A., Willard, M. N., & Meidell, R. S. (1998). Overexpression of cholesterol 7alphahydroxylase (CYP7A) in mice lacking the low density lipoprotein (LDL) receptor gene. LDL transport and plasma LDL concentrations are reduced. The Journal of Biological Chemistry, 273, 126
132. Stampfer, M. J., Hu, F. B., Manson, J. E., Rimm, E. B., & Willett, W. C. (2000). Primary prevention of coronary heart disease in women through diet and lifestyle. The New England Journal of Medicine, 343, 16
22. Vittek, J. (1995). Effect of royal jelly on serum lipids in experimental animals and humans with atherosclerosis. Experientia, 51, 927
935. Wang, J., Shimada, M., Kato, Y., Kusada, M., & Nagaoka, S. (2015). Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins. Bioscience, Biotechnology, and Biochemistry, 79, 456
461. Wang, Y.-M., Xi, P., He, Y., Chi, C.-F., & Wang, B. (2020). Applied sciences. MDPI. Yamauchi, R., Ohinata, K., & Yoshikawa, M. (2003). Beta-lactotensin and neurotensin rapidly reduce serum cholesterol via NT2 receptor. Peptides, 24, 1955
1961. Yamauchi, R., Wada, E., Yamada, D., Yoshikawa, M., & Wada, K. (2006). Effect of beta-lactotensin on acute stress and fear memory. Peptides, 27, 3176
3182. Yeh, L. A., Lee, K. H., & Kim, K. H. (1980). Regulation of rat liver acetyl-CoA carboxylase. Regulation of phosphorylation and inactivation of acetyl-CoA carboxylase by the adenylate energy charge. The Journal of Biological Chemistry, 255, 2308
2314. Zanoni, C., Aiello, G., Arnoldi, A., & Lammi, C. (2017a). Hempseed peptides exert hypocholesterolemic effects with a statin-like mechanism. Journal of Agricultural and Food Chemistry, 65, 8829
8838. Zanoni, C., Aiello, G., Arnoldi, A., & Lammi, C. (2017b). Investigations on the hypocholesterolaemic activity of LILPKHSDAD and LTFPGSAED, two peptides from lupin beta-conglutin: Focus on LDLR and PCSK9 pathways. J Funct Foods, 32, 1
8.

568 Chapter 22 Zhang, H., Bartley, G. E., Zhang H., Jing, W., Fagerquist, C. K., Zhang, H., . . . Yokoyama, W. (2013). Peptides identified in soybean protein increase plasma cholesterol in mice on hypercholesterolemic diets. Journal of Agricultural and Food Chemistry, 61, 8389
8395. Zhang, H., & Yokoyama, W. H. (2012). Concentration-dependent displacement of cholesterol in micelles by hydrophobic rice bran protein hydrolysates. Journal of the Science of Food and Agriculture, 92, 1395
1401. Zhang, X., & Beynen, A. C. (1993). Influence of dietary fish proteins on plasma and liver cholesterol concentrations in rats. The British Journal of Nutrition, 69, 767
777. Zhang, X., Shu, X. O., Gao, Y. T., Yang, G., Li, Q., Li, H., . . . Zheng, W. (2003). Soy food consumption is associated with lower risk of coronary heart disease in Chinese women. The Journal of Nutrition, 133, 2874
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CHAPTER 23

Applications in nutrition: Peptides as taste enhancers Yu Fu1, Mohammad Sadiq Amin1, Qian Li2, Kathrine H. Bak3 and Rene´ Lametsch2 1

College of Food Science, Southwest University, Chongqing, China, 2Department of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark, 3Institute of Food Safety, Food Technology and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria

23.1 Introduction Taste is one of the most crucial attributes of food palatability, which influences food selection, intake, absorption, and digestion. The five basic tastes, including bitter, salty, sour, sweet, and umami, serve as the most important factors influencing an individual’s preference toward foods. In recent years, kokumi has been incorporated into taste sensations to describe the characteristics of richness, mouthfulness, and continuity of food (Feng, Zhang, Zhuang, Zhou, & Xu, 2016). The taste of food is the perception produced or stimulated in the mouth when food ingredients interact with taste receptors located on taste buds on the surface of the tongue (Liu, Deng, Sha, Hashem, & Gai, 2017). The attractive taste of food can not only give people a pleasant enjoyment but also contribute to nutrition status. In addition to imparting biological and functional properties to food, some food-derived peptides have been found to enhance the basic taste characteristics of food (Zhao & Ga¨nzle, 2016), which has attracted widespread attention in the field of food science. The taste of peptides covers all five basic tastes as well as kokumi. However, the sweet and sour tastes of peptides are not very pronounced or even weak in the taste perception of taste-active peptides (Temussi, 2012). At present, there are no known natural sweet peptides. The most renowned sweet peptide is aspartame (L-Asp-L-Phe-OMe), which was discovered during the synthesis of dipeptide analogs (Temussi, 2012). Aspartame has been employed as the noncaloric sweetener, which drives people to design and synthesize the novel artificial sweet peptide to improve its characteristics. The recent development in molecular neurobiology enables the characterization and identification of the specific taste receptors (see Table 23.1). G protein-coupled receptors (GPCRs) designated as T2Rs have been identified as the receptors for a variety of bitter taste compounds (Avau & Depoortere, 2016; Mueller et al., 2005). In contrast, the receptors Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00014-5 © 2021 Elsevier Inc. All rights reserved.

569

570 Chapter 23 Table 23.1: Taste attributes and their corresponding taste receptors. Taste attributes

Reported taste receptors

References

Bitter

T2Rs, a family of GPCRs that detect bitter compounds

Salty

ENaC, TRPV1

Sour

The polycystic kidney disease 1-like 3 (PKD1L3)
polycystic kidney disease 2-like 1 (PKD2L1) complex T1Rs, a family of taste GPCRs that detect sweet compounds and function as heterodimers, for example, T1R2 1 T1R3 Metabotropic glutamate receptors and T1R2/T1R3 Calcium-sensing receptor

Behrens and Meyerhof (2011) Chandrashekar et al. (2010) Ishimaru et al. (2006) Mueller et al. (2005) Temussi (2012) Maruyama et al. (2012)

Sweet Umami Kokumi

ENaC, Epithelium sodium channel; GPCR, G protein-coupled receptors.

of sweet and umami are dimers of GPCRs (Spaggiari, Di Pizio, & Cozzini, 2019; Zhang, Sun-Waterhouse, Su, & Zhao, 2019), including T1R2/T1R3 and T1R1/T1R3, respectively. Several recent studies have revealed that γ-glutamyl peptides can exhibit a kokumi taste, which is perceived through a calcium-sensing receptor (CaSR) in humans (Amino et al., 2016; Yamamoto et al., 2020). On the other hand, even though the receptor for saltiness has not been fully elucidated, epithelium sodium channels (ENaCs) and transient receptor potential vanilloid 1 (TRPV1) channels have been suggested to be potential salty taste receptors (Chandrashekar et al., 2010). This chapter focuses on the taste-active peptides of umami, bitter inhibitory, salt, and kokumi tastes, their respective taste receptors and structural characteristics.

23.2 Umami and umami-enhancing peptides 23.2.1 Umami taste The term “umami” is derived from a Japanese word (うま味), which was proposed by Ikeda to express the savory taste in 1909 (Ikeda, 1909; Lindemann, Ogiwara, & Ninomiya, 2002). In recent years, “umami” has gradually been recognized around the globe as the fifth basic taste (Kong et al., 2017; Zhao, Zhang, Devahastin, & Liu, 2019). In general, umami substances can be categorized into two types. The first one is umami agents, which bind to umami receptors to show umami taste and the second one is umami enhancer that improves umami taste. L-glutamic acid and monosodium glutamate (MSG) elicit umami taste when they bind to umami receptors (Zhao et al., 2019). In addition to MSG, a wide range of substances as the second type have been found to elicit umami taste, including some free L-amino acids, ribonucleotides, peptides, and their derivatives or Maillard reaction products (MRPs) (Suess, Festring, & Hofmann, 2015). Umami peptides are also promising for

Applications in nutrition: Peptides as taste enhancers 571 utilization as taste enhancers in foods (Zhao et al., 2019). It has been shown that pH can exert a significant impact on umami taste. The intensity of umami taste becomes the strongest from pH 5.5 to 8.0, whereas its intensity decreases when pH is below 4.0 (Zhao et al., 2019).

23.2.2 Umami taste receptors Umami taste receptors belong to a family of GPCR. At present, three major receptors for umami taste have been already proposed (Zhang, Sun-Waterhouse, et al., 2019). The first umami receptor, discovered in the year 2000, is known as metabotropic glutamate 4 (mGluR4), the second umami receptor T1R1 1 T1R3 was discovered in 2002, and the third receptor, the uncommon mGlu receptor that belongs to brain glutamate receptor mGluR1, was discovered in 2005. In addition, several receptors including CaSR, GPRC6A, and GPR92 expressed in gustatory T1R1-expressing cells have been suggested to act as umami taste receptors, which are involved in umami taste transduction induced by different sorts of umami tastants (Zhang, Zhang, et al., 2019). Some recently published articles have summarized the sensing mechanisms of umami taste stimulated by the interaction between umami or umami-enhancing peptides and umami taste receptors via orthosteric or allosteric binding sites (Zhang, Sun-Waterhouse, et al., 2019; Zhang, Zhao, Su, & Lin, 2019). However, sufficient understanding of the sensing mechanisms is still lacking due to limitations and difficulties in characterization and analysis of the receptor crystal structure (Liu, Da, & Liu, 2019; Zhang, Sun-Waterhouse, et al., 2019; Zhang, Zhao, et al., 2019).

23.2.3 Structural characteristics of umami and umami-enhancing peptides Recently, a large number of umami/umami-enhancing peptides derived from fermented food or protein hydrolysates have been reported (Zhang, Venkitasamy, Pan, Liu, & Zhao, 2017). Umami and umami-enhancing peptides are a group of peptides with specific structural features, which can elicit umami taste or impart umami-enhancing properties. In general, amino acid composition and molecular weight of umami peptides can exert an impact on the umami taste (Fu, Liu, Hansen, Bredie, & Lametsch, 2018; Zhuang et al., 2016). Umami peptides from different plant or animal protein sources usually have a molecular weight distribution of less than 5000 Da. Apart from short-chain peptides, several long-chain peptides have been reported to possess strong umami intensity (Zhang, Sun-Waterhouse, et al., 2019; Zhuang et al., 2016). Rhyu and Kim (2011) reported that small peptides with acidic residues (Glu and Asp) may play an important role in the perception of umami taste. It has been shown that peptides with more Glu residues tend to exhibit higher umami intensity, but Arg residues indicate weak intensity (Zhang, Zhao, et al., 2019). Compared with short-chain peptides, amino acid composition is less important for long-chain umami peptides, while

572 Chapter 23 spatial composition, surface charge distribution, as well as hydrophilic and hydrophobic amino acids are responsible for their umami taste (Zhang, Sun-Waterhouse, et al., 2019). Some tasteless or slightly bitter umami-enhancing peptides are capable of increasing the umami intensity of solutions containing umami substances, such as MSG, inosine monophosphate, and guanosine monophosphate (Zhang, Sun-Waterhouse, et al., 2019). The relationship between structure and umami-enhancing capacity of the umami-enhancing peptides is sophisticated. Dang et al. (2019) recently suggested a novel two-step model for elucidation of potentiating effect of umami-enhancing peptides. Specifically, MSG may firstly bind to T1R1 by enlarging the size of the binding cavity of T1R3 due to dimerization, which makes it easier for peptides to bind with T1R3. However, this interaction process is more complicated than that of umami peptides and remains to be investigated to fully understand the binding mechanism of umami-enhancing peptides.

23.3 Bitter and bitter inhibitory peptides 23.3.1 Bitter taste Bitterness is generally deemed an undesirable attribute of foods. Protein hydrolysates/ peptides derived from different sources have been reported to exhibit notorious bitterness, which impedes their further application in the food industry (Fu, Chen, Bak, & Lametsch, 2019; Fu, Bak, et al., 2019). It is well documented that many sorts of peptides with hydrophobic amino acids, such as Leu, Ile, Val, Phe, Tyr, and Trp, can elicit bitterness (Zhu, Sun-Waterhouse, Chen, Cui, & Wang, 2020). In addition to hydrophobicity of peptides, peptide length, amino acid sequence, and spatial structure can influence bitter taste perception (Idowu & Benjakul, 2019). Although elimination or reduction in bitterness in protein hydrolysates is challenging, a wide array of strategies has been utilized. Some efficient methods of debittering and taste enhancement of taste-active peptides have been well summarized in a recent review article (Fu, Chen, et al., 2019).

23.3.2 Bitter taste receptor The sensation of bitterness is associated with the binding of bitter peptides with a series of bitter taste receptors (T2Rs) (Behrens & Meyerhof, 2011). T2Rs are a class of GPCRs that can be activated by binding of bitter compounds (Maehashi & Huang, 2009). Among different receptors, T2R1, T2R4, T2R14, and T2R16 can preferably recognize most of bitter peptides (Maehashi et al., 2009), whereas T2R1 shows specificity toward bitter di- and tri-peptides (Upadhyaya, Pydi, Singh, Aluko, & Chelikani, 2010; Xu et al., 2019). The binding mechanism of bitter peptides to their specific bitter receptors involves two functional units (binding unit and stimulating unit) of bitter peptides (Fu, Chen, et al., 2019). The hydrophobic side chains of bitter peptides can provide a binding unit recognized

Applications in nutrition: Peptides as taste enhancers 573 by bitter taste receptors, whereas a bulky basic or hydrophobic group of bitter peptides can serve the stimulating unit. It has been documented that bitter intensity depends on the hydrophobic recognition region of bitter taste receptors, and the simultaneous presence of two functional units in the steric conformation of bitter peptides contributes to a more potent bitter taste (Fu, Chen, et al., 2019).

23.3.3 Bitter taste inhibitory peptides Currently, increasing attention has been paid to searching for bitter taste modulators that help block bitter taste receptors after discovery of bitter taste receptors and elucidation of their transduction mechanisms (Jaggupilli, Howard, Aluko, & Chelikani, 2019; Xu et al., 2019; Zhang, Alashi, et al., 2018; Zhang, Alashi, Singh, Chelikani, & Aluko, 2019). The Maillard reaction between peptides and reducing sugar can improve the taste of protein hydrolysates as well as decrease bitterness of peptides (Fu, Chen, et al., 2019; Fu, Zhang, Soladoye, & Aluko, 2019). The resultant MRPs produced from peptides are conducive to the generation of color, taste, aroma, and biological activity (Fu, Zhang, et al., 2019). Due to the reduced extent of bitter amino acids and peptide modification, the Maillard reaction can decrease bitter taste, and MRPs can act as bitterness masking agents (Fu, Chen, et al., 2019; Fu et al., 2020). The MRPs of soy protein hydrolysates elicit a powerful caramel-like aroma and a particularly weak bitter taste (Hong, Ndagijimana, & Betti, 2016). Recently, a wide range of nonbitter peptides and their advanced glycation end-products (AGEs) were shown to serve as potential bitter taste inhibitors. Peptides from enzymatic beef protein hydrolysates and their AGEs could act as bitter taste T2R4 receptor blockers and play a role in a blocking of bitter taste (Zhang, Elfalleh, et al., 2018; Zhang, Zhang, et al., 2019). However, the underlying mechanism responsible for the inhibition of bitterness requires further investigation. More recently, Fu et al. (2020) demonstrated that exopeptidase treatment combined with the Maillard reaction can result in less bitter protein hydrolysates derived from porcine muscle and plasma, which is attributed to the generation of Maillard-reacted peptides and peptide glycation modification.

23.4 Salt taste-enhancing peptides 23.4.1 Salt taste Salty taste, one of the five elementary taste attributes, can be evoked by some cationic salts, such as sodium chloride (NaCl), potassium chloride (KCl), and calcium chloride (CaCl2) (Frankowski, Miracle, & Drake, 2014). The intensity of salty taste is mainly dependent on the cations, whereas the anions can only modify the salty taste. Even though dietary salt intake takes a pivotal part in homeostatic regulation of water, pH, osmotic pressure as well as nerve conductance in our body, there is a close connection between a high salt intake

574 Chapter 23 and metabolic disorders, such as cardiovascular diseases, stroke, hypertension, stomach cancer, Ca21 deficiency, and osteoporosis (DiNicolantonio & O’Keefe, 2018; Do et al., 2020). Therefore it is imperative to explore and develop salty taste enhancers to reduce salt consumption in foods (Harth et al., 2016; Inguglia, Zhang, Tiwari, Kerry, & Burgess, 2017; Schindler et al., 2011).

23.4.2 Salty taste receptors Salty taste is mainly produced by the interaction of cations with the salty taste receptors on the tongue. The cations can be enriched in the cell, leading to depolarization of the cell membrane and changes in intracellular calcium ion concentration, generating nerve signal transduction and finally sensing the salty taste (Kinnamon & Finger, 2019). Two main salty taste receptors of humans are ENaC subunits and transient receptor potential vanillin acid receptor 1 (TRPV1), respectively (Chandrashekar et al., 2010). Among them, ENaC is the salty taste receptor with a specific response. When Na1 accumulates to a certain concentration in the mouth, ENaC can specifically respond to a low concentration of Na1 and respond only to Na1, which can be inhibited by amiloride. By contrast, the nonspecific response of the salty receptor TRPV1 has no special requirements for cationic species and responds not only to Na1 but also to K1, but this response cannot be inhibited by amiloride (DeSimone & Lyall, 2006; Gilbertson, Damak, & Margolskee, 2000; Munger, 2016). Structurally, the human ENaC salty receptor is composed of four subunits (α, β, γ, and δ subunit) (Kasahara et al., 2019; Xu et al., 2017), which are mainly distributed in the salty receptor cells of the contour papilla, lobular papilla, and bacteriform papilla on the tongue, whereas the salty receptor TRPV1 is mainly expressed in the central and terminal nervous systems (Kim et al., 2014).

23.4.3 Structural characteristics of salty taste-enhancing peptides A number of recent studies have shown that some food-derived peptides and MRPs have salty taste-enhancing effects (Harth et al., 2016; Katsumata et al., 2008). Tada, Shinoda, and Okai (1984) originally discovered that the salty taste of ornithyl peptides (Orn-β-Ala • HCl and Orn-Tau • HCl) can elicit a salty taste that is similar to sodium chloride during the synthesis process of N-terminal analogs of casein hydrolysates. Since these salty peptides do not contain sodium ions, they can be used as a substitute for sodium chloride. In the process of studying unsalted soy sauce, Zhu et al. (2008) found three salty peptides, namely Phe-Ile, Ala-Phe, and Ile-Phe. These peptides not only have a salty taste but also can reduce blood pressure by the inhibition of angiotensin-I converting enzyme. Harth et al. (2016) studied the taste-active components of Gouda cheese and found that the peptides containing L-arginine have the ability to enhance salty taste. It has been reported that Ala-Arg can improve salty taste intensity of 50 mmol/L NaCl in aqueous solution by 14% and 19% in model broth.

Applications in nutrition: Peptides as taste enhancers 575 A series of arginyl dipeptides Arg-Pro, Arg-Ala, Ala-Arg, Arg-Gly, Arg-Ser, Arg-Val, Val-Arg, and Arg-Met serve as salty taste improving molecules in fermented fish sauces and fish protamine digests (Schindler et al., 2011). Apart from Arg-containing peptides, Ogasawara, Katsumata, and Egi (2006) reported that Maillard-reacted peptides also have a salt-enhancing effect. A Maillard peptide is a glycated peptide formed by the reaction between peptides of 1
5 kDa with a reducing sugar at high temperatures (Fu et al., 2020; Kang, Alim, & Song, 2019; Ogasawara et al., 2006). Maillard-reacted peptides from soybean protein can effectively increase the saltiness of fresh soup (Zhang et al., 2018). Recently, the MRPs derived from turkey protein peptide and glucosamine can significantly improve the saltiness of thick soup (Hong et al., 2016). Nevertheless, the exact molecular mechanisms for the improved salty taste by these peptides still need to be elucidated.

23.5 Kokumi peptides 23.5.1 Kokumi taste Nowadays, consumers are not satisfied with a bland taste of food, so there is a growing desire to experience food with more complex taste. Kokumi is defined as a taste sensation associated with continuity, mouthfulness, thickness, taste-enhancing, and long-lasting characteristics (Feng et al., 2016). The kokumi taste was originally characterized in a water extract of garlic, which could enhance continuity, mouthfulness, and thickness when added to an umami solution (Ueda, Sakaguchi, Hirayama, Miyajima, & Kimizuka, 1990). The main active ingredients of kokumi are sulfur-containing components, amino acids, peptides, and their derivatives. Kokumi-active compounds are normally not taste-active and they sometimes exhibit an astringent or bitter taste (Yang, Bai, Zeng, & Cui, 2019). However, when they are added to a solution containing basic taste-active substances, kokumi taste can be perceived.

23.5.2 Kokumi taste receptors It has been proposed that CaSR is the potential receptor of kokumi compounds (Maruyama, Yasuda, Kuroda, & Eto, 2012; Ohsu et al., 2010). CaSR belongs to the family of GPCRs, which can sense the change of extracellular calcium ion concentration, which is conducive to maintaining the calcium ion balance in vivo. It has been found in the human tongue, which can be activated by kokumi-active compounds, for example, γ-glutamyl peptides, giving rise to a strong sensation and intensifying the five basic tastes (Dunkel, Ko¨ster, & Hofmann, 2007; Feng et al., 2016).

23.5.3 The characteristics of kokumi peptides The γ-glutamyl peptides serve as key contributors to the kokumi taste in diverse foods. Currently, a large number of kokumi-imparting peptides have been identified to excite

576 Chapter 23 CaSR (Yang et al., 2019). An array of γ-glutamyl peptides has been isolated and identified in some fermented products (cheese, soy sauce, and fish sauce), scallops, and yeast extract, etc. These peptides can be generated by a γ-glutamyl transferase (GGT)-catalyzed reaction with glutamine as the γ-glutamyl donor and an amino acid as the γ-glutamyl acceptor (Hillmann, Behr, Ehrmann, Vogel, & Hofmann, 2016; Kuroda et al., 2013). The formation of kokumi-active γ-glutamyl peptides in ripened cheese has been attributed to the presence of GGT activity in starter cultures or raw milk. It has been shown that Penicillium roquefortii employed during cheese ripening possesses GGT activity. The amount of kokumi-active γ-glutamyl peptides was found to increase during the ripening period in Parmesan and Gouda cheeses (Hillmann et al., 2016). It is worth noting that the increased content of γ-glutamyl peptides in Parmesan is attributed to GGT activity in raw milk rather than microbial enzyme activity. In addition, prolonged fermentation promoted the formation of beta-glutamyl peptides in Parmesan and Cheddar cheeses (Hillmann et al., 2016). Recently, the modified gluten hydrolysates catalyzed by γ-glutamyl transpeptidase have been reported to exhibit strong kokumi taste characteristics (Suzuki, Nakafuji, & Tamura, 2017). A series of γ-Glu(n .1)-Val and γ-Glu (n .1)-Met can be generated by glutaminase which originates from Bacillus amyloliquefaciens via γ-glutamyl transpeptidation. The γ-glutamyl dipeptides, which can be prepared by γ-glutamyl transpeptidase in the presence of Gln and amino acids, are γ-Glu-Val, γ-Glu-Phe, γ-Glu-Tau, γ-Glu-Leu, γ-Glu-Met, γ-Glu-Val, γ-Glu-Phe, γ-Glu-Leu, γ-Glu-Gln, and γ-Glu-Glu (Yang et al., 2019). These γ-glutamyl peptides are found in edible beans, cheese, yeast extract, or biosynthesized with pronounced kokumi taste. Kokumi is a relatively new aspect in the scientific field of sensory science and there are numerous aspects to be further explored.

23.6 Summary This chapter summarized a wide range of food-derived peptides that are associated with umami, bitter, salty, and kokumi taste. The molecular mechanisms of binding for these taste-active peptides to their respective taste receptors were overviewed, including some GPCRs for the taste of kokumi, umami, and bitter as well as salty taste for ENaC1 and TRPV1. The peptide characteristics in relation to their taste properties were also discussed, indicating that both the peptide sequences and steric structures are crucial for the taste properties. Further studies will focus on clarification of the taste
structure relationship of these taste-active peptides as well as the enzymatic and metabolic pathways responsible for the formation of taste-active peptides. In addition, it is expected that the advance in biotechnology will enable the preparation of novel taste-active proteins and peptides. The improved knowledge of molecular mechanisms for the interactions between taste-active peptides and their receptors will contribute to the development of peptides as taste enhancers.

Applications in nutrition: Peptides as taste enhancers 577

Acknowledgments The authors gratefully acknowledge the financial support by Fundamental Research Funds for the Central Universities, P.R. China (SWU 019009) and Innovation Program for Chongqing’s Overseas Returnees (cx2019072).

References Amino, Y., Nakazawa, M., Kaneko, M., Miyaki, T., Miyamura, N., Maruyama, Y., & Eto, Y. (2016). Structure
CaSR
activity relation of kokumi γ-glutamyl peptides. Chemical and Pharmaceutical Bulletin, 64(8), 1181
1189. Avau, B., & Depoortere, I. (2016). The bitter truth about bitter taste receptors: Beyond sensing bitter in the oral cavity. Acta Physiologica, 216(4), 407
420. Behrens, M., & Meyerhof, W. (2011). Oral and extraoral bitter taste receptors. In Sensory and metabolic control of energy balance (pp. 87-99): Springer. Chandrashekar, J., Kuhn, C., Oka, Y., Yarmolinsky, D. A., Hummler, E., Ryba, N. J., & Zuker, C. S. (2010). The cells and peripheral representation of sodium taste in mice. Nature, 464(7286), 297. Dang, Y., Hao, L., Cao, J., Sun, Y., Zeng, X., Wu, Z., & Pan, D. (2019). Molecular docking and simulation of the synergistic effect between umami peptides, monosodium glutamate and taste receptor T1R1/T1R3. Food Chemistry, 271, 697
706. DeSimone, J. A., & Lyall, V. (2006). Taste receptors in the gastrointestinal tract III. Salty and sour taste: Sensing of sodium and protons by the tongue. American Journal of Physiology-Gastrointestinal and Liver Physiology, 291(6), G1005
G1010. DiNicolantonio, J. J., & O’Keefe, J. H. (2018). Salt and hypertension: What do we know? Current Opinion in Cardiology, 33(4), 377
381. Do, M. H., Lee, H.-B., Oh, M.-J., Jhun, H., Ha, S. K., & Park, H.-Y. (2020). Consumption of salt leads to ameliorate symptoms of metabolic disorder and change of gut microbiota. European Journal of Nutrition, 1
12. Dunkel, A., Ko¨ster, J., & Hofmann, T. (2007). ). Molecular and sensory characterization of γ-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.). Journal of Agricultural and Food Chemistry, 55(16), 6712
6719. Feng, T., Zhang, Z., Zhuang, H., Zhou, J., & Xu, Z. (2016). Effect of peptides on new taste sensation: Kokumi-review. Mini-Reviews in Organic Chemistry, 13(4), 255
261. Frankowski, K. M., Miracle, R., & Drake, M. (2014). The role of sodium in the salty taste of permeate. Journal of Dairy Science, 97(9), 5356
5370. Fu, Y., Liu, J., Hansen, E. T., Bredie, W. L. P., & Lametsch, R. (2018). Structural characteristics of low bitter and high umami protein hydrolysates prepared from bovine muscle and porcine plasma. Food Chemistry, 257, 163
171. Fu, Y., Chen, J., Bak, K. H., & Lametsch, R. (2019). Valorisation of protein hydrolysates from animal by-products: Perspectives on bitter taste and debittering methods: A review. International Journal of Food Science and Technology, 54(4), 978
986. Fu, Y., Bak, K. H., Liu, J., De Gobba, C., Tostesen, M., Hansen, E. T., . . . Lametsch, R. (2019). Protein hydrolysates of porcine hemoglobin and blood: Peptide characteristics in relation to taste attributes and formation of volatile compounds. Food Research International, 121, 28
38. Fu, Y., Zhang, Y., Soladoye, O. P., & Aluko, R. E. (2019). Maillard reaction products derived from food protein-derived peptides: Insights into flavor and bioactivity. Critical Reviews in Food Science and Nutrition, 1
14. Fu, Y., Liu, J., Zhang, W., Wæhrens, S. S., Tøstesen, M., Hansen, E. T., . . . Lametsch, R. (2020). Exopeptidase treatment combined with Maillard reaction modification of protein hydrolysates derived from porcine muscle and plasma: Structure
taste relationship. Food Chemistry, 306, 125613.

578 Chapter 23 Gilbertson, T. A., Damak, S., & Margolskee, R. F. (2000). The molecular physiology of taste transduction. Current Opinion in Neurobiology, 10(4), 519
527. Harth, L., Krah, U., Linke, D., Dunkel, A., Hofmann, T., & Berger, R. G. (2016). Salt taste enhancing L-arginyl dipeptides from casein and lysozyme released by peptidases of Basidiomycota. Journal of Agricultural and Food Chemistry, 66(10), 2344
2353. Hillmann, H., Behr, J., Ehrmann, M. A., Vogel, R. F., & Hofmann, T. (2016). Formation of kokumi-enhancing γ-glutamyl dipeptides in Parmesan cheese by means of γ-glutamyltransferase activity and stable isotope double-labeling studies. Journal of Agricultural and Food Chemistry, 64(8), 1784
1793. Hong, P. K., Ndagijimana, M., & Betti, M. (2016). Glucosamine-induced glycation of hydrolysed meat proteins in the presence or absence of transglutaminase: Chemical modifications and taste-enhancing activity. Food Chemistry, 197, 1143
1152. Idowu, A. T., & Benjakul, S. (2019). Bitterness of fish protein hydrolysate and its debittering prospects. Journal of Food Biochemistry, 43(9), e12978. Ikeda, K. (1909). On a new seasoning. Journal of Tokyo Chemical Society, 30, 820
836. Inguglia, E. S., Zhang, Z., Tiwari, B. K., Kerry, J. P., & Burgess, C. M. (2017). Salt reduction strategies in processed meat products
A review. Trends in Food Science & Technology, 59, 70
78. Ishimaru, Y., Inada, H., Kubota, M., Zhuang, H., Tominaga, M., & Matsunami, H. (2006). Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour taste receptor. Proceedings of the National Academy of Sciences, 103(33), 12569
12574. Jaggupilli, A., Howard, R., Aluko, R. E., & Chelikani, P. (2019). Advanced glycation end-products can activate or block bitter taste receptors. Nutrients, 11(6), 1317. Kang, L., Alim, A., & Song, H. (2019). Identification and characterization of flavor precursor peptide from beef enzymatic hydrolysate by Maillard reaction. Journal of Chromatography B, 1104, 176
181. Kasahara, Y., Sakurai, T., Matsuda, R., Narukawa, M., Yasuoka, A., Mori, N., . . . Asakura, T. (2019). Novel indole and benzothiophene ring derivatives showing differential modulatory activity against human epithelial sodium channel subunits, ENaC β and γ. Bioscience, Biotechnology, and Biochemistry, 83(2), 243
250. Katsumata, T., Nakakuki, H., Tokunaga, C., Fujii, N., Egi, M., Phan, T.-H. T., . . . Lyall, V. (2008). Effect of Maillard reacted peptides on human salt taste and the amiloride-insensitive salt taste receptor (TRPV1t). Chemical Senses, 33(7), 665
680. Kim, M. J., Son, H. J., Kim, Y., Kweon, H.-J., Suh, B.-C., Lyall, V., & Rhyu, M.-R. (2014). Selective activation of hTRPV1 by N-geranyl cyclopropylcarboxamide, an amiloride-insensitive salt taste enhancer. PloS One, 9(2), e89062. Kinnamon, S., & Finger, T. (2019). Recent advances in taste transduction and signaling. F1000Research, 8(2117). Kong, Y., Yang, X., Ding, Q., Zhang, Y.-Y., Sun, B.-G., Chen, H.-T., & Sun, Y. (2017). Comparison of non-volatile umami components in chicken soup and chicken enzymatic hydrolysate. Food Research International, 102, 559
566. Kuroda, M., Kato, Y., Yamazaki, J., Kai, Y., Mizukoshi, T., Miyano, H., & Eto, Y. (2013). Determination and quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in commercial soy sauces. Food Chemistry, 141(2), 823
828. Lindemann, B., Ogiwara, Y., & Ninomiya, Y. (2002). The discovery of umami. Chemical Senses, 27(9), 843
844. Liu, D., Deng, Y., Sha, L., Hashem, M. A., & Gai, S. (2017). Impact of oral processing on texture attributes and taste perception. Journal of Food Science and Technology, 54(8), 2585
2593. Liu, H., Da, L.-T., & Liu, Y. (2019). Understanding the molecular mechanism of umami recognition by T1R1-T1R3 using molecular dynamics simulations. Biochemical and Biophysical Research Communications, 514(3), 967
973. Maehashi, K., & Huang, L. (2009). Bitter peptides and bitter taste receptors. Cellular and Molecular Life Sciences, 66(10), 1661
1671.

Applications in nutrition: Peptides as taste enhancers 579 Maruyama, Y., Yasuda, R., Kuroda, M., & Eto, Y. (2012). Kokumi substances, enhancers of basic tastes, induce responses in calcium-sensing receptor expressing taste cells. PloS One, 7(4), e34489. Mueller, K. L., Hoon, M. A., Erlenbach, I., Chandrashekar, J., Zuker, C. S., & Ryba, N. J. (2005). The receptors and coding logic for bitter taste. Nature, 434(7030), 225
229. Munger, S. D. (2016). The mechanisms of salty and sour taste. In F. Zufall, & S. D. Munger (Eds.), Chemosensory transduction (pp. 287
297). Academic Press, Chapter 16 -. Ogasawara, M., Katsumata, T., & Egi, M. (2006). Taste properties of Maillard-reaction products prepared from 1000 to 5000Da peptide. Food Chemistry, 99(3), 600
604. Ohsu, T., Amino, Y., Nagasaki, H., Yamanaka, T., Takeshita, S., Hatanaka, T., . . . Eto, Y. (2010). Involvement of the calcium-sensing receptor in human taste perception. Journal of Biological Chemistry, 285(2), 1016
1022. Rhyu, M.-R., & Kim, E.-Y. (2011). Umami taste characteristics of water extract of Doenjang, a Korean soybean paste: Low-molecular acidic peptides may be a possible clue to the taste. Food Chemistry, 127(3), 1210
1215. Schindler, A., Dunkel, A., Sta¨hler, F., Backes, M., Ley, J., Meyerhof, W., & Hofmann, T. (2011). Discovery of salt taste enhancing arginyl dipeptides in protein digests and fermented fish sauces by means of a sensomics approach. Journal of Agricultural and Food Chemistry, 59(23), 12578
12588. Spaggiari, G., Di Pizio, A., & Cozzini, P. (2019). Sweet, umami and bitter taste receptors: State of the art of in silico molecular modeling approaches. Trends in Food Science & Technology, 96, 21
29. Suess, B., Festring, D., & Hofmann, T. (2015). Umami compounds and taste enhancers. In Flavour development, analysis and perception in food and beverages (pp. 331
351): Elsevier. Suzuki, H., Nakafuji, Y., & Tamura, T. (2017). New method to produce kokumi seasoning from protein hydrolysates using bacterial enzymes. Journal of Agricultural and Food Chemistry, 65(48), 10514
10519. Tada, M., Shinoda, I., & Okai, H. (1984). L-Ornithyltaurine, a new salty peptide. Journal of Agricultural and Food Chemistry, 32(5), 992
996. Temussi, P. A. (2012). The good taste of peptides. Journal of Peptide Science, 18(2), 73
82. Ueda, Y., Sakaguchi, M., Hirayama, K., Miyajima, R., & Kimizuka, A. (1990). Characteristic flavor constituents in water extract of garlic. Agricultural and Biological Chemistry, 54(1), 163
169. Upadhyaya, J., Pydi, S. P., Singh, N., Aluko, R. E., & Chelikani, P. (2010). Bitter taste receptor T2R1 is activated by dipeptides and tripeptides. Biochemical and Biophysical Research Communications, 398(2), 331
335. Xu, J.-J., Elkaddi, N., Garcia-Blanco, A., Spielman, A. I., Bachmanov, A. A., Chung, H. Y., & Ozdener, M. H. (2017). Arginyl dipeptides increase the frequency of NaCl-elicited responses via epithelial sodium channel alpha and delta subunits in cultured human fungiform taste papillae cells. Scientific Reports, 7(1), 7483. Xu, Q., Singh, N., Hong, H., Yan, X., Yu, W., Jiang, X., . . . Wu, J. (2019). Hen protein-derived peptides as the blockers of human bitter taste receptors T2R4, T2R7 and T2R14. Food Chemistry, 283, 621
627. Yamamoto, M., Terada, Y., Motoyama, T., Shibata, M., Saito, T., & Ito, K. (2020). N-terminal [Glu] 3 moiety of γ-glutamyl peptides contributes largely to the activation of human calcium-sensing receptor, a kokumi receptor. Bioscience, Biotechnology, and Biochemistry, 1
4. Yang, J., Bai, W., Zeng, X., & Cui, C. (2019). Gamma glutamyl peptides: The food source, enzymatic synthesis, kokumi-active and the potential functional properties
a review. Trends in Food Science & Technology., 91, 339
346. Zhang, C., Alashi, A. M., Singh, N., Chelikani, P., & Aluko, R. E. (2019). Glycated beef protein hydrolysates as sources of bitter taste modifiers. Nutrients, 11(9), 2166. Zhang, C., Alashi, A. M., Singh, N., Liu, K., Chelikani, P., & Aluko, R. E. (2018). Beef protein-derived peptides as bitter taste receptor T2R4 blockers. Journal of Agricultural and Food Chemistry, 66(19), 4902
4912. Zhang, J., Sun-Waterhouse, D., Su, G., & Zhao, M. (2019). New insight into umami receptor, umami/umamienhancing peptides and their derivatives: A review. Trends in Food Science & Technology, 88, 429
438. Zhang, J., Zhao, M., Su, G., & Lin, L. (2019). Identification and taste characteristics of novel umami and umami-enhancing peptides separated from peanut protein isolate hydrolysate by consecutive chromatography and UPLC
ESI
QTOF
MS/MS. Food Chemistry, 278, 674
682.

580 Chapter 23 Zhang, Y., Venkitasamy, C., Pan, Z., Liu, W., & Zhao, L. (2017). Novel umami ingredients: Umami peptides and their taste. Journal of Food Science, 82(1), 16
23. Zhang, Y., Zhang, L., Venkitasamy, C., Pan, Z., Ke, H., Guo, S., . . . Zhao, L. (2019). Potential effects of umami ingredients on human health: Pros and cons. Critical Reviews in Food Science and Nutrition, 1
9. Available from https://doi.org/10.1080/10408398.2019.1633995. Zhang, Z., Elfalleh, W., He, S., Tang, M., Zhao, J., Wu, Z., . . . Sun, H. (2018). Heating and cysteine effect on physicochemical and flavor properties of soybean peptide Maillard reaction products. International Journal of Biological Macromolecules, 120, 2137
2146. Zhao, C. J., & Ga¨nzle, M. G. (2016). Synthesis of taste-active γ-glutamyl dipeptides during sourdough fermentation by Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 64(40), 7561
7568. Zhao, Y., Zhang, M., Devahastin, S., & Liu, Y. (2019). Progresses on processing methods of umami substances: A review. Trends in Food Science & Technology, 93, 125
135. Zhu, X.-L., Watanabe, K., Shiraishi, K., Ueki, T., Noda, Y., Matsui, T., & Matsumoto, K. (2008). Identification of ACE-inhibitory peptides in salt-free soy sauce that are transportable across Caco-2 cell monolayers. Peptides, 29(3), 338
344. Zhu, X., Sun-Waterhouse, D., Chen, J., Cui, C., & Wang, W. (2020). Bitter-tasting hydrophobic peptides prepared from soy sauce using aqueous ethanol solutions influence taste sensation. International Journal of Food Science & Technology, 55(1), 146
156. Zhuang, M., Lin, L., Zhao, M., Dong, Y., Sun-Waterhouse, D., Chen, H., . . . Su, G. (2016). Sequence, taste and umami-enhancing effect of the peptides separated from soy sauce. Food Chemistry, 206, 174
181.

CHAPTER 24

Cardiovascular benefits of food proteinderived bioactive peptides Rotimi E. Aluko The Richardson Centre for Functional Foods and Nutraceuticals, Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada

24.1 Introduction The World Health Organization has recognized cardiovascular disease (CVD) as a leading cause of mortality with global death of B18 million people in 2016, which represents 31% of all deaths (WHO, 2017). The term “CVD” encompasses several heart and blood vessel disorders that include congenital heart disease, peripheral arterial disease, cerebrovascular disease, deep vein thrombosis and pulmonary embolism, rheumatic heart disease, and coronary heart disease (WHO, 2017). Therefore heart attack and stroke are responsible for majority (B85%) of the fatalities associated with CVD. Hypertension or raised blood pressure has been recognized as a major contributor to CVD and an estimated 1.13 billion people suffer from this condition as of 2015 data (NCD Risk Factor Collaboration, 2017; Pickering et al., 2005). Thus one of the main approaches to reducing CVDs is adoption of strategies that lower blood pressure and a target of 25% reduction in global rate of hypertension has been suggested (NCD Risk Factor Collaboration, 2017). Hypertension is a condition where the systolic blood pressure (SBP) and diastolic blood pressure (DBP) are .140 and 90 mmHg, respectively, which has a negative effect on the ability of the heart to pump blood efficiently (Carretero and Oparil, 2000; Pickering et al., 2005; Sundstrom et al., 2015). The reduced blood flow can cause underperformance of several organs and gradually could limit blood flow to levels that cause organ failure. Apart from changes in lifestyle, various blood pressure-lowering agents have been successfully used to treat hypertension globally. These antihypertensive agents consist mainly of synthetic compounds (drugs) such as lisinopril, captopril, elanapril, aliskiren, and ramipril that have been approved for therapeutic interventions (Aluko, 2019). However, even though these drugs are very effective in reducing blood pressure, their continued uses have been shown to lead to the development of unwanted health conditions. Typical serious negative side effects of drugs include persistent dry cough (Yesil, Yesil, Bayata, & Postaci, 1994), Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00009-1 © 2021 Elsevier Inc. All rights reserved.

581

582 Chapter 24 congenital malformations (Cooper et al., 2006; Pryde, Sedman, Nugent, & Barr, 1993), erectile dysfunction (Blumentals, Brown, & Gomez-Caminero, 2003; Fogari and Zoppi, 2002), and angioedema (Gunkel, Thurner, Kanonier, Sprinzl, & Thumfart, 1996; Vleeming, van Amsterdam, Stricker, & de Wildt, 1998). These negative events can lead to patient noncompliance with physician recommended drug doses, which reduces efficacy of the treatment regimen and could actually exacerbate the hypertension and associated cardiovascular disorders (Bangalore, Kamalakkannan, Parkar, & Messerli, 2007; Flack, Novikov, & Ferrario, 1997). Therefore research into nondrug alternatives to current antihypertensive agents have intensified in the past 20 years with food protein-derived peptides at the forefront of these efforts. Typically, food proteins consist of certain inactive amino acid sequences that have been encrypted within the primary structure and are referred to as “cryptides.” However, optimal proteolysis can release these cryptides, which then become active with potential positive impact on several human physiological systems. The released cryptides are called “bioactive peptides” because of their intrinsic property of being able to impart positive health benefits, including blood pressure attenuation and overall reduction in the pathological intensity of CVDs. In addition, food protein-derived peptides are deemed to be less toxic and with fewer negative side effects as drugs, which could enhance patient tolerance and adherence to recommended therapeutic doses. For example, excessive (five fold) oral administration of casein-derived peptides to normal and hypertensive human volunteers reduced blood pressure as expected but did not lead to any observable negative event and was well tolerated (Ishida et al., 2011). Generally, bioactive peptides consist of 2
20 amino acids that have been released from food proteins through in vivo or in vitro enzymatic hydrolysis, including microbial fermentation that involves protease secretion (Aluko, 2019). However, for regulatory purposes, the US Food and Drug administrations considers polymers with up to 40 amino acids as peptides (Herzig, 2019). Therefore the amino acid composition and sequence of bioactive peptides will vary depending on the catalytic specificity of the protease used during digestion. The initial product of protein hydrolysis is called “protein hydrolysate” and consists of several peptides of various length and bioactive efficiency. In order to enhance bioactive strength, the protein hydrolysate is usually subjected to fractionation and purification protocols that separate active from inactive peptides. Ultrafiltration membrane, gel chromatography, and HPLC methods are the most common separation protocols used to enrich protein hydrolysates and produce purified peptide sequences. Irrespective of the amino acid sequence, bioactive peptides with CVD benefits generally work through several mechanisms, most of which are similar to the mode of action of drugs. However, due to the expensive nature of peptide purification, the use of protein hydrolysates or minimally processed equivalents are most commonly used for therapeutic interventions. Peptide purification is used mainly to enable determination of amino acid sequence, which provides useful information to carry out structure
function studies. Fundamental scientific

Cardiovascular benefits of food protein-derived bioactive peptides 583 information from structure
function studies could enable development of better enzyme hydrolysis tools or hydrolysis conditions that release desired peptide sequences from food proteins. Knowledge of peptide amino acid sequence is also useful for the development of peptidomimetics that could have greater potency than the original sequence. In this chapter, the focus will be on peptides that modulate the renin
angiotensin
aldosterone system (RAAS) to reduce blood pressure and ameliorate associated cardiovascular disorders.

24.2 Inhibition of the renin
angiotensin
aldosterone system: antihypertensive peptides The mammalian blood pressure is controlled mainly by activities of the RAAS, which consists of enzymatic reaction cascades that lead to the production of a vasoconstrictive agent or inactivation of the vasorelaxative compound (Aluko, 2019). The rate-determining step in this cascade is catalyzed by renin (EC 3.4.23.15), which converts angiotensinogen into the inactive angiotensin I (Ang I), a decapeptide. A second enzyme called angiotensinconverting enzyme (ACE; EC 3.4.15.1) then acts on Ang I to remove a dipeptide and produce the highly vasoconstrictive angiotensin II (Ang II), which binds to the Ang II receptors on blood vessels to cause constriction and ensure blood flow. In addition, Ang II stimulates the release of aldosterone from the adrenal cortex. The resultant high concentration of aldosterone leads to increased potassium excretion and sodium reabsorption. High blood sodium enhances water retention that leads to increased blood volume and hence high blood pressure. Ang II binds to the brain to increase thirst and hence water intake in addition to enhanced production of the antidiuretic hormone (vasopressin), which stimulates the kidney to absorb more water into blood circulation. ACE also catalyzes the hydrolysis and inactivation of bradykinin (a vasodilating peptide), which leads to increased vasoconstriction but reduced relaxation and ultimately the development of high blood pressure. Therefore the overall effect of Ang II is to increase blood sodium and water as well as reduce vasodilatory ability of blood vessels, all of which lead to increased blood pressure. Thus under disease conditions (MacMachon et al., 1990) or as a result of aging (Allen et al., 2012), where the normal physiological levels of renin and/or ACE are exceeded, there is excessive production of Ang II. When uncontrolled, the excessive Ang II level will continue to exacerbate the high blood pressure until the pathological condition of hypertension is established. Therefore peptide interventions to reduce renin and ACE activities or prevent binding of Ang II to its receptors are demonstrated means of attenuating blood pressure and treating hypertension. The animal model that is most commonly used to evaluate the cardiovascular benefits of bioactive peptides is the spontaneously hypertensive rat (SHR), which was developed from the normotensive Wistar-Kyoto (WKY) rat. The SHR is the closest model to human essential hypertension with rapid hypertension development after the first 6 weeks of life in addition

584 Chapter 24 to elevated levels of ACE and renin activities (Feld, van Liew, Brentjens, & Boylan, 1981). Male SHRs grow bigger (up to 300 g) and develop hypertension more rapidly (SBP up to 203 mmHg), which makes them the favorite choice over the females. Therefore in the following sections, emphasis will be on the use of SHRs to test the potential cardiovascular benefits of food protein hydrolysates because these experiments provide a more realistic efficacy evaluation than simple in vitro assays.

24.2.1 ACE- and renin-inhibitory peptides 24.2.1.1 Animal protein-derived hydrolysates and peptides Various food proteins have been subjected to enzymatic hydrolysis to produce hydrolysates and peptides with ability to interfere with ACE activity. Two of the first demonstrated food protein-derived peptides are the famous milk protein-derived tripeptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), which were shown to inhibit ACE activity with IC50 values of 9 and 5 μM, respectively (Nakamura et al., 1995). The peptides were confirmed to be the main active hypotensive agents present in Lactobacillus helveticus fermented milk. Single oral administration of 5 mL of the fermented milk to SHRs, which is equivalent to 0.6 and 0.3 mg VPP and IPP, respectively, per kilogram of rat body weight (BW) led to a maximum 20 mmHg decrease in SBP (Nakamura, Yamamoto, Sakai, & Takano, 1995). Oral administration of individual peptides to the SHRs at 0.6 and 0.3 mg/kg BW for VPP and IPP resulted in maximum SBP decreases of 32.1 and 28.3 mmHg, respectively. It was further demonstrated that the tripeptides inhibited ACE activity in the SHR aorta as part of the blood pressure-lowering mechanism (Masuda, Nakamura, & Takano, 1996). In elderly hypertensive men, daily oral administration of 95 mL of the fermented milk led to 9.4 and 14.1 mmHg decreases in SBP after 4 and 8 weeks, respectively, whereas there was a 6.9 mmHg decrease in DBP after 8 weeks (Hata et al., 1996). However, in another human intervention trial, the oral administration of an enzymatic digest of casein containing VPP and IPP reduced only SBP after 6 weeks but not DBP (Mizuno et al., 2005). The work showed that daily consumption of 1.8, 2.5, and 3.6 mg doses of VPP 1 IPP led to SBP decreases of 26.3, 26.7, and 210.1 mmHg, respectively, after 6 weeks. In contrast, the work of Aihara et al. (Aihara, Kajimoto, Hirata, Takahashi, & Nakamura, 2005) suggests that the antihypertensive effect of the peptides is dependent on the blood pressure of human volunteers. This is because in subjects with high-normal blood pressure, daily consumption of 12 g of the dry fermented milk for 4 weeks resulted only in significant DBP decreases (25 mmHg) but not the SBP. On the other hand, SBP decreased by 11.2 mmHg in the mildly hypertensive group, whereas there was no significant difference in the DBP changes when compared to the placebo. A recent work reported the isolation and ACE-inhibitory effects of several peptides from bovine bone gelatin hydrolysate. The most active peptides were RGM-Hyp-GF and RGL-Hyp-GL with IC50 values of 10.23 and 1.44 μM, respectively (Cao, Wang, Hao, Zhang, & Zhou, 2020). After

Cardiovascular benefits of food protein-derived bioactive peptides 585

Figure 24.1 Effect of single oral administration of physiological saline alone, captopril (10.0 mg/kg), and RGM and RGL (10 and 30 mg/kg) on systolic blood pressure in SHRs. Note: RGL-Hyp-GL: RGL and RGM-Hyp-GF: RGM; the single asterisk (*) indicates a significant difference from the negative control group (P , 0.05); the double asterisks (**) indicate a significant difference from the negative control group (P , 0.01). Source: Reprinted with permission from Cao, S., Wang, Y., Hao, Y., Zhang, W., & Zhou, G. (2020). Antihypertensive effects in vitro and in vivo of novel angiotensin-converting enzyme inhibitory peptides from bovine bone gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 68, 759
768. Copyright (2020) American Chemical Society.

oral administration to SHRs (30 mg/kg BW), there were 38.6 and 31.3 mmHg decreases in SBP for RGM-Hyp-GF and RGL-Hyp-GL, respectively (Fig. 24.1). The inverse relationship between SBP depression and the IC50 values indicates that in vitro ACE-inhibitory activity may not necessarily be useful in predicting in vivo efficacy of bioactive peptides. Contrasting results were reported for a human feeding trial where consumption of a whey protein hydrolysate (WPH) led to plasma decreases in ACE activity but with no effect on blood pressure (Martin et al., 2020). It should be noted that the WPH study was conducted using normotensive human volunteers, which could have been responsible for the lack of significant depressions in blood pressure even when blood ACE activity was decreased. RGL-Hyp-GL had a faster action with maximum SBP depression after 4 h, which indicates a more rapid absorption in comparison to the 6 h for RGM-Hyp-GF. Since the SBP-lowering effects of the two peptides are similar to that of captopril (antihypertensive drug), it could be concluded that the peptides may serve as therapeutic tools against hypertension. Similarly, three active peptides (WYK, IVDR, and VASVI) isolated from fish (olive flounder) surimi (protein gel) exhibited strong in vitro ACEinhibitory activity in addition to producing reduced blood pressure after oral administration to SHRs (Oh et al., 2020). WYK, IVDR, and VASVI had ACE-inhibitory IC50 values of 32.97,

586 Chapter 24 46.90, and 32.66 μM, respectively. Treatment of human umbilical vein endothelial cells (HUVEC) with the peptides resulted in increased nitric oxide (NO) production and the tripeptide (WYK) was most potent in this regard. The increased NO production was positively related to increased activation of the Akt/eNOS (endothelial nitric oxide synthase) signaling pathway. Thus in addition to ACE inhibition, the peptides produced hypotensive effects by enhancing vasodilation through increased eNOS activity and hence higher plasma NO levels when compared to untreated cells. The data are similar to those reported for ACE-inhibitory umami peptides (CC, HCHT, CCNK, and AHSVRF), which also enhanced NO production in HUVECs (Hao et al., 2020). The WYK was the most effective in reducing SBP of SHRs after 3 h with up to 30 mmHg at 50 mg/kg BW dose; however, all the peptides had similar SBP depression after 6 h. The faster-acting effect of WYK may be due to the smaller size and suggests better initial absorption rate from the rat gastrointestinal tract (GIT) when compared to IVDR and VASVI. The three peptides lost their antihypertensive effects after 6 h with SBP returning to normal high levels at 9 h. Therefore the results suggest rapid inactivation through peptidase degradation of the peptides after 6 h. LSGYGP is a peptide isolated from tilapia skin gelatin hydrolysate and was shown to be a noncompetitive ACE inhibitor, which indicates binding to the enzyme’s nonactive side amino acid residues (Tianrui, Bingtong, Ling, Liping, & Yongliang, 2019). LSGYGP had an IC50 of 25.74 μM and interacted with ACE protein molecule through seven hydrogen bonds to nonactive site amino acid residues (Arg522, Phe527, Gly414, and Asp415), which confirm the kinetic data indicating a noncompetitive inhibitor. When administered to SHRs at a 20 mg/kg BW, LSGYGP produced maximum significant SBP and DBP reductions of about 25 and 35 mmHg, respectively, after 3 h. The target organ was the lung where the peptide significantly reduced ACE activity but not the kidney and serum. Cell culture tests indicated that the peptide was absorbed but was susceptible to cellular peptidases, which produced different degradation products such as GYGP, LSGY, and SGYGP as detected in the basolateral compartment. Results of the cell culture and SHR experiments suggest that the breakdown products of LSGYGP may also exert blood pressure-reducing effects. This is consistent with a pro-drug type of peptide whereby breakdown products have bioactive effects just as the parent sequence (Fujita, Yokoyama, & Yoshikawa, 2000). Tilapia skin gelatin was also hydrolyzed and QAGLSPVR identified as the major active ACE-inhibitory peptide (Sun, Wu, Yan, Hou, & Zhuang, 2019). QAGLSPVR at an oral dose of 20 mg/kg BW produced up to 30 mmHg reduction in SBP and DBP after 3 h, which coincided with the point at which plasma ACE activity was most depressed in the SHRs. Therefore the results confirm that the BP-lowering effect of QAGLSPVR is due to in vivo inhibition of ACE activity, which means the peptide was absorbed from the GIT. Cell culture experiments showed that QAGLSPVR was resistant to structural degradation by cellular proteases and absorption was independent of cellular peptide transporters because Gly-Sar and wortmannin (inhibitors of peptide transporters) had minimal effect on rate of

Cardiovascular benefits of food protein-derived bioactive peptides 587 translocation into the cell. However, QAGLSPVR might have been absorbed through the paracellular pathway because of the observed inhibitory effect of cytochalasin. In a separate work, the pepsin hydrolysate of tilapia gelatin and membrane ultrafiltration peptide fractions were evaluated for their in vitro ACE- and renin-inhibitory activities in addition to in vivo testing for antihypertensive effect after oral administration to SHRs (Lin, Alashi, Aluko, Pan, & Chang, 2017). Results showed IC50 values of 0.57, 0.41, 0.55, 0.79, and 0.83 mg/mL for the pepsin hydrolysate, ,1 kDa, 1
3 kDa, 3
5 kDa, and 5
10 kDa peptides, respectively, which indicate minimal beneficial effect on ACE-inhibitory potency as a result of the membrane separation. Similarly, the pepsin hydrolysate with a renininhibitory value of 53% was better than the peptide fractions (B10%). Only the ,1 kDa peptide fraction was evaluated for antihypertensive effects at 100 mg/kg SHR BW with 33, 24, and 28 mmHg reductions in SBP, DBP, and mean arterial pressure (MAP), respectively. Heart rate of the SHR was also reduced 58 beats/min by the ,1 kDa peptide fraction, which in addition to the blood pressure effects suggests potential use as a positive modulator of the cardiovascular system. An endoproteinase porcine gelatin hydrolysate (PGH) with ACE-inhibitory IC50 value of 220 μg/mL also showed some cardiovascular benefits after oral administration to SHRs (O’Keeffe, Norris, Alashi, Aluko, & FitzGerald, 2017). Oral administration of 50 mg/kg BW of gelatin and PGH resulted in 10.5 and 28.9 mmHg SBP decreases as well as 5.8 and 22.9 mmHg DBP decreases, respectively. The PGH also produced significantly better decreases in MAP and heart rate of the rats when compared to the unhydrolyzed gelatin. The results confirmed that the endoproteinase hydrolysis released physiologically active ACE-inhibitory peptides from gelatin. A previous work also examined the antihypertensive efficacy of another marine product, the protease AP hydrolysate of boarfish, which had about 70% inhibition of ACE in vitro activity (Hayes, Mora, Hussey, & Aluko, 2016). SHRs were orally administered (200 mg/kg BW) with the boarfish hydrolysate followed by SBP measurement. Results showed a rapid B15 mmHg decrease in SBP 2 h after oral administration of the hydrolysate, which was followed by 37 mmHg decrease after 8 h. The SBP was depressed by B15 mmHg after 24 h, which shows not only the potency but persistency of the boarfish hydrolysate as an antihypertensive agent. Other marine protein sources that have been studied for their ability to release cardioprotective peptides after enzymatic hydrolysis include cod and salmon. Cod muscle proteins were subjected to consecutive hydrolysis, first with pepsin and then followed by a mixture of trypsin and chymotrypsin. The resultant cod protein hydrolysate was passed through a 1 kDa membrane to collect the permeate (,1 kDa peptides), which was then tested for blood pressure-reducing effects in SHRs (Girgih et al., 2015). The ,1 kDa peptides inhibited the in vitro activities of ACE and renin with IC50 values of 0.13 and 0.28 mg/mL, respectively. The ,1 kDa peptides were then separated into four fractions (CF1
CF4) by RP-HPLC and CF3 chosen for further evaluation due to the superior

588 Chapter 24 inhibitions of ACE and renin activities. CF3 was more potent than the ,1 kDa peptides as ACE and renin inhibitor with IC50 values of 0.11 and 0.16 mg/mL, respectively. CF3 and the ,1 kDa peptides inhibited ACE activity in an uncompetitive manner, which means that the peptides were mostly bound to the enzyme
substrate complex. In contrast, the CF3 and ,1 kDa peptides inhibited renin activity through noncompetitive mode, indicating peptide binding to nonactive sites on the enzyme protein. The CF3 and ,1 kDa peptides were orally administered to SHRs at doses of 30 and 200 mg/kg BW, respectively, and blood pressure monitored for 24 h. Results showed that both peptides produced the most reduction in SBP after only 2 h with values of 240 and 218 mmHg for CF3 and ,1 kDa peptides, respectively. Since the in vitro ACE inhibition was similar for the two peptide samples, the stronger SBP-lowering effect of CF3 was attributed to the higher renin inhibition. The authors estimated that a 70-kg adult will required B350 mg daily dose to benefit from the blood pressure-lowering ability of CF3. A similar work was performed for salmon muscle proteins with results also showing improved renin inhibition by SF3 fraction from RPHPLC separation of the ,1 kDa peptides (Girgih et al., 2016). Kinetics of enzyme inhibition also showed that the salmon peptides inhibited ACE activity in an uncompetitive manner but noncompetitive for renin. The SF3 also produced B43 mmHg reduction in SBP in comparison to the 21 mmHg for the ,1 kDa peptides, 2 h after oral administration to SHRs using similar doses as the cod peptides. Egg protein hydrolysates have been studied extensively for their cardiovascular protection effects, especially blood pressure reduction. One of the earlier studies examined the blood pressure-modulating ability of enzymatically hydrolyzed fried whole egg as well as hydrolyzed egg white (Jahandideh et al., 2014). SHRs were fed 1 g/kg BW dose daily for 18 days accompanied with continuous monitoring of blood pressure and heart rate by telemetry. Results showed that the fried whole egg hydrolysate reduced SBP by about 20 mmHg at the end of the study. Similar decreases in MAP and DBP were also observed for the rats that consumed the hydrolysate but not the unhydrolyzed protein. In addition, the egg hydrolysate significantly reduced plasma Ang II levels with concomitant enhanced relaxation of the mesenteric arteries, which was attributed to increased contribution from NO. Therefore the authors concluded that the observed hypotensive activity of the hydrolysate was due to the digested peptides, which were absent from the unhydrolyzed proteins. A tripeptide sequence (IRW) present in egg white protein ovotransferrin was identified as a potent ACE inhibitor and its antihypertensive effects demonstrated in SHR (Majumder et al., 2013). Using 2 IRW doses (3 and 15 mg/kg BW per day), which were given orally to SHR for 18 days, the work reported 10 and 40 mmHg decreases in SBP, respectively, at the end of the study but without a significant effect on the heart rate. Mechanistic investigations revealed that IRW reduced Ang II in the plasma and collagen type I in aorta and kidneys, whereas there were enhanced levels of eNOS and bradykinin (Fig. 24.2). The low levels of Ang II plus the high levels of eNOS and bradykinin promote

Cardiovascular benefits of food protein-derived bioactive peptides 589

Figure 24.2 IRW treatment attenuates plasma Ang II levels through possible ACE-inhibitory effects. (A) Plasma Ang II (pg/mL) levels from untreated and high dose (15 mg/kg BW) IRW-treated SHRs are shown. (B) Plasma bradykinin (ng/mL) levels from untreated and high dose (15 mg/kg BW) IRW-treated SHRs. Data represented as mean 6 SEM from n 5 6 animals per treatment group. The single asterisk (*) and double asterisks (**) indicate P , 0.05 and P , 0.01, respectively, as compared to the untreated group. Source: Reprinted with authors’ permission from Majumder K., Chakrabarti, S., Morton, J. S., Panahi, S., Kaufman, S., Davidge, S. T., & Wu, J. (2013). Egg-derived tri-peptide IRW exerts antihypertensive effects in spontaneously hypertensive rats. PLoS ONE, 8(11), e82829.

vasorelaxation, whereas the reduced collagen level has a negative effect on fibrosis development within the organs, all of which contribute to a healthy cardiovascular system. RVPSL is another peptide isolated from egg white protein hydrolysate and shown to prevent SBP elevation in SHR with the 50 mg/kg BW dose being most effective (Yu, Yin, Zhao, Chen, & Liu, 2014). Gene expression data showed that RVPSL suppressed renin, ACE, and angiotensin type 1 (AT1) receptor synthesis in the kidneys. In the blood, RVPSL also suppressed the level of renin activity in addition to reduced concentrations of aldosterone and Ang II. These mechanistic data confirm ability of the peptide to positively influence cellular molecular pathways that control the RAAS with concomitant significant downregulation of blood pressure.

590 Chapter 24 Chicken products have also been used as raw materials to generate protein hydrolysates with potential cardioprotective effects. Spent hen meat was hydrolyzed with pepsin (SPH-P) or a pepsin 1 pancreatin (SPH-PPc) combination and the blood pressure-reducing effects determined after oral administration (200 mg/kg BW) to SHRs (Udenigwe et al., 2017). Results showed ACE or renin-inhibitory IC50 values of 0.42 and 0.65 mg/mL or 0.52 and 0.34 mg/mL for SPH-PPc and SPH-P, respectively. During the in vivo tests, SPH-PPc was more effective in reducing SBP of SHRs (36 mmHg) when compared to SPH-P (27 mmHg), which suggests a positive relationship with the observed in vitro ACE inhibition but not renin. The authors suggested that the stronger blood pressure-reducing ability of SPH-PPc might be due to the smaller peptide sizes, which could have enhanced the GIT absorption when compared to SPH-P. Chicken skin is considered a waste product of the poultry processing industry but conversion to bioactive protein hydrolysates could enhance valueaddition, especially in the functional foods sector. Onuh et al. (Onuh, Girgih, Malomo, Aluko, & Aliani, 2015) used alcalase and pepsin 1 pancreatin to convert chicken skin proteins into hydrolysates that inhibited ACE and renin activities. The work showed lower ACE-inhibitory IC50 values (,0.7 mg/mL) than renin inhibition (1.6
2.6 mg/mL), which is consistent with most reports of stronger ACE inhibition by food protein-derived peptides. Interestingly, kinetic analysis revealed mixed-type inhibition by the chicken skin protein hydrolysates (CSPH), which indicates binding of the peptides to the free enzyme in addition to the enzyme
substrate complex. Such ability to bind to all forms of the enzyme could make the CSPH a very potent therapeutic agent against hypertension. Using an oral dose of 100 mg/kg BW, it was shown that the CSPH reduced SBP by a maximum of 31 mmHg after 6 h in SHR when compared to 14 mmHg for the unhydrolyzed chicken skin protein extract. Separation of the CSPH into peptide fractions of defined sizes (,1, 1
3, 3
5, and 5
10 kDa) resulted in significant 50% loss in SBP reduction. The loss in antihypertensive potency suggest that the peptide synergistic effects in the CSPH were stronger than those within the peptide fractions. The results support the use of CSPH as a potential therapeutic tool against hypertension, which is actually cheaper to produce than the peptide fractions. During long-term (6 weeks) oral feeding of CSPH to rats, SBP was also significantly depressed (up to 38 mmHg) with lower plasma ACE activity when compared to the 26 mmHg reduction for unhydrolyzed protein (Onuh et al., 2016). However, there was no measurable effect on plasma renin activity, which indicates that one of the main mechanisms of CSPH antihypertensive activity is through ACE inhibition. Metabolomics analysis indicated that rats fed the CSPH diet had downregulated RAAS and oxidative stress, which is consistent with the reduced ACE activity. Another product that has been evaluated for antihypertensive effects is chicken foot protein hydrolysate (CFPH). After hydrolysis with protamex, the CFPH exhibited 96% inhibition of ACE activity and kinetic data indicated competitive inhibition, that is, peptides are able to compete with substrates for the enzyme active site (Mas-Capdevila, Pons, Aleixandre, Bravo, & Muguerza, 2018). This is in contrast to the CSPH that showed mixed-type inhibition. At CFPH oral doses of

Cardiovascular benefits of food protein-derived bioactive peptides 591 55 and 85 mg/kg BW, SBP of SHRs decreased by 26.3 and 30.5 mmHg, respectively, but the 25 mg dose had no effect. The 55 mg CFPH dose reduced plasma ACE activity by 21% but had no vasorelaxation effect on the aorta blood vessels and did not change SBP of WKY, the normotensive rats, which is consistent with several other reports on antihypertensive food protein hydrolysates. One of the earlier works examined the blood pressure-reducing ability of a porcine muscle hydrolysate (ACE-inhibitory IC50 5 130 μM), which was generated with pepsin hydrolysis (Katayama et al., 2008). Two peptides, KRQKYDI and EKERERQ with IC50 values of 26.2 and 552.5 μM, respectively, were identified to be the main active compounds. KRQKYDI inhibited ACE activity in a competitive manner, and an oral dose of 10 mg/kg BW suppressed SBP of SHRs by B10 mmHg after 4
6 h. The same research group also reported the isolation of another porcine muscle-derived peptide, VKKVLGNP with ACEinhibitory IC50 value of 28.5 μM, which is similar in potency to KRQKYDI (Katayama et al., 2007). However, VKKVLGNP acted as a noncompetitive inhibitor and reduced SBP of SHRs by 24 mmHg after 3 h using 10 mg/kg BW dose, which indicates stronger antihypertensive potency than KRQKYDI. VKKVLGNP was highly resistant to ACEinduced proteolysis, which might have been responsible for the stronger antihypertensive effect when compared to KRQKYDI, a more readily hydrolyzed peptide. Another group reported isolation of two other porcine muscle-derived peptides, KRVIQY and VKAGF with ACE-inhibitory IC50 values of 6.1 and 20.3 μM, respectively (Muguruma et al., 2009). At an oral dose of 10 mg/kg BW, KRVIQY and VKAGF reduced SBP of SHRs by 23 and 17 mmHg, respectively, after 6 h, which is consistent with the differences in IC50 values. Because both peptides inhibited ACE activity in a competitive, the stronger antihypertensive effect of KRVIQY was attributed to its behavior as a pro-drug (i.e., in vivo conversion into more potent peptide fragments) because incubation with ACE led to a lower IC50 value in contrast to the higher value for VKAGF. In a separate work, three novel peptides RPR, KAPVA, and PTPVP identified from pork meat protein were synthesized and evaluated for their antihypertensive properties after oral administration to SHRs (Wang et al., 2008). Results showed that RPR, KAPVA, and PTPVP had ACE-inhibitory IC50 values of 382.0, 46.6, and 256.4 μM with SBP reductions of 33, 33, and 35 mmHg, respectively, at 1 mg/kg BW dose each. Pepsin hydrolysis of oyster proteins was also shown to yield a hydrolysate from which a nonapeptide (VVYPWTQRF) was isolated (Escudero, Toldra´, Sentandreu, Nishimura, & Arihara, 2012). VVYPWTQRF had ACEinhibitory IC50 value of 66 μM, and the activity was not significantly changed after incubation at different temperatures (up to 100 C), pH 2
12 and with ACE. VVYPWTQRF inhibited ACE activity in a competitive manner and oral administration of up to 100 mg/kg dose to SHRs led to significant reductions in SBP with the 20 mg dose being the most effective (B27 mmHg after 2 h). In a long-term study, the 20 mg dose also reduced SBP of the SHRs by up to 31 mmHg after 30 days. The stability of VVYPWTQRF to various heat

592 Chapter 24 and pH conditions could make it a suitable ingredient to formulate various foods that retain antihypertensive property after undergoing food preparation and processing treatments. 24.2.1.2 Plant protein-derived hydrolysates and peptides In a recent work, it was shown that the enzymatic digest of moringa seed as well as the ultrafiltration membrane fractions inhibited in vitro activity of ACE with the 1
3 kDa peptides having the strongest effect (Aderinola et al., 2019). Oral administration of the moringa protein hydrolysate and peptide fractions to SHR at 200 mg/kg BW showed that the 1
3 kDa acted fastest with 35 mmHg reduction in SBP after only 2 h, which may be due to the smaller size when compared to the .3 kDa peptide fractions. However, the ability of the protein hydrolysate to produce the most persistent SBP reduction (34 mmHg 24 h after the oral administration) may be due to the presence of a wider variety of active peptides when compared to the isolated fractions. DBP reductions were most in rats that consumed the 5
10 kDa peptides, though the 3
5 kDa fraction had the most persistent effect with 30 mmHg at 24 h. The moringa hydrolysate and peptide fractions were also effective in depressing MAP as well as heart rate, which suggest multifunctional ability of these products. The heart rate depression ability is particularly a benefit for maintaining optimum heart health conditions and reducing the risk of negative cardiovascular events associated with excessive levels of heart beats. Shih et al. (Shih, Chen, Wang, & Hsu, 2019) reported the isolation and antihypertensive effects of FHAPWK, a novel ACE-inhibitory peptide from the thermolysin hydrolysate of Cassio obtusifolia seeds. FHAPWK had IC50 value of 16.83 μM and acted as a competitive inhibitor of ACE, which indicates peptide binding to the enzyme active site in competition with the substrate. Molecular docking studies indicate that the peptide acted as a competitive inhibitor through electrostatic interactions of its C-terminal lysine residue with the active site zinc and glutamic acids in addition to hydrophobic interactions of tryptophan with the enzyme histidine residue. The peptide was shown to be a true inhibitor because incubation with ACE did not lead to significant changes in IC50 neither was there any evidence of structural fragmentation after liquid chromatography-mass spectrometry (LC/MS) analysis of the incubated solution. When orally administered at 2 mg/kg BW to SHRs, FHAPWK reduced SBP by 17 mmHg after 6 h in comparison to 25 mmHg for a similar dose of captopril. The low FHAPWK dose required for such a significant SBP depression suggest that this peptide could serve as a suitable agent in ameliorating human hypertension. Two noncompetitive ACE-inhibitory peptides (FQINMCILR and TGAPCR) were isolated from red algae and shown to reduce blood pressure in SHRs (Deng et al., 2018). FQINMCILR and TGAPCR had ACE-inhibitory IC50 values of 9.64 and 23.94 μM, respectively. However, molecular docking data revealed that the two peptides formed hydrogen bonds with the amino acids in the ACE active site, which contradicts the noncompetitive behavior from kinetic analysis. It is possible that the active site interactions

Cardiovascular benefits of food protein-derived bioactive peptides 593 are not strong enough to cause inhibition and the two peptides have stronger interactions with nonactive sites on the ACE molecule, which are responsible for the inhibitory effects. FQINMCILR and TGAPCR were resistant against GIT enzymes and ACE because there were no significant reductions in inhibitory activity after incubation with these enzymes. Oral administration (10 mg/kg BW) of FQINMCILR and TGAPCR to SHRs resulted in 34 and 28 mmHg decreases in SBP after 2 h, respectively, as well as B28 mmHg decrease in DBP. It should be noted that in order to enhance stability and solubility of FQINMCILR, the methionine sulfoxide form (FQIN [M(O)] CILR) was synthesized and used for the experiments. Similalry, a tridecapeptide IRLIIVLMPILMA with in vitro renin-inhibitory property was identified from the papain hydrolysate of red seaweed and its blood pressurelowering effect demonstrated in SHRs. Oral administration of the papain hydrolysate (50 mg/kg BW) and IRLIIVLMPILMA (3 mg/kg BW) resulted in similar (B33 mmHg) reductions in SBP (Fitzgerald, Aluko, Hossain, Rai, & Hayes, 2014). However, the lower dose of IRLIIVLMPILMA suggests it might have stronger blood pressure-reducing ability than the seaweed papain hydrolysate. Thermolysin was also used to generate a pea protein hydrolysate (PPH) with cardioprotective and renoprotective functions. PPH inhibited ACE and renin activities with IC50 values of 0.10 and 0.57 mg/mL, respectively, and short-term oral administration (100 mg/kg BW) to SHRs led to significant reductions in SBP with a maximum of 36 mmHg at 2
4 h, whereas long-term feeding (0.5% and 1.0%, wt./wt. of feed) reduced SBP by 26 mmHg after 3 weeks (Girgih, Nwachukwu, Onuh, Malomo, & Aluko, 2016). In an 8-week oral feeding study, inclusions of 0.5% and 1.0% PPH in the diet of chronic kidney disease (CKD) rats resulted in 29 and 25 mmHg reductions in SBP and DBP, respectively (Li et al., 2011). The PPH-containing diets reduced plasma Ang II concentration and renin gene expression in the CKD rats, which supports the observed blood pressure reductions (Fig. 24.3). The results are significant because the ability to control blood pressure in CKD conditions is a critical approach to reducing cardiovascular damages and associated heart failure. The results suggest that PPH might have acted in vivo as a renin inhibitor but with negligible effect on ACE activity. In a placebo-controlled human intervention trial, daily consumption of 3 g PPH over a 3-week period led to 6 mmHg decrease in SBP, which indicates potential use of this product for blood pressure control in people with hypertension (Li et al., 2011). Rapeseed and hemp seed protein enzymatic hydrolysates have been investigated for potential blood pressure-lowering effects. Four antihypertensive peptides (LY, TF, RALP, and GHS) have been isolated and characterized from rapeseed enzymatic protein hydrolysates (He et al., 2013; He, Malomo, Girgih, Ju, & Aluko, 2013). LY, TF, RALP, and GHS inhibited ACE activity with IC50 values of 0.11, 0.81, 0.65, and 1.74 mM in addition to renin-inhibitory values of 1.87, 3.06, 0.97, and 1.09 mM, respectively. Oral administration to SHRs at 30 mg/kg BW dose showed that LY was the most potent with

594 Chapter 24 A

Angiotensin II concentration (µmol/L)

16 14 12 10

*

*

PPH (0.5% of diet)

PPH (1.0% of diet)

8 6 4 2 0

Kidney relative renin mRNA expression

Case in

B

0.8

0.6

0.4

* * 0.2

0.0 Case in

PPH (0.5% of diet)

PPH (1.0% of diet)

Figure 24.3 Effect of pea protein hydrolysate (PPH) on plasma level of angiotensin II (A) and renal expression of renin mRNA (B) in Han:SPRD-cy (chronic kidney disease) rats. Diets were formulated to contain 20% protein (casein) in the control rats but partially substituted with PPH (19.5% casein 1 0.5% PPH or 19% casein 1 1% PPH) in treated rats. *Significantly different from casein at P , 0.05. Source: Reprinted with permission from Li, H., Prairie, N., Udenigwe, C. C., Adebiyi, A. P., Tappia, P., Aukema, H. M., Jones, P. J. H., & Aluko, R. E. (2011). Blood pressure lowering effect of a pea protein hydrolysate in hypertensive rats and humans. Journal of Agricultural and Food Chemistry, 59, 9854
9860. Copyright (2011) American Chemical Society.

maximum SBP depression of B27 mmHg after 2 h in comparison to B17 mmHg for RALP and GHS, whereas TF had the weakest effect (12 mmHg). The stronger effect of LY is consistent with the higher activities against ACE and renin, which suggest that dual inhibition of both enzymes could provide better blood pressure control. Evaluation of hemp

Cardiovascular benefits of food protein-derived bioactive peptides 595 seed has involved the protein hydrolysates as well as various peptide sequences. Malomo et al. (Malomo, Girgih, Onuh, & Aluko, 2015) reported the antihypertensive effects of hemp seed protein hydrolysates (HPH) produced with different enzymes. At 200 mg/kg BW dose, the alcalase HPH produced the most SBP reduction (33 mmHg), but the pepsin HPH had the most persistent effect with 23 mmHg reduction at 24 h. Initial short-term evaluation of another HPH obtained from simulated in vitro GIT digestion revealed that membrane separation weakened ACE and renin-inhibitory activities as well as the SBP-lowering ability (Girgih, Udenigwe, Li, Adebiyi, & Aluko, 2011). Therefore the unfractionated HPH was evaluated for long-term antihypertensive effects in SHRs, especially the ability to prevent and treat high blood pressure. Results showed that during an 8-week feeding trial, inclusion of 0.5% or 1.0% (wt./wt. of feed) HPH prevented hypertension development with a final SBP of B112 mmHg in the eighth week in comparison to 147 mmHg for the control rats (Girgih, Alashi, He, Malomo, & Aluko, 2014). Treatment of hypertensive rats over another 4-week period also showed that the HPH reduced SBP from B145 to 117 mmHg. The HPH-containing diet significantly reduced plasma ACE and renin activities to levels that were similar to or even lower than the levels in normotensive rats (Fig. 24.4). The preventive and treatment effects of the HPH might have been due to the dual ability to suppress plasma levels of ACE and renin, two of the most important factors in mammalian blood pressure regulation. Thus the HPH could be a suitable candidate for therapeutic management of hypertension if similar effects observed in the SHR study can be duplicated in a human intervention trial. The HPH was subjected to peptide purification, which led to identification of five main sequences that inhibited ACE and renin activities in addition to reducing SBP of SHRs using 30 mg/kg BW dose (Girgih et al., 2014). WYT had the weakest effect with a maximum 13 mmHg SBP reduction after 2 h. In contrast, WVYY produced the fastest decrease in SBP (234 mmHg) after 2 h but subsequently, this effect gradually faded over the 24 h period. However, the second tetrapeptide (SVYT) produced a gradual decrease in SBP that reached a peak of 224 mmHg at 6 h, after which the effect decreased to 212 mmHg at 24 h. The two pentapeptides, PSLPA and IPAGV, were the most effective in lowering SBP with maximum effects of 40 and 36 mmHg, respectively, after 4 h of oral administration before subsequently decreasing by B13 mmHg at 24 h. In another work, peach seed protein hydrolysate was hydrolyzed with thermolysin followed by membrane ultrafiltration and peptide sequencing to identify the most active sequence (Va´squez-Villanueva, Orellana, Marina, & Garcı´a, 2019). Results show that the ,3 kDa peptides were the most active with ACE-inhibitory IC50 value of 16.4 μg/mL. RP-HPLC separation of the 3 kDa fraction led to identification of IYSPH as the most active peptide sequence (IC50 5 24.0 μg/mL), which slightly increased to 35.0 μg/mL after simulated GIT digestion. Oral administration of the ,3 kDa peptide fraction (10 mg/kg BW) and IYSPH (1.5 mg/kg BW) to SHRs resulted in about 30 mmHg reduction in SBP after 3
6 h. Based on the SHR dosage, the authors suggested equivalent human doses of 0.243 mg/kg and

596 Chapter 24 A

Plasma ACE activity (U/mL)

0.150 a

0.125 0.100 b

0.075 c d

0.050 0.025 0.000 Control

0.5% HMH

1% HMH

1% HPI

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Plasma renin level (µg/mL)

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0.10 0.08 c

0.06

d

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0.5% HMH

1% HMH

1% HPI

Diet group

Figure 24.4 Effect of the long-term feeding of young growing spontaneously hypertensive rats (SHRs) with caseinonly diet or casein diet that contained hemp seed meal protein hydrolysate (HMH) or protein isolate (HPI) on plasma ACE (A) and renin (B) activities. Bars with different letters have mean values that are significantly different (P , 0.05). Source: Reprinted by permission from: Springer European Journal of Nutrition. Girgih, A. T., Alashi, A. M., He, R., Malomo, S. A., & Aluko, R. E. (2014). Preventive and treatment effects of hemp seed (Cannabis sativa L.) meal protein hydrolysate against high blood pressure in spontaneously hypertensive rats, European Journal of Nutrition, 53, 1237
1246. Copyright 2014.

1.62 mg/kg for the ,3 kDa fraction and IYSPH, respectively. These values translate to B17 and 113 mg doses of the ,3 kDa and IYSPH, respectively, for a 70 kg adult, which are definitely within the range for drugs. Additionally, the , 3 kDa peptide fraction and IYSPH showed no toxicity toward different cell lines such as the HeLa (cervix), HT-29

Cardiovascular benefits of food protein-derived bioactive peptides 597 (colon) and HK-2 (kidney), which indicate they are potentially safe antihypertensive therapeutic agents. A recent report showed that protein hydrolysate fractions from an alcalase digest of corn produced using enzymatic membrane reactor system (EMR) exhibited both in vitro and in vivo ACE-inhibitory activity (Guo et al., 2020). The corn protein hydrolysate was tested at three doses of 250, 500, and 1000 mg/kg BW using shortterm (12 h) and long-term (35 days) oral feeding. During the short-term experiment, the 1000 mg/kg BW dose was the most effective by producing the maximum SBP reduction (218.22 mmHg) at 6 h. Interestingly, the SBP-lowering effects of the three doses lasted for 9 h but became ineffective after 12 h. During the long-term oral feeding experiment, the three doses became effective from the 14th day and lasted until the 35th day with maximum SBP reduction (226.7 mmHg) recorded for the 100 mg/kg BW dose. Analysis of various organs revealed that the 500 and 1000 mg/kg BW doses were the most effective in producing decreased ACE activity in the kidney, lung, and heart but not in the arteries. Plasma levels of renin and Ang II were significantly reduced, whereas NO increased in rats fed the 500 and 1000 mg/kg BW doses. Therefore it is evident from these data that the corn peptides were absorbed into blood circulation and various organs where they reduced renin and ACE activities (hence decreased Ang II) and increased NO production to enhance vasodilation, which translated to SBP decreases. A similar work with rice bran protein hydrolysates showed that low-molecular weight (,3 kDa) peptides could effectively reduce SBP in SHRs (Piotrowicz et al., 2020). Rice bran proteins were digested with alcalase or flavourzyme followed by ultrafiltration using a 3 kDa membrane to collect the permeates (,3 kDa peptides). ACE-inhibitory IC50 values for the permeates were in the 0.15
1.70 mg/mL range with the alcalase digests having lower values (higher potency) than the flavourzyme digests. Oral administration (80 mg/kg BW) of the ,3 kDa peptides to rats led to significant decreases (up to 30 mmHg) in SBP. The strong SBP-reducing effect of the peptides suggests synergistic interactions of the peptides to enhance in vivo ACE inhibition. The peptides had no blood pressure-reducing effect in normotensive (WKY) rats, which is an indication that the peptides target excessive ACE activity in the body and will not produce unsafe levels of blood pressure even when ingested by people with normal blood pressure. Enzymatic hydrolysis of pigeon pea proteins with pepsin or pancreatin as well as a combination of both enzymes have been shown to produce hydrolysates with ability to inhibit ACE and renin activities and reduce SHR blood pressure at 100 mg/kg BW oral dose (Olagunju, Omoba, Enujiugha, Alashi, & Aluko, 2018). In vitro tests showed that the pigeon PPHs were stronger inhibitors of ACE (71%
75%) than renin (7%
14%). The pepsin hydrolysate had the fastest effect with B31 mmHg reduction in SBP after 2 h, whereas pancreatin and pepsin 1 pancreatin hydrolysates had similar effects after 4
6 h. However, the pepsin 1 pancreatin hydrolysate had the highest SBP-reducing effect and was the most persistent with B38 mmHg reduction after 12 h. All the hydrolysates had superior

598 Chapter 24 SBP-reducing effect than the unhydrolyzed pigeon pea protein, which supports the rationale for predigestion as a means of producing peptides with potent cardiovascular benefits. Another product with ACE and renin-inhibitory activities as well as antihypertensive effect is the mung bean protein hydrolysate (MPH), which was obtained following bromelain digestion (Sonklin, Alashi, Laohakunjit, Kerdchoechuen, & Aluko, 2020). Active peptide sequences present in the MPH were identified and also tested for in vitro and in vivo activities. The MPH had ACE-inhibitory IC50 values of 0.69 mg/mL, whereas those of the peptides ranged from 5.4 μM for LRLESF to 1912 μM for LPRL. The MPH had B34% renin-inhibitory activity, whereas the peptides had 30%
97% inhibition with YADLVE being the most active. Oral administration (20 mg/kg BW) to SHRs showed that the peptides were more potent antihypertensive agents with up to 36 mmHg reduction in SBP when compared to a maximum 15 mmHg for MPH. YADLVE produced the most persistent effect with 27 mmHg reduction in SBP when compared to ,18 mmHg for MPH and other peptides. YADLVE also produced the strongest effects in lowering MAP and DBP. The strong blood pressure-reducing ability of YADLVE was attributed to the high renin inhibition because the peptide had no measurable ACE inhibition. Therefore the results support the concept that renin, which catalyzes the rate-determining step could be the best target for antihypertensive agents. Interestingly, the mung bean peptides also reduced heart rate of the rats but this effect was lost after 6 h. PGSGCAGTDL, which was the longest mung bean protein-derived peptide had the poorest blood pressure and heart rate-reducing effects. This could be attributed to susceptibility of the long chain to in vivo proteolysis to produce inactive peptide fragments. Other plant sources of cardioprotective peptides include canola, flaxseed, and seaweeds. Alashi et al. (2014) utilized several proteases to produce protein hydrolysates that inhibited in vitro ACE and renin activities. Alcalase-digested canola protein hydrolysate was the most effective ACE inhibitor while pancreatin produced the most effective renin-inhibitory hydrolysate. The canola protein hydrolysates reduced SBP, but alcalase hydrolysate produced the most reduction (34 mmHg at 4 h) when 200 mg/kg BW dose was orally provided to SHRs. Flaxseed was hydrolyzed with 2.5% and 3% thermoase followed by ultrafiltration membrane separation of the hydrolysates. The flaxseed protein hydrolysates had higher ACE inhibition (up to 90%) than renin inhibition (up to 40%), and the 3% thermoase digest was generally more effective than the 2.5% (Nwachukwu, Girgih, Malomo, Onuh, & Aluko, 2014). However, both the 2.5% and 3% hydrolysates produced significant SBP depressions (max. of 228 mmHg at 4 h), and was persistent with 213 mmHg at 24 h.

24.2.2 Foods formulated with antihypertensive protein hydrolysates and peptides In addition to the use of protein hydrolysates and peptides as intervention tools, some reports have shown that foods formulated with these products can also serve as

Cardiovascular benefits of food protein-derived bioactive peptides 599 antihypertensive agents. Amaranth protein was digested with alcalase and the resultant hydrolysate incorporated (4%) into pasta, which was then fed to SHRs (Valdez-Meza et al., 2019). Initial analysis showed that the amaranth protein hydrolysate was a potent ACE inhibitor with an IC50 of 14 μg/mL. The rats were fed 8 g of the formulated pasta products followed by SBP measurements at hourly intervals up to 9 h. In addition to the 4% amaranth protein hydrolysate, one pasta contained 8.5% of the unhydrolyzed amaranth protein concentrate (B), whereas the other had 43% (C). The results showed minimal SBP reductions (max. of 220 mmHg after 5
6 h) in rats that consumed pasta formulated with 100% semolina (A). Rats that consumed pasta B or C had additional significant SBP reductions (215 mmHg) after 3 h in comparison to rats that consumed pasta A. Maximum SBP reductions of up to 245 and 260 mmHg were achieved after 5
6 h in rats that consumed pasta B and C, respectively. The stronger SBP-reducing effect of pasta C was attributed to the higher protein concentrate content, which may have trapped the antihypertensive peptides to enhance bioavailability. However, it is also possible that additional antihypertensive peptides were generated during GIT digestion of the amaranth protein concentrate, which will supplement the added protein hydrolysate peptides. This is because a previous work has demonstrated the release of ACE-inhibitory peptides during simulated GIT digestion of amaranth protein concentrate (Tiengo, Faria, & Netto, 2009). Therefore it can be suggested that digestion of pasta C generated more antihypertensive peptides due to the higher protein concentrate content and hence produced stronger SBPreducing effects when compared to pasta B with less amounts of the protein concentrate. Another work examined the influence of alcalase-generated amaranth protein hydrolysatefortified cookies on blood pressure of SHR (Ontiveros et al., 2020). In vitro analysis revealed an IC50 value of B30 μg/mL for this amaranth protein hydrolysate, which is less active than the previously reported value of 14 μg/mL for another amaranth hydrolysate (Valdez-Meza et al., 2019). The SHRs consumed 10 g of cookies, which supplied a hydrolysate dose of 1.2 g/kg BW. Significant reductions in SBP (up to 260 mmHg) were observed in rats fed the hydrolysate-fortified cookies up to 7 h. In contrast, the regular cookies with no added hydrolysate produced no significant changes in SBP of the SHR. The plasma of SHRs that consumed the hydrolysate-fortified cookies also inhibited ACE activity during in vitro tests, which suggest that the amaranth peptides were absorbed from the GIT and present in the active form within the blood circulatory system. Plasma from SHR that consumed regular cookies did not inhibit ACE activity during the in vitro test, which confirms the bioactive effect of the cookies was due to the incorporated amaranth protein hydrolysate. A similar work demonstrated that bread containing 4% bovine globulin hydrolysate (generated through papain hydrolysis) could reduce blood pressure when consumed by SHR (Lafarga, Gallagher, Aluko, Auty, & Hayes, 2016). The globulin hydrolysate inhibited ACE activity with an IC50 value of 0.95 mg/mL and produced no significant change in the sensory and physical properties of the bread. Oral administration of an aqueous extract of the bread to SHR at a dose equivalent to 200 mg/kg BW led to

600 Chapter 24 36 mmHg decrease in SBP after 8 h, which is comparable to the effect produced by the globulin hydrolysate alone at the same dosage. Results from these formulated baked products indicate that the peptides are stable to the levels of heat applied during regular food processing, which is encouraging for future formulations of functional food products that contain bioactive protein hydrolysates. A recent study examined the effect of rice bran fortified with an antihypertensive peptide (LRA) on blood pressure of human subjects (Ogawa et al., 2019). This was based on an earlier work that showed a thermolysin digest of rice bran contained LRA as the main active peptide with an ACE-inhibitory IC50 value of 62 μg/mL (Shobako et al., 2018). Oral administration of LRA alone to SHRs at a dose of 0.25 mg/kg BW produced up to B22 mmHg reduction in SBP after 4
6 h (Shobako et al., 2018). Therefore the latest work incorporated the LRA into rice bran and was fed to human volunteers at a dose of 172 μg LRA/day for 12 weeks against a placebo that contained only the rice bran (Ogawa et al., 2019). SBP in the group that consumed the LRA-fortified rice bran was 4.3 mmHg less than that of the placebo group (P , 0.05). However, the treatment produced no significant changes in DBP. LRA is also known to enhance NO production (Shobako et al., 2018), and this effect seems to benefit SBP reduction but not DBP, which could explain the lack of significant changes in DBP. Analysis of the trial population indicated that the blood pressure-reducing effect was significant within the group with high-normal SBP ( , 135 mmHg) but not those with grade one hypertension (SBP .135 mmHg). However, the authors suggested that the wide fluctuations in measured SBP might be responsible for the lack of a measurable significant effect of the LRA treatment within the hypertensive group. For all the participants, the LRA treatment did not produce any adverse effects and there were no abnormal changes in BW, liver functions, lipid profiles, and fasting blood glucose. Data from this human intervention trial confirm the presumed safety of bioactive food protein-derived peptides during long-term management of hypertension. Another rice product is the genetically engineered seed that contained multiple (10) sequences of antihypertensive peptides (MAHP) within the protein primary structure (Qian et al., 2020). These peptides are DKIHPF, YQQPVL, IPP, VPP, LKPNM, RPLKPW, KVLPVPE, SKVYPFPGPI, YLAHKALPMHIR, and FFVAPFPEVFGK. The SHRs were fed with rice products containing the MAHP at 50
200 μg/kg BW in comparison to similar doses of regular rice. During short-term tests, the MAHP rice reduced SBP significantly (up to 260 mmHg) within 8 h of consumption whereas the regular rice produced no effect. During the long-term (5 weeks) test, daily oral administration of 50 μg/kg BW of the MAHP rice led to significant SBP decreases with a maximum of about 240 mmHg in the fifth week (Fig. 24.5). In contrast, the normal rice produced no significant changes in SBP throughout the 5 weeks. After the fifth week, the MAHP rice was removed and all the rats switched to regular diets, which led to increased SBP and confirmed that the MAHP diet was responsible for the observed blood pressure-lowering effects. The MAHP rice diet

Cardiovascular benefits of food protein-derived bioactive peptides 601

Figure 24.5 SBP change of SHRs after long-term intragastric administration of pulverized transgenic rice seeds equivalent to 50 μg/kg of MAHP (S-50MAHP) and the same amount of seed flour from WT rice (S-50WT). Source: Reprinted with permission from Qian, D., Qiu, B., Zhou, N., Takaiwa, F., Yong, W., & Qu, L.Q. 2020. Hypotensive activity of transgenic rice seed accumulating multiple antihypertensive peptides. Journal of Agricultural and Food Chemistry, 68, 7162
7168. Copyright (2020) American Chemical Society.

produced no adverse effects on rat BW or serum biochemistry, which indicate safety of the product. Interestingly, the MAHP rice diet had no effect on the SBP of the WKY rats during short- and long-term tests. Therefore consumption of this rice product by normotensive people may not produce undesirable blood pressure depression, and the results suggest no safety issues that will require partitioning of the MAHP rice from normal rice products.

24.3 Conclusions There is now abundant evidence from scientific literature that bioactive peptides generated from food proteins through enzymatic hydrolysis can efficiently modulate RAAS and ameliorate high blood pressure conditions. Previous works have also established a range of doses based on animal and human trials that indicate these peptides could function at comparable levels as drugs. However, in most cases, these peptides have weaker blood pressure effects, but since they consist of natural amino acid sequences, they could be used as therapeutic agents at several fold higher doses than drugs. The food protein-derived peptides have demonstrated better safety use and with less negative side effects than drugs. Therefore hypertensive people may be more compliant with recommended peptide doses, which could ensure a reliable and effective blood pressure control. While there is not much information on peptide absorption from the GIT, the detection of significant changes in the blood concentration of blood pressure control-related compounds such as ACE, renin, Ang II, and NO provides solid support that the peptides eventually reach blood circulation and various organs. Lastly, a prominent advantage of peptides is their demonstrated use for food

602 Chapter 24 formulation, which provides a familiar medium for ingestion as a regular food. Therefore people who may be averse to pills (usual form of drugs) intake will find the formulated foods highly acceptable products that can be ingested like regular foods but with the added cardiovascular health protection benefits.

24.4 Future trends Recent advances in computer technology could provide significant electronic power to assist in developing in silico tools that will help identify bioactive peptide sequences and the optimization of enzymatic methods for efficient release from parent food proteins. This will facilitate production of bioactive protein hydrolysates with customized content of desirable peptides while minimizing unwanted sequences. There is continuing interest in the development of multifunctional peptides and knowledge of their structure
function relationships. The multiple-acting peptides are effective against several targets, which could provide a more efficient therapeutic delivery by minimizing or even eliminating the use of separate treatment agents for each disease. For example, a protein hydrolysate that possess antioxidant properties, in addition to inhibiting renin and ACE activities could be used to treat hypertension and associated renal or organ damage. Similarly, a single-protein hydrolysate that blocks Ang II receptors while enhancing ACE-2 activity will also provide significant benefits in maintaining normal blood pressure and an overall normal cardiovascular health. There is also increasing interest in the use of foods rather than pills to treat human disease conditions, which is expected to propel the food industry to greater heights with respect to novel product development and ingredient technology.

References Aderinola, T. A., Fagbemi, T. N., Enujiugha, V. N., Alashi, A. M., Nwachukwu, I. D., & Aluko, R. E. (2019). Antihypertensive and antioxidant properties of Moringa oleifera seed enzymatic protein hydrolysate and ultrafiltration fractions. Current Topics in Nutraceutical Research, 17, 437
444. Aihara, K., Kajimoto, O., Hirata, H., Takahashi, R., & Nakamura, Y. (2005). Effect of powdered fermented milk with Lactobacillus helveticus on subjects with high-normal blood pressure or mild hypertension. Journal of the American College of Nutrition, 24, 257
265. Alashi, A. M., Blanchard, C. L., Mailer, R. J., Agboola, S. O., Mawson, A. J., He, R., . . . Aluko, R. E. (2014). Blood pressure lowering effects of Australian canola protein hydrolysates in spontaneously hypertensive rats. Food Research International, 55, 281
287. Allen, N., Berry, J. D., Ning, H., Van Horn, L., Dyer, A., & Lloyd-Jones, D. M. (2012). Impact of blood pressure and blood pressure change during middle age on the remaining lifetime risk for cardiovascular disease. The cardiovascular lifetime risk pooling project. Circulation, 25, 37
44. Aluko, R. E. (2019). Food protein-derived renin-inhibitory peptides: in vitro and in vivo properties. Journal of Food Biochemistry, 43, e12648. Bangalore, S., Kamalakkannan, G., Parkar, S., & Messerli, F. H. (2007). Fixed-dose combinations improve medication compliance: A meta analysis. American Journal of Medicine, 120, 713
719. Blumentals, W. A., Brown, R. R., & Gomez-Caminero, A. (2003). Antihypertensive treatment and erectile dysfunction in a cohort of type II diabetes patients. International Journal of Impotence Research, 15, 314
317.

Cardiovascular benefits of food protein-derived bioactive peptides 603 Cao, S., Wang, Y., Hao, Y., Zhang, W., & Zhou, G. (2020). Antihypertensive effects in vitro and in vivo of novel angiotensin-converting enzyme inhibitory peptides from bovine bone gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 68, 759
768. Carretero, O. A., & Oparil, S. (2000). Essential hypertension. Part I: definition and etiology. Circulation, 101, 329
335. Cooper, W. O., Hernandez-Diaz, S., Arbogast, P. G., Dudley, J. A., Dyer, S., Gideon, P. S., . . . Ray, W. A. (2006). Major congenital malformations after first-trimester exposure to ACE inhibitors. New England Journal of Medicine, 354, 2443
2451. Deng, Z., Liu, Y., Wang, J., Wu, S., Geng, L., Sui, Z., & Zhang, Q. (2018). Antihypertensive effects of two novel angiotensin I-converting enzyme (ACE) inhibitory peptides from Gracilariopsis lemaneiformis (Rhodophyta) in spontaneously hypertensive rats (SHRs). Marine Drugs, 16, 299. Escudero, E., Toldra´, F., Sentandreu, M. A., Nishimura, H., & Arihara, K. (2012). Antihypertensive activity of peptides identified in the in vitro gastrointestinal digest of pork meat. Meat Science, 91, 382
384. Feld, L. G., van Liew, B., Brentjens, J. R., & Boylan, J. W. (1981). Renal lesions and proteinuria in the spontaneously hypertensive rats made normotensive by treatment. Kidney International, 20, 606
614. Fitzgerald, C., Aluko, R. E., Hossain, M., Rai, D. K., & Hayes, M. (2014). The potential of a renin inhibitory peptide from the red seaweed Palmaria palmata as functional food ingredient following confirmation and characterization of a hypotensive effect in spontaneously hypertensive rats (SHRs). Journal of Agricultural and Food Chemistry, 62, 8352
8356. Flack, J. M., Novikov, S. V., & Ferrario, C. M. (1997). Benefits of adherence to anti-hypertensive therapy. Blood Pressure, 1(Suppl.), 47
51. Fogari, R., & Zoppi, A. (2002). Effects of antihypertensive therapy on sexual activity in hypertensive men. Current Hypertension Reports, 4, 202
210. Fujita, H., Yokoyama, K., & Yoshikawa, M. (2000). Classification and antihypertensive activity of angiotensin Iconverting enzyme inhibitory peptides derived from food proteins. Journal of Food Science, 65, 564
569. Girgih, A. T., Alashi, A. M., He, R., Malomo, S. A., & Aluko, R. E. (2014). Preventive and treatment effects of hemp seed (Cannabis sativa L.) meal protein hydrolysate against high blood pressure in spontaneously hypertensive rats. European Journal of Nutrition, 53, 1237
1246. Girgih, A. T., He, R., Malomo, S. A., Offengenden, M., Wu, J., & Aluko, R. E. (2014). Structural and functional characterization of hemp seed (Cannabis sativa L.) protein-derived antioxidant and antihypertensive peptides. Journal of Functional Foods, 6, 384
394. Girgih, A. T., Nwachukwu, I. D., Hasan, F. M., Fagbemi, T. N., Gill, T. A., & Aluko, R. E. (2015). Kinetics of the inhibition of renin and angiotensin I-converting enzyme by Cod (Gadus morhua) protein hydrolysates and their antihypertensive effects in spontaneously hypertensive rats. Food and Nutrition Research, 59, 29788. Girgih, A. T., Nwachukwu, I. D., Hasan, F. M., Fagbemi, T. N., Malomo, S. A., Gill, T. A., & Aluko, R. E. (2016). Kinetics of in vitro enzyme inhibition and blood pressure lowering effects of salmon (Salmo salar) protein hydrolysates in spontaneously hypertensive rats. Journal of Functional Foods, 20, 43
53. Girgih, A. T., Nwachukwu, I. D., Onuh, J. O., Malomo, S. A., & Aluko, R. E. (2016). Antihypertensive properties of a pea protein hydrolysate during short and long term oral administration to spontaneously hypertensive rats. Journal of Food Science, 81, H1281
H1287. Girgih, A. T., Udenigwe, C. C., Li, H., Adebiyi, A. P., & Aluko, R. E. (2011). Kinetics of enzyme inhibition and antihypertensive effects of hemp seed (Cannabis sativa L.) protein hydrolysates. Journal of the American Oil Chemists’ Society, 88, 1767
1774. Gunkel, A. R., Thurner, K. H., Kanonier, G., Sprinzl, G. M., & Thumfart, W. F. (1996). Angioneurotic edema as a reaction to angiotensin-converting enzyme inhibitors. American Journal of Otolaryngology, 17, 87
91. Guo, Y., Wang, K., Wu, B., Wu, P., Duan, Y., & Ma, H. (2020). Production of ACE inhibitory peptides from corn germ meal by an enzymatic membrane reactor with a novel gradient diafiltration feeding workingmode and in vivo evaluation of antihypertensive effect. Journal of Functional Foods, 64, 103584.

604 Chapter 24 Hao, L., Gao, X., Zhou, T., Cao, J., Sun, Y., Dang, Y., & Pan, D. (2020). Angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activity of umami peptides after in vitro gastrointestinal digestion. Journal of Agricultural and Food Chemistry, 68, 8232
8241. Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y., & Takano, T. (1996). A placebo controlled study of the effect of sour milk on blood pressure in hypertensive subjects. American Journal of Clinical Nutrition, 64, 767
771. Hayes, M., Mora, L., Hussey, K., & Aluko, R. E. (2016). Boarfish protein recovery using the pH-shift process and generation of protein hydrolysates with ACE-I and antihypertensive bioactivities in spontaneously hypertensive rats. Innovative Food Science and Emerging Technologies, 37, 253
260. He, R., Malomo, S. A., Alashi, A., Girgih, A. T., Ju, X., & Aluko, R. E. (2013). Purification and hypotensive activity of rapeseed protein-derived renin and angiotensin converting enzyme inhibitory peptides. Journal of Functional Foods, 5, 781
789. He, R., Malomo, S. A., Girgih, A. T., Ju, X., & Aluko, R. E. (2013). Glycinyl-Histidinyl-Serine (GHS), a novel rapeseed protein-derived peptide has blood pressure-lowering effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 61, 8396
8402. Herzig, K.-H. (2019). Peptides and their receptors: treasure for pharmacological interventions. Peptides, 122, 170198. Ishida, Y., Shibata, Y., Fukuhara, I., Yano, Y., Takehara, I., & Kaneko, K. (2011). Effect of an excess intake of casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro in subjects with normal blood pressure, highnormal blood pressure, or mild hypertension. Bioscience, Biotechnology and Biochemistry, 75, 427
433. Jahandideh, F., Majumder, K., Chakrabarti, S., Morton, J. S., Panahi, S., Kaufman, S., . . . Wu, J. (2014). Beneficial effects of simulated gastro-intestinal digests of fried egg and its fractions on blood pressure, plasma lipids and oxidative stress in spontaneously hypertensive rats. PLoS ONE, 9(12), e115006. Katayama, K., Anggraeni, H. E., Mori, T., Ahhmed, A. M., Kawahara, S., Sugiyama, M., . . . Muguruma, M. (2008). Porcine skeletal muscle troponin is a good source of peptides with angiotensin-I converting enzyme inhibitory activity and antihypertensive effects in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 56, 355
360. Katayama, K., Jamhari., Mori, T., Kawahara, S., Miake, K., Kodama, Y., . . . Muguruma, M. (2007). Angiotensin-I converting enzyme inhibitory peptide derived from porcine skeletal muscle myosin and its antihypertensive activity in spontaneously hypertensive rats. Journal of Food Science, 72, S702
S706. Lafarga, T., Gallagher, E., Aluko, R. E., Auty, M. A. E., & Hayes, M. (2016). Addition of bovine globulins to bread and determination of hypertensive effects in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 64, 1741
1750. Li, H., Prairie, N., Udenigwe, C. C., Adebiyi, A. P., Tappia, P., Aukema, H. M., . . . Aluko, R. E. (2011). Blood pressure lowering effect of a pea protein hydrolysate in hypertensive rats and humans. Journal of Agricultural and Food Chemistry, 59, 9854
9860. Lin, H.-C., Alashi, A. M., Aluko, R. E., Pan, B. S., & Chang, Y.-W. (2017). Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates. Food and Nutrition Research, 61, 1391666. MacMachon, S., Peto, R., Cutler, J., Collins, R., Sorlie, P., Neaton, J., & Stamler, J. (1990). Blood pressure, stroke and coronary heart disease. Part 1, prolonged differences in blood pressure: Prospective observational studies corrected for the regression dilution bias. Lancet, 335, 765
774. Majumder, K., Chakrabarti, S., Morton, J. S., Panahi, S., Kaufman, S., Davidge, S. T., & Wu, J. (2013). Eggderived tri-peptide IRW exerts antihypertensive effects in spontaneously hypertensive rats. PLoS ONE, 8 (11), e82829. Malomo, S. A., Girgih, A. T., Onuh, J. O., & Aluko, R. E. (2015). Structural and antihypertensive properties of enzymatic hemp seed protein hydrolysates. Nutrients, 7, 7616
7632. Martin, M., Hagemann, D., Nguyen, T. T., Schwarz, L., Khedr, S., Moskopp, M. L., . . . Deussen, A. (2020). European Journal of Nutrition, 59, 1135
1147. Mas-Capdevila, A., Pons, Z., Aleixandre, A., Bravo, F. I., & Muguerza, B. (2018). Dose-related antihypertensive properties and the corresponding mechanisms of a chicken foot hydrolysate in hypertensive rats. Nutrients, 10, 1295.

Cardiovascular benefits of food protein-derived bioactive peptides 605 Masuda, O., Nakamura, Y., & Takano, T. (1996). Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides to spontaneously hypertensive rats. Journal of Nutrition, 126, 3063
3068. Mizuno, S., Matsuura, K., Gotou, T., Nishimura, S., Kajimoto, O., Yabune, M., . . . Yamamoto, N. (2005). Antihypertensive effect of casein hydrolysate in a placebo controlled study in subjects with high-normal blood pressure and mild hypertension. British Journal of Nutrition, 94, 84
91. Muguruma, M., Ahhmed, A. M., Katayama, K., Kawahara, S., Maruyama, M., & Nakamura, T. I. (2009). Identification of pro-drug type ACE inhibitory peptide sourced from porcine myosin B: Evaluation of its antihypertensive effects in vivo. Food Chemistry, 114, 516
522. Nakamura, Y., Yamamoto, N., Sakai, K., & Takano, T. (1995). Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors of angiotensin converting enzyme. Journal of Dairy Science, 78, 1253
1257. Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., & Takano, T. (1995). Purification and characterization of angiotensin converting enzyme inhibitors from sour milk. Journal of Dairy Science, 78, 777
783. NCD Risk Factor Collaboration. (2017). Worldwide trends in blood pressure from 1975 to 2015: a pooled analysis of 1479 population based measurement studies with 19.1 million participants. Lancet, 389, 37
55. Nwachukwu, I. D., Girgih, A. T., Malomo, S. A., Onuh, J., & Aluko, R. E. (2014). Thermoase-derived flaxseed protein hydrolysates and membrane ultrafiltration peptide fractions have systolic blood pressure-lowering effects in spontaneously hypertensive rats. International Journal of Molecular Sciences, 15, 18131
18147. O’Keeffe, M. B., Norris, R., Alashi, M. A., Aluko, R. E., & FitzGerald, R. J. (2017). Peptide identification in a porcine gelatin prolyl endoproteinase hydrolysate with angiotensin converting enzyme (ACE) inhibitory and hypotensive activity. Journal of Functional Foods, 34, 77
88. Ogawa, Y., Shobako, N., Fukuhara, I., Satoh, H., Kobayashi, E., Kusakari, T., . . . Ishikado, A. (2019). Rice bran supplement containing a functional substance, the novel peptide Leu-Arg-Ala, has anti-hypertensive effects: A double-blind, randomized, placebo-controlled study. Nutrients, 11, 726. Oh, J.-Y., Je, J.-G., Lee, H.-G., Kim, E.-A., Kang, S. I., Lee, J.-S., & Jeon, Y.-J. (2020). Anti-hypertensive activity of novel peptides identified from olive flounder (Paralichthys olivaceus) surimi. Foods, 9, 647. Olagunju, A. I., Omoba, O. S., Enujiugha, V. N., Alashi, A. M., & Aluko, R. E. (2018). Antioxidant properties, ACE/renin inhibitory activities of pigeon pea hydrolysates and effects on systolic blood pressure of spontaneously hypertensive rats. Food Science and Nutrition, 6, 1879
1889. Ontiveros, N., Lo´pez-Teros, V., Vergara-Jime´nez, M. J., Islas-Rubio, A. R., Ca´rdenas-Torres, F. I., CuevasRodrı´guez, E.-O., . . . Cabrera-Cha´vez, F. (2020). Amaranth-hydrolyzate enriched cookies reduce the systolic blood pressure in spontaneously hypertensive rats. Journal of Functional Foods, 64, 103613. Onuh, J. O., Girgih, A. T., Malomo, S. A., Aluko, R. E., & Aliani, M. (2015). Kinetics of in vitro renin and angiotensin converting enzyme inhibition by chicken skin protein hydrolysates and their blood pressure lowering effects in spontaneously hypertensive rats. Journal of Functional Foods, 14, 133
143. Onuh, J. O., Girgih, A. T., Nwachukwu, I. D., Levari-Shariati, S., Raj, P., Netticadan, T., . . . Aliani, M. (2016). A metabolomics approach for investigating urinary and plasma changes in spontaneously hypertensive rats (SHR) fed chicken skin protein hydrolysates diets. Journal of Functional Foods, 22, 20
33. Pickering, T. G., Hall, J. E., Appel, L. J., Falkner, B. E., Graves, J., Hill, M. N., . . . Roccella, E. J. (2005). Recommendations for blood pressure measurement in humans and experimental animals. Part 1: Blood pressure measurement in humans. Circulation, 111, 697
716. Piotrowicz, I. B. B., Garce´s-Rimo´n, M., Moreno-Ferna´ndez, S., Aleixandre, A., Salas-Mellado, M., & MiguelCastro, M. (2020). Antioxidant, angiotensin-converting enzyme inhibitory properties and blood-pressurelowering effect of rice bran protein hydrolysates. Foods, 9, 812. Pryde, P. G., Sedman, A. B., Nugent, C. E., & Barr, M., Jr. (1993). Angiotensin-converting enzyme inhibitor tetopathy. American Society of Nephrology, 3, 1575
1582. Qian, D., Qiu, B., Zhou, N., Takaiwa, F., Yong, W., & Qu, L. Q. (2020). Hypotensive activity of transgenic rice seed accumulating multiple antihypertensive peptides. Journal of Agricultural and Food Chemistry, 68, 7162
7168.

606 Chapter 24 Shih, Y.-H., Chen, F.-A., Wang, L. F., & Hsu, J.-L. (2019). Discovery and study of novel antihypertensive peptides derived from Cassia obtusifolia seeds. Journal of Agricultural and Food Chemistry, 67, 7810
7820. Shobako, N., Ogawa, Y., Ishikado, A., Harada, K., Kobayashi, E., Suido, H., . . . Ohinata, K. (2018). A novel antihypertensive peptide identified in thermolysin-digested rice bran. Molecular Nutrition and Food Research, 62, 1700732. Sonklin, C., Alashi, A. M., Laohakunjit, L., Kerdchoechuen, O., & Aluko, R. E. (2020). Identification of antihypertensive peptides from mung bean protein hydrolysate and their effects in spontaneously hypertensive rats. Journal of Functional Foods, 64, 103635. Sun, L., Wu, B., Yan, M., Hou, H., & Zhuang, Y. (2019). Antihypertensive effect in vivo of QAGLSPVR and its transepithelial transport through the caco-2 cell monolayer. Marine Drugs, 17, 288. Sundstrom, J., Arima, H., Jackson, R., Turnbull, F., Rahimi, K., Chalmers, J., & Neal, B. (2015). Effects of blood pressure reduction in mild hypertension: A systematic review and meta-analysis. Annals of Internal Medicine, 162, 184
191. Tianrui, Z., Bingtong, L., Ling, Y., Liping, S., & Yongliang, Z. (2019). ACE inhibitory activity in vitro and antihypertensive effect in vivo of LSGYGP and its transepithelial transport by Caco-2 cell monolayer. Journal of Functional Foods, 51, 103488. Tiengo, A., Faria, M., & Netto, F. M. (2009). Characterization and ACE-inhibitory activity of amaranth proteins. Journal of Food Science, 74, H121
H126. Udenigwe, C. C., Girgih, A. T., Mohan, A., Gong, M., Malomo, S. A., & Aluko, R. E. (2017). Antihypertensive and bovine plasma oxidation-inhibitory activities of spent hen meat protein hydrolysates. Journal of Food Biochemistry, 41, e12378. Valdez-Meza, E. E., Raymundo, A., Figueroa-Salcido, O. G., Ramı´rez-Torres, G. I., Fradinho, P., Oliveira, S., . . . Cabrera-Cha´vez, F. (2019). Pasta enrichment with an amaranth hydrolysate affects the overall acceptability while maintaining antihypertensive properties. Foods, 8, 282. Va´squez-Villanueva, R., Orellana, J. M., Marina, M. L., & Garcı´a, M. C. (2019). Isolation and characterization of angiotensin converting enzyme inhibitory peptides from peach seed hydrolysates: In vivo assessment of antihypertensive activity. Journal of Agricultural and Food Chemistry, 67, 10313
10320. Vleeming, W., van Amsterdam, J. G. C., Stricker, B. H. C., & de Wildt, D. J. (1998). ACE inhibitor
induced angioedema incidence, prevention and management. Drug Safety, 18, 171
188. Wang, J., Hu, J., Cui, J., Bai, X., Du, Y., Miyaguchi, Y., & Lin, B. (2008). Purification and identification of a ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in spontaneously hypertensive rats. Food Chemistry, 111, 302
308. WHO. (2017). World Health Organization Fact Sheet on Cardiovascular Diseases. https://www.who.int/en/newsroom/fact-sheets/detail/cardiovascular-diseases-(cvds). Accessed September 10 2020. Yesil, S., Yesil, M., Bayata, S., & Postaci, N. (1994). ACE inhibitors and cough. Angiology, 45, 805
808. Yu, Z., Yin, Y., Zhao, W., Chen, F., & Liu, J. (2014). Antihypertensive effect of angiotensin-converting enzyme inhibitory peptide RVPSL on spontaneously hypertensive rats by regulating gene expression of the renin 2 angiotensin system. Journal of Agricultural and Food Chemistry, 62, 912
917.

CHAPTER 25

Applications in medicine: hypoglycemic peptides Forough Jahandideh and Jianping Wu Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada

25.1 Introduction Diabetes mellitus is a complex chronic disease characterized by persistent hyperglycemia. If not treated properly, diabetes generates serious microvascular and macrovascular complications that reduce the patient’s quality of life. The global prevalence of diabetes was 425 million adults in 2017, and this number is predicted to rise to 629 million people by 2045 (Nita Gandhi Forouhi, 2019). Diabetes is classified into three major categories of type 1 diabetes which is an autoimmune disease, type 2 diabetes in which a progressive insulin secretory defect occurs on the background of insulin resistance, and gestational diabetes occurring during pregnancy. Type 2 diabetes accounts for about 95% of all diabetic cases (Thomas and Philipson, 2015), and its incidence is rapidly increasing in association with aging and increased obesity worldwide. The development of insulin resistance in addition to the insufficient insulin secretion which occurs in later stages of this disease, leads to hyperglycemia and dyslipidemia in type 2 diabetes (Hirano, 2018). Uncontrolled hyperglycemia damages vascular system. The microvascular lesions can cause nephropathy, retinopathy, and neuropathy (Labazi and Trask, 2017). Chronic hyperglycemia also increases the risk of macrovascular complications including cardiovascular and cerebrovascular diseases. Effective management of type 2 diabetes requires continuous glycaemic control, along with the management of comorbidities such as hypertension and hyperlipidemia. The required life-long therapy along with the adverse side effects of antidiabetic drugs negatively impact therapy adherence by patients (Garcia-Perez, Alvarez, Dilla, Gil-Guillen, & Orozco-Beltran, 2013). Considerable research in recent decades has been conducted toward identifying naturally derived compounds without side effects or toxicity. This chapter deals with the reported studies on the antidiabetic properties of food-derived protein hydrolysates and bioactive peptides mainly through animal studies and human clinical trials.

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00023-6 © 2021 Elsevier Inc. All rights reserved.

607

608 Chapter 25

25.2 Carbohydrate digestion and glucose homeostasis α-Amylase and α-glucosidase are the two enzymes required for carbohydrate digestion. While α-amylase breaks down large carbohydrates, α-glucosidase facilitates the production of absorbable monosaccharides. Inhibition of these enzymes leads to delayed carbohydrate digestion, and absorption leading to reduced postprandial blood glucose and insulin peaks (Ross, Gulve, & Wang, 2004). D-Glucose is the primary source of energy production for cells. Blood glucose level needs to be controlled tightly for a normal body function. This is achieved through a complex network of various hormones and neuropeptides released mainly from the brain, pancreas, liver, intestine, adipose, and muscle tissues (Roder, Wu, Liu, & Han, 2016). The pancreas is the key player within this network by secreting various hormones including glucagon, insulin, amylin, C-peptide, pancreatic polypeptide, somatostatin, and ghrelin. Each of these hormones has distinct functions in regulating blood glucose levels. Glucagon increases blood glucose level, whereas insulin decreases it. Somatostatin inhibits glucagon and insulin release. Pancreatic polypeptide regulates the exocrine and endocrine secretion activity of the pancreas. Altogether, these hormones regulate glucose homeostasis in the body. Glucagon and insulin are the major hormones to maintain blood glucose levels within the very narrow range of 4
6 mm, referred to as glucose homeostasis. A low glucose level stimulates glucagon promoting hepatic glycogenolysis during sleep or in between meals. In prolonged fasting, glucagon activates hepatic and renal gluconeogenesis to increase endogenous blood glucose levels. In the fed state, ingested nutrients stimulate the release of incretins, glucagon like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP), from intestinal endocrine cells. Secretion of these hormones along with the rise in blood glucose stimulate insulin release from the pancreatic β-cells. After binding to its receptor on muscle and adipose tissue, insulin stimulates glucose uptake into these tissues and hence lowers blood glucose levels. Being an anabolic hormone, insulin also promotes glycogenesis, lipogenesis, and the incorporation of amino acids into proteins. Currently, the most used antidiabetic drugs include α-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose, and emiglitate), insulin secretagogues (e.g., sulfonylureas), incretin mimetics, and insulin sensitizers (e.g., metformin or thiazolidinediones). By addressing different problems and stages of type 2 diabetes, these drugs can exert synergistic effects when prescribed in combination. Although oral antidiabetic agents have therapeutic benefits for the treatment of type 2 diabetes, most of these drugs are associated with various side effects including hypoglycemia, fluid retention, osteoporosis, and heart failure (Ghosh and Parida, 2016; Phung, Scholle, Talwar, & Coleman, 2010). Therefore there has been much interest in identifying naturally derived products for the management of hyperglycemia in diabetic patients (Rios, Francini, & Schinella, 2015) as they are usually safer, cheaper, more accessible, and sometimes more efficacious than synthetic ones. Medicinal plants have been explored extensively for this purpose. Flavonoids, alkaloids, saponins, coumarins, glycosides,

Applications in medicine: hypoglycemic peptides 609 phenolics, terpens, polysaccharides, and polypeptides are plant constituents with antidiabetic properties (Qi et al., 2010; Xu, Li, Dai, & Peng, 2018). Food proteins and bioactive peptides have also shown potential in the management of diabetes and its complications.

25.3 Pathophysiology of type 2 diabetes As mentioned before, normal glucose metabolism is obtained through a feedback loop involving pancreatic β-cells and insulin-sensitive tissues. Indeed, tissue insulin sensitivity determines the magnitude of the β-cell response in secreting insulin (Kahn, Cooper, & Del Prato, 2014). Impairment of insulin sensitivity and β-cell function leads to complete insulin deficiency in type 2 diabetes. In the presence of insulin resistance, β-cells maintain normal glucose levels by increasing insulin output. However, prolonged chronic stimulation with glucose leads to glucotoxicity, β-cells exhaustion, and diminished insulin secretion resulting in the manifestation of hyperglycemia (Kahn et al., 2014). While β-cell dysfunction has a distinct genetic element, environmental factors also play an important role. The association between certain hexoses, branched-chain, and aromatic amino acids, as well as fatty acids with obesity, β-cell dysfunction, and type 2 diabetes have been reported in literature (Floegel et al., 2013; Newgard et al., 2009; Wurtz et al., 2012). Glucose and lipid overloading, oxidative stress, inflammation, adipokines, autophagy, and perturbed insulin secretion also affect insulin sensitivity (Fletcher, Gulanick, & Lamendola, 2002). Obesity plays a crucial role in the pathogenesis of type 2 diabetes, which can result in cellular oxidative stress and insulin resistance, cytokine release, lipid-induced impairment, and dysfunctional protein tyrosine phosphatase signaling. With the overloaded nutrient intake, excess calories are stored in adipose tissue leading to the appearance of hypoxia and inflammation in adipose tissue (Girgis, Cheng, Scott, & Gunton, 2012; He et al., 2011). With the aggravation of inflammation, various inflammation cytokines are released to exacerbate insulin resistance (Feng et al., 2016) and lipolysis (Nieto-Vazquez et al., 2008). In addition, inflammatory markers can further reduce the activity of peroxisome proliferator-activated receptor γ (PPARγ) and accelerate adipocyte death and inflammation. With development of insulin resistance, the activity of insulin as an antilipolytic hormone decreases (Sears and Perry, 2015). Hyperinsulinemia can activate the lipoprotein lipase and release free-fatty acids from lipoprotein triglycerides hydrolysis. Accordingly, insulin resistance and increased flux of free-fatty acids in adipose tissue can form a vicious cycle; high level of adipocyte-derived free-fatty acids is released into circulation, transported, and accumulated in other organs to further induce the lipotoxicity and accelerate systemic insulin resistance (Sears and Perry, 2015). Liver playing a critical role in maintaining stable blood glucose level through establishing a balance between glycogenesis and glycolysis of stored glycogen (Samuel and Shulman, 2012), is affected by free-fatty acid overload. High level of free-fatty acids causes accumulation of diacylglycerol and ceramide. Diacylglycerol can inhibit insulin action through activating the protein kinase C isoforms

610 Chapter 25 (PKC) and interfering insulin signal transduction by serine phosphorylation of insulin receptor (IRS) (Amati, 2012; Turban and Hajduch, 2011). Moreover, ceramide, as a potent activating agent of inflammation, can activate inflammatory pathways including JNK and NF-κB/IKK, which are closely related to insulin resistance (Kuzmenko and Klimentyeva, 2016). Insulin resistance also develops in skeletal muscle with a major role in glucose uptake (Wu and Ballantyne, 2017). Furthermore, the lipotoxicity of high-level free-fatty acids can cause β-cells dysfunction. B-cells are also susceptible to the damage mediated by inflammation cytokines (Kahn, 2003). Dysfunctional β-cells cannot keep the stable insulin level in a compensatory way, eventually leading to hyperinsulinemia and hyperglycemia, and accelerates the development of type 2 diabetes.

25.4 Clinical diagnosis of diabetes Diabetes diagnostic criteria are based on thresholds of glycemia associated with retinopathy. Fasting glucose, oral glucose tolerance test (OGTT), and hemoglobin A 1c (HbA 1c) are parameters/tests used for the diagnosis of diabetes while the decision of the test to be used is left to clinical judgement (Harreiter and Roden, 2019). Each diagnostic test has advantages and disadvantages (Christophi et al., 2013). Table 25.1 summarizes the diagnosis criteria for diabetes and prediabetes, a practical term referring to impaired fasting glucose (IFG), impaired glucose tolerance (IGT), or a glycated HbA 1c of 6.0%
6.4% placing individuals at high risk of developing diabetes and its complications. The diagnosis based on plasma glucose (fasting or 2 h plasma glucose in OGTT) is made regardless of age and gender (Harreiter and Roden, 2019). HbA 1c as a diagnostic criterion can be used only when measured by a validated assay which is standardized to the National Glycohemoglobin Standardization Program-Diabetes Control and Complications Trial reference (Diabetes Canada Clinical Practice Guidelines Expert C, Punthakee, Goldenberg, & Katz, 2018). HbA 1c may not be used in individuals with various health conditions such as hemoglobinopathies, hemolytic anemia, iron deficiency, iron-deficiency anemias, and severe hepatic and renal diseases (Attard et al., 2015). If one laboratory test result is positive for diabetes and no symptomatic hyperglycemia is present, a repeat confirmatory laboratory test (preferably the same test) must be done on another day for confirmation. Table 25.1: Diagnosis criteria for diabetes and prediabetes (Diabetes Canada Clinical Practice Guidelines Expert C et al., 2018).

Random plasma glucose Fasting plasma glucose 2-h plasma glucose after 75 g OGTT HbA 1c

Manifested diabetes mellitus

Prediabetes

$ 200 mg/dL (11.1 mmol/L) $ 126 mg/dL (7.0 mmol/L) $ 200 mg/dL (11.1 mmol/L) $ 6.5% (48 mmol/mol)

— 6.1
6.9 (IFG) 7.8
11.0 (IGT) 6.0%
6.4%

HbA 1c, hemoglobin A 1c; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; OGTT, oral glucose tolerance test.

Applications in medicine: hypoglycemic peptides 611 A random plasma glucose in the diabetes range in an asymptomatic individual, however, should be confirmed with another test. When symptomatic hyperglycemia is present, diabetes is diagnosed, and there is no need for a confirmatory test before treatment is initiated. When the results of two different tests are above the diagnostic thresholds, the diagnosis of diabetes is confirmed (Diabetes Canada Clinical Practice Guidelines Expert C et al., 2018).

25.5 Diverse physiological properties of protein hydrolysates and bioactive peptides The role of diet in the etiology of type 2 diabetes has been known for a long time. An unhealthy diet is one of the main behavioral risk factors for development of noncommunicable diseases including cardiovascular diseases, diabetes, cancer, and chronic respiratory diseases (WHO, 2011). Adopting a healthy diet and physical activity is considered as the integral part of the WHO’s action plan for the prevention and control of noncommunicable diseases (WHO, 2011). Diabetes can be controlled through improvement in patient’s dietary knowledge, attitudes, and practices which are considered as an integral part of comprehensive diabetes care (Islam et al., 2015). With the enhanced consumers’ awareness about food and health, development of functional foods for specific health effects is on the rise. Bioactive peptides are one category of functional foods with proven efficiency in the prevention or treatment of many chronic diseases. These peptides can exert their biological effects after being released from their parent proteins. Bioactive peptides have been mostly identified using empirical and bioinformatic approaches (Gu and Wu, 2013; Li-Chan, 2015; Nongonierma and FitzGerald, 2014; Wu, Aluko, & Nakai, 2006) or an integrated approach (Ngoh and Gan, 2018; Siow, Lim, & Gan, 2017) to obtain in vitro data. While enzymatic hydrolysis is the main method for the production of bioactive peptides, microbial fermentation and food processing also have the potential to release such peptides (Korhonen and Pihlanto, 2006; Wu, Jahandideh, Yu, & Majumder, 2015). Owing to their inherent amino acid composition and sequence, bioactive peptides exert diverse physiological effects on body systems such as cardiovascular (Aluko, 2015; Jahandideh et al., 2014; Udenigwe and Rouvinen-Watt, 2015Chakrabarti, Jahandideh, & Wu, 2014; Esfandi, Walters, & Tsopmo, 2019), digestive (Caron, Domenger, Dhulster, Ravallec, & Cudennec, 2017; Walters, Esfandi, & Tsopmo, 2018), nervous (Liu and Udenigwe, 2019; Tyagi, Daliri, Kwami Ofosu, Yeon, & Oh, 2020), and immune (Dziuba and Dziuba, 2014; Ledesma-Martinez, Aguiniga-Sanchez, Weiss-Steider, & Rivera-Martinez, 2019; Salas, Badillo-Corona, Ramirez-Sotelo, & Oliver-Salvador, 2015; Wilson, Buchanan, Allan, & Tikoo, 2012) systems. Bioactive peptides may have a specific physiological effect or exert multifunctional physiological effects through acting upon different systems in the body (Jahandideh and Wu, 2020).

612 Chapter 25

25.6 Antidiabetic properties of protein hydrolysates/peptides (in vivo studies) Dietary proteins have a great satiety effect (Veldhorst et al., 2008; Westerterp-Plantenga, Lemmens, & Westerterp, 2012). Protein intake also positively affects blood glucose and insulin as well as body fat (Layman et al., 2003). Beneficial effects of protein intake on energy and glucose homeostasis have been attributed to amino acid composition (Westerterp-Plantenga et al., 2012) as well as bioactive peptides generation (Caron et al., 2017). The past decade has seen a growing body of literature on food-derived protein hydrolysates and bioactive peptides with glucoregulatory properties. Protein sources of antidiabetic peptides include dairy, fish, shellfish, egg, soy, oat, wheat, and among others. Protein hydrolysates/bioactive peptides potentially improve glucose homeostasis through inhibiting carbohydrate digestion and absorption, augmented gut hormone and insulin secretion, enhanced insulin function and sensitivity, increased peripheral glucose uptake, and modification of the adipose tissue. While antidiabetic effects of food-derived peptides might be less compared to synthetic drugs, there is less chance for their tissue accumulation or serious side effects since nature has provided the mechanism for their metabolism and utilization or excretion (Li-Chan, 2015). Antidiabetic effects of bioactive peptides have been mainly explored through in vitro assays. Animal experimentation is necessary for validation purposes. Examples of antidiabetic protein hydrolysates/bioactive peptides in animal studies along with their mechanism of action are presented in Table 25.2.

25.7 Antidiabetic properties of protein hydrolysates/peptides (clinical studies) Clinical trials carried out in humans involving bioactive peptides/protein hydrolysates are very limited but necessary in determining physiological properties of these compounds as functional foods/nutraceuticals. Evidence from literature shows that enzymatic hydrolysis of food proteins generates a mixture of bioactive peptides with potential glucoregulatory properties in human subjects. Table 25.3 has summarized recent clinical studies on the antidiabetic effects of food-derived bioactive peptides/protein hydrolysates. Dairy proteins especially casein (Drummond et al., 2018; Geerts et al., 2011; Jonker et al., 2011; Manders et al., 2014; Saleh et al., 2018; Horner, Drummond, O’Sullivan, Scsh, & Brennan, 2019) followed by fish proteins (Dale et al., 2018; Hovland et al., 2020; Zhu et al., 2010) are among the most studied protein sources for generation of bioactive peptides with glucoregulatory properties in humans. A twice-daily dose of 8.5 g of casein hydrolysate (before breakfast and before dinner) for 8 days in patients with gestational diabetes, moderately reduced plasma glucose levels without insulinotropic effects, suggesting an increase in insulin sensitivity in these patients (Saleh et al., 2018). In another study,

Table 25.2: Examples of animal studies on antidiabetic properties of food-derived protein hydrolysates and bioactive peptides. Peptide or protein hydrolysate

Preparation

Milk protein (MP) or milk protein hydrolysate (MPH)

MPH: Protein solutions (2% wt./ wt.) were hydrated and hydrolyzed by trypsin enzyme solution (80 mg/mL) at 0.5%-2% vol./vol. protein, 2 pH levels of 6.9 and 7.5 and 3, 6, 9, 20, and 24 h of hydrolysis. MP: Protein solutions (2% wt./ wt.) were hydrated overnight

Casein hydrolysate

Sodium caseinate suspension (10% wt./wt. in water) was hydrolyzed with food-grade gastrointestinal enzymes

LPQNIPPL (derived from Gouda cheese)

Dry-salted Gouda cheese was ripened for 0
12 months at 10 C and in vitro DPP-IV activity of the water-soluble extracts of the cheeses were measured. The sample with the highest inhibitory effect was fractionated. Several peptides were identified and tested for DPP-IV inhibitory activity. LPQNIPPL was chosen to be tested in vivo

Animal model/treatment groups

Route of administration and frequency

Observed effect

References

Male albino rats treated with alloxan (120 mg/kg BW) Groups: diabetic and nondiabetic rats receiving 0 (control) or 800 mg/kg BW/day of MP or MPH Selected MPH treatment: milk protein hydrolyzed by trypsin at 2% vol./ vol. protein for 20 h at pH 5 7.5 based on the hydrolysate HPLC chromatogram ob/ob and C57BL/6 male mice Acute study: Oral gavage of 100 μL of casein hydrolysate at 100 mg/kg BW 1 h prior to the GTT; control mice received 100 μL of distilled water Chronic study: Oral gavage of 100 μL of 100 mg/kg BW casein hydrolysate or sterile distilled water in control mice Female Sprague Dawley rats Crossover experimental design with two groups (active and control) with a wash-out period of 6 days. Active group received 300 mg/kg BW peptide

Intragastric/ daily for 6 weeks after diabetes establishment (for 1 week)

Treatments of diabetic rats by MP or MPH reduced the concentration of plasma glucose, total lipid, triglycerides, total cholesterol, LDL and VLDL. MPH also reduced the concentrations of urea, creatinine, and bilirubin

El-Sayed et al. (2016)

Oral gavage, every second day for 12 weeks

Acute and chronic administration of casein hydrolysate in ob/ob mice revealed a glucose-lowering effect and a lipid reducing effect (43% reduction in overall liver fat and a 28% reduction in the average fat globule size compare to the control mice). Islets isolated from ob/ob mice treated chronically with casein hydrolysate were significantly more responsive to glucose in an ex vivo glucose-stimulated insulin secretion assay

Drummond et al. (2018)

Used once in the oral glucose tolerance test (OGTT)

The area under the blood glucose curve was reduced (P , 0.02) in the LPQNIPPL-administered group compared with the placebo-treated group without any differences in insulin secretion between groups. The amount of LPQNIPPL peptide increased 4.3-fold during ripening between 1 and 12 months

Uenishi, Kabuki, Seto, Serizawa and Nakajima (2012)

(Continued)

Table 25.2: (Continued) Peptide or protein hydrolysate

Preparation

Animal model/treatment groups

Route of administration and frequency

Sea cucumber (Holothuria nobilis) hydrolysates (SCH)

Sea cucumber was first hydrated through several steps and hydrolyzed for 4 h with enzyme mixture of papain and protamex (at a protease to substrate ratio of 1% wt./wt.), centrifuged and supernatant was collected and lyophilized. Mostly contained peptides with MW , 3 kDa and ,1 kDa

High-fat diet (HFD) 1 streptozotocin injection (male Sprague Dawley rats) Groups: Normal control; diabetic control; low-dose SCH (200 mg/kg/day); high-dose SCH (400 mg/ kg/day); positive control (metformin 250 mg/kg/ day)

Oral gavage, once a day for 8 weeks

Halibut skin gelatin hydrolysate (HSGH) and tilapia skin gelatin hydrolysate (TSGH)

Hydrolysis of extracted gelatin form fish skin by Flavourzyme at E/S ratio of 1, 3, and 5% wt./wt.

Oral gavage, once a day for 30 days

Atlantic salmon skin gelatin hydrolysate

The extracted gelatin from Atlantic salmon skin was hydrolyzed with flavourzyme with an E/S ratio of 6% for 4 h

Streptozotocin-induced diabetic rats (male Sprague Dawley) Groups: normal control rats; normal rats 1 TSGH (750 mg/kg/day); diabetic control rats; diabetic rats 1 HSGH (750 mg/kg/day); diabetic rats 1 TSGH (750 mg/kg/day); and diabetic rats 1 sitagliptin (120 mg/kg/day; positive control) Streptozotocin-induced diabetic rats (male Sprague Dawley) Groups: normal control rats; diabetic rats; diabetic rats 1 fish skin gelatin hydrolysates (FSGH; 300 mg per day)

Oral gavage, once a day for 5 weeks

Observed effect

References

SCH alleviated body weight loss, oral glucose tolerance, and insulin resistance in diabetic rats. Fasting blood glucose level was decreased by 40.39% after SCH intake. Lipid metabolism disorders in diabetic rats were attenuated to near normal after SCH treatment. SCH triggered PI3K/Akt signaling pathway (ex vivo study); the expressions of PI3K, p-Akt, p-GSK3β, and GLUT2/GLUT4 were increased and the expression of pIRS1 was decreased after SCH treatment in liver and skeletal muscle tissues of diabetic rats Inhibition of plasma DPP-IV activity, enhancement of GLP1, and insulin secretion as well as improved glucose tolerance in diabetic rats administered with TSGH. IPGDPGPPGPPGP, LPGERGRPGAPGP, GPKGDRGLPGPPGRDGM were identified from TSGH as the DPP-IV inhibitory peptides

Wang et al. (2020)

Reduced blood glucose levels of diabetic rats during an OGTT, inhibition of plasma DPP-IV activity, enhanced plasma activity of GLP-1, insulin, and the insulin-to-glucagon ratio in FSGH-treated diabetic rats

Hsieh, Wang, Hung, Chen and Hsu (2015)

Wang et al. (2015)

Goby fish (Zosterisessor ophiocephalus) muscle hydrolysates (GPHs) and Undigested goby fish protein (UGP)

Goby fish protein extract was hydrolyzed using enzyme preparations from Bacillus mojavensis A21 (GPH-A) and gray triggerfish (GPH-TF) at pH 10, 50 C, and 1:3 (U/mg) enzyme/protein ratio

High-fat-high-fructose diet (HFFD)-fed rats (male Wistar rats) Groups: control group; HFFD group; HFFD 1 UGP; HFFD 1 GPH-A (400 mg/kg BW); HFFD 1 GPH-TF (400 mg/kg BW)

Blue whiting hydrolysate

6% wt./vol. blue whiting protein suspension in water was hydrolyzed with Alcalase 2.4 L and Flavourzyme 500 L at an E:S ratio of 0.74% v/w for both enzyme at 50 C for 4 h. Hydrolysate was filtered to remove insoluble material. Part of the hydrolysate was digested with pepsin (E:S of 2.5% wt./wt. for 90 min) and Corolase PP (E:S of 1% wt./wt. for 150 min) to simulate gastrointestinal digestion (SGID) process

Healthy male NIH Swiss mice

Amaranth grain protein hydrolysates (Albumin 1, Globulin, and Glutelin hydrolysates)

Albumin 1 (Alb-1), globulin (Glo), and glutelin (Glu) proteins were extracted form defatted amaranth flour and hydrolyzed by Alcalase at E/S 5 0.8 UA/g protein for 1
48 h

Streptozotocin-induced diabetic mice (CD1 male mice) Two administration protocols were used: a single-administration study or a chronic daily dosing study Groups: diabetic mice; Sitagliptin as positive control; Alb1H48, GloH48, and GluH24 (chosen because of their higher in vitro DPP-IV inhibitory activity)

Oral gavage, once a day for 10 weeks

Administration of GPHs to HFFDfed rats decreased α-amylase activity, blood glucose and hepatic glycogen. UGP and GPHs administration improved redox status in liver and kidney of HFFDrats. GPHs exhibited a renal protective role by reversing the HFFD-induced decease of uric acid and increase of creatinine levels in serum Oral gavage of either Acute and persistent glucoseglucose alone or in lowering effects of the blue whiting combination with the hydrolysate were observed in mice. blue whiting hydrolysate In addition to inhibiting DPP-IV, the (100 mg/kg BW) to test hydrolysates mediated insulin and glucose-lowering and GLP-1 release from BRIN-BD11 and insulin releasing GLUTag cells, respectively. SGID properties of the enhanced GLP-1 secretion hydrolysates significantly. SGID resulted in a significant increase in membrane potential, intracellular calcium and cyclic AMP concentration versus a glucose control, indicating that insulin secretion may be mediated by the KATP channel-dependent and the protein kinase A pathways Oral administration of GluH24 improved glucose tolerance hydrolysates at 300 mg/ significantly (P , 0.05), with kg BW once for the remarkable increments in plasma OGTT (acute) or once a insulin in both single administration day for 4 weeks (chronic) and chronic study (1.25 and 2.25 mg/mL, respectively). This effect was comparable to the one obtained from the mice group that was administered Sitagliptin

Nasri et al. (2015)

Harnedy et al. (2018)

Jorge et al. (2015)

(Continued)

Table 25.2: (Continued) Peptide or protein hydrolysate

Preparation

Oat protein hydrolysate

A 5% (wt./wt.) suspension of the defatted oat flour was hydrolyzed by 4% Alcalase 2.4/L at 55 C, pH 7.5 for 4 h. Fraction with ,5 kDa was collected for experiments

Meju (unsalted fermented soybeans)

Cooked soybeans were fermented outdoors by micro-organisms naturally present in the environment (mainly Bacillus species for early stages and Aspergillus oryzae for late stages) for 60 days. Standardized meju: cooked soybeans were inoculated with Bacillus subtilis, dried, inoculated with Aspergillus oryzae, and fermented in a fermentation chamber at 30 C for 6 days

Aglycin (ASCNGV CSPFEMPPCGSS ACRCIPVGLVV GYCRHPSG)

—

Animal model/treatment groups

Route of administration and frequency

Streptozotocin-induced diabetic mice (ICR male mice) Groups: model control (water); positive control (metformin 0.6 g/kg BW); low oat peptide (0.25 g/kg BW); medium oat peptide (0.5 g/kg BW); and high oat peptide (1 g/kg BW) 90% pancreatectomized (Px) diabetic rats (male Sprague Dawley rats) leading to 50% decrease in insulin secretory capacity. Groups: unfermented cooked soybeans (CSB); traditional fermentation (60 days, TMS); standard fermentation (6days, MMS); casein (control)

Orally once a day for 4 weeks

HFD 1 streptozotocin injection (male BALB/c mice) Groups: nondiabetic control; diabetic (vehicle); diabetic 1 aglycin (50 mg/kg/d); diabetic 1 metformin (100 mg/kg/d)

Orally with ad libitum access to a HFD supplemented with 10% wt./wt. meju treatments for 8 weeks

Orally, once a day for 4 weeks

Observed effect

References

Zhang, Diabetic mice receiving high oat Wang, Liu peptide (1 g/kg BW) had lower and Sun total food intake and fasting blood (2015) glucose, higher food efficiency, elevated serum fasting insulin and insulin activity index, and hepatic glycogen content. FLQPNLDEH, DLELQNNVFPH, and TPNAGVSGAAAGAGAGGKH were identified as potent peptides in the oat hydrolysate Yang, Kwon, CBS, TMS, and MMS improved Kim, Kang glucose tolerance in diabetic rats. and Park CSB-enhanced peripheral insulin (2012) sensitivity including hepatic insulin sensitivity better than the control. TMS and MMS enhanced only hepatic insulin sensitivity through activating insulin signaling in diabetic rats. TMS and MMS, but not CSB, potentiated glucosestimulated insulin secretion and β-cell mass. Changes in isoflavonoid aglycones and peptide profiles according to fermentation was attributed to the observed antidiabetic effects of treatments Lu et al. Aglycin was effective in controlling (2012) hyperglycemia and improving oral glucose tolerance. Aglycin exerts its effect by enhancing gene expression levels of IR and IRS1, as well as improving p-IR, p-IRS1, and p-Akt levels. Aglycin also increased the number of GLUT4 at the cell surface, thereby increasing glucose uptake in peripheral tissues (tested using C2C12 muscle cells)

Pea oligopeptides

Rice albumin hydrolysate

Pea protein was suspended in distilled water, hydrolyzed by Alcalase and neutrase for 4 h, centrifuged, and supernatant was passed through 10 and 1 kDa MW cut off membrane successively, the permeate was desalted, condensed, decolored, and spray dried to yield pea oligopeptides powder

Alkali-treated rice albumin (RA) powder was dissolved in water and hydrolyzed with 50 mM ammonium bicarbonate solution and hydrolyzed with trypsin from bovine pancreas (180 U/mg) for 6 h (RAH). Trypsin-treated RA was fractionated into highmolecular weight peptide (HMP,14 kDa) and low molecular weight peptides (LMP, ,2 kDa)

HFD 1 streptozotocin injection (male Kunming mice) Groups: normal control; diabetic control; diabetic 1 metformin (185 mg/ kg BW); diabetic 1 metformin (185 mg/kg BW) 1 medium dose of pea oligopeptide (1600 mg/kg BW); diabetic 1 metformin (92.5 mg/kg BW) 1 medium dose of pea oligopeptide (800 mg/kg BW); diabetic with a low dose of pea oligopeptide (800 mg/kg BW); diabetic with a medium dose pea oligopeptide (1600 mg/ kg BW); diabetic with a high dose of pea oligopeptide (3200 mg/ kg BW) Male Wistar rats To study effects of RAH on glucose tolerance, three groups were used: control; RA (200 mg/kg BW), RAH (200 mg/kg BW) To study effects of peptides on glucose tolerance, four groups were used: control; RA (200 mg/ kg BW), HMP (133 mg/kg BW), or LMP (67 mg/kg BW)

Pea oligopeptides significantly reduced blood glucose, lipids, and liver fat in diabetic mice. Pea oligopeptide and metformin exhibited synergistic effects and improved glucose tolerance, promoted glycogen synthesis, insulin secretion, and protected the liver and kidney structures in diabetic mice

Wei et al. (2019)

Oral gavage, once for the Both RA and RAH reduced the area OGTT; PBS solution under the curve of OGTT without containing 1 g/kg BW of changes in insulin secretion. HMP glucose with/without RA, and LMP suppressed the increase in RAH, HMP, or LMP blood glucose. HMP adsorbed glucose and retarded its diffusion rate (in vitro diffusion test). LMP suppressed the expression of sodium-dependent glucose transporter-1 in STC-1 cells

Ina et al. (2020)

Orally, once a day for 28 days

(Continued)

Table 25.2: (Continued) Peptide or protein hydrolysate

Preparation

Animal model/treatment groups

Route of administration and frequency

Egg white hydrolysate (EWH)

Observed effect

References

Commercial product-production condition was not mentioned

Male Wistar and GotoKakizaki rats Groups: Wistar Casein (200 g/kg diet); Wistar EWH (267 g/kg diet); Goto-Kakizaki Casein; Goto-Kakizaki EWH

Orally for 6 weeks

EWH improved serum glucose level and HOMA-IR but not insulin secretion. EWH also decreased TG accumulation especially in the soleus muscle in Goto-Kakizaki rat

Ochiai, Kuroda and Matsuo (2014)

Egg white hydrolysate (EWH)

Egg white slurry (5% wt./vol.) was hydrolyzed by thermoase PC10F (0.1% wt./wt. for 90 min) followed by pepsin (10,000 U/mg at 1% wt./ wt. for 180 min)

HFD induced insulin resistant rats (male Sprague Dawley rats) Groups: HFD 1 casein; HFD 1 EWH (1% wt./wt. food); HFD 1 EWH (2% wt./wt. food); HFD 1 EWH (4% wt./wt. food)

Orally, ad libitum access to the HFD supplemented with egg white hydrolysate for 6 weeks

EWH (4% wt./wt.) increased insulin sensitivity, improved oral glucose tolerance, and reduced systemic inflammation. EWH potentiated insulin-induced muscle and adipose tissue p-Akt, reduced adipocyte, and increased PPARγ2 protein abundance and activity

Jahandideh et al. (2019), De Campus Zani, Jahandideh, Wu and Chan (2019)

Table 25.3: Examples of recent clinical studies on antidiabetic and glucoregulatory properties of food-derived protein hydrolysates and bioactive peptides (2010
2020). Source Whey protein

Casein

Enzymatic hydrolysis Flavourzyme

Treatment groups

Study design and intervention regimens

Key findings

Whey protein isolate Double-blind randomised study; Ingestion of 0.2 g/kg WPH or (WPI) and whey protein after an overnight fast, male type 0.4 g/kg WPI enhanced insulin hydrolysate (WPH) at 0.1, 2 diabetic patients (n 5 10) were secretion leading to a major 0.2, and 0.4 g/kg BW and served whey protein beverages. reduction in postchallenge control (distilled water) Blood samples were taken plasma glucose response. Plasma immediately after treatment. insulin concentration increased 30 min after the ingestion of only in the 0.4 g/kg WPH group, beverages, and subjects were fed and glucose levels returned to the a meal of 12 kcal/kg energy and normal range 2 h after the meal allowed to eat within another 30 min. Blood samples were collected at 30 min intervals from 0 to 180 min and analyzed for glucose and insulin concentration Food-grade gastrointestinal Casein hydrolysate and Randomized, controlled, A significant increase in insulin enzymes (pepsin and sodium caseinate crossover design; healthy secretion concomitant with a pancreatin) (control) at 10% wt./vol. overweight/obese Caucasian reduction in glucose. No effect adults (40
65 years). Recruited on c-peptide or GIP secretion subjects (n 5 62) attended the study center on two separate occasions, at least 7 days apart. Subjects fasted for 12 h prior to each visit and were fitted with a cannula before giving fasting blood samples. They then consumed a prescribed breakfast (74 g of carbohydrates) along with a 10% (wt./vol.) solution (100 mL) of casein treatments in a randomized, crossover fashion. Blood was drawn at t 5 15, 30, 60, 90, and 120 min

References Goudarzi and Madadlou (2013)

Drummond et al. (2018)

(Continued)

Table 25.3: (Continued) Source

Enzymatic hydrolysis

Pep2Dia A proprietary milk protein containing alaninehydrolysate from native proline (AP) whey protein containing AP dipeptide with dipeptide (between 0.15% α-glucosidase and 0.4%) inhibitory properties

Black soy

—

Treatment groups Maltodextrin with dextrose equivalent of 9 as placebo, low-dose (1400 mg) or high-dose (2800 mg) milk protein hydrolysate

Black soy peptide or placebo control

Study design and intervention regimens

Key findings

A randomized, double-blind, The low-dose milk peptides placebo-controlled, three-wayreduced postprandial blood crossover study with a 7 days glucose with more glycemic than wash-out period between the insulinotropic properties in study days. Prediabetic subjects prediabetic subjects after a (n 5 21 adults, 30
70 years old) challenge meal in a single dose received a single dose of placebo, intake and after a 6-week low-dose, or high-dose milk intervention period, whereas protein hydrolysate prior to a impacts on postprandial effects challenge meal high in were not strengthened by carbohydrates. Within the intervention over a longer period. crossover study design, subjects A slight increase of the Matsuda received all interventions index and reduction of HbA 1c randomly allocated to three levels (measures of whole-body sequence groups. Also, an openinsulin sensitivity) were label single-arm phase was demonstrated after the 6-week performed with a daily intake of intervention period. the low-dose milk peptide for 6 weeks to estimate effects over a longer period. Then the postprandial assessment after the intake of 1400 mg bioactive peptides from milk protein hydrolysate 15 min prior to a challenge meal was repeated comparable to the crossover phase Double-blind, placebo-controlled Subjects with fasting glucose study, subjects with prediabetes $ 110 mg/dL consuming black and type 2 diabetes were soy peptides tended to have randomly assigned to the placebo lower fasting glucose levels and control or the black soy peptide had a significant reduction in 2-h intervention groups. Fasting PG level compared with baseline serum glucose, HbA 1c, insulin, levels. The 2-h PG levels were also and free-fatty acids, 2-h postload reduced in the intervention group glucose (2-h PG) test, and serum compared with the placebo lipid profiles before and after the group. HbA 1c levels were not 12-week supplementation were improved by the dietary performed. intervention

References Sartorius et al. (2019), Sartorius et al. (2020)

Kwak et al. (2010)

Egg protein (lysozyme)

Marine collagen peptides (MCP)

Fish protein hydrolysates

Alcalase

Lyzozyme hydrolysate or maize starch (control)

Mixed proteases (25% pepsin, 35% trypsin, 35% chymotrypsin, and 5% pancreatic lipase)

Control (carboxymethylcellulose) and MCP (6.5 g) twice per day for 3 months

Papain and Bromelain for hydrolysis of herring and salmon. Cod protein and casein-whey mixture were not hydrolyzed prior to use

2.5 g protein/day from herring, salmon, cod, or milk (a casein-whey mixture as positive control) for 8 weeks

A randomized, placeboDecrease in triacylglycerol (shortcontrolled, double-blind term trial), glucose (acute trial), crossover design to assess acute and insulin (acute trial) (2 h) and short-term (2 days) effects of 5 g/d egg protein hydrolysate in 40 overweight and obese subjects (consumed for 3 days) with impaired glucose tolerance or type 2 diabetes. The study had a 2-week wash-out period Chinese patients with type 2 MCPs reduced levels of fasting diabetes. A total of 100 diabetic blood glucose and insulin, HbA patients and 50 healthy controls 1c, total triglycerides, were recruited. Blood samples cholesterol, LDL, and free-fatty were collected before, and 1.5, acids, but increased levels of and 3 months after, treatment to insulin sensitivity index and evaluate glucose and lipid HDL in diabetic patients. Also. metabolism levels of hs-CRP and NO reduced but levels of bradykinin, PGI2, and adiponectin were increased A double-blind, randomized, HER and SAL samples did not intervention study with a parallel affect glucose and insulin group design and four concentrations. COD reduced intervention arms; herring protein postprandial glucose levels, hydrolysate (HER), salmon but changes were not different protein hydrolysate (SAL), cod from HER and SAL groups. protein (COD), and casein-whey CAS supplementation mixture as positive control (CAS) reduced the area under the in overweight adults of Caucasian curve for glucose origin (n 5 93) concentrations, especially when compared to SAL group, and reduced postprandial insulin c-peptide levels. Serum lipid concentrations were not affected in any of the intervention groups

Plat et al. (2019)

Zhu et al. (2010)

Hovland et al. (2020)

(Continued)

Table 25.3: (Continued) Source

Enzymatic hydrolysis

Treatment groups

Cod protein hydrolysate

Protamex (Novozymes AS)

Fish protein hydrolysate (25%
30% of the peptides have a MW , 200 Da) or casein (control) at 20 mg/kg BW

Cod protein hydrolysate (CPH)

Protamex (Novozymes AS)

Four different doses: 10, 20, 30, or 40 mg/kg BW of CPH daily for 7 days

Study design and intervention regimens

Key findings

References

Dale et al. A double-blind crossover trial, A single dose of cod protein (2018) including two different study hydrolysate before a breakfast days, with a 4
7 days wash-out meal reduced postprandial period. Healthy and active insulin secretion, without individuals (n 5 41, 41
64 years affecting blood glucose response old). Blood samples were taken, or GLP-1 levels in healthy and subjects were served a drink individuals with the fish protein hydrolysate or control, before a breakfast meal (500 kcal energy) was given. The first postmeal sample (0 min sample) was taken 15 min after the breakfast was served. Blood samples were taken every 20 min until 120 min and a final sample at 180 min The study was a double-blind Serum glucose and insulin levels Jensen et al. (2019) crossover trial. Participants tend to decrease with increasing (healthy older adults, n 5 31, amounts of CPH. No differences 60
78 years old) received in estimated maximum value of different doses of CPH daily for 7 glucose, insulin, or GLP-1 were days with 1-week wash-out observed when comparing the periods in between. After baseline lowest dose of CPH with higher blood sampling, the last dosage doses as tested by a mixed-model of CPH was given followed by a statistical analysis standardised breakfast meal (455 kcal energy) 10 min later. 25 min after the CPH drink, postprandial blood samples were taken in 20 min intervals until 120 min

Applications in medicine: hypoglycemic peptides 623 however, casein hydrolysate increased insulin secretion in the early postprandial period in obese or overweight subjects and affected glucose levels compared to the native casein (Horner et al., 2019). The effect of milk protein hydrolysates on gastric emptying as a major determinant of postprandial glycemia has been studied in healthy and obese adults (Calbet and Holst, 2004; Horner et al., 2019). It appears that hydrolysis of casein or whey proteins does not affect the rate of gastric emptying in overweight and obese or healthy subjects (Calbet and Holst, 2004; Horner et al., 2019). Moreover, the rate of plasma GLP-1 and peptide YY responses to feeding with whey and casein protein solutions in healthy humans are independent of the protein hydrolysis (Calbet and Holst, 2004). Gastric secretion, on the other hand, is affected by the protein hydrolysis; milk protein hydrolysates elicited about 50% more gastric secretion than the native protein. Higher plasma GIP levels are also observed during the first 20 min of the gastric emptying process in healthy subjects (Calbet and Holst, 2004). Soy and egg protein hydrolysates have also been reported to exert positive effects on postprandial blood glucose in subjects with prediabetes and type 2 diabetes (Kwak et al., 2010; Plat, Severins, & Mensink, 2019).

25.8 Conclusions Bioactive peptides have shown potential in improving health and human well being and studies on their physiological functionalities to develop functional foods/nutraceuticals are on the rise. Despite the great potential, several hurdles exist in commercialization and widespread application of functional foods from protein hydrolysates and bioactive peptides. While antidiabetic properties of some protein hydrolysates have been confirmed in human studies, there are hundreds of peptides/protein hydrolysates with reported antidiabetic properties through in vitro and animal studies awaiting clinical confirmation. Moreover, precision nutrition and metabotyping, the classification of individuals in subgroups according to their metabolic profile, needs to be employed to successfully identify differential response to dietary interventions (Curran et al., 2019; Hillesheim and Brennan, 2020). Other aspects to consider regarding development of functional foods include scalable production methods, identifying mechanisms of action, and protection of bioactive peptides against the gastrointestinal digestion. Finally, regulatory requirements of food-derived protein hydrolysates and bioactive peptides which vary in different countries are also important in this field (Chalamaiah, Ulug, Hong, & Wu, 2019).

References Aluko, R. E. (2015). Antihypertensive peptides from food proteins. Annual Review of Food Science and Technology, 6, 235
262. Amati, F. (2012). Revisiting the diacylglycerol-induced insulin resistance hypothesis. Obesity Reviews: An Official Journal of the International Association for the Study of Obesity, 13(Suppl 2), 40
50.

624 Chapter 25 Attard, S. M., Herring, A. H., Wang, H., Howard, A. G., Thompson, A. L., Adair, L. S., et al. (2015). Implications of iron deficiency/anemia on the classification of diabetes using HbA1c. Nutrition & Diabetes, 5, e166. Calbet, J. A., & Holst, J. J. (2004). Gastric emptying, gastric secretion and enterogastrone response after administration of milk proteins or their peptide hydrolysates in humans. European Journal of Nutrition, 43 (3), 127
139. Caron, J., Domenger, D., Dhulster, P., Ravallec, R., & Cudennec, B. (2017). Protein digestion-derived peptides and the peripheral regulation of food intake. Frontiers in Endocrinology (Lausanne), 8, 85. Chakrabarti, S., Jahandideh, F., & Wu, J. P. (2014). Food-derived bioactive peptides on inflammation and oxidative stress. BioMed Research International. Chalamaiah, M., Ulug, S. K., Hong, H., & Wu, J. (2019). Regulatory requirements of bioactive peptides (protein hydrolysates) from food proteins. Journal of Functional Foods, 58, 123
129. Christophi, C. A., Resnick, H. E., Ratner, R. E., Temprosa, M., Fowler, S., Knowler, W. C., et al. (2013). Confirming glycemic status in the diabetes prevention program: Implications for diagnosing diabetes in high risk adults. Journal of Diabetes and its Complications, 27(2), 150
157. Curran, A. M., Horner, K., O’Sullivan, V., Nongonierma, A. B., Le Maux, S., Murphy, E., et al. (2019). Variable glycemic responses to intact and hydrolyzed milk proteins in overweight and obese adults reveal the need for precision nutrition. The Journal of Nutrition, 149(1), 88
97. Dale, H. F., Jensen, C., Hausken, T., Lied, E., Hatlebakk, J. G., Bronstad, I., et al. (2018). Effect of a cod protein hydrolysate on postprandial glucose metabolism in healthy subjects: A double-blind cross-over trial. Journal of Nutritional Science, 7, e33. De Campus Zani, S. C., Jahandideh, F., Wu, J., & Chan, C. B. (2019). 122-Egg white hydrolysate effects in adipose tissue of high fat diet-induced insulin resistant rats. Canadian Journal of Diabetes, 43(7), S42. Diabetes Canada Clinical Practice Guidelines Expert C., Punthakee, Z., Goldenberg, R., & Katz, P. (2018). Definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome. Canadian Journal of Diabetes, 42(Suppl 1), S10
S15. Drummond, E., Flynn, S., Whelan, H., Nongonierma, A. B., Holton, T. A., Robinson, A., et al. (2018). Casein hydrolysate with glycemic control properties: Evidence from cells, animal models, and humans. Journal of Agricultural and Food Chemistry, 66(17), 4352
4363. Dziuba, B., & Dziuba, M. (2014). New milk protein-derived peptides with potential antimicrobial activity: An approach based on bioinformatic studies. International Journal of Molecular Sciences, 15(8), 14531
14545. El-Sayed, M. I., Awad, S., Wahba, A., El-Attar, A., Yousef, M. I., & Zedan, M. (2016). In vivo anti-diabetic and biological activities of milk protein and milk protein hydrolyaste. Advances in Dairy Research, 4(1), 6. Esfandi, R., Walters, M. E., & Tsopmo, A. (2019). Antioxidant properties and potential mechanisms of hydrolyzed proteins and peptides from cereals. Heliyon, 5(4), e01538. Feng, Y. M., Zhao, D., Zhang, N., Yu, C. G., Zhang, Q., Thijs, L., et al. (2016). Insulin resistance in relation to lipids and inflammation in type-2 diabetic patients and non-diabetic people. PLoS ONE, 11(4), e0153171. Fletcher, B., Gulanick, M., & Lamendola, C. (2002). Risk factors for type 2 diabetes mellitus. The Journal of Cardiovascular Nursing, 16(2), 17
23. Floegel, A., Stefan, N., Yu, Z., Muhlenbruch, K., Drogan, D., Joost, H. G., et al. (2013). Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach. Diabetes, 62(2), 639
648. Garcia-Perez, L. E., Alvarez, M., Dilla, T., Gil-Guillen, V., & Orozco-Beltran, D. (2013). Adherence to therapies in patients with type 2 diabetes. Diabetes Therapy, 4(2), 175
194. Geerts, B. F., van Dongen, M. G., Flameling, B., Moerland, M. M., de Kam, M. L., Cohen, A. F., et al. (2011). Hydrolyzed casein decreases postprandial glucose concentrations in T2DM patients irrespective of leucine content. Journal of Dietary Supplements, 8(3), 280
292. Ghosh, D., & Parida, P. (2016). Drug discovery and development of type 2 diabetes mellitus: Modernintegrative medicinal approach. Current Drug Discovery Technologies, 13(2), 60
67.

Applications in medicine: hypoglycemic peptides 625 Girgis, C. M., Cheng, K., Scott, C. H., & Gunton, J. E. (2012). Novel links between HIFs, type 2 diabetes, and metabolic syndrome. Trends in Endocrinology and Metabolism: TEM, 23(8), 372
380. Goudarzi, M., & Madadlou, A. (2013). Influence of whey protein and its hydrolysate on prehypertension and postprandial hyperglycaemia in adult men. International Dairy Journal, 33(1), 62
66. Gu, Y., & Wu, J. (2013). LC-MS/MS coupled with QSAR modeling in characterising of angiotensin Iconverting enzyme inhibitory peptides from soybean proteins. Food Chemistry, 141(3), 2682
2690. Harnedy, P. A., Parthsarathy, V., McLaughlin, C. M., O’Keeffe, M. B., Allsopp, P. J., McSorley, E. M., . . . FitzGerald, R. J. (2018). Blue whiting (Micromesistius poutassou) muscle protein hydrolysate with in vitro and in vivo antidiabetic properties. Journal of Functional Foods, 40, 137
145. Harreiter, J., & Roden, M. (2019). Diabetes mellitus-Definition, classification, diagnosis, screening and prevention (Update 2019). Wiener klinische Wochenschrift, 131(Suppl 1), 6
15. He, Q., Gao, Z., Yin, J., Zhang, J., Yun, Z., & Ye, J. (2011). Regulation of HIF-1{alpha} activity in adipose tissue by obesity-associated factors: Adipogenesis, insulin, and hypoxia. American Journal of Physiology. Endocrinology and Metabolism, 300(5), E877
E885. Hillesheim, E., & Brennan, L. (2020). Metabotyping and its role in nutrition research. Nutrition Research Reviews, 33(1), 33
42. Hirano, T. (2018). Pathophysiology of diabetic dyslipidemia. Journal of Atherosclerosis and Thrombosis, 25(9), 771
782. Horner, K., Drummond, E., O’Sullivan, V., Scsh, P., & Brennan, L. (2019). Effects of a casein hydrolysate versus intact casein on gastric emptying and amino acid responses. European Journal of Nutrition, 58(3), 955
964. Hovland, I. H., Leikanger, I. S., Stokkeland, O., Waage, K. H., Mjos, S. A., Brokstad, K. A., et al. (2020). Effects of low doses of fish and milk proteins on glucose regulation and markers of insulin sensitivity in overweight adults: A randomised, double blind study. European Journal of Nutrition, 59(3), 1013
1029. Hsieh, C. H., Wang, T. Y., Hung, C. C., Chen, M. C., & Hsu, K. C. (2015). Improvement of glycemic control in streptozotocin-induced diabetic rats by Atlantic salmon skin gelatin hydrolysate as the dipeptidyl-peptidase IV inhibitor. Food & Function, 6(6), 1887
1892. Ina, S., Hamada, A., Nakamura, H., Yamaguchi, Y., Kumagai, H., & Kumagai, H. (2020). Rice (Oryza sativa japonica) albumin hydrolysates suppress postprandial blood glucose elevation by adsorbing glucose and inhibiting Na 1 -d-glucose cotransporter SGLT1 expression. Journal of Functional Foods, 64(103603), 1
8. Islam, S. M., Niessen, L. W., Seissler, J., Ferrari, U., Biswas, T., Islam, A., et al. (2015). Diabetes knowledge and glycemic control among patients with type 2 diabetes in Bangladesh. Springerplus, 4, 284. Jahandideh, F., Majumder, K., Chakrabarti, S., Morton, J. S., Panahi, S., Kaufman, S., et al. (2014). Beneficial effects of simulated gastro-intestinal digests of fried egg and its fractions on blood pressure, plasma lipids and oxidative stress in spontaneously hypertensive rats. PLoS ONE, 9(12). Jahandideh, F., & Wu, J. (2020). Perspectives on the potential benefits of antihypertensive peptides towards metabolic syndrome. International Journal of Molecular Sciences, 21(6). Jahandideh, F., Zani, S. C. C., Son, M., Proctor, S. D., Davidge, S. T., Chan, C. B., et al. (2019). Egg white hydrolysate enhances insulin sensitivity in high-fat diet-induced insulin-resistant rats via Akt activation. The British Journal of Nutrition, 122(1), 14
24. Jensen, C., Dale, H. F., Hausken, T., Lied, E., Hatlebakk, J. G., Bronstad, I., et al. (2019). Supplementation with cod protein hydrolysate in older adults: A dose range cross-over study. Journal of Nutritional Science, 8, e40. Jonker, J. T., Wijngaarden, M. A., Kloek, J., Groeneveld, Y., Gerhardt, C., Brand, R., et al. (2011). Effects of low doses of casein hydrolysate on post-challenge glucose and insulin levels. European Journal of Internal Medicine, 22(3), 245
248. Jorge, S. S., Rau´l, R. B., Isabel, G. L., Edith, P. A., Bernardo, E. B. H., Ce´sar, A. P. J., . . . Rube´n, R. R. (2015). Dipeptidyl peptidase IV inhibitory activity of protein hydrolyzates from (Amaranthus hypochondriacus L.) grain and their influence on postprandial glycemia in streptozotocin-induced diabetic mice. African Journal of Traditional, Complementary, and Alternative Medicines, 12(1), 90
98.

626 Chapter 25 Kahn, S. E. (2003). The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia, 46(1), 3
19. Kahn, S. E., Cooper, M. E., & Del Prato, S. (2014). Pathophysiology and treatment of type 2 diabetes: perspectives on the past, present, and future. Lancet, 383(9922), 1068
1083. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16(9), 945
960. Kuzmenko, D. I., & Klimentyeva, T. K. (2016). Role of ceramide in apoptosis and development of insulin resistance. Biochemistry (Mosc), 81(9), 913
927. Kwak, J. H., Lee, J. H., Ahn, C. W., Park, S. H., Shim, S. T., Song, Y. D., et al. (2010). Black soy peptide Supplementation improves glucose control in subjects with prediabetes and newly diagnosed type 2 diabetes mellitus. Journal of Medicinal Food, 13(6), 1307
1312. Labazi, H., & Trask, A. J. (2017). Coronary microvascular disease as an early culprit in the pathophysiology of diabetes and metabolic syndrome. Pharmacological Research: The Official Journal of the Italian Pharmacological Society, 123, 114
121. Layman, D. K., Boileau, R. A., Erickson, D. J., Painter, J. E., Shiue, H., Sather, C., et al. (2003). A reduced ratio of dietary carbohydrate to protein improves body composition and blood lipid profiles during weight loss in adult women. The Journal of Nutrition, 133(2), 411
417. Ledesma-Martinez, E., Aguiniga-Sanchez, I., Weiss-Steider, B., Rivera-Martinez, A. R., & Santiago-Osorio, E. (2019). Casein and peptides derived from casein as antileukaemic agents. Journal of Oncology, 2019, 8150967. Li-Chan, E. C. Y. (2015). Bioactive peptides and protein hydrolysates: Research trends and challenges for application as nutraceuticals and functional food ingredients. Current Opinion in Food Science, 1, 28
37. Liu, Z., & Udenigwe, C. C. (2019). Role of food-derived opioid peptides in the central nervous and gastrointestinal systems. Journal of Food Biochemistry, 43(1), e12629. Lu, J. L., Zeng, Y., Hou, W. R., Zhang, S. S., Li, L. L., Luo, X., et al. (2012). The soybean peptide aglycin regulates glucose homeostasis in type 2 diabetic mice via IR/IRS1 pathway. The Journal of Nutritional Biochemistry, 23(11), 1449
1457. Manders, R. J., Hansen, D., Zorenc, A. H., Dendale, P., Kloek, J., Saris, W. H., et al. (2014). Protein coingestion strongly increases postprandial insulin secretion in type 2 diabetes patients. Journal of Medicinal Food, 17(7), 758
763. Nasri, R., Abdelhedi, O., Jemil, I., Daoued, I., Hamden, K., Kallel, C., et al. (2015). Ameliorating effects of goby fish protein hydrolysates on high-fat-high-fructose diet-induced hyperglycemia, oxidative stress and deterioration of kidney function in rats. Chemico-biological Interactions, 242, 71
80. Newgard, C. B., An, J., Bain, J. R., Muehlbauer, M. J., Stevens, R. D., Lien, L. F., et al. (2009). A branchedchain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metabolism, 9(4), 311
326. Ngoh, Y. Y., & Gan, C. Y. (2018). Identification of Pinto bean peptides with inhibitory effects on alphaamylase and angiotensin converting enzyme (ACE) activities using an integrated bioinformatics-assisted approach. Food Chemistry, 267, 124
131. Nieto-Vazquez, I., Fernandez-Veledo, S., Kramer, D. K., Vila-Bedmar, R., Garcia-Guerra, L., & Lorenzo, M. (2008). Insulin resistance associated to obesity: The link TNF-alpha. Archives of Physiology and Biochemistry, 114(3), 183
194. Nita Gandhi Forouhi, N. J. W. (2019). Epidemiology of diabetes. Medicine, 47(1), 22
27. Nongonierma, A. B., & FitzGerald, R. J. (2014). An in silico model to predict the potential of dietary proteins as sources of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. Food Chemistry, 165, 489
498. Ochiai, M., Kuroda, T., & Matsuo, T. (2014). Increased muscular triglyceride content and hyperglycemia in Goto-Kakizaki rat are decreased by egg white hydrolysate. International Journal of Food Sciences and Nutrition, 65(4), 495
501. Phung, O. J., Scholle, J. M., Talwar, M., & Coleman, C. I. (2010). Effect of noninsulin antidiabetic drugs added to metformin therapy on glycemic control, weight gain, and hypoglycemia in type 2 diabetes. JAMA: The Journal of the American Medical Association, 303(14), 1410
1418.

Applications in medicine: hypoglycemic peptides 627 Plat, J., Severins, N., & Mensink, R. P. (2019). Improvement of pulse wave velocity and metabolic cardiovascular risk parameters through egg protein hydrolysate intake: A randomized trial in overweight or obese subjects with impaired glucose tolerance or type 2 diabetes. Journal of Functional Foods, 52, 418
423. Qi, L. W., Liu, E. H., Chu, C., Peng, Y. B., Cai, H. X., & Li, P. (2010). Anti-diabetic agents from natural products—An update from 2004 to 2009. Current Topics in Medicinal Chemistry, 10(4), 434
457. Rios, J. L., Francini, F., & Schinella, G. R. (2015). Natural products for the treatment of type 2 diabetes mellitus. Planta Medica, 81(12-13), 975
994. Roder, P. V., Wu, B., Liu, Y., & Han, W. (2016). Pancreatic regulation of glucose homeostasis. Experimental & Molecular Medicine, 48, e219. Ross, S. A., Gulve, E. A., & Wang, M. (2004). Chemistry and biochemistry of type 2 diabetes. Chemical Reviews, 104(3), 1255
1282. Salas, C. E., Badillo-Corona, J. A., Ramirez-Sotelo, G., & Oliver-Salvador, C. (2015). Biologically active and antimicrobial peptides from plants. BioMed Research International, 2015, 102129. Saleh, L., Schrier, N. L., Bruins, M. J., Steegers, E. A. P., van den Meiracker, A. H., & Visser, W. (2018). Effect of oral protein hydrolysate on glucose control in patients with gestational diabetes. Clinical Nutrition (Edinburgh, Scotland), 37(3), 878
883. Samuel, V. T., & Shulman, G. I. (2012). Mechanisms for insulin resistance: Common threads and missing links. Cell, 148(5), 852
871. Sartorius, T., Weidner, A., Dharsono, T., Boulier, A., Wilhelm, M., & Schon, C. (2019). Postprandial effects of a proprietary milk protein hydrolysate containing bioactive peptides in prediabetic subjects. Nutrients, 11(7). Sartorius, T., Weidner, A., Dharsono, T., Boulier, A., Wilhelm, M., & Schon, C. (2020). Correction: Sartorius et al. “Postprandial effects of a proprietary milk protein hydrolysate containing bioactive peptides in prediabetic subjects” Nutrients 2019, 11, 1700. Nutrients, 12(5). Sears, B., & Perry, M. (2015). The role of fatty acids in insulin resistance. Lipids in health and disease, 14, 121. Siow, H. L., Lim, T. S., & Gan, C. Y. (2017). Development of a workflow for screening and identification of alpha-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study—Cumin seed. Food Chemistry, 214, 67
76. Thomas, C. C., & Philipson, L. H. (2015). Update on diabetes classification. The Medical Clinics of North America, 99(1), 1
16. Turban, S., & Hajduch, E. (2011). Protein kinase C isoforms: Mediators of reactive lipid metabolites in the development of insulin resistance. FEBS Letters, 585(2), 269
274. Tyagi, A., Daliri, E. B., Kwami Ofosu, F., Yeon, S. J., & Oh, D. H. (2020). Food-derived opioid peptides in human health: A review. International Journal of Molecular Sciences, 21(22). Udenigwe, C. C., & Rouvinen-Watt, K. (2015). The role of food peptides in lipid metabolism during dyslipidemia and associated health conditions. International Journal of Molecular Sciences, 16(5), 9303
9313. Uenishi, H., Kabuki, T., Seto, Y., Serizawa, A., & Nakajima, H. (2012). Isolation and identification of caseinderived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL from gouda-type cheese and its effect on plasma glucose in rats. International Dairy Journal, 22(1), 24
30. Veldhorst, M., Smeets, A., Soenen, S., Hochstenbach-Waelen, A., Hursel, R., Diepvens, K., et al. (2008). Proteininduced satiety: Effects and mechanisms of different proteins. Physiology & Behavior, 94(2), 300
307. Walters, M. E., Esfandi, R., & Tsopmo, A. (2018). Potential of food hydrolyzed proteins and peptides to chelate iron or calcium and enhance their absorption. Foods, 7(10). Wang, T., Zheng, L., Zhao, T., Zhang, Q., Liu, Z., Liu, X., et al. (2020). Anti-diabetic effects of sea cucumber (Holothuria nobilis) hydrolysates in streptozotocin and high-fat-diet induced diabetic rats via activating the PI3K/Akt pathway. Journal of Functional Foods, 75. Wang, T. Y., Hsieh, C. H., Hung, C. C., Jao, C. L., Chen, M. C., & Hsu, K. C. (2015). Fish skin gelatin hydrolysates as dipeptidyl peptidase IV inhibitors and glucagon-like peptide-1 stimulators improve glycaemic control in diabetic rats: A comparison between warm and cold-water fish. Journal of Functional Foods, 19, 330
340.

628 Chapter 25 Wei, Y., Zhang, R., Fang, L., Qin, X., Cai, M., Gu, R., et al. (2019). Hypoglycemic effects and biochemical mechanisms of Pea oligopeptide on high-fat diet and streptozotocin-induced diabetic mice. Journal of Food Biochemistry, 43(12), e13055. Westerterp-Plantenga, M. S., Lemmens, S. G., & Westerterp, K. R. (2012). Dietary protein—its role in satiety, energetics, weight loss and health. The British Journal of Nutrition, 108(Suppl 2), S105
S112. WHO. (2011). Global Status Report on Noncommunicable Diseases 2010. World Health Organization. Wilson, H. L., Buchanan, R. M., Allan, B., & Tikoo, S. K. (2012). Milk-derived antimicrobial peptides to protect against neonatal diarrheal disease: An alternative to antibiotics. Procedia in Vaccinology, 6, 21
32. Wu, H., & Ballantyne, C. M. (2017). Skeletal muscle inflammation and insulin resistance in obesity. The Journal of Clinical Investigation, 127(1), 43
54. Wu, J., Jahandideh, F., Yu, W., & Majumder, K. (2015). Bioactive peptides from meat proteins as functional food components. In G. Cirillo, U. Gianfranco Spizzirri, & F. Lemma (Eds.), Functional polymers in food science (pp. 181
208). . Wu, J. P., Aluko, R. E., & Nakai, S. (2006). Structural requirements of angiotensin I-converting enzyme inhibitory peptides: Quantitative structure-activity relationship study of di- and tripeptides. Journal of Agricultural and Food Chemistry, 54(3), 732
738. Wurtz, P., Makinen, V. P., Soininen, P., Kangas, A. J., Tukiainen, T., Kettunen, J., et al. (2012). Metabolic signatures of insulin resistance in 7,098 young adults. Diabetes, 61(6), 1372
1380. Xu, L., Li, Y., Dai, Y., & Peng, J. (2018). Natural products for the treatment of type 2 diabetes mellitus: Pharmacology and mechanisms. Pharmacological Research: The Official Journal of the Italian Pharmacological Society, 130, 451
465. Yang, H. J., Kwon, D. Y., Kim, M. J., Kang, S., & Park, S. (2012). Meju, unsalted soybeans fermented with Bacillus subtilis and Aspergilus oryzae, potentiates insulinotropic actions and improves hepatic insulin sensitivity in diabetic rats. Nutrition & Metabolism, 9. Zhang, H., Wang, J., Liu, Y., & Sun, B. (2015). Peptides derived from oats improve insulin sensitivity and lower blood glucose in streptozotocin-induced diabetic mice. Journal of Biomedical Science, 4(1). Zhu, C. F., Li, G. Z., Peng, H. B., Zhang, F., Chen, Y., & Li, Y. (2010). Treatment with marine collagen peptides modulates glucose and lipid metabolism in Chinese patients with type 2 diabetes mellitus. Applied Physiology, Nutrition, and Metabolism (Physiologie appliquee, nutrition et metabolisme), 35(6), 797
804.

CHAPTER 26

Application in medicine: obesity and satiety control Alina Kondrashina1, Shauna Heffernan2, Nora O’Brien2 and Linda Giblin1 1

Food Biosciences Department, Teagasc Food Research Centre, Cork, Ireland, 2School of Food and Nutritional Sciences, University College Cork, Cork, Ireland

Abbreviations AA AMPK BMI bw CCK CMP CSPHP DPP-4 FA FDA FOSHU GI GLP-1 GMP GRAS HDL HMGR I.c.v. I.p. i.v. LXRα NEP PI2 PPARγ PYY s.c. T2DM WHO

amino acid AMP-activated protein kinase body mass index body weight cholecystokinin caseinomacropeptide C-fraction soy protein hydrolyzate with bound phospholipids dipeptidyl peptidase-4 fatty acid Food and Drug Administration Foods for Specified Health Uses gastrointestinal glucagon-like peptide-1 glycomacropeptide granted generally recognized as safe high-density lipoprotein 3-hydroxy-3-methylglutaryl-CoA reductase intracerebroventricular intraperitoneal intravenous liver X receptor alpha neutral endopeptidase proteinase inhibitor II peroxisome proliferator-associated receptor gamma peptide YY subcutaneous type 2 diabetes mellitus World Health Organization

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00005-4 © 2021 Elsevier Inc. All rights reserved.

629

630 Chapter 26

26.1 Introduction Obesity is a worldwide epidemic, prevalent in low- and high-income populations, in urbanized and rural areas, and in developed and developing countries (Collaboration, 2019). Generally, a body mass index (BMI) of .25 kg/m2 is classified as overweight and .30 kg/m2 as obese. According to the World Health Organization (WHO) in March 2020, obesity has tripled over the last 45 years with 39% of the world population now overweight and 13% of the population obese. Increased BMI index is a major risk factor for cardiovascular disease, diabetes, cancer, and musculoskeletal disorders. Pertinently for 2020, it also increases the risk of severe illness from Covid-19, as in the study by Cai et al. (2020), out of 91 patient in critical care 29.3% were overweight and 39.0% were obese (Cai et al., 2020). Increased BMI therefore places a huge burden on the world’s health systems and accounts for over 4 million deaths every year (Moini, Ahangari, Miller, & Samsam, 2020). The reasons for developing obesity vary from diet to genetics, physical activity, and medical factors (Heymsfield & Wadden, 2017). Prevention of obesity is more effective than treatment with a major focus on obesity prevention programs in school settings, emphasizing healthy eating, lifestyle choices, and balancing food intake with energy output (physical exercise) (Birch & Ventura, 2009). Treatment of obesity includes (1) dietary intervention/caloric restriction, (2) structured physical activity, (3) behavioral therapy, (4) pharmacotherapy, (5) bariatric surgery, and (6) combinations of the above (Wadden & Bray, 2018). In severely and morbidly obese adult patients, pharmacotherapy and bariatric surgeries are routinely employed. Bariatric surgery is the most effective treatment for obesity but it is highly invasive and imposes a considerable risk of harmful side effects (Buchwald et al., 2004). Pharmacotherapy provides a less invasive, potentially safer option with peptides making up a large proportion of available pharmacotherapeutic options. This chapter summarizes peptides, both synthetic and food derived, capable of reducing appetite and promoting weight loss in vivo to treat obesity. In vivo regulation of appetite primarily occurs via satiety peptides, such as Glucagon-like peptide-1 (GLP-1, 29/30-AA) (amino acids), Peptide YY (PYY, 33AA), Cholecystokinin (CCK, 8/22/33/58-AA), Oxyntomodulin (37AA), and the hunger hormone, ghrelin (28AA) (Hellstro¨m, 2013). These peptides are secreted from specialized enteroendocrine cells in the gastrointestinal (GI) tract in response to the presence or absence of food and bind to their receptors in the central nervous system, signaling either satiation or hunger. For example, CCK intravenously infused at 2.7 pmol/kg/min to healthy subjects resulted in a 29% food intake reduction ad libitum after 140 minutes, while 1.5 pmol/kg/min of GLP-1active and 0.8 pmol/kg/min of PYY resulted in 32% (t 5 60 minutes) and 23% (t 5 210 minutes) reduction of food intake, respectively (Lim & Poppitt, 2019). In contrast, ghrelin intravenously infused at 5.0 pmol/kg/min led to a 28% increase of ad libitum food intake after 240 minutes (Wren et al., 2001). It is important to note that the circulating lifespan of

Application in medicine: obesity and satiety control 631 endogenous satiety hormones is minutes. The bioactive form of GLP-1 has a circulation half-life ,2 minutes, after which it is cleaved to an inactive form by the endogenous proteolytic enzyme dipeptidyl peptidase-4 (DPP-4) (Baggio & Drucker, 2007). Satiety signaling can be modulated with bioactive peptides by two different approaches: (1) altering production/release/circulation time of the hormone or (2) interfering with its corresponding receptor. Other endogenous GI peptides that are worthwhile pharmacological targets include serotonin and opioids as they also suppress appetite and food intake (Adan, 2013). In addition, the hormone glucagon (29AA), produced in the pancreas, stimulates energy expenditure and possesses hypolipidemic and satiating effects (Habegger et al., 2010). Another approach to combat obesity has been the development of bioactive peptides that inhibit GI digestive enzymes, slow down hydrolysis of foods, and reduce their absorption by the body (Cristina Oliveira de Lima, Piuvezam, Leal Lima Maciel, & Heloneida de Arau´jo Morais, 2019). Targeting lipid metabolism and processes in adipose tissue is also an emerging area of research, employing bioactive peptides that activate lipolysis leading to decreased fat storage (Torres-Fuentes, Schellekens, Dinan, & Cryan, 2015) or inhibition of adipocyte proliferation and differentiation (adipogenesis) (Torres-Fuentes et al., 2015). Lastly, bioactive peptides that act on the quantity and composition of circulating lipoproteins in the blood can also address dyslipidemia, associated with obesity (Dias, Paredes, & Ribeiro, 2018).

26.2 Synthetic peptides All currently approved peptidic drugs for the treatment of obesity are mimetics of GI satiety hormones with the added benefit of prolonged circulation time (Bray & Ryan, 2014). Therefore these drugs mediate their effect by binding to receptors of satiety hormones. A detailed comparison of all approved GLP-1 analogs was recently published (Nauck & Meier, 2019). Even though many of these are used for the regulation of glucose homeostasis in type 2 diabetes mellitus (T2DM) patients (McBrayer & Tal-Gan, 2017), this chapter focuses specifically on their efficacy for obesity treatment. Table 26.1 details the structure, mechanism, dosage, and efficacy for each individual mimetic. To compare and contrast from an obesity perspective, Table 26.1 details the largest weight loss reported in the literature for different populations and doses studied.

26.2.1 Synthetic peptides: glucagon-like peptide-1 mimetics The delivery route of a GLP-1 mimetic depends on its stability in the GI tract, its bioavailability postdigestion, and its affinity to serum proteins. For the patient the most preferred route is oral administration, but until recently, this was rarely available due to the proteolytic conditions of the GI tract. Even though these mimetics are designed with

Table 26.1: Synthetic glucagon-like peptide-1 (GLP-1) analogs and multiagonists approved for the use or undergoing clinical development. Peptide

Approval

Structure/mechanism

Half-life

Dose

Obesity/weight loss effect

References

Exenatide

Approved by FDA (2005) and by the European Medicines Agency (2011)

Appetite-suppressing synthetic mimetic of exendin-4, has 53% AA homology to GLP-1. Binds to GLP-1 receptor. Slows down gastric emptying.

2.4 h, app. 2 weeks (for once weekly)

S.c. injection: 10 μg twice daily; 2 mg once weekly (sustained release)

Buse et al. (2007), Cvetkovi´c and Plosker (2007), Gupta (2013), Su et al. (2016)

Lixisenatide

Approved by FDA (2016)

3h

Once-daily s.c. injection, 20 μg

Taspoglutide

Halted in 2010

Appetite-suppressing synthetic mimetic of exendin-4 with one AA substitution and 6 lysine residues. Binds to GLP-1 receptor. Slows down gastric emptying. Appetite-suppressing synthetic GLP-17
36 analog with AA substitutions at positions 8, 35, and 36. Binds to GLP-1 receptor.

2 2.1 kg over 30 weeks; 24.7 kg over 2 years versus baseline (T2DM, n 5 283, BMI 5 34 6 6 kg/m2) 2 4.47 kg versus control group in nondiabetic overweight and obese volunteers over 12
24 weeks (n 5 362) 21.76 kg over 24 weeks versus baseline in T2DM patients (n 5 859, BMI 5 30 6 7 kg/m2)

85 h

Once-weekly s. c. injection, 10
20 mg

Liraglutide

Approved by FDA (2014) and by the European Medicines Agency (2015)

Appetite-suppressing synthetic GLP-1 analog (Arg34, C16 FA chain at position 26). Binds to GLP-1 receptor. Slows down gastric emptying.

13
15 h

Once-daily s.c. injection, 3.0 mg

Dulaglutide

Approved by FDA and by the European Medicines Agency (2014)

Synthetic GLP-1 analog, where GLP-17
37 covalently linked to an Fc fragment of human IgG4. Binds to GLP-1 receptor. Slows gastric emptying, promotes weight loss or reduces weight gain.

4.7 days

Once-weekly s. c. injection, 1.5 mg

Rosenstock et al. (2014), Werner, Haschke, Herling, and Kramer (2010)

22.0 kg at 10 mg weekly, Retterstøl 22.8 kg at 20 mg weekly and (2009), 21.9 kg at 20 mg biweekly Rosenstock et al. over 8 weeks versus baseline (2013), in T2DM patients (n 5 1189, Sebokova et al. BMI 5 32.7 6 5 kg/m2) (2010) 25.6 kg versus placebo and Drucker, 28.4 kg versus baseline in Dritselis, and nondiabetic subjects Kirkpatrick (n 5 3731, BMI . 30 kg/m2) (2010), over 56 weeks Manigault and Thurston (2016) 22.29 kg (monotherapy, Barrington et al. n 5 807) to 23.18 kg (2011), Scheen (combined with metformin, (2016), n 5 1098) in T2DM Umpierrez et al. patients (BMI . 30 kg/m2) (2016) over 26
52 weeks

Albiglutide

Approved by FDA and by the European Medicines Agency (2014).

Semaglutide

Approved by FDA (2017), European Medicines Agency, Health Canada and the Japanese Ministry of Health, Labour and Welfare (2018)

Epfeglenatide (HM 11260C)

III stage of clinical development (to be finished in 2021)

Appetite-suppressing synthetic GLP-1 analog, with one AA substitution and covalent link to human albumin. Binds to GLP-1 receptor. Slows down gastric emptying. Appetite-suppressing synthetic GLP-1 analog (Aib8, Arg34, C18 FA chain at position 26). Binds to GLP-1 receptor. Slows down gastric emptying, reduces preference for high-fat food, improves lipid metabolism.

Appetite-suppressing synthetic GLP-1 analog, single AA modification in Exendin-4 and IgG4 Fc fragment. Binds to GLP-1 receptor. Slows gastric emptying.

6
7 days

Once-weekly s. c. injection, 30 or 50 mg

20.6 kg over 32 weeks monitoring in T2DM patients (n 5 841, av. BMI 5 32.8 kg/m2)

Matthews et al. (2008), Pratley et al. (2014)

7 days

Once-weekly s.c. injection, 0.5
1.0 mg Once-daily oral dose, 7
14 mg

2 5 kg (1 mg) versus baseline over 12 weeks in nondiabetic obese adults (n 5 30, BMI 5 30
45 kg/m2) Up to 25 kg (0.5 mg) and up to 27 kg (1.0 mg) over 56 weeks in T2DM patients (av. BMI 5 39.3 kg/m2) Weight loss for oral dose: 2 0.9 kg (2.5 mg), 21.5 kg (5 mg), 23.6 kg (10 mg), 25.0 kg (20 mg), 25.7 kg (40 mg) versus baseline over 26 weeks in T2DM patients (n 5 632, BMI 5 25
40 kg/m2) 2 2.34 kg versus baseline and 22.00 kg versus placebo group over 4 months in obese (n 5 156, BMI 5 32.1 6 4.4 kg/m2) T2DM adults (8
16 mg once monthly) -7.3 kg (6 mg once weekly), 27.0 kg (8 mg biweekly) versus baseline in obese (n 5 237, BMI 5 35.4 6 4.2 kg/m2) nondiabetic adults

Blundell et al. (2017), Christou et al. (2019), Davies et al. (2017), Lau et al. (2015)

160
170 h Once-monthly s.c. injection, 8
16 mg; once weekly s. c. injection, 2
8 mg

Del Prato et al. (2020), Pratley et al. (2019)

(Continued)

Table 26.1: (Continued) Peptide Cotadutide (MED10382)

Approval

Structure/mechanism

Half-life

Dose

Obesity/weight loss effect

References

II stage of clinical development

Dual receptor GLP-1 and glucagon agonist. Delays gastric emptying, reduces body weight through appetite suppression and enhanced energy expenditure. Dual receptor GLP-1 and glucagon agonist. Delays gastric emptying, reduces body weight through appetite suppression and enhanced energy expenditure.

8.4
9.4 h

Once-daily s.c. 50
300 μg dose

23.41% body weight in overweight/obese (n 5 63, BMI 5 27
40 kg/m2) adults over 50 days versus baseline

Parker et al. (2020)

12
14 h

Once-daily s.c. 0.06
0.18 mg dose

2 5.32 kg over 21 days in nondiabetic subjects (n 5 40, BMI 5 20
30 kg/ m2) versus baseline 2 5.46 kg over 28 days in T2DM subjects (n 5 36, BMI 5 28
42 kg/m2) versus baseline 20.9 to 211.3 kg at 1
15 mg dose over 26 weeks in T2DM patients (n 5 318, BMI 5 25
50 kg/m2) versus baseline

Tillner et al. (2019)

SAR425899

II stage of clinical development.

Tirzepatide (LY3298176)

III stage of clinical development

Dual receptor GLP-1 and GIP 106
123 h Once-weekly s. c. injection agonist. Delays gastric (1
15 mg) emptying, reduces body weight through appetite suppression and effect on lipid metabolism.

AA, Amino acid; Aib, 2-aminoisobutyric acid; BMI, body mass index; s.c., subcutaneous; T2DM, type 2 diabetes mellitus.

Frias et al. (2018)

Application in medicine: obesity and satiety control 635 sequence modifications to resist DPP-4 degradation, they can be hydrolyzed by other proteases and peptidases, resulting in reduced half-lives (Gupta, 2013). The first-approved GLP-1 mimetic drug was exenatide which has a 53% AA homology to GLP-1 and is a synthetic analog of a naturally occurring venom (exenatide-4) from the Heloderma lizard, capable of resisting DPP-4 inactivation (Gupta, 2013). With a half-life of 2.4 hours, exenatide requires twice-daily subcutaneous (s.c.) administration. To reduce frequency of injections a slow-release version, exenatide LAR, was developed by encapsulation within biodegradable polymeric microspheres of polylactic-co-glycolic acid, which is suitable for once-weekly s.c. injection (Jose, Tahrani, Piya, & Barnett, 2010). The body weight reduction of either twice-daily exenatide or once-weekly exenatide LAR in T2DM patients was similar over 30 weeks (Drucker et al., 2008). In addition, a metaanalysis of randomized controlled trials has demonstrated a significant reduction in body weight (24.47 kg), BMI (20.86 kg/m2), and waist circumferences (21.78 cm) in obese and overweight nondiabetic volunteers over 12
24 weeks (Su et al., 2016). An AA derivative of exenatide, lixisenatide, has a half-life of 3h, and as such was the first once-daily GLP-1 mimetic administered by s.c. injection (Dicembrini, Bigiarini, & Mannucci, 2014). Lixisenatide provided significant weight loss in overweight/obese T2DM patients with 14.4% patients losing $ 5% weight over 24 weeks compared to the baseline (Rosenstock et al., 2014). Both exenatide and lixisenatide reach maximum plasma concentration at approximately 2 hour postadministration, are primarily degraded by metalloproteases into small peptides, and eventually excreted in urine (Cvetkovi´c & Plosker, 2007; Dicembrini et al., 2014). Taspoglutide features 93% homology to human GLP-1 and fully resists cleavage by DPP-4, resulting in a plasma half-life of 9.8 hours at 10 mg dosage (Sebokova et al., 2010). At the higher concentration of 20 mg, it is suitable for once-weekly s.c. injection due to its increased half-life of up to 85 hours (Christou, Katsiki, Blundell, Fruhbeck, & Kiortsis, 2019). Taspoglutide caused significant (P , .05) body weight reduction in overweight/obese T2DM patients over 8 weeks compared to baseline (Retterstøl, 2009; Rosenstock et al., 2013). One of the most successful synthetic GLP-1 mimetics to date is liraglutide (Manigault & Thurston, 2016). The attachment of a C16 fatty acid (FA) chain to its structure, hugely increased its affinity to albumin (up to 98%), resulting in a slow release of liraglutide from albumin with a half-life of 8
10 hours after intravenous (i.v.) injection and 13
15 hours after s.c. injection (Lau et al., 2015). It has reduced renal excretion (6%) compared to endogenous GLP-1 (B70%), indicating complete degradation in the body by DPP-4 and neutral endopeptidases (NEPs) (Malm-Erjefa¨lt et al., 2010; Manigault & Thurston, 2016; Meier et al., 2004). Once-daily s.c. administration of 3 mg liraglutide for 5 weeks to nondiabetic obese subjects (BMI 5 30
40 kg/m2), slowed down gastric emptying of a standardized breakfast by 23% (P 5 .007) compared to vehicle placebo (Van Can et al., 2014) and reduced body weight (up to 28.4 kg in 56 weeks), compared to baseline

636 Chapter 26 (Manigault & Thurston, 2016). Moreover, in the clinical trial for FDA approval, there was further weight loss 12 weeks after liraglutide treatment in those individuals who followed a restricted calories diet with exercise plan compared to individuals who received a vehicle placebo (6.2% vs 0.2%) (Manigault & Thurston, 2016). Dulaglutide is synthetic GLP-1 covalently linked to the Fc fragment of human IgG4 and exhibits a prolonged circulating half-life of up to 4.7 days (Jendle et al., 2016). Maximum concentration of dulaglutide occurred in plasma 24
48 hours post s.c. injection with bioavailability of 47% for 1.5 mg and 65% for 0.75 mg doses, after which it was hydrolyzed by peptidases (Sanford, 2014). Efficacy of dulaglutide was significantly improved when coadministrated with T2DM drug metformin (Table 26.1) with 24%
34% participants losing .5% weight over 26
52 weeks (Umpierrez et al., 2016). The attachment of human albumin to the GLP-1 sequence resulted in another mimetic, albiglutide, with a half-life of 6
7 days (Matthews et al., 2008). Catabolism of albiglutide occurs similarly to the usual route of human albumin with maximum concentration in plasma achieved on day 3. However, in a 32 weeks phase III study with overweight/obese T2DM patients, 50 mg albiglutide administered once-weekly by s.c. injection produced a weight loss of only 20.64 kg, what was significantly lower than other GLP-1 mimetics and ultimately led to its withdrawal from the market (Madsbad, 2016; Rendell, 2016). The longest half-life for a GLP-1 mimetic is .7 days and was achieved with the GLP-1 analog semaglutide which has an albumin-binding domain, an AA substitution and a C18 FA attached (Goldenberg & Steen, 2019; Lau et al., 2015). Similar to other GLP-1 mimetics, it binds to its receptor, GLP-1R, and displays all the expected outcomes of endogenous GLP-1, including increased secretion of insulin, lowered blood glucose levels, slowing of gastric emptying, and weight loss due to the suppression of appetite (Lovshin & Drucker, 2009). Moreover, it can interfere with neural pathways involved in appetite regulation, without crossing the blood
brain barrier or binding to GLP-1R, by central c-Fos activation. This leads to neurons in the lateral parabrachial nucleus signaling meal termination (Gabery et al., 2020). More importantly an oral formulation of semaglutide has been developed by the addition of the absorption enhancer, sodium N-(8-[2-hydroxybenzoyl] amino) caprylate, which protects the compound from proteolytic degradation by locally increasing pH (Granhall, Søndergaard, Thomsen, & Anderson, 2018) and increasing its permeability across the intestinal barrier. Oral semaglutide is .99% bound to human albumin and achieves maximum circulation concentration 1 hour postadministration (Hedrington & Davis, 2019). It is catabolized by cleavage of the peptide backbone with peptidases and NEP in the kidneys, followed by oxidation of the FA side chain (Christou et al., 2019). Approximately 70%
80% of the intact semaglutide form is present in plasma and about 3% of the absorbed dose is eliminated via the renal route. Semaglutide s.c. administrated once weekly at 1.0 mg dose to 30 obese (BMI 5 30
45 kg/m2) nondiabetic subjects for 12 weeks significantly delayed gastric emptying by over 1 hour, decreased fasting levels of total

Application in medicine: obesity and satiety control 637 cholesterol, high-density lipoprotein (HDL) cholesterol, very low
density lipoprotein (LDL) and triglycerides, suppressed subjective appetite ratings, lowered preference for high-fat food and reduced energy intake by 24% (P # .01) (Blundell et al., 2017; Hjerpsted et al., 2018). A recent review of human trials suggests that s.c. injected 0.5
1 mg semaglutide can reduce body weight up to 25 kg in nondiabetic and up to 27 kg in T2DM patients with obesity, while oral formulation at 40 mg dosage produced a 25.7 kg reduction of body weight in T2DM patients over 12
56 weeks, compared to the baseline (Christou et al., 2019; Davies et al., 2017). Indeed, daily oral doses of 20 or 40 mg resulted in equivalent body weight reduction to once-weekly s.c. administration of 1 mg semaglutide over a 26 week period. Recent developments of synthetic satiety peptides have focused on (1) oral formulations, (2) prolonging time active, and/or (3) improving efficacy. One of the GLP-1 mimetics currently in development is efpeglenatide (HM 11260C), which has half-life of 160
170 hours and is suitable for the once-monthly s.c. administration (Davies, Thiman, & Kugler, 2016; Yoon et al., 2020). Efpeglenatide achieves maximum circulation concentration 2
3 days if administered once weekly or 3
5 days if administered once monthly. It has a demonstrated weight loss efficacy at 8
16 mg doses in T2DM obese subjects over 4 months (P , .01) (Del Prato et al., 2020) and at 6
8 mg doses in obese nondiabetic subjects over 20 weeks (Pratley et al., 2019). Efpeglenatide fragments are excreted in the urine, with ,10% excreted in the feces. Also of note is that lixisenatide, liraglutide, dulaglutide, and semaglutide have lower dissociation constants, higher binding affinities, and longer residence times on the GLP-1R than endogenous GLP-1, which may suggest a lower GLP-1R activation capacity of these mimetics (Choi, Moon, Trautmann, Hompesch, & Sorli, 2018; Jones et al., 2018). In contrast, efpeglenatide demonstrated a higher dissociation constant (360.7 vs 58.7 nmol/L) and 6.1-fold lower GLP-1R binding affinity compared to liraglutide resulting in higher availability of receptor for signaling, even compared to endogenous GLP-1 (Choi et al., 2018). In agreement with this, efpeglenatide demonstrated the greatest efficiency among synthetic mimetics tested for once-monthly s.c. administration. Mimetics of other satiety hormones are rare but the PYY analog, NNC0165-1562, has entered phase I clinical trials with healthy, overweight/obese, and T2DM cohorts to investigate safety, effective dose, side effects, and route of body elimination (Rebello & Greenway, 2020).

26.2.2 Synthetic peptides: multiple actions mimetics A peptide targeting multiple satiety pathways may be the most potent in terms of body weight reduction (Zhou et al., 2017). Cotadutide (31-AA) acts as an agonist to both GLP-1 and glucagon, similar to endogenous oxyntomodulin (Parker et al., 2020). However, its half-life is only 8.4
9.4 hours requiring daily s.c. injection. In clinical trials, it reduced

638 Chapter 26 appetite and weight over 50 days, compared to the baseline and placebo group (Ambery et al., 2018). Another dual GLP-1 and glucagon agonist, SAR425899, has a half-life of 12
14 hours in steady state upon daily s.c. administration (Tillner et al., 2019). In nondiabetic normal/overweight and in T2DM overweight/obese patients, it reduced body weight by .5 kg in 4 weeks. Tirzepatide (LY3298176) was developed as a dual agonist to both GLP-1 and gastric inhibitory polypeptide (GIP) receptors (Frias et al., 2018). Similar to GLP-1, GIP is an incretin hormone that functions to induce insulin secretion. Activation of both GLP-1 and GIP receptors by s.c. injection of this unimolecular agonist decreased weight by up to 211.3 kg (15 mg), waist circumference by 2.1
10.2 cm, and total cholesterol by 0.2
0.3 mmol/L in overweight/obese T2DM subjects over 26 weeks (Frias et al., 2018). In fact, a dosage-dependent (1
15 mg) decrease of .5% weight was achieved in 14%
71% of subjects and .10% weight loss in 6%
39% of subjects, with Tirzepatide. Interestingly, a triagonist (GIP-Glucagon-GLP-1) HM15211 has recently entered phase I clinical trials with reports indicating that a single dose is well tolerated by overweight subjects (Choi et al., 2019). It reaches a maximum circulating concentration in plasma 31
68 hours post s.c. administration and has half-life of 72
142 hours. The antiobesity potential of C2816 a synthetic peptide that targets both GLP-1 and CCK receptors was recently evaluated in mice (Hornigold et al., 2018). C2816 was constructed from an exenatide analog and a selective CCK-1R agonist Hpa-Nle-Gly-Trp-Lys(Tac)-AspNMePhe-NH2. C2816 intraperitoneally (I.p.) injected at a dose of 10 nmol/kg body weight in C57/BL6J mice reduced food intake in a 24 hour period by 45% compared to the vehicle control, and resulted in significant weight loss (Hornigold et al., 2018). Moreover, in dietinduced obese C57/BL6J mice, C2816 I.p. injected at 50 nmol/kg, significantly reduced levels of plasma triglycerides, cholesterol, and body weight by 228.4% over 10 days, compared to the control group. Several other multiagonist peptides are at various stages of preclinical and clinical evaluations including peptides targeting GLP-1-gastrin, GLP-1xenin, and GLP-1-amylin (Hasib, 2020).

26.2.3 Safety considerations and limitations for synthetic peptides All of the GLP-1 mimetics appear to cause GI disorders of different severity, including nausea, vomiting, diarrhea, abdominal pain, and constipation. In addition, some mimetics have contraindications with certain medical conditions. For example, exenatide is contraindicated for patients with type 1 diabetes, kidney, and liver diseases. The most common side effect for exenatide was nausea, which was reported by up to 27% patients treated once a week with exenatide LAR (Colagiuri, 2010). The rare side effects of acute kidney injury and pancreatitis have been reported for lixisenatide (Bolli & Owens, 2014). Dulaglutide is generally well tolerated at doses up to 12 mg with no withdrawal from studies (Barrington et al., 2011). Albigutide was well tolerated up to 32 mg with the rare

Application in medicine: obesity and satiety control 639 side effects of nausea and vomiting (Matthews et al., 2008) but was withdrawn from the market in 2018 for lack of efficacy. Liraglutide, 3.0 mg administrated s.c., is well tolerated with the most common side effects being nausea and diarrhea (Lovshin & Drucker, 2009). It is currently recommended for the treatment of patients with a BMI $ 30 kg/m2 or $ 27 kg/m2 with accompanying comorbidities (Manigault & Thurston, 2016). Semaglutide, 1.0 mg administrated s.c. is generally well tolerated with transient side effects of diarrhea, constipation, nausea, and vomiting (Christou et al., 2019). Oral administration of semaglutide (10 mg) was proven to be safe for patients with renal and hepatic impairment (Bækdal, Thomsen, Kupˇcova´, Hansen, & Anderson, 2018; Granhall et al., 2018). However, it is currently contraindicated for people with a family history of oncology as an association with thyroid cancer is under investigation. Nausea is the most common side effect for efpeglenatide with 33%
59% of subjects reporting this effect on once-weekly administration and 30%
67% on once-monthly doses (Davies et al., 2016). SAR425899 administrated at a single dose caused side effects in 67% of subjects (Tillner et al., 2019). Participants withdrew from studies due to dyspepsia, nausea, vomiting, increased heart rate, and elevated lipase levels. Currently, SAR425899 production is halted by the developer until adverse effects are resolved. Treatment with LY3298176 for 26 weeks resulted in 4% of participants experiencing dose-dependent side effects; however, most of them were mild with transient nausea, diarrhea, and vomiting (Frias et al., 2018). Antibody response to bioactive peptides is commonplace with 57% of patients producing antibodies to exenatide (once weekly), 70% for lixisenatide, 9% for liraglutide, 3% for albiglutide, 2% for dulaglutide, 4% for semaglutide (once weekly) and 13% for efpeglenatide (Bolli & Owens, 2014; Christou et al., 2019; Davies et al., 2016). High antibody titer can reduce the efficiency of treatment. Indeed, taspoglutide production was halted due to immunogenicity and hypersensitivity issues (Christou et al., 2019).

26.2.4 Other synthetic peptides in preclinical trials and in vitro development Several other synthetic peptides have been identified that target other pathways to reduce weight. Adipotide (CKGGRAKDC-GG-KLAKLAKKLAKLAK) (Kolonin, Saha, Chan, Pasqualini, & Arap, 2004) is a chimeric peptide containing sequence CKGGRAKDC which targets the receptor of the scaffold protein, prohibtin, on endothelial cells of white fat adipose tissue and KLAKLAKKLAKLAK peptide, which functions to distort mitochondrial membranes. The result of this dual action is targeted apoptosis and disruption of the vascular supply to white adipose tissue. Adipotide administrated s.c. at 100 nM daily to diet-induced obese C57BL/6 mice resulted in weight loss of 15 g ( . 30%) over 4 weeks (Kolonin et al., 2004). Epididymal fat pad was significantly decreased compared to the vehicle control (0.6 vs 2.1 g, P , .001) and liver fat deposition

640 Chapter 26 was reduced twofold. Obese rhesus macaques s.c. injected with adipotide at 0.43 mg/kg body weight daily for 4 weeks had a decrease in body weight of 7%
15%, a decrease in BMI by 4%
17%, and a reduction in abdominal circumferences by 6%
14% compared to baseline (Barnhart et al., 2011). Moreover, total body fat reduction was 38.7% (P , .0001) 4 weeks after the end of treatment compared to the vehicle control group. No side effects of GI disorder or changes in primate behavior were observed (Barnhart et al., 2011). However, further development of adipotide as an antiobesity drug appears to have been discontinued. Kumar et al. reported in 2019 that peptides that activate lipid metabolism, mimic adiokines (apolipoprotein and amylin), and inhibit fat and glucose absorption are also currently under development (Kumar, 2019; Rebello & Greenway, 2020). In vitro screening has revealed tetrapeptidic sequences with demonstrated antiobesity potential (Le Neve´ & Daniel, 2011). GGGG at 20 mM stimulated 1.5-fold increased secretion of GLP-1 from human enteroendocrine cell line NCI-H716 over 2 hours, compared to the Krebs buffer control (P , .05). In addition, peptides AAAA (10 mM) and GWGG (10 mM) stimulated .1.5-fold increase of GLP-1 release in NCI-H716 cells (P , .05). This stimulation is mediated via calcium signaling, as intracellular Ca21 was dose dependently (3
10 mM) increased by tetrapeptides, while no effect on cAMP levels was observed. The length of peptides was suggested as an important parameter for bioactivity, as GG, GGG, and GGGGG did not stimulate intracellular Ca21 flux (Le Neve´ & Daniel, 2011). In another study, a range of synthetic di- and tripeptides were screened by oral administration at a dose 1 mg/g body weight to C57BL/6J mice and oral glucose tolerance test performed to estimate GLP-1 secretagogue properties (Zhang et al., 2013). Among them, tripeptides were most efficient with sequence GGL identified as a frontrunner. GGL (diapin) demonstrated 2.4-fold elevation of total GLP-1 plasma levels 30 minute postadministration, compared to glucose alone. However, further studies are required to estimate potential of GGL for appetite-suppressing and weight-lowering applications.

26.3 Food-derived peptides As dietary protein consumption is a major inducer of satiety response in the GI tract (Rolls, Hetherington, & Burley, 1988), food scientists initially looked to identify food peptides capable of increasing secretion of satiety hormones in the gut. Table 26.2 lists the foodderived peptides that have demonstrated antiobesity bioactivity in vivo but to date none have been approved for obesity treatment. Food-derived peptides are most likely to find their application in long-term weight management (i.e., obesity prevention) rather than as a treatment for obesity. As such, food-derived peptides will probably be consumed as an ingredient in a food matric rather than delivered by injection.

Table 26.2: Food-derived peptides with antiobesity potential demonstrated in vivo. Antiobesity effect Peptide/source VRIRLLQRFNKRS from soybean β-conglycinin

Administration Intraduodenal infusion in rats

RF from rice glutelin

I.p. injection for food intake, oral gavage for gut transit

IHRF from rice glutelin

Oral gavage for food intake and gut transit

Potide-G (POT II (Proteinase Inhibitor II)/ PPIC/Potein/Slendesta) from potato

GMP and CMP from κ-casein β-casomorphin-7 (YPFPGPI) from β-casein

LIVTQTMKG (lactoghrestatin) from β-LG

Oral gavage/ intraduodenal to rats Preload of 1.5 g POT II with soup in lean men Oral preload in whey drink (25% of energy from protein) Oral gavage

Oral gavage

Mechanism of action Appetite suppressing: stimulates CCK secretion via Ca21 signaling. Appetite suppressing: stimulates CCK secretion via intracellular Ca21 flux.

In vitro

In vivo

References

Dose-dependent CCK release in STC-1 cells (0.3
2.4 mM, 1 h).

Increased plasma CCK levels threefold after Nakajima et al. 45 min (at 3 μmol/L) and suppressed 1 h (2010), Nishi food intake (at 3
12 μmol/L) in Spragueet al. (2003) Dawley rat (n 5 10). .100-fold increase in intracellular Ca21 Kontani et al. Significantly suppressed food intake 1 h (2014) flux and .2-fold increase in CCK (P , .05) and 2 h (P , .01) post I.p. injection secretion (P , .05) over 1 h incubation of 10 mg/kg in 18 h fasted ddY mice with 3 mM, compared to PBS control. (n 5 5
8), compared to saline. Suppressed gut transit postoral delivery of 100 mg/mL (P , .05).  50-fold increase in intracellular Ca21 Appetite suppressing: Kagebayashi et al. Significantly suppressed food intake 1 and stimulates CCK (2012) flux and  3-fold increase in CCK 2 h (P , .05, n 5 4) postoral gavage of secretion via intracellular secretion (P , .05) over 1 h incubation 100 mg/kg in 18 h fasted ddY mice, Ca21 flux. with 3 mM, compared to PBS control. compared to saline. Suppressed gut transit postoral delivery of 3
100 mg/mL (P , .05, n 5 10
18). Chen et al. In lean men suppressed ad libitum food Appetite suppressing: Inhibits trypsin and chimotrypsn with (2012), Hill et al. intake by 17.5% compared to soup stimulates CCK IC50 of 2.5
5 mg/mL. At concentration preload without POT II (n 5 11). (1990), Kim et al. secretion, acts as tripsin 5 mg/mL increases CCK secretion from (2006) STC-1 cells .2-fold versus Hepes buffer At 0.5 g/kg bw increases plasma CCK 2and chymotrypsin over 1 h. fols and at doses 1
1.5 g/kg bw inhibitor. significantly suppresses food intake in Sprague-Dawley rats over 6 h (P , .05, n 5 12) compared to water. Appetite suppressing: Increased secretion of CCK from isolated Reduced ad libitum food intake by 10% in 25 Beucher et al. stimulates CCK duodenojejunum of rat threefold versus healthy subjects (1994), Veldhorst et al. (2009) secretion. basal, when treated with 187.5 mg/mL of digested κ-casein. Appetite suppressing: Stimulated up to 2.4-fold increase in Delayed gastric transit in Wistar rats (oral Daniel et al. stimulates CCK CCK secretion from STC-1 cells versus gavage of 179 mg/kg bw)  4-fold compared (1990), Osborne et al. (2014), secretion, acts as opioid vehicle control (P , .05). to whey preload (P , .05, n 5 10) Pupovac and receptor agonist, delays Anderson (2002) gastric transit. Appetite suppressing: Dose-dependently (10
100 μM) Reduced plasma ghrelin (1 h Aoki et al. (2017) suppresses secretion of decreased ghrelin secretion over 4 h in postadministration) and food intake (over acylated ghrelin. MGN3-1 cells. 4 h) at a dose 1 mg/kg bw in fasted dYY mice, compared to the saline control (P , .05, n 5 17)

(Continued)

Table 26.2: (Continued) Antiobesity effect Peptide/source

Administration

In vitro

In vivo

PVNFKFLSH (hemopressin) from hemoglobin

I.p. (500 nmol/kg) and I.c.v. (1, 5, or 10 nmol)

Mechanism of action Appetite suppressing: signaling through CB1 cannabinoid receptor.

Receptor specificity was studied in ELISA assay with conformation-sensitive antibodies.

Soybean β-conglycinin YPFVV (soymorphin5), YPFVVN (soymorphin-6) and YPFVVNA (soymorphin-7) HIRL (β-lactotensin) from β-LG

Oral injection to mice

Appetite suppressing: stimulates μ-opioid receptor and slows down gut transit.

IC50 of 6.0, 9.2, and 13 μM, respectively, in GPI assay. Affinity to μ-opioid receptor with IC50 of 17, 39, and 47 μM.

Significantly reduced cumulative food intake 1 h post I.c.v. administration and 2 h post I. p. injection in CD1 mice, Sprague-Dawley rats and ob/ob mice (P , .05, n 5 6
7). SM-5 and 7 at 48 μmol/kg suppressed 2 h food intake in 18-h fasted mice. At 16 μmol/ kg bw SM-5, 6, and 7 suppressed small intestinal transit (P , .05, n 5 6
8)

I.c.v., I.p. injections, oral consumption

Appetite suppressing: stimulates ileum contractions, delays gut transit hypocholesterolemic. Appetite suppressing: stimulates ileum contractions, delays gut transit.

Stimulated contraction in longitudinal muscle of GPI assay.

Inhibits HMGR in HepG2 cells, enhances LDL uptake by hepatic cells, increases LDLR expression.

AFKAWAVAR (albutensin A) from serum albumin

I.c.v., I.p. injections

Soybean lunasin SKWQHQQDSCRKQKQ GVNLTPCEKHIMEKIQ GRGDDDDDDDDD Soybean glycinin LPYPR

Oral injection to mice

Affects lipid metabolism. Hypocholesterolemic, triglyceride-lowering.

Oral injection to mice

Affects lipid metabolism. 33.7% inhibition of HMGR at 200 μM in Hypocholesterolemic. HepG2 cells.

Soybean peptides VAWWMY—soystatin

GPETAFLR from lupine (Lupinus angustifolius L.)

Oral gavage

Oral administration with drinking water

Affects lipid metabolism. Inhibitor of cholesterol absorption, binds bile salts. Affects lipid metabolism. Triglyceride-lowering

Stimulated contraction in longitudinal muscle of GPI assay.

References Dodd et al. (2010), Gomes et al. (2010) Kaneko et al. (2010), Ohinata et al. (2007)

Reduced food intake in fasted dYY mice 20 min post 40 nmol/mouse via i.c.v. and 100 mg/ kg via I.p. injection (P , .05, n 5 4
8). Oral dose of 500 mg/kg reduced food intake 20
60 min postadministration (n 5 7). Delayed gastric emptying in ddY mice 2 h after I.c.v. administration of 50 nmol/mouse and significantly reduced 20 min food intake ( . 4-fold). I.p. injection of 0.3
1 μmol/ mouse reduced 20 min food intake (P , .05, n 5 5
8). Reduced serum TG levels by 27%, after 3 times a week 3 mg dose for 25 weeks in C57BL/6 mice (n 5 8).

Hou et al. (2009), Pihlanto¨ et al. Leppa¨la (1997)

Reduced total cholesterol by 25.4% and LDLcholesterol by 30.6% compared to saline control in mice fed 2 days with 50 mg/kg.

Pak, Koo, Lee, Kim, and Kwon (2005), Yoshikawa et al. (2000) Nagaoka et al. (2010), Yoshikawa (2015)

Binds over 94% of taurocholate over 2 h at 25 mg/mL concentration.

30 mg dose reduced serum, liver, and intestine cholesterol levels compared to casein hydrolyzate with trypsin and soybean hydrolyzate with pepsin over 1 h (P , .05, n 5 9)




Reduced body weight 1.3-fold, blood TG 2.1fold and serum leptin 2.7-fold when fed at a dose 1 mg/kg/day to HFD-induced obese C57BL/6J mice for 8 weeks compared to HFD control (n 5 10).

Ohinata et al. (2002), Yoshikawa and Chiba (1992)

Drori et al. (2017), Gu et al. (2019)

Lemus-Conejo et al. (2020)

Soybean ILL, LLL, VHVV

I.p. injected

Enhance lipolysis of adipose tissue

VFVRN from chickpea

Oral gavage

HMGR inhibitor, promotes preadipocytes apoptosis

VAGTWY from β-LG

I.p. injection in zebra fish

AGFAGDDAPR from black tea

Oral gavage to hyperglycemic mice

Appetite suppressing: DPP-4 inhibitor. Affects lipid metabolism. Appetite suppressing: DPP-4 inhibitor, increases GLP-1 secretion. Appetite suppressing: DPP-4 inhibitor.

RRDY from yam dioscorin

Oral gavage to normal ICR mice

Increased glycerol release in differentiated mouse 3T3-L1 adipocytes from 300 mmol/mg for vehicle control to 548, 579, and 687 mmol/mg protein, respectively. Reduced HMGR and LXRα expressions in HepG2 cells more than 40% at concentration 1 mM. Suppressed 24 h grows of 3T3-L1 preadipocytes at 0.2
3 mM and induced 58.75% apoptosis at 3 mM. DPP-4 inhibition with IC50 5 174 μM.

VHVV at concentration 25 mg/kg/day reduced LDL-cholesterol, TG levels and size of adipocytes over 6 weeks in diet-induced obese mice compared to the saline control (n 5 8). Significantly reduced total cholesterol, LDLcholesterol, TG levels and increase HDLcholesterol when administrated at 50 mg/kg bw dose to HFD-induced obese C57BL/6J mice for 10 weeks, compared to HFD control (n 5 10).

DPP-4 inhibition with IC50 5 1.02 mM.

At 400 mg/day increased blood GLP-1 levels  2-fold after 57 days (n 5 6
10).

DPP-4 inhibition with IC50 5 0.93 mM.

Decreased blood DPP-4 concentration over 120 min (n 5 4).

In zebra fish at 200 μg/g dose reduced triglycerides and free cholesterol levels over 5 days versus water (P , .05, n 5 5)

Chiang et al. (2014), Tsou et al. (2013)

Shi et al. (2019)

MohammedGeba et al. (2016), Uchida et al. (2011) Lu et al. (2019)

Lin et al. (2016)

bw, Body weight; CCK, cholecystokinin; CMP, caseinomacropeptide; DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; GMP, glycomacropeptide; GPI, guinea pig ileum; HMGR, 3-hydroxy-3-methylglutarylCoA reductase; I.c.v., intracerebroventricular; I.p., intraperitoneal; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; PBS, phosphate-buffered saline.

644 Chapter 26

26.3.1 Food-derived peptides targeting CCK and GI enzymes with proven in vivo efficacy Peptide VRIRLLQRFNKRS, derived from soy β-conglycinin, increased CCK blood levels .3-fold and significantly reduced food intake 1 hour postintraduodenal infusion at 3 μmol/L in Sprague-Dawley rats, compared to water (P , .05) (Table 26.2) (Nishi, Hara, Asano, & Tomita, 2003). Appetite-suppressing properties of this particular sequence were not studied in human trials; however, 3 g hydrolyzate of the parental protein, β-conglycinin, produced with pineapple proteases, significantly suppressed appetite in 30 healthy volunteers at 1 hour postoral administration (Hira et al., 2011). In agreement a pepsin hydrolyzate of β-conglycinin significantly slowed down gastric transit twofold, stimulated CCK release .4-fold, and suppressed food intake by 17% in Sprague-Dawley rats compared to the vehicle control when administered by duodenal infusion (Nishi et al., 2003). Surprisingly, in this experiment, the hydrolyzate was as effective as the VRIRLLQRFNKRS peptide in reducing food intake. In vitro this peptide dose dependently (0.3
2.4 mM) promotes secretion of satiety hormone CCK from STC-1 cells over 1 hour (Nakajima, Hira, Eto, Asano, & Hara, 2010). A mechanistic study revealed that CCK release occurs via stimulation of calcium-sensing receptor (CaR) followed by intracellular Ca21 mobilization. Rice glutelin peptide RF and its parental IHRF demonstrated appetite-suppressing potential in murine models. At a dose 10 mg/kg, RF significantly suppressed food intake at 1 hour (P , .05) and 2 hours (P , .01) post I.p. injection to 18 hours fasted ddY mice compared to saline; however, this effect was not long standing with no suppression observed at a 4 hour time point (Kontani et al., 2014). Intestinal transit was significantly delayed 35 minute postoral delivery of 30 mg/kg (P , .05) and 100 mg/mL (P , .01) of this peptide. Peptide IHRF (100 mg/mL) could significantly suppress food intake 1 and 2 hours (P , .05) when administered by oral gavage (Kagebayashi et al., 2012). In vitro both peptides (3 mM) exhibited .50-fold increase of intracellular Ca21 flux and .2-fold increase of CCK secretion (P , .05) in STC-1 cells over 1 hour incubation, compared to phosphate buffer saline control. A 5578.9 Da protein from potato, potide-G (also known as POT II/PPIC/Potein), dose dependently inhibited GI proteases trypsin and chymotrypsin with an IC50 5 2.5
5 mg/mL (Kim et al., 2006). In 11 lean subjects, a preload soup with 1.5 g of Potein resulted in a 17.5% reduction of energy intake ad libitum 5 minutes later, compared to soup alone (Hill, Peikin, Ryan, & Blundell, 1990). In 18 hour fasted Sprague-Dawley rats, administration of Potein 1
1.5 g/kg bw by oral gavage significantly suppressed food intake for 6 hours compared to water (Table 26.2) (Chen et al., 2012). This effect was due to stimulation of the CCK receptor, as the addition of the CCK receptor antagonist, devazepide, blocked Potein ability to suppress food intake. Indeed, direct infusion of 0.5 g/kg Potein into duodenum of anesthetized rats resulted in a twofold increase of plasma CCK levels over

Application in medicine: obesity and satiety control 645 90 minutes compared to water. Moreover, Potein (5 mg/mL) increased CCK secretion in murine enteroendocrine cell line STC-1 more than twofold over a 1 hour period, compared to Hepes buffer (Chen et al., 2012). This can be explained by trypsin inhibition slowing the deactivation of luminal CCK-releasing factor cumulating in increased circulating CCK levels (Staljanssens, Smagghe, & Van Camp, 2012). Trypsin-inhibiting properties have also been identified in fractions of soluble peptides from legumes, corn, tamarind seed, and peanut; however, studies on their in vivo efficacy are still on-going (Cristina Oliveira de Lima et al., 2019). Dairy proteins are rich in bioactive peptides capable of stimulating satiety hormones production or release (Kondrashina, Brodkorb, & Giblin, 2020; Santos-Herna´ndez, Tome´, Gaudichon, & Recio, 2018). Glycomacropeptide (GMP) and its carbohydrate-free form, caseinomacropeptide (CMP), are cleaved from parental κ-casein in the course of microbial fermentation or GI transit. In 25 healthy subjects, consumption of whey preload supplemented with GMP lowered ad libitum food intake by 10% (2877 kJ vs 3208 kJ, P , .05), compared to a whey only preload (Veldhorst et al., 2009). However, in overweight and obese subjects, ad libitum food intake and subjective appetite ratings were similar in GMP supplemented and control groups (Clifton et al., 2009; Keogh et al., 2010). Indeed, when fed to Wistar rats for 7 weeks at 200 g GMP/kg bw, there was no significant reduction in body weight (Royle, McIntosh, & Clifton, 2008). This lack of efficacy is likely due to the variation in glycosylated forms present. Both peptide and carbohydrate chains are important for exhibiting appetite-suppressing bioactivity (Madureira, Tavares, Gomes, Pintado, & Malcata, 2010), while degree and sites of CMP glycosylation significantly affect its digestibility and cleavage by brush border membrane enzymes (Boutrou, Jardin, Blais, Tome,́ & Leó nil, 2008). GMP satiety bioactivity appears to be mediated via CCK secretion. In isolated rat duodenojejunum, there was a threefold increase in CCK levels after treatment with 187.5 mg/mL GMP (Beucher, Levenez, Yvon, & Corring, 1994). β-Casomorphins (β-CMs) are a group of peptides derived from β-casein with demonstrated opioid bioactivities. As such, these peptides have appetite-suppressing capabilities via activation of opioid receptors but there is also evidence that they stimulate CCK production (Pupovac & Anderson, 2002). Mixtures of β-CMs (179 mg/kg bw), administered by oral gavage, significantly delayed gastric transit in Wistar rats resulting in T50% of 78 6 9 minutes compared to 21 6 3 minutes for a whey control group (Table 26.2) (Daniel, Vohwinkel, & Rehner, 1990). In STC-1 cells, 125
1000 μM of peptide YPFPGPI (β-CM7) stimulated up to 2.4-fold higher secretion of CCK compared to the HBSS vehicle control (P , .05) (Osborne et al., 2014). However, in broiler chickens, s.c. injection of 1.0 mg/kg bw β-CM for 7 days actually increased feed intake, fat deposition, and daily weight gain (Chang et al., 2019). Mechanistic follow-up experiments revealed substantial modulation of lipid metabolism on a transcription level, which may have outweighed appetite-suppressing properties. Other casein peptides capable of exhibiting ileum-contracting bioactivity ex vivo

646 Chapter 26 include Casoxin C (YIPIQYVLSR) and casoxin D (YVPFPPF), which have been comprehensively reviewed previously (Teschemacher, 2003).

26.3.2 Food-derived peptides targeting ghrelin, opioid receptor, and GI transit with proven in vivo efficacy Lacto-ghrestatin (LIVTQTMKG) derived from the dairy protein β-lactoglobulin was identified as the first ghrelin suppressing sequence (Table 26.2) (Aoki et al., 2017). In fasted ddY mice orally administrated at a dose 1 mg/kg bw LIVTQTMKG significantly reduced plasma ghrelin levels 1 hour later and reduced food intake over 4 hours, compared to the saline control (P , .05). Interestingly, this effect could not be replicated in nonfasted mice. In the murine gastric cell line, MGN3-1, this peptide dose dependently decreased ghrelin secretion and suppressed mRNA transcript levels of preproghrelin. Appetite lowering mechanism of the hemoglobin alpha-chain fragment, PVNFKFLSH (hemopressin), is mediated via selective binding to CB1 cannabinoid receptor to act as an antagonist (Gomes et al., 2010). Intracerebroventricular (I.c.v.) injection of PVNFKFLSH at 10 nmol dose significantly suppressed night time food intake in mice 1, 2, and 4 hour postinjection (P , .05), however cumulative food intake normalized to the levels of saline control after 12 hours (Dodd, Mancini, Lutz, & Luckman, 2010). In Sprague-Dawley rats the same dose of PVNFKFLSH suppressed cumulative food intake for only 1 hour post i.c. v. administration. PVNFKFLSH administration by I.p. (500 nmol/kg bw) in mice suppressed food intake for 2 hours, probably due to the time taken to cross the blood
brain barrier and access the CB1 cannabinoid receptor. This 2 hour suppression of food intake (P , .05 compared to saline) was also replicated in leptin-deficient obese ob/ob mice who received PVNFKFLSH by I.p. (Table 26.2). Peptides YPFVV (soymorphin-5), YPFVVN (soymorphin-6), and YPFVVNA (soymorphin-7) from soy protein β-conglycinin demonstrated opioid activity with specificity to μ-opioid receptor. These peptides were studied ex vivo in guinea pig ileum assay, where the inhibitory effect of electrically stimulated contractions was reported with IC50 of 6.0, 9.2, and 13 μM, respectively (Ohinata, Agui, & Yoshikawa, 2007). The small intestinal transit in mice was most inhibited by soymorphin-7, followed by soymorphin-5 and soymorphin-6. In 18 hour fasted mice soymorphin-5 and soymorphin-7, orally administrated at 48 μmol/kg, significantly suppressed food intake for 2 hours (Kaneko, Iwasaki, Yoshikawa, & Ohinata, 2010). Dairy whey peptides HIRL (β-lactotensin) and AFKAWAVAR (albutensin A) also delay gastric emptying (Pihlanto-Leppa¨la¨, Paakkari, Rinta-Koski, & Antila, 1997; Yoshikawa & Chiba, 1992). HIRL is known to act via neurotensin receptor (Yoshikawa, 2015), while AFKAWAVAR has demonstrated an affinity for the C3a receptor (Ohinata et al., 2002). HIRL administrated either by i.c.v. at 40 nmol/mouse, I.p. at 100 mg/kg or orally at

Application in medicine: obesity and satiety control 647 500 mg/kg, significantly suppressed food intake in fasted ddY mice compared to saline (Hou, Yoshikawa, & Ohinata, 2009) (Table 26.2). Surprisingly, the oral route was most efficient with .4 and .2-fold suppression of food intake after 20 and 60 minutes, respectively (P , .05). However, 2 hour postadministration, none of the treatments had a significant effect on food intake, compared to saline. AFKAWAVAR also delayed gastric emptying in ddY mice 2 hour post I.c.v. administration, compared to artificial cerebrospinal fluid (P , .05) (Ohinata et al., 2002). This resulted in .4-fold decrease of food intake at 20 minutes with significant differences compared to the control group observed up to 60 minutes. I.p. injection was less effective with significant differences in food intake limited to 20 minutes (P , .05) (Ohinata et al., 2002).

26.3.3 Food-derived peptides targeting lipid metabolism with proven in vivo efficacy A group of soy peptides, YVVNPDNDEN and YVVNPDNNEN from soy β-conglycinin, IAVPGEVA, IAVPTGVA, SFGVAE, LPYPR, and LPYP from glycinin, lunasin (SKWQHQQDSCRKQKQGVNLTPCEKHIMEKIQGRGDDDDDDDDD), and other soy peptides WGAPSL, VAWWMY, and FVVNATSN, were shown to suppress biosynthesis of cholesterol in vitro via inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity in the human liver hepatoma cell line, HepG2 (Chatterjee, Gleddie, & Xiao, 2018). In addition, lunasin enhanced LDL uptake by hepatic cells (Gu et al., 2017). In follow-up murine studies, lunasin (3 mg) administered by oral gavage 3 times per week for 25 weeks resulted in a significant reduction of serum triglycerides from 103 mg/L in the vehicle control group to 75 mg/L (P , .05) (Drori, Rotnemer-Golinkin, Zolotarov, & Ilan, 2017). However, this effect did not translate into reduction of body fat with 33.5% body fat in the lunasin treated group versus 37.2% in the control group. LPYPR orally administrated to mice at 50 mg/kg bw for 2 days reduced total cholesterol by 25.4% and LDL-cholesterol by 30.6% compared to saline control (Yoshikawa et al., 2000). Oral administration of VAWWMY, labeled as soystatin, at 30 mg/rat reduced serum, liver, and intestine cholesterol levels at 1 hour compared to casein hydrolyzate or soybean hydrolyzate (P , .05) (Nagaoka, Nakamura, Shibata, & Kanamaru, 2010; Yoshikawa, 2015). Its bioactivity is related to the taurocholate binding ability (25 mg/mL soystatin bound .94% taurocholate in 2 hours) with an efficacy comparable to the hypocholesterolemic drug, cholestyramine at the same dosage (Nagaoka et al., 2010). Peptide GPETAFLR from the edible seeds of the flowering plant lupine reduced body weight by 1.3-fold in an 8 week study with high-fat diet (HFD) induced obese mice (del Carmen Milla´n-Linares, Milla´n, Pedroche, & del Mar Yust, 2015), when fed at 1 mg/kg bw/day in drinking water, compared to the HFD control (P , .05) (Lemus-Conejo et al., 2020). In addition, triglyceride blood levels were reduced 2.1-fold and serum leptin levels 2.7-fold, compared to the HFD control. This led the authors to suggest that weight-lowering modulation of GPETAFLR is mediated via lipid metabolism regulation.

648 Chapter 26 When soy peptide VHVV (25 mg/kg bw/day) was I.p. administered to obese mice for 6 weeks it significantly reduced serum LDL-cholesterol, serum TG, and adipocytes size but not body weight compared to control animals (Chiang et al., 2014). In vitro experiments revealed that VHVV (4 ppm) and 2 other soybean peptides ILL and LLL significantly increased glycerol release in differentiated mouse 3T3-L1 adipocytes from 300 mmol/mg for vehicle control to 548, 579, and 687 mmol/mg protein for ILL, LLL, and VHVV, respectively (Tsou, Kao, Lu, Kao, & Chiang, 2013). Peptide VFVRN, identified in chickpea, significantly reduced body weight in obese C57BL/6J mice over a 10-week study when administered at 10 and 50 mg/kg bw. The higher dose also significantly increased HDL-cholesterol, reduced levels of serum triglycerides, total cholesterol, and LDL-cholesterol compared to control animals (Zhang, Shi, He, Cao, & Hou, 2020). In vitro VFVRN inhibited the activity of HMGR enzyme with IC50  0.3 mM, indicating suppression of lipid biosynthesis (Shi, Hou, Guo, & He, 2019). Moreover, this peptide regulates lipid metabolism as evidenced by a reduction of HMGR and Liver X receptor alpha (LXRα) mRNA transcripts by 40% in HepG2 cells when treated with 1mM VFVRN. At concentrations of 0.2
3 mM, this peptide suppressed growth of 3T3-L1 preadipocytes and at concentration of 3 mM induced apoptosis in 59% of these preadiopyctes (Shi, He, Ruge, & Tao, 2019).

26.3.4 Food-derived peptides inhibiting protease dipeptidyl peptidase-4 DPP-4 inhibitors naturally occur in food products (Nongonierma & FitzGerald, 2019) or are encrypted in food protein sequences (Nielsen, Beverly, Underwood, & Dallas, 2018; Nongonierma & FitzGerald, 2016; Tulipano, Sibilia, Caroli, & Cocchi, 2011). Food peptides that inhibit DPP-4 could prolong the circulating life of active GLP-1 and thereby suppress food intake. DPP-4 inhibitory peptides derived from meat, fish, eggs, wheat, oat, pea, bean, bovine, and camel milk have been recently reviewed (Nongonierma & FitzGerald, 2019; Zambrowicz et al., 2015), but only a limited number have been tested in vivo for reduction of food intake or body weight. Dairy peptide VAGTWY, identified in β-Lactoglobulin hydrolyzate, demonstrated DPP-4 inhibition in vitro with IC50 5 174 μM (Uchida, Ohshiba, & Mogami, 2011). Interestingly, VAGTWY also significantly reduced levels of triglycerides and free cholesterol, as well as improved ratio of HDL/LDLcholesterol over 5 days, in the liver of zebra fish after I.p. injection of 200 μg/g bw, compared to a water control (Mohammed-Geba et al., 2016). Peptide AGFAGDDAPR from black tea extract demonstrated DPP-4 inhibition in vitro of IC50 5 1.02 mM (Lu et al., 2019). Oral gavage of 400 mg of synthetized AGFAGDDAPR per day in streptozotocininduced hyperglycemic mice resulted in the significant increase of blood GLP-1 levels from 9.85 6 1.96 pmol/L to 19.22 6 6.79 pmol/L after 57 days. However, blood DPP-4 concentration was similar to the vehicle control group (P , .05). Yam dioscorin peptide

Application in medicine: obesity and satiety control 649 RRDY inhibited DPP-4 activity in vitro (IC50 5 0.93 mM) (Lin, Han, Lin, & Hou, 2016). RRDY administrated by oral gavage at 100 mg/kg dose to mice decreased blood DPP-4 levels after 30 minutes and at 1 and 2 hours (postglucose load) (P , .05). In a follow-up study administration of dioscorin (80 mg/kg bw) to HFD-induced obese C57BL/6 mice resulted in a significant weight reduction after 135 days compared to the control group (31.75 6 2.6 vs 34.97 6 2.1 g, P , .05) (Wu, Lin, Liang, & Hou, 2018).

26.3.5 In vitro evidence of food-derived peptides Several food-derived peptides have exhibited antiobesity potential in vitro but to date have not been tested in vivo (Table 26.3). Sources of these peptides include dairy, hemoglobin, soybean, hazelnut, egg, and fish. Peptide sequences and in vitro effects are summarized in Table 26.3, but without confirmed efficacy in vivo they currently have no application in foods or medicines for weight management. In addition, hydrolyzates produced from dietary proteins have exhibited antiobesity activity in vitro and in vivo (Hira et al., 2011; Kondrashina et al., 2018; Nobile et al., 2016; O’Halloran et al., 2018). However these hydrolyzates contain as yet unidentified bioactive peptides and as such are not detailed in this chapter but are reviewed elsewhere (Faridy, Stephanie, Gabriela, & Cristian, 2020; Gevaert, Veryser, Verbeke, Wynendaele, & De Spiegeleer, 2016; Gewehr et al., 2020; Kondrashina et al., 2020; Manikkam, Vasiljevic, Donkor, & Mathai, 2016).

26.3.6 Limitations: survival of food-derived peptides during gut transit Similar to synthetic peptides, food-derived peptides must contend with the proteolytic conditions of the GI tract. But unlike synthetic peptides, these peptides are not bound to albumin or FAs to resist degradation. GI proteolysis can enhance bioactivity by releasing these peptides from dietary proteins or may destroy bioactivity by hydrolyzing the peptides into free AAs. In general, plant proteins are more resistant to digestion than animal proteins in the human gut (Tsou et al., 2013). Soybean-conglycinin peptide, Soymorphin-5, but not its relatives soymorphins-6 and -7, was produced from soybean by GI proteases, pancreatic elastase, and leucine aminopeptidase (36.5% yield) (Ohinata et al., 2007). The satiety effect of soymorphins was observed only after oral administration and not I.p. injection, suggesting their resistance to further digestion and importance of direct delivery to target site (i.e., enteroendocrine cells) (Kaneko et al., 2010). Soybean peptide VHVV resisted a 4 hour digestion with pepsin, followed by a 4 hour digestion with pancreatin, as determined by maintenance of its glycerol releasing bioactivity (P..05). However, soy peptide LLL lost  20% of glycerol releasing bioactivity after pepsin digestion (Tsou et al., 2013). Unfortunately, soy peptide IAVPGEVA was digested by brush border enzymes as it traveled across differentiated Caco-2 intestinal barrier with only 0.05% appearing on the basolateral side. Instead

650 Chapter 26 Table 26.3: Food-derived peptides with antiobesity potential demonstrated in vitro. Peptide/source LLVVYPW, LVVYPW, VVYPW, VVYPWQRF, and YPWQRF from hemoglobin

ALPMH from β-LG

GPVRGPFPIIV from β-casein YPWT, LVVYPWTQRF, VVYPWTQRF, LVVYPWT, and LVVYPWTQR from bovine hemoglobin Soybean IAVPGEVA, IAVPTGVA, and LPYP

Mechanism of action

YPWQRF inhibits DPP-4 in vitro with IC50 5 117 μM. Other peptides at concentration 100 μM increase secretion of GLP-1 and CCK from STC-1 cells 1.5
2.5fold over 2 h versus vehicle control. Appetite suppressing: Stimulated 20-fold increase in stimulates CCK secretion. CCK secretion from STC-1 cells over 12 h incubation at 2 mM concentration, versus vehicle control (P , .01). Appetite suppressing: Increased GLP-1 secretion from stimulates GLP-1 secretion. GLUTag cells by 30% at 5 mM concentration versus Hepes. Opioid bioactivity, slow down Inhibited electrically stimulated gut transit. contractions in GPI bioassay. Appetite suppressing: stimulate active GLP-1 and CCK release, DPP-4 inhibitors.

Affect lipid metabolism. Hypocholesterolemic, DPP-VI inhibitors.

Soybean KA, VK, and SY

Triglyceride-lowering.

Soybean IQN

Inhibits adipogenesis.

RLLPH from hazelnut

In vitro effect

Reduces adipogenesis via AMPK pathway and slows down digestion via inhibition of pancreatic lipase.

Competitive inhibitors of HMGR in HepG2 cells with IC50 of 222, 274, and 300 μM; DPP-VI inhibitors with IC50 of 94.6, 106, and 164.3 μM. Reduced TG synthesis by 25%
50% (at 5 mg/mL) in HepG2 cells over 24 h compared to vehicle. SY as well reduced apolipoprotein B secretion by  30%. Inhibited adipogenesis in differentiated mouse 3T3-L1 adipocytes over 8 days with IC50 value of 0.014 mg protein/mL. Significantly reduced adipogenic transcription factors HMGR, PPARg, C/EBPa, aP2, SREBP-1c, FAS, and ACC1 (P , .05) in differentiated 3T3L1 adipocytes over 8 days; inhibited .40% of pancreatic lipase activity and reduced accumulation of TGs and total cholesterol by .30% at concentration 80 μM, compared to the vehicle control.

References Domenger et al. (2017)

Tulipano et al. (2011)

Wren et al. (2001)

Lafarga and Hayes (2014), Xing, Liu, Cao, Zhang, and Guanghong (2019) Aiello et al. (2018), Chatterjee et al. (2018)

Inoue et al. (2011)

Kim, Bae, Ahn Lee, and Lee (2007)

Wang, Zhou, et al. (2020)

(Continued)

Application in medicine: obesity and satiety control 651 Table 26.3: (Continued) Peptide/source

Mechanism of action

In vitro effect

References

Upregulated PPARγ expression Jahandideh, Liu, 1.5
2-fold (P , .05) at 100 μM and Wu (2018) in 3T3-F442A preadipocytes over 72 h versus vehicle. FLV from soy Improves adipose tissue Reduced levels of inflammation Kwak et al. (2016) functionality. biomarkers (TNFa, MCP-1, and IL-6) in 3T3-L1 adipocytes and improved insulin sensitivity. Treatment with 500 and DIVDKIEI from Downregulation of Kim, Kim, Kim, and 1000 ng/mL (48 h) reduced TG Nam (2015), Kim, desalinated boiled tuna adipogenesis transcription accumulation in differentiated Kim, Choi, Lee, and factors via activation of the 3T3-L1 cells by 35% and 57%, Wnt/β-catenin signaling Nam (2015) respectively, and reduced pathway. glucose uptake by 33% and 63%, respectively, compared with MDI-treated group. AP, VAP, and AKK (100 M) VW, VY, KY, KW, IY, AP, Downregulated the expression Henda et al. (2015) reduced ell viability of HWP of adipocyte markers and VIY, LKP, GPL, AKK, and cells [46.4% 6 3.9%, 59.5% 6 adipocyte-specific VAP from various marine 7.3% and 24.5% 6 5.3%, transcription factors. fish respectively (24 h treatment)] and [55.8% 6 4.5%, 81.1% 6 4.3% and 22.8% 6 4.3%, respectively (72 h treatment)] compared with nontreated cells. GPL and IY (100 M) reduced final lipid content in differentiated HWPs [13.5% 6 3.6% and 8.3% 6 0.3%, respectively (8 days)]. VIAPW and IRWWW (100 μM) VIAPW and IRWWW Downregulated expression Wang, Pan, et al. reduced TG levels from from Miiuy croaker levels of lipogenesis genes (2020) 0.247 6 0.004 mM (OA model and upregulated the control) to 0.201 6 0.006 and expression levels of lipolysis 0.186 6 0.005 mm, respectively genes. and TC levels from 53.45 6 0.10 μg/mg protein (OA model group) to 45.88 6 0.74 μg/mg protein and 41.02 6 0.14 μg/mg protein, respectively, in OAchallenged HepG2 cells QAMPFRVTEQE and VFKGL from egg ovalbumin

Improves adipogenic differentiation.

CCK, Cholecystokinin; DPP, dipeptidyl peptidase; GLP-1, glucagon-like peptide-1; GPI, guinea pig ileum; OA, oleic acid.

breakdown products were observed in both the apical (13.5%) and basolateral (17.5%) compartments (Aiello et al., 2018). Interestingly Cruz-Huerta et al. (2015) observed that the soy peptide lunasin when consumed as a food was protected from trypsin and chymotrypsin degradation by the action of Bowman
Birk protease inhibitors, naturally found in soy

652 Chapter 26 (Cruz-Huerta et al., 2015). A 5 hour simulated GI digestion of rice glutelin (DEHQKIHRFRQGDV) produced bioactive peptides RF and IHRF at 6.1 and 2.3 mol%, respectively (Kontani et al., 2014). While hazelnut peptide RLLPH was sensitive to intestinal rather than gastric digestion, with degradation of 31.67% 6 2.48% observed after 3 hours (Wang, Zhou, et al., 2020). Bioactive peptides from animal food sources can also be efficiently released during gut transit but they are more easily degraded. The GLP-1 secretagogue peptide GPVRGPFPIIV from β-casein appears during gastric (simulated and in vivo) digestion of skim milk powder. However, it is rapidly degraded in the duodenal phase (Egger et al., 2017). In contrast, Cattaneo, Stuknyt˙e, Masotti, and De Noni (2017) reported that β-CM7 (YPFPGPI) was released from β-casein during simulated GI digestion of infant formulas and appeared resistant to further gastric and intestinal digestion (Cattaneo et al., 2017). Similar to synthetic peptides, food-derived peptides may require encapsulation if delivered orally (Mohan, Rajendran, He, Bazinet, & Udenigwe, 2015). In addition, food matric and processing method will no doubt alter bioactivity.

26.4 Commercial dietary protein hydrolyzates with antiobesity potential Dietary protein hydrolyzates offer a commercially viable alternative to purified peptides, for food companies to compete in the weight management market. Several commercial products containing food-derived antiobesity peptides and hydrolyzates are already available (Table 26.4). These hydrolyzates are usually produced by microbial fermentation or food-grade proteolytic enzymes, such as alcalase, neutrase, flavourzyme, or protamex. Industrial purification is performed by ultrafiltration, liquid chromatography, or membrane separation systems, including microfiltration, gel filtration, and reverse osmosis filtration (Shimizu & Hettiarachchy, 2012). If required, bioactive peptides from the hydrolyzed fraction can then be identified by chromatography and mass spectrometry (Kagawa, Matsutaka, Fukuhama, Watanabe, & Fujino, 1996). LunaRich X is a purified and encapsulated form of soy peptide lunasin (Lule, Garg, Pophaly, & Tomar, 2015). This dietary supplement claims to lower cholesterol levels and attenuate lipid metabolism, supporting weight loss when taken regularly up to 5 capsules/ day (Galvez, Matel, Ivey, & Bowles, 2013). When administrated to obese LDL-receptor mutant pigs, it resulted in a 74% reduction of free FAs and a 52% increase in circulating levels of the energy balance regulator, leptin (Galvez et al., 2013). This led to a significantly slower weight gain compared to the control group on a standard diet. The cholesterol-lowering fraction of soy protein hydrolyzate CSPHP (C-fraction soy protein hydrolyzate with bound phospholipids) is a GRAS dietary supplement, marketed as SuperSoy, and is suitable for adults with slightly elevated serum cholesterol levels (265 6 7.7 mg/dL) (Shimizu & Hettiarachchy, 2012). A potato protein dietary supplement

Table 26.4: Commercial proteins/hydrolyzates/extracts/peptides with antiobesity claims. Product LunaRich X

Source

Claimed application

Soy

Reduced cholesterol levels and lipid metabolism, thereby supporting weight loss Cholesterol-lowering effect in persons with a slightly elevated serum cholesterol level Enhances the release of CCK, prolonging feeling of fullness, resulting in weight loss

SuperSoy

Soy protein

Slendesta

Potato

Ovamine

Egg

Napple drink

Slimpro

Peptide N

Red blood cells of bovine or swine Blue whiting

Fish

Suppresses appetite by inhibiting ghrelin and secreting leptin, resulting in weight loss Reduce postprandial serum triglyceride levels of those whose diets are rich in fat Increases secretion of satiety hormones (CCK and GLP-1), reduces food intake and weight gain

Promotes production of satiety hormones, thereby reducing appetite

Type of fraction Lunasinenriched soy extract CSPHP

Regulation

GRAS

Potato protein extract containing PI2 Purified ovalbumin

GRAS

Peptide (VVYP)

FOSHU

Whole hydrolyzate

Whole Regulation hydrolyzate (EEC) No. 2092/ 91 and DIN EN ISO.1400

Form according to supplier

Manufacturer

Reference

Dietary supplement, 5 capsules/day Dietary supplement, 3 g/ day for 3 months

Reliv International (United States) Kyowa Hakko (United States)

Reliv.com. 2020 Kyowa-usa. com. 2020

Dietary ingredient, 1 capsule (15 mg) taken 1 h before meal

Kemin (Portugal)

Kemin.com. 2020

Dietary supplement, taken 2
3 times a day for 1
3 months Dietary supplement, 50 mL serving size with meals Dietary supplement taken before meals

Nutreven laboratories (Paris)

Nutreven. com. 2020

MG Pharma Inc (Japan)

Mgpharma. co.jp. 2020

Compagnie des peĉ hes Saint-Malo Santé (France) Nutraceuticals International Group (United States) Celergen (Switzerland)

SlimPro.eu. 2020

Dietary supplement

Swisscelergen. com. 2020

(Continued)

Table 26.4: (Continued) Product Nutripeptin

Naticol

Source

Claimed application

Cod

Reduces glycemic index in foods and reduces fat deposition Reduced weight gain and fat mass Reduced plasma glucose and plasma total cholesterol

Fish collagen

Type of fraction

Regulation

Whole hydrolyzate Peptides

Form according to supplier Food ingredient

ISO 9001 and FSSC 22000 standards

Food ingredient

Manufacturer

Reference

NutriMarine Life Science AS (Norway) Weishardt International (France)

Copalis.fr. 2020 Weishardt. com. 2020

CCK, Cholecystokinin; CSPHP, C-fraction soy protein hydrolyzate with bound phospholipids; FOSHU, Foods for Specified Health Uses; GLP-1, glucagon-like peptide-1; GRAS, granted generally recognized as safe.

Application in medicine: obesity and satiety control 655 Slendesta, which contains Proteinase Inhibitor II, enhances the release of CCK (Ku et al., 2016). It is marketed as (1) boosting satiety by helping to feel full sooner and longer; (2) controlling hunger and suppressing appetite; and (3) inducing weight loss. A purified egg protein Ovamine is suggested to induce immediate satiety by suppressing ghrelin and increasing leptin secretion and serotonin signaling. The health claim states increased satiety and decreased food intake if consumed 2
3 times a day. The hemoglobin peptide (VVYP) is derived from bovine or swine red blood cells by hydrolysis with an Aspergillus niger acidic protease (Kagawa et al., 1996). It has been added as an ingredient to a soft drink and has received approval from the Japanese government (FOSHU) to reduce postprandial serum triglyceride levels. Several marine-derived peptide mixtures and hydrolyzates are sold in Europe and North America as food supplements without approved health claims including Slimpro, Peptide N, Nutripeptin, and Naticol. Slimpro is a branch chain AAenriched hydrolyzate from blue whiting fish produced by enzymatic hydrolysis (Nobile et al., 2016). The product is suggested to limit calorie intake and control body weight, as clinical studies have demonstrated its ability to increase CCK and GLP-1 secretion while reducing food intake and weight gain without side effects. Celergen claims that Peptide N, a marine protein hydrolyzate, has a potential to reduce dietary glycemic index and prevent body fat accumulation in healthy adults. Peptide N is a component of Hydro MN Peptide, a marine cartilage extract composed of collagen hydrolyzates, glycosaminoglycan, and chondroitin sulfate. Nutripeptin, a codfish protein hydrolyzate, is reported to reduce glycemic index of food, in turn reducing fat deposition, thereby acting as a potential weight management solution (Cheung, Ng, & Wong, 2015). Naticol contains collagen peptides (including di- and tripeptides) and is sold as an effective weight management solution via fat mass reduction (Bonnet). Protein hydrolyzates for weight management face many challenges not least of which is the absolute requirement for scientifically substantiated claims. In addition, there are large-scale production issues, compatibility with various food matrices, bitterness, shelf life, GI stability, and bioavailability concerns awaiting resolution (Harnedy & FitzGerald, 2012).

26.5 Summary In summary, synthetic antiobesity peptides predominantly rely on mimicking endogenous satiety hormones with the additional attributes of extended circulation times and proteaseresistant abilities. Many of these drugs are well tolerated and effective in reducing weight gain for the obese individual. On the other hand, bioactive peptides from food may never be employed to treat obesity but may instead help to manage weight gain over time for the healthy weight individual. There is still a large pool of identified peptides from various food sources with antiobesity potential awaiting confirmation in vivo and even more unknown but potentially efficient peptides in protein hydrolyzates awaiting discovery.

656 Chapter 26

Acknowledgments LG receives financial support from the Science Foundation Ireland (SFI) and the Department of Agriculture, Food and the Marine on behalf of the Government of Ireland under Grant Number 16/RC/3835. SH and NOB received funding from the Department of Agriculture, Food and the Marine (FIRM project 17 F 260).

References Adan, R. A. (2013). Mechanisms underlying current and future anti-obesity drugs. Trends in Neurosciences, 36 (2), 133
140. Aiello, G., Ferruzza, S., Ranaldi, G., Sambuy, Y., Arnoldi, A., Vistoli, G., & Lammi, C. (2018). Behavior of three hypocholesterolemic peptides from soy protein in an intestinal model based on differentiated Caco-2 cell. Journal of Functional Foods, 45, 363
370. Ambery, P., Parker, V. E., Stumvoll, M., Posch, M. G., Heise, T., Plum-Moerschel, L., & Petrone, M. (2018). MEDI0382, a GLP-1 and glucagon receptor dual agonist, in obese or overweight patients with type 2 diabetes: A randomised, controlled, double-blind, ascending dose and phase 2a study. The Lancet, 391 (10140), 2607
2618. Aoki, H., Nakato, J., Mizushige, T., Iwakura, H., Sato, M., Suzuki, H., & Ohinata, K. (2017). Lacto-ghrestatin, a novel bovine milk-derived peptide, suppresses ghrelin secretion. FEBS Letters, 591(14), 2121
2130. Bækdal, T. A., Thomsen, M., Kupˇcova´, V., Hansen, C. W., & Anderson, T. W. (2018). Pharmacokinetics, safety, and tolerability of oral semaglutide in subjects with hepatic impairment. The Journal of Clinical Pharmacology, 58(10), 1314
1323. Baggio, L. L., & Drucker, D. J. (2007). Biology of incretins: GLP-1 and GIP. Gastroenterology, 132(6), 2131
2157. Barnhart, K. F., Christianson, D. R., Hanley, P. W., Driessen, W. H., Bernacky, B. J., Baze, W. B., & Kolonin, M. G. (2011). A peptidomimetic targeting white fat causes weight loss and improved insulin resistance in obese monkeys. Science Translational Medicine, 3(108), 108ra112. Barrington, P., Chien, J., Tibaldi, F., Showalter, H., Schneck, K., & Ellis, B. (2011). LY2189265, a long-acting glucagon-like peptide-1 analogue, showed a dose-dependent effect on insulin secretion in healthy subjects. Diabetes, Obesity and Metabolism, 13(5), 434
438. Beucher, S., Levenez, F., Yvon, M., & Corring, T. (1994). Effects of gastric digestive products from casein on CCK release by intestinal cells in rat. The Journal of Nutritional Biochemistry, 5(12), 578
584. Birch, L. L., & Ventura, A. K. (2009). Preventing childhood obesity: What works? International Journal of Obesity, 33(1), S74
S81. Blundell, J., Finlayson, G., Axelsen, M., Flint, A., Gibbons, C., Kvist, T., & Hjerpsted, J. B. (2017). Effects of once-weekly semaglutide on appetite, energy intake, control of eating, food preference and body weight in subjects with obesity. Diabetes, Obesity and Metabolism, 19(9), 1242
1251. Bolli, G., & Owens, D. (2014). Lixisenatide, a novel GLP-1 receptor agonist: Efficacy, safety and clinical implications for type 2 diabetes mellitus. Diabetes, Obesity and Metabolism, 16(7), 588
601. Bonnet, C. (2015). Innovative fish collagen peptide products–directed to functional food and beverage applications, cosmetofoods and nutraceutical products. Wellness Foods and Supplements, 3, 4
6. Boutrou, R., Jardin, J., Blais, A., Tome,́ D., & Leó nil, J. l (2008). Glycosylations of κ-casein-derived caseinomacropeptide reduce its accessibility to endo-but not exointestinal brush border membrane peptidases. Journal of Agricultural and Food Chemistry, 56(17), 8166
8173. Bray, G. A., & Ryan, D. H. (2014). Update on obesity pharmacotherapy. Annals of the New York Academy of Sciences, 1311(1), 1
13. Buchwald, H., Avidor, Y., Braunwald, E., Jensen, M. D., Pories, W., Fahrbach, K., & Schoelles, K. (2004). Bariatric surgery: A systematic review and meta-analysis. The Journal of the American Medical Association, 292(14), 1724
1737.

Application in medicine: obesity and satiety control 657 Buse, J. B., Klonoff, D. C., Nielsen, L. L., Guan, X., Bowlus, C. L., Holcombe, J. H., & Wintle, M. E. (2007). Metabolic effects of two years of exenatide treatment on diabetes, obesity, and hepatic biomarkers in patients with type 2 diabetes: An interim analysis of data from the open-label, uncontrolled extension of three double-blind, placebo-controlled trials. Clinical Therapeutics, 29(1), 139
153. Cai, Q., Chen, F., Wang, T., Luo, F., Liu, X., Wu, Q., & Liu, L. (2020). Obesity and COVID-19 severity in a designated hospital in Shenzhen, China. Diabetes Care, 43. Cattaneo, S., Stuknyt˙e, M., Masotti, F., & De Noni, I. (2017). Protein breakdown and release of β-casomorphins during in vitro gastro-intestinal digestion of sterilised model systems of liquid infant formula. Food Chemistry, 217, 476
482. Chang, W., Zheng, A., Chen, Z., Zhang, S., Cai, H., & Liu, G. (2019). β-Casomorphin increases fat deposition in broiler chickens by modulating expression of lipid metabolism genes. Animal, 13(4), 777
783. Chatterjee, C., Gleddie, S., & Xiao, C.-W. (2018). Soybean bioactive peptides and their functional properties. Nutrients, 10(9), 1211. Chen, W., Hira, T., Nakajima, S., Tomozawa, H., Tsubata, M., Yamaguchi, K., & Hara, H. (2012). Suppressive effect on food intake of a potato extract (Potein®) involving cholecystokinin release in rats. Bioscience, Biotechnology, and Biochemistry, 76(6), 1104
1109. Cheung, R. C. F., Ng, T. B., & Wong, J. H. (2015). Marine peptides: Bioactivities and applications. Marine Drugs, 13(7), 4006
4043. Chiang, W.-D., Shibu, M. A., Lee, K.-I., Wu, J.-P., Tsai, F.-J., Pan, L.-F., & Lin, W.-T. (2014). Lipolysisstimulating peptide-VHVV ameliorates high fat diet induced hepatocyte apoptosis and fibrosis. Journal of Functional Foods, 11, 482
492. Choi, I. Y., Moon, M. J., Trautmann, M. E., Hompesch, M., & Sorli, C. H. (2018). In vitro studies to evaluate the receptor kinetics of efpeglenatide vs. other glucagon-like peptide-1 receptor (GLP-1R) agonists. American Diabetes Association. Choi, J. D., Baek, S., Kim, Y., Eun, K., Kwon, S. C., Morrow, L., & Kang, J. (2019). 982-P: A double-blinded, placebo controlled, single ascending dose study for safety, tolerability, pharmacokinetics, and pharmacodynamics after subcutaneous administration of novel long-acting GLP-1/GIP/glucagon triple agonist (HM15211) in healthy obese subjects. American Diabetes Association. Christou, G. A., Katsiki, N., Blundell, J., Fruhbeck, G., & Kiortsis, D. N. (2019). Semaglutide as a promising antiobesity drug. Obesity Reviews, 20(6), 805
815. Clifton, P., Keogh, J., Woonton, B., Taylor, C., Janakievski, F., & De Silva, K. (2009). Effect of glycomacropeptides (GMP) on satiety hormones and food intake. Australian Journal of Dairy Technology, 64(1), 29. Colagiuri, S. (2010). Diabesity: Therapeutic options. Diabetes, Obesity and Metabolism, 12(6), 463
473. Collaboration, N. R. F. (2019). Rising rural body-mass index is the main driver of the global obesity epidemic in adults. Nature, 569(7755), 260. Cristina Oliveira de Lima, V., Piuvezam, G., Leal Lima Maciel, B., & Heloneida de Arau´jo Morais, A. (2019). Trypsin inhibitors: Promising candidate satietogenic proteins as complementary treatment for obesity and metabolic disorders? Journal of Enzyme Inhibition and Medicinal Chemistry, 34(1), 405
419. Cruz-Huerta, E., Ferna´ ndez-Tome´ , S., Arques, M. C., Amigo, L., Recio, I., Clemente, A., & Herna´ ndezLedesma, B. (2015). The protective role of the Bowman-Birk protease inhibitor in soybean lunasin digestion: The effect of released peptides on colon cancer growth. Food & Function, 6(8), 2626
2635. Cvetkovi´c, R. S., & Plosker, G. L. (2007). Exenatide. Drugs, 67(6), 935
954. Daniel, H., Vohwinkel, M., & Rehner, G. (1990). Effect of casein and β-casomorphins on gastrointestinal motility in rats. The Journal of Nutrition, 120(3), 252
257. Davies, M., Pieber, T. R., Hartoft-Nielsen, M.-L., Hansen, O. K., Jabbour, S., & Rosenstock, J. (2017). Effect of oral semaglutide compared with placebo and subcutaneous semaglutide on glycemic control in patients with type 2 diabetes: A randomized clinical trial. The Journal of the American Medical Association, 318 (15), 1460
1470.

658 Chapter 26 Davies, M., Thiman, M., & Kugler, A. (2016). Efpeglenatide: A once monthly GLP-1 RA in the pipeline. Austin Journal of Endocrinology and Diabetes, 3(4), 1053. del Carmen Milla´n-Linares, M., Milla´n, F., Pedroche, J., & del Mar Yust, M. (2015). GPETAFLR: A new antiinflammatory peptide from Lupinus angustifolius L. protein hydrolysate. Journal of Functional Foods, 18, 358
367. Del Prato, S., Kang, J., Trautmann, M. E., Stewart, J., Sorli, C. H., Derwahl, M., & Yoon, K. H. (2020). Efficacy and safety of once-monthly efpeglenatide in patients with type 2 diabetes: Results of a phase 2 placebo-controlled, 16-week randomized dose-finding study. Diabetes, Obesity and Metabolism, 22. Dias, S., Paredes, S., & Ribeiro, L. (2018). Drugs involved in dyslipidemia and obesity treatment: Focus on adipose tissue. International Journal of Endocrinology, 2018. Dicembrini, I., Bigiarini, M., & Mannucci, E. (2014). Lixisenatide: Clinical profile and available evidence. Diabetes Management, 4(1), 71. Dodd, G. T., Mancini, G., Lutz, B., & Luckman, S. M. (2010). The peptide hemopressin acts through CB1 cannabinoid receptors to reduce food intake in rats and mice. Journal of Neuroscience, 30(21), 7369
7376. Domenger, D., Caron, J., Belguesmia, Y., Lesage, J., Dhulster, P., Ravallec, R., & Cudennec, B. (2017). Bioactivities of hemorphins released from bovine haemoglobin gastrointestinal digestion: Dual effects on intestinal hormones and DPP-IV regulations. Journal of Functional Foods, 36, 9
17. Drori, A., Rotnemer-Golinkin, D., Zolotarov, L., & Ilan, Y. (2017). Oral administration of CardioAid and lunasin alleviates liver damage in a high-fat diet nonalcoholic steatohepatitis model. Digestion, 96(2), 110
118. Drucker, D. J., Buse, J. B., Taylor, K., Kendall, D. M., Trautmann, M., Zhuang, D., & Group, D.-S. (2008). Exenatide once weekly versus twice daily for the treatment of type 2 diabetes: A randomised, open-label, non-inferiority study. The Lancet, 372(9645), 1240
1250. Drucker, D. J., Dritselis, A., & Kirkpatrick, P. (2010). Liraglutide. Nature Publishing Group. Egger, L., Schlegel, P., Baumann, C., Stoffers, H., Guggisberg, D., Bru¨gger, C., & Portmann, R. (2017). Physiological comparability of the harmonized INFOGEST in vitro digestion method to in vivo pig digestion. Food Research International, 102, 567
574. Faridy, J.-C. M., Stephanie, C.-G. M., Gabriela, M.-M. O., & Cristian, J.-M. (2020). Biological activities of chickpea in human health (Cicer arietinum L.). A review. Plant Foods for Human Nutrition, 1
12. Frias, J. P., Nauck, M. A., Van, J., Kutner, M. E., Cui, X., Benson, C., & Robins, D. (2018). Efficacy and safety of LY3298176, a novel dual GIP and GLP-1 receptor agonist, in patients with type 2 diabetes: A randomised, placebo-controlled and active comparator-controlled phase 2 trial. The Lancet, 392(10160), 2180
2193. Gabery, S., Salinas, C. G., Paulsen, S. J., Ahnfelt-Rønne, J., Alanentalo, T., Baquero, A. F., & Frederiksen, K. S. (2020). Semaglutide lowers body weight in rodents via distributed neural pathways. JCI Insight, 5(6). Galvez, A. F., Matel, H., Ivey, J., & Bowles, D. (2013). Lunasin-enriched soy extract (LunaRich Xt), in combination with the dietary supplement reliv now, reduces free fatty acid by increasing plasma leptin and adiponectin levels in LDL-receptor mutant pigs. wealththrunutrition.com, 1, 1
14. Gevaert, B., Veryser, L., Verbeke, F., Wynendaele, E., & De Spiegeleer, B. (2016). Fish hydrolysates: A regulatory perspective of bioactive peptides. Protein and Peptide Letters, 23(12), 1052
1060. Gewehr, M. C., Silverio, R., Rosa-Neto, J. C., Lira, F. S., Reckziegel, P., & Ferro, E. S. (2020). Peptides from natural or rationally designed sources can be used in overweight, obesity, and type 2 diabetes therapies. Molecules, 25(5), 1093. Goldenberg, R. M., & Steen, O. (2019). Semaglutide: Review and place in therapy for adults with type 2 diabetes. Canadian Journal of Diabetes, 43(2), 136
145. Gomes, I., Dale, C. S., Casten, K., Geigner, M. A., Gozzo, F. C., Ferro, E. S., & Devi, L. A. (2010). Hemoglobinderived peptides as novel type of bioactive signaling molecules. The AAPS Journal, 12(4), 658
669. Granhall, C., Søndergaard, F. L., Thomsen, M., & Anderson, T. W. (2018). Pharmacokinetics, safety and tolerability of oral semaglutide in subjects with renal impairment. Clinical Pharmacokinetics, 57(12), 1571
1580.

Application in medicine: obesity and satiety control 659 Gu, L., Gong, Y., Zhao, C., Wang, Y., Tian, Q., Lei, G., & Tan, S. (2019). Lunasin improves the LDL-C lowering efficacy of simvastatin via inhibiting PCSK9 expression in hepatocytes and ApoE 2 / 2 mice. Molecules, 24(22), 4140. Gu, L., Wang, Y., Xu, Y., Tian, Q., Lei, G., Zhao, C., & Nong, L. (2017). Lunasin functionally enhances LDL uptake via inhibiting PCSK9 and enhancing LDLR expression in vitro and in vivo. Oncotarget, 8(46), 80826. Gupta, V. (2013). Glucagon-like peptide-1 analogues: An overview. Indian Journal of Endocrinology and Metabolism, 17(3), 413. Habegger, K. M., Heppner, K. M., Geary, N., Bartness, T. J., DiMarchi, R., & Tscho¨p, M. H. (2010). The metabolic actions of glucagon revisited. Nature Reviews Endocrinology, 6(12), 689. Harnedy, P. A., & FitzGerald, R. J. (2012). Bioactive peptides from marine processing waste and shellfish: A review. Journal of Functional Foods, 4(1), 6
24. Hasib, A. (2020). Multiagonist unimolecular peptides for obesity and type 2 diabetes: Current advances and future directions. Clinical Medicine Insights: Endocrinology and Diabetes, 13 , 1179551420905844. Hedrington, M. S., & Davis, S. N. (2019). Oral semaglutide for the treatment of type 2 diabetes. Expert Opinion on Pharmacotherapy, 20(2), 133
141. Hellstro¨m, P. M. (2013). Satiety signals and obesity. Current Opinion in Gastroenterology, 29(2), 222
227. Henda, Y. B., Laamari, M., Lanneluc, I., Travers, M.-A., Agogue´, H., Arnaudin, I., & Sannier, F. (2015). Di and tripeptides from marine sources can target adipogenic process and contribute to decrease adipocyte number and functions. Journal of Functional Foods, 17, 1
10. Heymsfield, S. B., & Wadden, T. A. (2017). Mechanisms, pathophysiology, and management of obesity. New England Journal of Medicine, 376(3), 254
266. Hill, A. J., Peikin, S. R., Ryan, C. A., & Blundell, J. E. (1990). Oral administration of proteinase inhibitor II from potatoes reduces energy intake in man. Physiology & Behavior, 48(2), 241
246. Hira, T., Mori, N., Nakamori, T., Furuta, H., Asano, K., Chiba, H., & Hara, H. (2011). Acute effect of soybean beta-conglycinin hydrolysate ingestion on appetite sensations in healthy humans. Appetite, 57(3), 765
768. Hjerpsted, J. B., Flint, A., Brooks, A., Axelsen, M. B., Kvist, T., & Blundell, J. (2018). Semaglutide improves postprandial glucose and lipid metabolism, and delays first-hour gastric emptying in subjects with obesity. Diabetes, Obesity and Metabolism, 20(3), 610
619. Hornigold, D. C., Roth, E., Howard, V., Will, S., Oldham, S., Coghlan, M. P., & Trevaskis, J. L. (2018). A GLP-1: CCK fusion peptide harnesses the synergistic effects on metabolism of CCK-1 and GLP-1 receptor agonism in mice. Appetite, 127, 334
340. Hou, I.-C., Yoshikawa, M., & Ohinata, K. (2009). β-Lactotensin derived from bovine β-lactoglobulin suppresses food intake via the CRF system followed by the CGRP system in mice. Peptides, 30(12), 2228
2232. Inoue, N., Nagao, K., Sakata, K., Yamano, N., Gunawardena, P. E. R., Han, S.-Y., & Takamatsu, K. (2011). Screening of soy protein-derived hypotriglyceridemic di-peptides in vitro and in vivo. Lipids in Health and Disease, 10(1), 1
10. Jahandideh, F., Liu, P., & Wu, J. (2018). Purification and identification of adipogenic-differentiating peptides from egg white hydrolysate. Food Chemistry, 259, 25
30. Jendle, J., Grunberger, G., Blevins, T., Giorgino, F., Hietpas, R. T., & Botros, F. T. (2016). Efficacy and safety of dulaglutide in the treatment of type 2 diabetes: A comprehensive review of the dulaglutide clinical data focusing on the AWARD phase 3 clinical trial program. Diabetes/Metabolism Research and Reviews, 32 (8), 776
790. Jones, B., Buenaventura, T., Kanda, N., Chabosseau, P., Owen, B. M., Scott, R., & Bosco, D. (2018). Targeting GLP-1 receptor trafficking to improve agonist efficacy. Nature Communications, 9(1), 1
17. Jose, B., Tahrani, A. A., Piya, M. K., & Barnett, A. H. (2010). Exenatide once weekly: Clinical outcomes and patient satisfaction. Patient Preference and Adherence, 4, 313. Kagawa, K., Matsutaka, H., Fukuhama, C., Watanabe, Y., & Fujino, H. (1996). Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect. Life Sciences, 58(20), 1745
1755.

660 Chapter 26 Kagebayashi, T., Kontani, N., Yamada, Y., Mizushige, T., Arai, T., Kino, K., & Ohinata, K. (2012). Novel CCK-dependent vasorelaxing dipeptide, A rg-P he, decreases blood pressure and food intake in rodents. Molecular Nutrition & Food Research, 56(9), 1456
1463. Kaneko, K., Iwasaki, M., Yoshikawa, M., & Ohinata, K. (2010). Orally administered soymorphins, soy-derived opioid peptides, suppress feeding and intestinal transit via gut μ1-receptor coupled to 5-HT1A, D2, and GABAB systems. American Journal of Physiology-Gastrointestinal and Liver Physiology, 299(3), G799
G805. Keogh, J. B., Woonton, B. W., Taylor, C. M., Janakievski, F., Desilva, K., & Clifton, P. M. (2010). Effect of glycomacropeptide fractions on cholecystokinin and food intake. British Journal of Nutrition, 104(2), 286
290. Kim, H. J., Bae, I. Y., Ahn, C.-W., Lee, S., & Lee, H. G. (2007). Purification and identification of adipogenesis inhibitory peptide from black soybean protein hydrolysate. Peptides, 28(11), 2098
2103. Kim, M.-H., Park, S.-C., Kim, J.-Y., Lee, S. Y., Lim, H.-T., Cheong, H., & Park, Y. (2006). Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety “Golden Valley”. Biochemical and Biophysical Research Communications, 346(3), 681
686. Kim, Y.-M., Kim, E.-Y., Kim, I.-H., & Nam, T.-J. (2015). Peptide derived from desalinated boiled tuna extract inhibits adipogenesis through the downregulation of C/EBP-α and PPAR-γ in 3T3-L1 adipocytes. International Journal of Molecular Medicine, 35(5), 1362
1368. Kim, Y. M., Kim, I. H., Choi, J. W., Lee, M. K., & Nam, T. J. (2015). The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/β-catenin signaling pathway. International Journal of Molecular Medicine, 36(2), 327
334. Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., & Arap, W. (2004). Reversal of obesity by targeted ablation of adipose tissue. Nature Medicine, 10(6), 625
632. Kondrashina, A., Brodkorb, A., & Giblin, L. (2020). Dairy-derived peptides for satiety. Journal of Functional Foods, 66, 103801. Kondrashina, A., Seratlic, S., Kandil, D., Treguier, N., Kilcawley, K., Schellekens, H., & Giblin, L. (2018). Irish cheddar cheese increases glucagon-like peptide-1 secretion in vitro but bioactivity is lost during gut transit. Food Chemistry, 265, 9
17. Kontani, N., Omae, R., Kagebayashi, T., Kaneko, K., Yamada, Y., Mizushige, T., & Ohinata, K. (2014). Characterization of I le-H is-A rg-P he, a novel rice-derived vasorelaxing peptide with hypotensive and anorexigenic activities. Molecular Nutrition & Food Research, 58(2), 359
364. Ku, S. K., Sung, S. H., Choung, J. J., Choi, J. S., Shin, Y. K., & Kim, J. W. (2016). Anti-obesity and antidiabetic effects of a standardized potato extract in ob/ob mice. Experimental and Therapeutic Medicine, 12(1), 354
364. Kumar, M. S. (2019). Peptides and peptidomimetics as potential antiobesity agents: Overview of current status. Frontiers in Nutrition, 6, 11. Kwak, S.-J., Kim, C.-S., Choi, M.-S., Park, T., Sung, M.-K., Yun, J. W., & Yu, R. (2016). The soy peptide Phe
Leu
Val reduces TNFα-induced inflammatory response and insulin resistance in adipocytes. Journal of Medicinal Food, 19(7), 678
685. Lafarga, T., & Hayes, M. (2014). Bioactive peptides from meat muscle and by-products: Generation, functionality and application as functional ingredients. Meat Science, 98(2), 227
239. Lau, J., Bloch, P., Schaf̈ fer, L., Pettersson, I., Spetzler, J., Kofoed, J., & Steensgaard, D. B. (2015). Discovery of the once-weekly glucagon-like peptide-1 (GLP-1) analogue semaglutide. Journal of Medicinal Chemistry, 58(18), 7370
7380. Le Neve´, B., & Daniel, H. (2011). Selected tetrapeptides lead to a GLP-1 release from the human enteroendocrine cell line NCI-H716. Regulatory Peptides, 167(1), 14
20. Lemus-Conejo, A., Grao-Cruces, E., Toscano, R., Varela, L. M., Claro, C., Pedroche, J., & Montserrat-de la Paz, S. (2020). A lupine (Lupinus angustifolious L.) peptide prevents non-alcoholic fatty liver disease in high-fat-diet-induced obese mice. Food & Function, 11(4), 2943
2952.

Application in medicine: obesity and satiety control 661 Lim, J. J., & Poppitt, S. D. (2019). How satiating are the ‘satiety’ peptides: A problem of pharmacology versus physiology in the development of novel foods for regulation of food intake. Nutrients, 11(7), 1517. Lin, Y.-S., Han, C.-H., Lin, S.-Y., & Hou, W.-C. (2016). Synthesized peptides from yam dioscorin hydrolysis in silico exhibit dipeptidyl peptidase-IV inhibitory activities and oral glucose tolerance improvements in normal mice. Journal of Agricultural and Food Chemistry, 64(33), 6451
6458. Lovshin, J. A., & Drucker, D. J. (2009). Incretin-based therapies for type 2 diabetes mellitus. Nature Reviews Endocrinology, 5(5), 262. Lu, Y., Lu, P., Wang, Y., Fang, X., Wu, J., & Wang, X. (2019). A novel dipeptidyl peptidase IV inhibitory tea peptide improves pancreatic β-cell function and reduces α-cell proliferation in streptozotocin-induced diabetic mice. International Journal of Molecular Sciences, 20(2), 322. Lule, V. K., Garg, S., Pophaly, S. D., & Tomar, S. K. (2015). Potential health benefits of lunasin: A multifaceted soy-derived bioactive peptide. Journal of Food Science, 80(3), R485
R494. Madsbad, S. (2016). Review of head-to-head comparisons of glucagon-like peptide-1 receptor agonists. Diabetes, Obesity and Metabolism, 18(4), 317
332. Madureira, A., Tavares, T., Gomes, A. M., Pintado, M., & Malcata, F. X. (2010). Invited review: Physiological properties of bioactive peptides obtained from whey proteins. Journal of Dairy Science, 93(2), 437
455. Malm-Erjefa¨lt, M., Bjørnsdottir, I., Vanggaard, J., Helleberg, H., Larsen, U., Oosterhuis, B., & Olsen, A. K. (2010). Metabolism and excretion of the once-daily human glucagon-like peptide-1 analog liraglutide in healthy male subjects and its in vitro degradation by dipeptidyl peptidase IV and neutral endopeptidase. Drug Metabolism and Disposition, 38(11), 1944
1953. Manigault, K. R., & Thurston, M. M. (2016). Liraglutide: A glucagon-like peptide-1 agonist for chronic weight management. The Consultant Pharmacist, 31(12), 685
697. Manikkam, V., Vasiljevic, T., Donkor, O., & Mathai, M. (2016). A review of potential marine-derived hypotensive and anti-obesity peptides. Critical Reviews in Food Science and Nutrition, 56(1), 92
112. Matthews, J. E., Stewart, M. W., De Boever, E. H., Dobbins, R. L., Hodge, R. J., Walker, S. E., & Group, A. S. (2008). Pharmacodynamics, pharmacokinetics, safety, and tolerability of albiglutide, a long-acting glucagon-like peptide-1 mimetic, in patients with type 2 diabetes. The Journal of Clinical Endocrinology & Metabolism, 93(12), 4810
4817. McBrayer, D. N., & Tal-Gan, Y. (2017). Recent advances in GLP-1 receptor agonists for use in diabetes mellitus. Drug Development Research, 78(6), 292
299. Meier, J. J., Nauck, M. A., Kranz, D., Holst, J. J., Deacon, C. F., Gaeckler, D., & Gallwitz, B. (2004). Secretion, degradation, and elimination of glucagon-like peptide 1 and gastric inhibitory polypeptide in patients with chronic renal insufficiency and healthy control subjects. Diabetes, 53(3), 654
662. Mohammed-Geba, K., Arrutia, F., Do-Huu, H., Borrell, Y., Galal-Khallaf, A., Ardura, A., & Garcia-Vazquez, E. (2016). VY6, a β-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver. Food & Function, 7(4), 1968
1974. Mohan, A., Rajendran, S. R., He, Q. S., Bazinet, L., & Udenigwe, C. C. (2015). Encapsulation of food protein hydrolysates and peptides: A review. RSC Advances, 5(97), 79270
79278. Moini, J., Ahangari, R., Miller, C., & Samsam, M. (2020). Global health complications of obesity. Elsevier. Nagaoka, S., Nakamura, A., Shibata, H., & Kanamaru, Y. (2010). Soystatin (VAWWMY), a novel bile acidbinding peptide, decreased micellar solubility and inhibited cholesterol absorption in rats. Bioscience, Biotechnology, and Biochemistry, 74(8), 1738
1741. Nakajima, S., Hira, T., Eto, Y., Asano, K., & Hara, H. (2010). Soybean β51
63 peptide stimulates cholecystokinin secretion via a calcium-sensing receptor in enteroendocrine STC-1 cells. Regulatory Peptides, 159(1
3), 148
155. Nauck, M. A., & Meier, J. J. (2019). Management of endocrine disease: Are all GLP-1 agonist equal in the treatment of type 2 diabetes? European Journal of Endocrinology, 1. (aop). Nielsen, S. D., Beverly, R. L., Underwood, M. A., & Dallas, D. C. (2018). Release of functional peptides from mother’s milk and fortifier proteins in the premature infant stomach. PLoS One, 13(11), e0208204.

662 Chapter 26 Nishi, T., Hara, H., Asano, K., & Tomita, F. (2003). The soybean β-conglycinin β 51
63 fragment suppresses appetite by stimulating cholecystokinin release in rats. The Journal of Nutrition, 133(8), 2537
2542. Nobile, V., Duclos, E., Michelotti, A., Bizzaro, G., Negro, M., & Soisson, F. (2016). Supplementation with a fish protein hydrolysate (Micromesistius poutassou): Effects on body weight, body composition, and CCK/ GLP-1 secretion. Food & Nutrition Research, 60(1), 29857. Nongonierma, A. B., & FitzGerald, R. J. (2016). Structure activity relationship modelling of milk proteinderived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Peptides, 79, 1
7. Nongonierma, A. B., & FitzGerald, R. J. (2019). Features of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from dietary proteins. Journal of Food Biochemistry, 43(1), e12451. O’Halloran, F., Bruen, C., McGrath, B., Schellekens, H., Murray, B., Cryan, J. F., & Giblin, L. (2018). A casein hydrolysate increases GLP-1 secretion and reduces food intake. Food Chemistry, 252, 303
310. Ohinata, K., Agui, S., & Yoshikawa, M. (2007). Soymorphins, novel μ opioid peptides derived from soy β-conglycinin β-subunit, have anxiolytic activities. Bioscience, Biotechnology, and Biochemistry, 71(10), 2618
2621. Ohinata, K., Inui, A., Asakawa, A., Wada, K., Wada, E., & Yoshikawa, M. (2002). Albutensin A and complement C3a decrease food intake in mice. Peptides, 23(1), 127
133. Osborne, S., Chen, W., Addepalli, R., Colgrave, M., Singh, T., Tran, C., & Day, L. (2014). In vitro transport and satiety of a beta-lactoglobulin dipeptide and beta-casomorphin-7 and its metabolites. Food & Function, 5(11), 2706
2718. Pak, V., Koo, M., Lee, N., Kim, M., & Kwon, D. (2005). Structure
activity relationships of the peptide Ile-AlaVal-Pro and its derivatives revealed using the semi-empirical AM1 method. Chemistry of Natural Compounds, 41(4), 454
460. Parker, V. E., Robertson, D., Wang, T., Hornigold, D. C., Petrone, M., Cooper, A. T., & Schlichthaar, H. (2020). Efficacy, safety, and mechanistic insights of cotadutide, a dual receptor glucagon-like peptide-1 and glucagon agonist. The Journal of Clinical Endocrinology & Metabolism, 105(3), 803
820. Pihlanto-Leppa¨la¨, A., Paakkari, I., Rinta-Koski, M., & Antila, P. (1997). Bioactive peptide derived from in vitro proteolysis of bovine β-lactoglobulin and its effect on smooth muscle. Journal of Dairy Research, 64(1), 149
155. Pratley, R. E., Kang, J., Trautmann, M. E., Hompesch, M., Han, O., Stewart, J., & Yoon, K. H. (2019). Body weight management and safety with efpeglenatide in adults without diabetes: A phase II randomized study. Diabetes, Obesity and Metabolism, 21(11), 2429
2439. Pratley, R. E., Nauck, M. A., Barnett, A. H., Feinglos, M. N., Ovalle, F., Harman-Boehm, I., & Stewart, M. (2014). Once-weekly albiglutide versus once-daily liraglutide in patients with type 2 diabetes inadequately controlled on oral drugs (HARMONY 7): A randomised, open-label, multicentre, non-inferiority phase 3 study. The Lancet Diabetes & Endocrinology, 2(4), 289
297. Pupovac, J., & Anderson, G. H. (2002). Dietary peptides induce satiety via cholecystokinin-A and peripheral opioid receptors in rats. The Journal of Nutrition, 132(9), 2775
2780. Rebello, C. J., & Greenway, F. L. (2020). Obesity medications in development. Expert Opinion on Investigational Drugs, 29(1), 63
71. Rendell, M. S. (2016). Albiglutide: A unique GLP-1 receptor agonist. Expert Opinion on Biological Therapy, 16(12), 1557
1569. Retterstøl, K. (2009). Taspoglutide: A long acting human glucagon-like polypeptide-1 analogue. Expert Opinion on Investigational Drugs, 18(9), 1405
1411. Rolls, B. J., Hetherington, M., & Burley, V. J. (1988). The specificity of satiety: The influence of foods of different macronutrient content on the development of satiety. Physiology & Behavior, 43(2), 145
153. Rosenstock, J., Balas, B., Charbonnel, B., Bolli, G. B., Boldrin, M., Ratner, R., & Group, T.-e S. (2013). The fate of taspoglutide, a weekly GLP-1 receptor agonist, versus twice-daily exenatide for type 2 diabetes: The T-emerge 2 trial. Diabetes Care, 36(3), 498
504. Rosenstock, J., Hanefeld, M., Shamanna, P., Min, K. W., Boka, G., Miossec, P., & Ratner, R. E. (2014). Beneficial effects of once-daily lixisenatide on overall and postprandial glycemic levels without significant

Application in medicine: obesity and satiety control 663 excess of hypoglycemia in type 2 diabetes inadequately controlled on a sulfonylurea with or without metformin (GetGoal-S). Journal of Diabetes and Its Complications, 28(3), 386
392. Royle, P. J., McIntosh, G. H., & Clifton, P. M. (2008). Whey protein isolate and glycomacropeptide decrease weight gain and alter body composition in male Wistar rats. British Journal of Nutrition, 100(1), 88
93. Sanford, M. (2014). Dulaglutide: First global approval. Drugs, 74(17), 2097
2103. Santos-Herna´ndez, M., Tome´, D., Gaudichon, C., & Recio, I. (2018). Stimulation of CCK and GLP-1 secretion and expression in STC-1 cells by human jejunal contents and in vitro gastrointestinal digests from casein and whey proteins. Food & Function, 9(9), 4702
4713. Scheen, A. J. (2016). Dulaglutide (LY-2189265) for the treatment of type 2 diabetes. Expert Review of Clinical Pharmacology, 9(3), 385
399. Sebokova, E., Christ, A. D., Wang, H., Sewing, S., Dong, J. Z., Taylor, J., & Culler, M. D. (2010). Taspoglutide, an analog of human glucagon-like peptide-1 with enhanced stability and in vivo potency. Endocrinology, 151(6), 2474
2482. Shi, W., Hou, T., Guo, D., & He, H. (2019). Evaluation of hypolipidemic peptide (Val-Phe-Val-Arg-Asn) virtual screened from chickpea peptides by pharmacophore model in high-fat diet-induced obese rat. Journal of Functional Foods, 54, 136
145. Shimizu, T., & Hettiarachchy, N. (2012). Food-derived bioactive peptides in the market. Food proteins and peptides—Chemistry, functionality, interactions, and commercialization (pp. 375
392). Boca Raton, FL: CRC Press. Staljanssens, D., Smagghe, G., & Van Camp, J. (2012). Protein-derived bioactives affecting CCK-induced satiety. Agro Food Industry Hi Tech, 23(2), 6. Su, N., Li, Y., Xu, T., Li, L., Kwong, J. S.-W., Du, H., & Sun, X. (2016). Exenatide in obese or overweight patients without diabetes: A systematic review and meta-analyses of randomized controlled trials. International Journal of Cardiology, 219, 293
300. Teschemacher, H. (2003). Opioid receptor ligands derived from food proteins. Current Pharmaceutical Design, 9(16), 1331
1344. Tillner, J., Posch, M. G., Wagner, F., Teichert, L., Hijazi, Y., Einig, C., & Bossart, M. (2019). A novel dual glucagon-like peptide and glucagon receptor agonist SAR425899: Results of randomized, placebo-controlled first-in-human and first-in-patient trials. Diabetes, Obesity and Metabolism, 21(1), 120
128. Torres-Fuentes, C., Schellekens, H., Dinan, T. G., & Cryan, J. F. (2015). A natural solution for obesity: Bioactives for the prevention and treatment of weight gain. A review. Nutritional Neuroscience, 18(2), 49
65. Tsou, M.-J., Kao, F.-J., Lu, H.-C., Kao, H.-C., & Chiang, W.-D. (2013). Purification and identification of lipolysis-stimulating peptides derived from enzymatic hydrolysis of soy protein. Food Chemistry, 138(2
3), 1454
1460. Tulipano, G., Sibilia, V., Caroli, A. M., & Cocchi, D. (2011). Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors. Peptides, 32(4), 835
838. Uchida, M., Ohshiba, Y., & Mogami, O. (2011). Novel dipeptidyl peptidase-4
inhibiting peptide derived from β-lactoglobulin. Journal of Pharmacological Sciences, 117(1), 63
66. Umpierrez, G., Pantalone, K. M., Kwan, A. Y., Zimmermann, A. G., Zhang, N., & Fernandez Lando, L. (2016). Relationship between weight change and glycaemic control in patients with type 2 diabetes receiving onceweekly dulaglutide treatment. Diabetes, Obesity and Metabolism, 18(6), 615
622. Van Can, J., Sloth, B., Jensen, C., Flint, A., Blaak, E., & Saris, W. (2014). Effects of the once-daily GLP-1 analog liraglutide on gastric emptying, glycemic parameters, appetite and energy metabolism in obese, nondiabetic adults. International Journal of Obesity, 38(6), 784
793. Veldhorst, M. A., Nieuwenhuizen, A. G., Hochstenbach-Waelen, A., Westerterp, K. R., Engelen, M. P., Brummer, R.-J. M., & Westerterp-Plantenga, M. S. (2009). Effects of complete whey-protein breakfasts versus whey without GMP-breakfasts on energy intake and satiety. Appetite, 52(2), 388
395. Wadden, T. A., & Bray, G. A. (2018). Handbook of obesity treatment. Guilford Publications.

664 Chapter 26 Wang, J., Zhou, M., Wu, T., Fang, L., Liu, C., & Min, W. (2020). Novel anti-obesity peptide (RLLPH) derived from hazelnut (Corylus heterophylla Fisch) protein hydrolysates inhibits adipogenesis in 3T3-L1 adipocytes by regulating adipogenic transcription factors and adenosine monophosphate-activated protein kinase (AMPK) activation. Journal of Bioscience and Bioengineering, 129(3), 259
268. Wang, Y.-M., Pan, X., He, Y., Chi, C.-F., & Wang, B. (2020). Hypolipidemic activities of two pentapeptides (VIAPW and IRWWW) from miiuy croaker (Miichthys miiuy) muscle on lipid accumulation in HepG2 cells through regulation of AMPK pathway. Applied Sciences, 10(3), 817. Werner, U., Haschke, G., Herling, A. W., & Kramer, W. (2010). Pharmacological profile of lixisenatide: A new GLP-1 receptor agonist for the treatment of type 2 diabetes. Regulatory Peptides, 164(2
3), 58
64. Wren, A. M., Seal, L. J., Cohen, M. A., Brynes, A. E., Frost, G. S., Murphy, K. G., ... & Bloom, S. R. (2001). Ghrelin enhances appetite and increases food intake in humans. The Journal of Clinical Endocrinology & Metabolism, 86(12), 5992
5995. Wu, G.-C., Lin, S.-Y., Liang, H.-J., & Hou, W.-C. (2018). 135-Day interventions of yam dioscorin and the dipeptide Asn-Trp (NW) to reduce weight gains and improve impaired glucose tolerances in high-fat diet-induced c57bl/6 mice. Journal of Agricultural and Food Chemistry, 66(3), 645
652. Xing, L., Liu, R., Cao, S., Zhang, W., & Guanghong, Z. (2019). Meat protein based bioactive peptides and their potential functional activity: A review. International Journal of Food Science & Technology, 54(6), 1956
1966. Yoon, K. H., Kang, J., Kwon, S. C., Trautmann, M. E., Hompesch, M., Stewart, J., & Sorli, C. H. (2020). Pharmacokinetic and dose-finding studies on efpeglenatide in patients with type 2 diabetes. Diabetes, Obesity and Metabolism, 22. Yoshikawa, M. (2015). Bioactive peptides derived from natural proteins with respect to diversity of their receptors and physiological effects. Peptides, 72, 208
225. Yoshikawa, M., & Chiba, H. (1992). Biologically active peptides derived from food and blood proteins (pp. 403
409). Amsterdam: Elsevier. Yoshikawa, M., Fujita, H., Matoba, N., Takenaka, Y., Yamamoto, T., Yamauchi, R., & Takahata, K. (2000). Bioactive peptides derived from food proteins preventing lifestyle-related diseases. BioFactors (Oxford, England), 12(1
4), 143
146. Zambrowicz, A., Pokora, M., Setner, B., Da˛browska, A., Szołtysik, M., Babij, K., & Chrzanowska, J. (2015). Multifunctional peptides derived from an egg yolk protein hydrolysate: Isolation and characterization. Amino Acids, 47(2), 369
380. Zhang, J., Xue, C., Zhu, T., Vivekanandan, A., Pennathur, S., Ma, Z. A., & Chen, Y. E. (2013). A tripeptide Diapin effectively lowers blood glucose levels in male type 2 diabetes mice by increasing blood levels of insulin and GLP-1. PLoS One, 8(12), e83509. Zhang, X., Shi, W., He, H., Cao, R., & Hou, T. (2020). Hypolipidemic effects and mechanisms of Val-Phe-ValArg-Asn in C57BL/6J mice and 3T3-L1 cell models. Journal of Functional Foods, 73, 104100. Zhou, J., Cai, X., Huang, X., Dai, Y., Sun, L., Zhang, B., & Qian, H. (2017). A novel glucagon-like peptide-1/glucagon receptor dual agonist exhibits weight-lowering and diabetes-protective effects. European Journal of Medicinal Chemistry, 138, 1158
1169.

CHAPTER 27

Food-derived osteogenic peptides towards osteoporosis Ming Du1, Zhe Xu1,2, Hui Chen3, Fengjiao Fan4, Pujie Shi1 and Di Wu1 1

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, P.R. China, 2College of Life Sciences, Key Laboratory of Biotechnology and Bioresources Utilization, Dalian Minzu University, Ministry of Education, Dalian, P.R. China, 3College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, P.R. China, 4College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, P.R. China

27.1 Introduction An increased human longevity represents a global phenomenon—an aging population with profound implications for health (Shapses, Pop, & Wang, 2017). Many changes associated with the process of aging increase the prevalence of chronic diseases. Aging is particularly involved in the decline in bone mass and strength leading to osteoporosis, which is the most common bone disease (Siddique, Odonoghue, Casey, & Walsh, 2017). It is estimated that over 200 million people worldwide have osteoporosis (Reginster & Burlet, 2006), and the number of hip fractures in men is expected to increase by 51.8% during the period between 2010 and 2030 (Stevens & Rudd, 2013). Osteoporosis is a public health threat. As the population is aging, the incidence of osteoporosis is on the rise. Therefore research on the treatment and prevention of osteoporosis is warranted. Normal bone metabolism is a complex process involving continuous remodeling of bone tissue, mediated by osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption. When bone formation is less than bone resorption, a decrease in bone density easily leads to the occurrence of osteoporosis. The basic characteristics of osteoporosis are: destruction of bone microstructure, decreased bone density, and increased bone fragility, which is a chronic disease prone to fracture. In the course of bone growth and development, OBs can regulate bone mineralization by secreting bone matrix and noncollagen proteins, and OCs are directly involved in the bone resorption process (Cann, Martin, Genant, & Jaffe, 1984) as shown in Fig. 27.1. Osteocytes can affect both

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00027-3 © 2021 Elsevier Inc. All rights reserved.

665

666 Chapter 27

Figure 27.1 The relationships of bone cells in bone matrix on regulation of bone mass.

bone formation and resorption, either directly or indirectly, and are closely linked with osteoporosis. Bone is a metabolically active and dynamic organ, which can continuously form and absorb bone through OBs and OCs on the surface of osteoid and trabecular bone. These two processes of bone formation and bone resorption are called bone remodeling and bone turnover, respectively. Since the surface area of cortical bone is smaller than trabecular bone, bone transformation often occurs in trabecular bone. OBs often synthesize new bone matrix, whereas OCs degrade the existing bone matrix to achieve a balance between the conversion and absorption of old and new bone. About 10% of the skeleton is reshaped every year (Manolagas, 2000), this allows the skeleton to adapt and repair the damage caused by some mechanical stress through this adjustment (Parfitt, 2002; Seeman, 2006). Maintaining calcium homeostasis and bone metabolism during bone remodeling is critical (Vaananen & Laitalaleinonen, 2008). During the growth period of children, bone formation takes the lead in the dynamic balance of bones. In this process, the strength and size of the

Food-derived osteogenic peptides towards osteoporosis 667 bones are continuously increased, reaching the peak bone mass of human body at the age of 20
30 (Ho & Kung, 2005; Matkovic et al., 1994). When this amount is reached, bone resorption and formation are in equilibrium. After 40 years of age, bone resorption gradually dominates, often accompanied by periosteal juxtaposition, endosteal resorption, increased in cortical porosity, and trabecularization of cortical bone. In women older than 40, decline in estrogen level due to menopause accelerates age-related deterioration (Kanis & Adami, 1994). Estrogen level is another risk factor for osteoporosis. Postmenopausal women are at greater risk of developing osteoporosis as the level of estrogen drops. After the age of 50 in some Western countries, 13% of men and 40% of women often suffer from fragility fractures (Melton, Chrischilles, Cooper, Lane, & Riggs, 2005). Osteoporosis is a systemic bone disease characterized by degeneration of the microstructure of bone tissue and reduced bone mass. There are many methods used to diagnose osteoporosis, such as bone mineral density (BMD) using dual energy X-ray absorptiometry. Generally, the value of BMD is defined as 2.5 standard deviations. Although osteoporosis is one of the most important factors affecting fractures, not all women with BMD below the diagnostic threshold are prone to fractures (Sanders et al., 2006; Schuit et al., 2004). Therefore judging the fracture risk of osteoporosis requires comprehensive consideration with other methods. In addition to judging osteoporosis by the value of BMD, bone formation markers and bone resorption markers in serum can usually be measured to reflect the rate of bone formation and bone resorption to determine osteoporosis. This method is mainly used in three areas: (1) identify the fracture risk of the population, (2) detection of antiabsorption therapy and anabolic therapy, and (3) the prediction of the risk of osteoporosis and the prediction of the potential occurrence of osteoporosis. This chapter first introduces the bone turnover and bone resorption markers relative to osteoporosis and then summarizes the preparation, characterization, absorption, and distribution methods of osteogenic peptides.

27.2 Evaluation and diagnosis of osteoporosis The impact of osteogenic peptides can be determined by observing whether they can promote or inhibit the proliferation and differentiation of OBs or OCs and can also be tested by measuring BMD of bones in animals or clinical trials. In addition, measuring the changes of biomarkers on cells or serum can also be used to characterize the osteogenic activity of peptides.

27.2.1 Bone formation and resorption biomarkers The marker of bone formation is an enzyme or bone tissue protein and fragments released in bone-related cells. The bone tissue protein can be bone salivary protein or osteocalcin or

668 Chapter 27 some collagen or its enzymatic hydrolysis product. The enzymes released by bone cells, such as antitartrate acid phosphatase 5b and bone-specific alkaline phosphatase, can be detected in the serum. The enzymatic activity of these enzymes can reflect the activity of OCs or OBs coupled in space and time, also correspond to signs of bone resorption and bone formation. Different stages of bone resorption and formation are reflected by different bone turnover markers, but some activities that distinguish cortical bone or trabecular bone and certain specific diseases cannot be reflected. Bone-specific alkaline phosphatase (Bone ALP), alkaline phosphatase (ALP), and total ALP are enzymes located on the surface of cells. Tissue nonspecific genes are generally expressed in bone and liver, whereas tissuespecific genes are expressed in placenta, intestine, and germline enzymes. Although tissue nonspecific ALP and tissue-specific ALP are expressed by the same ALP gene, their tissuespecific differences occur due to differences in carbohydrate chain translation and modification (Weiss et al., 1988). Usually the changes in bones and liver affect the serum ALP level. ALP exists on the surface of OBs in the bone and then dissolves through the membrane and is released into the blood circulation system. In a healthy adult human body, more than half of ALP is released from the bones into the serum. Therefore S-ALP is usually used as a bone turnover marker, but in some cases, it lacks specificity and sensitivity. Bone-specific ALP (S-Bone-ALP) is gradually applied to improve the sensitivity of the determination, but there is still a 15%
20% crossover between liver and bone ALP analysis (Garnero & Delmas, 1993). Bone Gla protein or osteocalcin is the most abundant noncollagen matrix protein derived from bones. It is a low-molecular-weight protein expressed by OBs, pre-OBs hypertrophic chondrocytes, and odontoblasts. Moreover, the three c-carboxyglutamate residues of osteocalcin have a very strong interaction force with the Ca21 in bone hydroxyapatite (Hoang, Sicheri, Howard, & Yang, 2003). A part of osteocalcin is distributed in the circulatory system and can be detected, and the other part is in the bone matrix. Therefore serum osteocalcin is one of the markers reflecting the bone formation rate and OB activity. There are one-third small fragments of osteocalcin in serum, one-third middle fragments composed of amino acids 1
43, and one-third complete osteocalcin (Garnero, Grimaux, Seguin, & Delmas, 2009). Bone matrix can also release osteocalcin during bone resorption (Ivaska et al., 2004), and it is rapidly degraded after entering the circulation. Some fragments of osteocalcin can be found in the kidney and liver (Ivaska et al., 2003). Although urinary osteocalcin (U-OC) has no effect on the production of type I bone formation collagen, it is related to the bone resorption process (Ivaska et al., 2005; Srivastava, Mohan, Singer, & Baylink, 2002). Type I collagen is produced extracellularly, as it cuts the carboxyl-terminal and amino-terminal extension peptides before forming fibrils (Szulc, Seeman, & Delmas, 2000). These peptides can guide collagen to form helically fold, and the C-terminal (PICP) and N-terminal peptides (PINP) released by type I collagen can be measured in the circulation. PINP and PICP have therefore become quantitative indicators of type I collagen. Type I collagen is mainly distributed in bones, but also in several soft tissues, the regeneration rate of collagen in bone is faster than

Food-derived osteogenic peptides towards osteoporosis 669 that of other tissues. Therefore S-PICP and S-PINP have become one of the main indicators reflecting bone collagen (Bilezikian et al., 2013; Szulc et al., 2000). Bone resorption markers mainly include tartrate-resistant acid phosphatase (TRAP), C, N-terminal cross-linked telopeptide of type I collagen, the carbon-terminal cross-linked telopeptide of type I collagen produced by metalloprotease, deoxypyridinoline, pyridinoline, and osteocalcin. Tartrate-resistant acid phosphatase 5b (TRACP5b) is a catalytic enzyme that acts on phosphate esters in acid environments. This enzyme is mainly expressed in alveoli, monocyte-derived macrophages, and OCs. There are two isozymes of TRACP5 in the circulatory system. TRACP5a is produced in macrophages, and TRACP5b is produced in OCs. The optimal pH of these two enzymes is different. The function of S-TRACP5b in OCs is still unknown. TRACP is engulfed into cells via vesicles and converted to matrix degradation products. TRACP assists in the degradation of the matrix in the vesicles through the generation of reactive oxygen species. Therefore TRACP can better reflect the number of OCs and the rate of bone resorption (Rissanen, Suominen, Peng, & Halleen, 2008). Since TRACP5b is less affected by kidney function, food intake cannot affect TRACP5b levels in the circulation (Hannon et al., 2004). In addition, TRACP5b is very stable in serum (Halleen, Tiitinen, Ylipahkala, Fagerlund, & Vaananen, 2006). The structure of collagen is very stable due to its intra- and intermolecular cross-linking. In bone, there is a cross-linking effect of deoxypyridoline (DPD) and pyridine (PYD). When collagen is degraded, the cross-linking bond between DPD and PYD is released. PYD predominates in collagen, and DPD, which has a higher content in dentin and bone, is considered to be cross-linked with bone characteristics (Seibel, Robins, & Bilezikian, 1992). This kind of cross-linking is degraded in the kidney. Free cross-links or cross-links combined with short-collagen peptides can be measured in serum or urine. Type I collagen cross-linked telopeptide (ICTP) includes cross-linked C-terminal peptide (CTX) and crosslinked N-terminal peptide (NTX). Different collagen breakdown pathways produce different fragments. NTX and CTX are produced by cathepsin K, whereas ICTP is produced by matrix metalloproteinases (Garnero et al., 2003). CTX exists in the form of nonisomerized α-CTX and isomerized β-CTX. The aging of bones is related to the isomerization of CTX. Therefore the degradation of older bones can be measured by the level of β-CTX (Fledelius, Johnsen, Cloos, Bonde, & Qvist, 1997). Currently, one of the most commonly used cross-linking measurement methods is the determination of β-CTX-1.

27.2.2 Computed tomography diagnosis BMD refers to the bone mineral content per unit volume (Vestergaard, 2007). Calcium and phosphorus are the main components of bone minerals. The more deposits in the bones, the stronger the bones. It is an important indicator of bone strength. It is an absolute value that is expressed in grams per cubic centimeter. When the BMD value is used clinically,

670 Chapter 27 because the absolute value of different BMD detectors is different, the T value is usually used to judge whether the BMD is normal (Kanis et al., 2001). The T value is a relative value for measuring bone density, and the normal reference value is between 21 and 11 (Lee et al., 2017). When the T value is lower than 22.5, it is abnormal. Bone density is an important sign of bone quality, reflecting the degree of osteoporosis and an important basis for predicting the risk of fracture. Due to the increasingly improved measurement method and the development of advanced software, the method can be used in different parts, and the measurement accuracy is significantly improved. In addition to the diagnosis of osteoporosis, it can also be used for clinical drug efficacy observations and epidemiological investigations and has significant advantages in predicting osteoporotic fractures. In addition to observing changes in cells and measuring changes in biomarkers, using BMD is one of the best ways to characterize osteoporosis. In animal study, osteoporosis is commonly induced by three methods: castration (removal of ovary or testis) (Min et al., 2018), intragastric retinoic acid (Orˇsoli´c et al., 2014), and glucocorticoid intramuscular injection (Ren et al., 2013). Other methods are also applied, such as attenuated mouse model (Huayue Chen, Emura, Yao, & Shoumura, 2004), low calcium feeding model (Lee et al., 2008), and parathyroid gland model (Shiraki & Orimo, 1991). Regardless of which model is used, the BMD is determined by micro-CT to characterize the activity of the osteogenic peptide. Micro-CT can also measure other related indicators to further determine the condition of bone, such as bone volume (BV), tissue volume (TV), BV/TV trabecular, trabecular thickness (Tb.Th), trabecular pattern factor (Tb.Pf), trabecular separation (Tb.Sp), trabecular number (Tb.N), and BMD (Yang, Yang, Pan, & Zhong, 2019).

27.3 Osteogenic agents 27.3.1 Drugs for osteoporosis Diseases that affect the skeletal system are generally difficult to treat. Most drugs for osteoporosis are mainly divided into two categories: antiresorption drugs and bone anabolic drugs (Riggs & Parfitt, 2004). Antiresorption drugs, such as denosumab and bisphosphonates, are commonly used in prescriptions. Hormone replacement therapy, selective estrogen receptor modulators, calcitonin, bisphosphonates, and the divalent cation strontium ranelate also belong to this class (Miller & Derman, 2010). There are many bisphosphonate drugs, including alendronate, risedronate, ebandronate, minodronate, and zoledronate. Bisphosphonates should inhibit isoprenylation of proteins in OBs, thereby reducing the ability of parathyroid hormone (PTH) to activate lining cells into active OBs. Bisphosphonates can effectively inhibit farnesyl pyrophosphate synthesis, the enzyme that produces isoprenoid lipids. It is required for the survival and function of OCs, reducing the rate of bone resorption, and reducing the risk of fracture (Ebetino et al., 2011; Plotkin & Bellido, 2013).

Food-derived osteogenic peptides towards osteoporosis 671 Treatment with antiabsorption agents can lead to filling of the remodeling space, increase in secondary mineralization and stabilization of the skeletal structure, thereby increasing bone strength and reducing the incidence of fractures. However, antiabsorption therapy cannot restore the bone and structure lost due to increased bone remodeling. Moreover, for patients with very severe osteoporosis, considering that the trabecular network has been thinned, the use of antiabsorption drugs may no longer prevent further fractures, and there are still some concerns about chronic liver disease. Bone anabolic drugs are commonly used in patients with osteoporosis, of which the main is teriparatide. Teriparatide refers to the 34 amino acid fragment of PTH (Saini et al., 2013). The main mechanism driving the anabolism of PTH is the activation of PTH1R by cyclic-AMP dependent protein kinase A (cAMP-PKA) (Kousteni & Bilezikian, 2008). PTH activate OBs’ receptors to transmit canonical Wingless/Integrated (Wnt) signals, which in turn promotes the proliferation and differentiation of OBs. In order to further amplify OB signals, Wnt antagonists inhibit sclerostin in bone cells. On the other hand, PTH can also regulate the expression of Wnt antagonist Dickkopf-related protein 1 (DKK1) (Anastasilakis et al., 2009). PTH 1
34 can promote the secretion of DKK1 (Gatti et al., 2011). Both the upregulation and downregulation of PTH may represent steady-state response, which also explains the decrease in bone formation markers in patients receiving PTH over time. The administration of PTH can also induce the expression of Runt-related transcription factor 2 (Runx2), which promotes the differentiation of OBs (Eriksen, 2010). In summary, intermittent PTH treatment can increase the number of OBs, induce the differentiation of committed OB precursors, and prolong the survival of OBs. OCs promote the anaerobic effect of PTH by releasing stored growth factors (IGF, BMP, and TGFβ) from bone matrix or by factors released by transiently activated OCs. Intermittent simultaneous rhPTH and bisphosphonate treatment will delay the bone’s anabolic response (Finkelstein, Wyland, Lee, & Neer, 2010). Bone formation caused by intermittent rhPTH administration includes not only an increase in BMD or bone mass, but also an increase in the number and connectivity of trabecular bones, thereby improving the microstructure of the bone. Intermittent rhPTH treatment can stimulate the juxtaposition of periosteum and cortical endometrium, which not only increases bone diameter and cortical thickness, but also facilitates intracortical porosity (Jilka et al., 2009). At the same time, the increase in bone strength also increases the mechanical strength against fracture. According to reports, teriparatide can reduce serum sclerostin levels in postmenopausal women with osteoporosis and improve bone fragility. However, long-term use of these drugs has serious side effects, such as uterine bleeding, carcinogenic effects, jaw bone necrosis, and cardiovascular disease (Uehara, Takahashi, Watanabe, & Nomura, 2014), and an increased risk of osteosarcoma (Guo et al., 2019). Therefore food-derived metabolizable bone-promoting active peptides have received more and more attention. Although various peptides or analogs (e.g., osteogenic growth peptides, calcitonin gene-related peptides) have advantages in bone formation, many of them are not applied for osteoporosis due to enzymatic degradation, immunogenicity, and clinical costs. These unmet needs have led to efforts to develop

672 Chapter 27 potential alternatives using food bioactives to prevent osteoporosis. At present, some biologically active peptides have been identified, and their effects on promoting bone metabolism have been confirmed.

27.3.2 Osteogenic peptides Due to the limitations of some drugs that contribute to bone activity, it is necessary to explore a new generation of safe and effective therapeutic alternatives to prevent osteoporosis. Therefore the development of new therapies that promote bone formation or prevent osteoporosis in the daily diet has attracted widespread interest. In recent years, researchers and consumers pay attention to the functional foods with many biological activities and therapeutic effects, including those prevent osteoporosis. Proteins or peptides are increasingly used in functional foods, nutritional supplements, and some drugs with potential to prevent diseases. The bioactive peptides have a beneficial effect of promoting diversification in proteins (Marcone, Belton, & Fitzgerald, 2017). Bioactive peptides have the advantages of low toxicity, good solubility, and good pharmacokinetic characteristics (Fosgerau & Hoffmann, 2015; Zvereva, Dudko, & Dikunets, 2017). Therefore this also makes it widely used in therapeutic drugs. In the past few decades, the market value of bioactive peptides has reached 25 billion US dollars (Daliri, Lee, & Oh, 2018). There are many peptides with osteoporosis prevention ability as shown in Table 27.1. Osteoporosis is caused by bone resorption more than bone formation, so it can be prevented by promoting the proliferation and differentiation of OBs or inhibiting the proliferation and differentiation of OCs. In Table 27.1 the peptides with serial numbers 1
28 are those that can promote the proliferation and differentiation of OBs or promote new bone formation, whereas the serial numbers 29
37 are those that inhibit the proliferation and differentiation of OCs or promote bone resorption. There is a protein called integrin in OBs and OCs. The combination of peptide and integrin can regulate the adhesion ability of OBs and OCs, thereby promoting or inhibiting the proliferation and differentiation of OBs or OCs. Both peptides RGD and DEGA bind well with integrins. In Table 27.1, peptides 38
44 contain RGD and peptide 47 contain DEGA, and most of these peptides have been proven effective in promoting bone formation or inhibiting bone resorption. The reason why peptides with sequence numbers 45
47 have a good effect on preventing osteoporosis is that they have a higher affinity with integrins. The essence of promoting bone formation is to promote the proliferation of OBs or inhibit the proliferation of OC. As shown in Fig. 27.2, osteogenic peptides can affect one pathway of OCs and inhibit OC proliferation and differentiation. OC activation occurs after RANKL binds to the receptor RANK, which is present on the membrane of OC progenitor cells. OCs are then polarized by cytoskeletal rearrangement. In Howshiplacuna (HL), which is

Table 27.1: Osteogenic peptides. No. Amino acid sequence

Origin

Model

1

ALKRQGRTLYGFGG

Histone H4

In vivo

2

ALKRQGRTLYGFGG/YGFGG/ GGFGY

Histone4

3

CPP

Casein

4

CGGKVGKACCVPTKLSPISVLYK

5

DVSTSQAVLPDDFPRYPVGKFFKFDTWRQSAGRL

Preptin

6

GFOGER

Collagen

7

GLRSKSKKFRRPDIQYPDATDEDITSHM

Osteopontin

8

GTPGPQGIAGQRGVV

Collagen type I

9

GVVPPQVLSQPNEEAGAALSPLPEVPPWTGEVSPAQR

C-Type Natriuretic Peptide

10

HSDGIPTDSYSRYRKQMAVKKYLAAVLGKQRVKNK

Pituitary Adenylate CyclaseActivating Polypeptide

11

HWAWFK

Hexarelin

pBone morphogenetic protein-9

Functions

Promote bone formation process and accelerate bone healing (Zhao et al., 2011) In vitro Induce MC3T3-E1 osteoblasts in vitro dose-dependent proliferation, differentiation and ALP activity, and increase the bone mass and trabecular bone density of rat model (Bab et al., 1992) In vivo Increase the BMD of OVX rats (Tsuchita, Goto, Shimizu, Yonehara, & Kuwata, 1996) In vitro Induced MC3T3-E1 differentiation (Bergeron, Marquis, Chretien, & Faucheux, 2007) In vitro and Stimulate the proliferation and in vivo differentiation of osteoblasts (Cornish et al., 2007) In vivo Increase bone formation (Wojtowicz et al., 2010) In vitro and Promote bone formation activity in vivo (Lee et al., 2007) In vitro Accelerate the process of bone formation on the surface of inorganic bone (Thorwarth et al., 2005) In vitro Promotes osteoblast proliferation (Lenz, Bennett, Skelton, & Vesely, 2010) In vitro Enhance osteoblast differentiation, increase expression of osterix, ALP, and collagen (Juhasz et al., 2014) In vivo Increase the bone mineral content and bone area of the femur (Sibilia et al., 2002) (Continued)

Table 27.1: (Continued) No. Amino acid sequence

Origin

Model

12

IAGVGGEKSGGP

Collagen type III

13

IAGVGGEKSGGF

Collagen IIIa

14

KPSSAPTQLN

Bone morphogenetic protein-7

15

KIPKASSVPTELSAISTLYL

Bone morphogenetic protein-2

16

LDLNLDLSKFRLPQPSSGRESPRH

Protein from rat stomach

17

NGLPGPIGP

Human collagen type I

18

NAVPITPTL

Buffalo casein

19

NGVFKYRPRYYLYKHAYFYPHLKRFPVQ

Bone sialoprotein

20

NSVNSKIPKACCVPTELSAI

Bone morphogenetic protein-2

21

PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC C-Type Natriuretic peptide

22

RKKNPNCRRH

In vitro and Promote osteoblast attachment and in vivo mineralization (Agrawal, Tottey, et al., 2011) In vitro and Increase the expression of osteogenic in vivo genes, ALP activity, and mineralization (Agrawal, Kelly, et al., 2011) In vitro Inhances the differentiation and density of human osteoblasts (Chen & Webster, 2009) In vitro Induce osteoblast precursor cells to differentiate into osteoblasts and activate osteoblasts (Saito, Suzuki, Ogata, Ohtsuki, & Tanihara, 2005) In vitro Promote osteoblast differentiation (Fukushima, Hiraoka, Shirachi, Kojima, & Nagata, 2010) In vitro Promote bone formation (Wang, Misra, & Amsden, 2008) In vitro Induction of osteoblast differentiation through PI3K signaling pathway (Reddi et al., 2016) In vitro and Stimulate human osteosarcoma cells in vivo to differentiate into osteoblasts and increase new bone area (Choi, Lee, Chung, & Park, 2013) In vivo Stimulate osteoblast differentiation and promote ectopic bone formation (Suzuki et al., 2000) In vivo Promote bone growth (Lorget et al., 2012) In vitro Stimulate osteoblast differentiation (Choi et al., 2010)

Bone morphogenetic protein-4

Functions

23

RQLKIWFQNRRMKWKKIPVGESLKDLIDQ

Casein kinase 2

24

SVSEIQLMHNLGKHLNSMERVGWLRKKLQDVHNF

PTH 1
34

25

SKIPKASSVPTELSAISTLDDD

26

VEIQLLHQXALWLHD

Bone morphogenetic protein-2 PTH(1
17)

27

VEHDKEFFHPRYHHR

28

YQPPSTNKNTKSQRRKGSTFEEHK

Insulin-like growth factor I

29

CGRP

Calcitonin

30

IPP, VPP, LKP

Chemical synthesis

31

KRQWAQFKIQWNQRWGRR

Human calcitonin receptor

32

KCNTATVATERLANPLVHSSNNPGAILSSTNVGSNYY

Amylin

33

SVVYGLR

Chemical synthesis

34

SVSEIQLMHNLGKHLNSMERVGWLRKKLQDV

rhPTH(1
31)

Bone morphogenetic protein-7

In vitro

Induce new bone formation (Bragdon et al., 2011) In vitro Osteoblast proliferation and differentiation, treatment of osteoporosis (Baron & Hesse, 2012) In vivo Induced ectopic bone formation (Li et al., 2011) In vivo Enhance bone density (Neerup et al., 2011) In vitro Promote CD44, CD51, CD45, ALP, mRNA expression and calcium upregulated deposition, enhance bone formation (Kim et al., 2017) In vitro Play an important role in the proliferation and differentiation of osteoblasts (Dai, Wu, Yeung, & Li, 2010) In vitro Inhibit bone resorption (Zaidi, Fuller, Bevis, Gainesdas, & Macintyre, 1987) In vitro Increase mineralization, increase Runx2 and decrease caspase-8 and RANKL/bone protein ratio (Huttunen, Pekkinen, Ahlstrom, & Lambergallardt, 2008) In vitro and Increase mineralization and prevent in vivo bone loss (Komatsu, Hadjiargyrou, Udin, Trasolini, & Pentyala, 2015) In vitro Silent osteoclasts, reducing bone resorption (COOPER & Garth) In vitro Promote the adhesion, proliferation, and proliferation of bone marrow mesenchymal stem cells and inhibit osteoclastogenesis (Egusa et al.) In vitro and Prevent the loss of cortical bone and in vivo cancellous bone (Henriksen et al., 2013) (Continued)

Table 27.1: (Continued) No. Amino acid sequence

Origin

Model

35

TRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSR

PTH-related protein (PTHrP)

In vitro

36

VLPVPQK

Buffalo milk casein

37

VTHRLAGLLSRSGGVVKNNFVPTNVGSKAF

Calcitonin

38

AGYKPDEGKRGDACEGDSGGPFV

Thrombin

39

DVDVPDGRGDSLAYG

Chemical synthesis

40

EEEEEEEPRGDT

Chemical synthesis

41

LVQPRGDTNGPGPWQGGRRKFRRQRPRLSHKGPMPF

Apelin

42

RGD

Fibronectin

43

TDLQERGDNDISPFSGDGQPFKD

Matrix extracellular phosphoglycoprotein

Functions

Inhibit bone resorption (J. et al., 1991) In vivo Promote femur bone density increased and trabecular microstructure improved in OVX rats; increased bone strength, glutathione levels, superoxide dismutase and catalase activity, and inhibit bone resorption (Mada, Reddi, Kumar, Kumar, & Ahmad, 2017) In vitro and Inhibit bone loss, but not bone in vivo formation (Valentijn et al.) In vivo Stimulates interosseous distraction and increases BMD (Li, Ryaby, Carney, & Wang, 2005) In vitro Enhance the differentiation and mineralization of rat mesenchymal stem cells (Shin, Zygourakis, Farachcarson, Yaszemski, & Mikos, 2004) In vitro Stimulates osseointegration of implants (Itoh et al., 2002) In vitro Stimulate MC3T3-E1 cell proliferation and inhibit apoptosis (Tang et al., 2007) In vitro Promote bone formation of bone marrow stromal cells (Yang et al., 2005) In vitro Affect the effective anabolic effects of human interstitial stem (Nagel et al., 2004)

44

YRGDVVPK

45

IEELEEELEAER

46

YPRKDETGAERT

47

DGEA

Tubulin Alpha-1C chain in Crassostrea gigas

In vitro

Promote osteoblast proliferation and differentiation (Chen et al., 2019) Pedal retractor muscle myosin heavy In vitro and Promote osteoblast proliferation chain in Mytilus edulis in vivo and differentiation, inhibit osteoclast proliferation, and differentiation (Xu, Chen, Fan, et al., 2019) Chitinase-like protein-3 in Mytilus In vitro Promote osteoblast proliferation edulis and differentiation (Xu, Chen, Wang, et al., 2019) Collagen type I In vitro Induces early osteogenic differentiation (Yoo, Kobayashi, Lee, & Lee, 2011)

678 Chapter 27

Figure 27.2 Schematic summary of bone tissue showing bone cells and the relationships among them and with bone matrix.

part of the OC, specialization of the membrane on the bone resorption surface and juxtaposed crease boundaries and clear zones are observed. When hydrogen ions (H1) are pumped to HL, hydroxyapatite is acidified and dissolves on the bone surface adjacent to the folding boundary (RF). H1 and ionic bicarbonate (HCO32) are derived from the cleavage of carbonate (H2CO3) under the action of carbonic anhydrase II (CAII). After mineral lysis, OCs release cathepsins (Cp), matrix metalloproteinase-9 (MMP-9), and TRAP to break down the organic matrix. The presence of ephrin B2 (Eph2) and ephrin B4 (Eph4) in the OC membrane promotes differentiation in the OC membrane, and reverse transcription signals (ephrin B4/ephrin B2) suppress the development of OCs. Sema4D produced by OCs inhibits OBs, and Sema3A secreted by OBs inhibits OCs. OBs also produce nuclear receptor activators κB (RANKL) and osteoprotegerin (OPG), increasing and decreasing OC production, respectively. OB contain collagen (Col1) and noncollagen proteins such as osteocalcin (OCN), osteopontin (OSP), osteonectin (OSN), bone saliva protein (BSP), and bone morphogenesis protein (BMP). Osteocytes (Ot) are located in the

Food-derived osteogenic peptides towards osteoporosis 679 bone matrix of the void surrounded by calcification. The intersection of the cytoplasmic processes can connect to other adjacent bone cell processes through the gap junction, which is mainly composed of Connexin 43, and the cytoplasmic processes of OBs and bone lining cells on the bone surface. RANKL secreted by osteoocytes stimulates OC production, and prostaglandin E2 (PGE2), nitric oxide (NO), and insulin-like growth factor (IGF) stimulate OB activity. Instead, osteoocytes produce OPGs that inhibit OCs. In addition, bone cells produce sclerostin and a Zickov WNT signaling pathway inhibitor (DKK1) that reduce OB activity. Therefore some bone-forming active peptides not only promote OB proliferation and inhibit OC proliferation but may also affect the activity of ALP or antitartrate acid phosphatase.

27.4 Characterization of osteogenic peptides 27.4.1 Preparation of osteogenic peptides Methods for preparing protein hydrolysates are generally acid, base, heat, or enzymatic hydrolysis. However, hydrolysis using heat, acid, or alkali hydrolysis can cause some amino acid damage and loss of nutritional value, thereby reducing consumer satisfaction. Therefore enzymatic hydrolysis is preferred. Food-grade enzymes are very effective in cutting specific peptides at specific sites (Cheng, Tu, Liu, Zhao, & Du, 2019; Tu, Cheng, Lu, & Du, 2018). The main sources of commercial enzymes commonly used for natural proteins are animals, plants, and microorganisms. These enzymes are protamex, bromelain, alkaline protease, flavourzyme, trypsin, pancreatin, pepsin, proteinase K, α-pancreas, chymotrypsin, neutral protease, and papain (Yathisha, Bhat, Karunasagar, & Mamatha, 2019). In the process of using enzymes to hydrolyze proteins, attention should be paid to the efficiency of enzymes, for instance which temperature and pH have the greatest influence on the reaction. Only at the optimum temperature and pH value can the maximum yield be achieved. In general, the optimal pH of neutral protease is around 7, whereas the optimal temperature is around 45 C (Xu, Chen, Wang, et al., 2019). Pepsin is an acidic protease so it works better at a pH of 2 (Chen et al., 2019), whereas trypsin is functional at a pH of 8. Pepsin and trypsin are digestive enzymes in the gastrointestinal tract, and their optimal temperature is generally about 37 C. Under optimal conditions, the degree of hydrolysis, the ratio of material to liquid, and the time of hydrolysis can also affect the composition of polypeptide and its osteogenic activity (Xu, Zhao, et al., 2019).

27.4.2 Identification of osteogenic peptides After various enzyme treatments, centrifugation is applied to obtain the hydrolysates that contain peptides with different molecular weights, different amino acid sequences, and different characteristics. In some research reports, peptides with low-molecular weight, or

680 Chapter 27 with the ability to chelate calcium ions, are more likely to have osteogenic activity. Therefore we aim to choose methods such as membrane filtration, membrane ultrafiltration, and column chromatography to further isolate peptides with potential osteogenic activity. In the process of separation and purification, because the peptide components will breed bacteria, each step of separation and purification may increase the number of microorganisms and cause deterioration.Therefore the resulting fractions are quickly freezedried at each step to prevent the growth of bacteria. The shortest peptide with osteogenesis-promoting activity contains only three amino acids such as IPP, VPP, LKP, and RGD (Huttunen et al., 2008; Yang et al., 2005). The longest peptide contains 34 amino acids such as SVSEIQLMHNLGKHLNSMERVGWLRKKLQDVHNF (Baron & Hesse, 2012). In order to further study the mechanism of promoting bone formation, it is crucial to be able to accurately identify the composition and sequence of the osteogenic peptide. The hydrolysate containing bioactive peptides after enzymolysis or fermentation is very complicated. This mixture composition cannot explain the mechanism of the peptide activity. Generally, the pure peptide is used to explore the mechanism of action. Therefore after the enzymatic hydrolysate, separation, and purification techniques including alcohol precipitation, membrane filtration, ion exchange chromatography, and gel chromatography are used to reduce the complexity of the enzymolysis solution. Then, the purpose of identifying the target peptide is achieved using some techniques such as HPLC-MS, UPLC-MS, UPLCTOF-MS, MADI-TOF-MS (Adoui et al., 2013), and CESI-TOF-MS (Chen et al., 2018). The schematic diagram of preparation, purification, and identification of osteopromoting peptides is shown in Fig. 27.3. Osteogenic peptides are important in regulating bone

Figure 27.3 Preparation, purification, identification, characterization, absorption, and structure
activity of osteogenic peptides.

Food-derived osteogenic peptides towards osteoporosis 681 turnover, and obtaining accurate peptide sequences is important for further evaluating their biological activities and exploring mechanisms in vivo or in vitro.

27.5 Bioavailability of osteogenic peptides 27.5.1 Absorption analysis Many factors affect the bioavailability of oral peptides. It is necessary to establish a suitable model to osteogenic peptides absorption in order to prove the mechanism of physical and biochemical barriers during osteogenic peptides absorption. There are two main absorption models, Caco-2 cell model (Hidalgo, Raub, & Borchardt, 1989) and turnover in the small intestine of mouse model (Neutze, Gooden, & Oddy, 1997). A Caco-2 cell model was established to simulate small intestinal epithelial cells to study these major absorption barriers. This model contains a variety of metabolic enzymes, which is close to the actual absorption in the body. This model can also determine the bioavailability and pathways of different drugs in the process of being absorbed and transported across the membrane. The Caco-2 cell line is derived from human colon cancer cells, and under certain culture conditions, it can spontaneously form polar, microvilli, and tight junctions that are similar to the differentiation characteristics of the brush border of the small intestine epithelial cells. It is an ideal model for studying drug transport mechanism and absorption characteristics in vitro. In addition, phospholipids are the main lipid form on mammalian epithelial cell membranes and can be used as a carrier to transport a variety of drugs. Some drugs wrapped with liposomes composed of phospholipids that can reduce the toxicity of the drugs so that the drugs act on the target site and enhance the therapeutic effect of the drugs. For permeability studies, an in vitro procedure utilizing everted rat intestinal sacs was valuable in predicting the permeability characteristics of various drug classes (Chowhan & Amaro, 1977). This model is more in line with the actual absorption of osteogenic peptides.

27.5.2 Pharmacokinetic analysis After confirming that the osteogenic peptides can be absorbed into the body, pharmacokinetics study is needed to further quantify the absorption, distribution, metabolism, and excretion of osteogenic peptides in vivo. The mathematical principles and methods are used to explain the blood drug concentration over time of change. The process is done by administrating osteogenic peptides orally to humans or animals, taking serum at different time periods, and finally determining the metabolism of peptides by liquid chromatography or mass spectrometry (Sturmer, Mehta, Giacchi, & Cagatay, 2013). In terms of distribution, metabolism, and excretion, fluorescently labeled peptides can be used. After feeding cells or animals with fluorescently labeled peptides, laser confocal

682 Chapter 27 (Xu, Yan, Liu, & Wu, 2018) or multifunctional imagers (Cong et al., 2019) can be used to further explore the distribution of bone peptides in cells or tissues.

27.6 Conclusions Food-derived osteogenic active peptides are of great significance. This chapter summarizes the preparation, separation, purification, and characterization methods of food-derived bioactive peptides. At the same time, this chapter also describes the pathogenesis and measurement methods of osteoporosis and discusses the biomarkers and osteogenic active peptides related to osteoporosis. It can be found that osteogenic peptides can be used to prevent osteoporosis by influencing the activity of OBs and OCs. Osteogenic peptides with RGD structure are more likely to interact with integrins to promote osteogenesis. Future studies are needed to develop nutritional and health products, osteoporosis drugs, and related bone materials using active peptides to promote osteogenesis.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31771926) and the State Key Research and Development Plan “Modern Food Processing and Food Storage and Transportation Technology and Equipment” (2017YFD0400201).

Reference Adoui, F., Boughera, F., Chataigne, G., Chihib, N. E., Hameur, H. E., Dhulster, P., & Nedjar-Arroume, N. (2013). A simple method to separate the antimicrobial peptides from complex peptic casein hydrolysate and identification of a novel antibacterial domains within the sequence of bovine αs-casein. International Review of Chemical Engineering—Rapid Communicati, 5(2), 179
187. Agrawal, V., Kelly, J., Tottey, S., Daly, K. A., Johnson, S. A., Siu, B. F., & Badylak, S. F. (2011). An isolated cryptic peptide influences osteogenesis and bone remodeling in an adult mammalian model of digit amputation. Tissue Engineering. Part A, 17, 3033
3044. Agrawal, V., Tottey, S., Johnson, S. A., Freund, J. M., Siu, B. F., & Badylak, S. F. (2011). Recruitment of progenitor cells by an extracellular matrix cryptic peptide in a mouse model of digit amputation. Tissue Engineering. Part A, 17, 2435
2443. Anastasilakis, A. D., Polyzos, S. A., Avramidis, A., Toulis, K. A., Papatheodorou, A., & Terpos, E. (2009). The effect of teriparatide on serum Dickkopf-1 levels in postmenopausal women with established osteoporosis. Clinical Endocrinology, 72(6), 752
757. Bab, I., Gazit, D., Chorev, M., Muhlrad, A., Shteyer, A., Greenberg, Z., & Kahn, A. (1992). Histone H4-related osteogenic growth peptide (OGP): A novel circulating stimulator of osteoblastic activity. The EMBO Journal, 11(5), 1867
1873. Baron, R., & Hesse, E. (2012). Update on bone anabolics in osteoporosis treatment: Rationale, current status, and perspectives. The Journal of Clinical Endocrinology and Metabolism, 97(2), 311
325. Bergeron, E., Marquis, M. E., Chretien, I., & Faucheux, N. (2007). Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres. Journal of Materials Science: Materials in Medicine, 18(2), 255
263.

Food-derived osteogenic peptides towards osteoporosis 683 Bilezikian, J. P., Bouillon, R., Clemens, T. L., Compston, J. E., Bauer, D. C., Ebeling, P. R., . . . Beur, S. M. J. (2013). Primer on the metabolic bone diseases and disorders of mineral metabolism. Journal of Bone and Joint Surgery-British Volume, 2, 1087. Bragdon, B., Thinakaran, S., Moseychuk, O., Gurski, L., Bonor, J., Price, C., & Nohe, A. (2011). Casein kinase 2 regulates in vivo bone formation through its interaction with bone morphogenetic protein receptor type Ia. Bone, 49(5), 944
954. Cann, C. E., Martin, M. C., Genant, H. K., & Jaffe, R. B. (1984). Decreased spinal mineral content in amenorrheic women. The Journal of the American Medical Association, 251(5), 626
629. Chen, H., Emura, S., Yao, X., & Shoumura, S. (2004). Morphological study of the parathyroid gland and thyroid C cell in senescence-accelerated mouse (SAMP6), a murine model for senile osteoporosis. Tissue & Cell, 36(6), 409
415. Chen, H., Shi, P., Fan, F., Tu, M., Xu, Z., Xu, X., & Du, M. (2018). Complementation of UPLC-Q-TOF-MS and CESI-Q-TOF-MS on identification and determination of peptides from bovine lactoferrin. Journal of Chromatography B, 1084, 150
157. Chen, H., Xu, Z., Fan, F., Shi, P., Tu, M., Wang, Z., & Du, M. (2019). Identification and mechanism evaluation of a novel osteogenesis promoting peptide from Tubulin Alpha-1C chain in Crassostrea gigas. Food Chemistry, 272, 751
757. Chen, Y., & Webster, T. J. (2009). Increased osteoblast functions in the presence of BMP-7 short peptides for nanostructured biomaterial applications. Journal of Biomedical Materials Research. Part A, 91(1), 296
304. Cheng, S., Tu, M., Liu, H., Zhao, G., & Du, M. (2019). Food-derived antithrombotic peptides: Preparation, identification, and interactions with thrombin. Critical Reviews in Food Science and Nutrition, 59, S81
S95. Choi, Y. J., Lee, J. Y., Chung, C., & Park, Y. J. (2013). Enhanced osteogenesis by collagen-binding peptide from bone sialoprotein in vitro and in vivo. Journal of Biomedical Materials Research Part A, 101(2), 547
554. Choi, Y. J., Lee, J. Y., Park, J. H., Park, J. B., Jin, S. S., Choi, Y. S., & Park, Y. J. (2010). The identification of a heparin binding domain peptide from bone morphogenetic protein-4 and its role on osteogenesis. Biomaterials, 31(28), 7226
7238. Chowhan, Z. T., & Amaro, A. A. (1977). Everted rat intestinal sacs as an in vitro model for assessing absorptivity of new drugs. Journal of Pharmaceutical Sciences, 66(9), 1249
1253. Cong, S., Wang, N., Wang, K., Wu, Y., Li, D., Song, Y., & Tan, M. (2019). Fluorescent nanoparticles in the popular pizza: Properties, biodistribution and cytotoxicity. Food & Function, 10(5), 2408
2416. Cooper, G. J. S. (1994). Amylin compared with calcitonin gene-related peptide: Structure, biology, and relevance to metabolic disease. Endocrine Reviews, 15(2), 163
201. Cornish, J., Callon, K., Bava, U., Watson, M., Xu, X., Lin, J., & Buchanan, C. (2007). Preptin, another peptide product of the pancreatic beta-cell, is osteogenic in vitro and in vivo. American Journal of Physiology. Endocrinology and Metabolism, 292(1), E117. Dai, Z., Wu, F., Yeung, E. W., & Li, Y. (2010). IGF-IEc expression, regulation and biological function in different tissues. Growth Hormone & IGF Research, 20(4), 275
281. Daliri, E. B., Lee, B. H., & Oh, D. H. (2018). Current trends and perspectives of bioactive peptides. Critical Reviews in Food Science and Nutrition, 58(13), 2273
2284. Ebetino, F. H., Hogan, A. M., Sun, S., Tsoumpra, M. K., Duan, X., Triffitt, J. T., & Oppermann, U. (2011). The relationship between the chemistry and biological activity of the bisphosphonates. Bone, 49(1), 20
33. Egusa, H., Kaneda, Y., Akashi, Y., Hamada, Y., Matsumoto, T., Saeki, M., & Yatani, H. (2009). Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts. Biomaterials, 30(27), 4676
4686. Eriksen, E. F. (2010). Cellular mechanisms of bone remodeling. Reviews in Endocrine & Metabolic Disorders, 11(4), 219
227. Finkelstein, J. S., Wyland, J. J., Lee, H., & Neer, R. M. (2010). Effects of teriparatide, alendronate, or both in women with postmenopausal osteoporosis. The Journal of Clinical Endocrinology and Metabolism, 95(4), 1838
1845.

684 Chapter 27 Fledelius, C., Johnsen, A. H., Cloos, P., Bonde, M., & Qvist, P. (1997). Characterization of urinary degradation products derived from type I collagen identification of a β-isomerized Asp-Gly sequence within the Cterminal telopeptide (α1) region. Journal of Biological Chemistry, 272(15), 9755
9763. Fosgerau, K., & Hoffmann, T. (2015). Peptide therapeutics: Current status and future directions. Drug Discovery Today, 20(1), 122
128. Fukushima, N., Hiraoka, K., Shirachi, I., Kojima, M., & Nagata, K. (2010). Isolation and characterization of a novel peptide, osteoblast activating peptide (OBAP), associated with osteoblast differentiation and bone formation. Biochemical and Biophysical Research Communications, 400(1), 157
163. Garnero, P., & Delmas, P. D. (1993). Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease. The Journal of Clinical Endocrinology and Metabolism, 77(4), 1046
1053. Garnero, P., Ferreras, M., Karsdal, M. A., Nicamhlaoibh, R., Risteli, J., Borel, O., & Delaisse, J. (2003). The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. Journal of Bone and Mineral Research, 18(5), 859
867. Garnero, P., Grimaux, M., Seguin, P., & Delmas, P. D. (2009). Characterization of immunoreactive forms of human osteocalcin generated in vivo and in vitro. Journal of Bone and Mineral Research, 9(2), 255
264. Gatti, D., Viapiana, O., Idolazzi, L., Fracassi, E., Rossini, M., & Adami, S. (2011). The waning of teriparatide effect on bone formation markers in postmenopausal osteoporosis is associated with increasing serum levels of DKK1. The Journal of Clinical Endocrinology and Metabolism, 96(5), 1555
1559. Guo, D., Liu, W., Zhang, X., Zhao, M., Zhu, B., Hou, T., & He, H. (2019). Duck egg white-derived peptide VSEE (Val-Ser-Glu-Glu) regulates bone and lipid metabolisms by Wnt/β-catenin signaling pathway and intestinal microbiota. Molecular Nutrition & Food Research, 63(24), 1900525. Halleen, J. M., Tiitinen, S. L., Ylipahkala, H., Fagerlund, K. M., & Vaananen, H. K. (2006). Tartrate-resistant acid phosphatase 5b (TRACP 5b) as a marker of bone resorption. Clinical Laboratory, 52, 499. Hannon, R. A., Clowes, J. A., Eagleton, A. C., Hadari, A. A., Eastell, R., & Blumsohn, A. (2004). Clinical performance of immunoreactive tartrate-resistant acid phosphatase isoform 5b as a marker of bone resorption. Bone, 34(1), 187
194. Henriksen, K., Andersen, J. R., Riis, B. J., Mehta, N., Tavakkol, R., Alexandersen, P., & Stern, W. (2013). Evaluation of the efficacy, safety and pharmacokinetic profile of oral recombinant human parathyroid hormone [rhPTH(1
31)NH2] in postmenopausal women with osteoporosis. Bone, 53(1), 160
166. Hidalgo, I. J., Raub, T. J., & Borchardt, R. T. (1989). Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology, 96(3), 736
749. Ho, A. Y. Y., & Kung, A. W. C. (2005). Determinants of peak bone mineral density and bone area in young women. Journal of Bone and Mineral Metabolism, 23(6), 470
475. Hoang, Q. Q., Sicheri, F., Howard, A., & Yang, D. S. C. (2003). Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature, 425(6961), 977
980. Huttunen, M. M., Pekkinen, M., Ahlstrom, M., & Lambergallardt, C. (2008). Long-term effects of tripeptide IlePro-Pro on osteoblast differentiation in vitro. Journal of Nutritional Biochemistry, 19(10), 708
715. Itoh, D., Yoneda, S., Kuroda, S., Kondo, H., Umezawa, A., Ohya, K., & Kasugai, S. (2002). Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro. Journal of Biomedical Materials Research, 62(2), 292
298. Ivaska, K. K., Hellman, J., Likojarvi, J., Kakonen, S., Gerdhem, P., Akesson, K., & Vaananen, H. K. (2003). Identification of novel proteolytic forms of osteocalcin in human urine. Biochemical and Biophysical Research Communications, 306(4), 973
980. Ivaska, K. K., Hentunen, T., Vaaraniemi, J., Ylipahkala, H., Pettersson, K., & Vaananen, H. K. (2004). Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro. Journal of Biological Chemistry, 279(18), 18361
18369. Ivaska, K. K., Kakonen, S., Gerdhem, P., Obrant, K., Pettersson, K., & Vaananen, H. K. (2005). Urinary osteocalcin as a marker of bone metabolism. Clinical Chemistry, 51(3), 618
628.

Food-derived osteogenic peptides towards osteoporosis 685 J., F. A., E., K. B., NEIL, K. G., M., M. J., MING-HAO, Z., J., R. D., & C., N. G. (1991). A carboxyl-terminal peptide from the parathyroid hormone-related protein inhibits bone resorption by osteoclasts. Endocrinology, 129(4), 1762
1768. Jilka, R. L., Obrien, C. A., Ali, A. A., Roberson, P. K., Weinstein, R. S., & Manolagas, S. C. (2009). Intermittent PTH stimulates periosteal bone formation by actions on post-mitotic preosteoblasts. Bone, 44 (2), 275
286. Juhasz, T., Matta, C., Katona, E., Somogyi, C., Takacs, R., Hajdu, T., & Toth, G. (2014). Pituitary adenylate cyclase-activating polypeptide (PACAP) signalling enhances osteogenesis in UMR-106 cell line. Journal of Molecular Neuroscience, 54(3), 555
573. Kanis, J. A., & Adami, S. (1994). Bone loss in the elderly. Journal of Prosthetic Dentistry, 4(1), S59
S65. Kanis, J. A., Johnell, O., Oden, A., Dawson, A., De Laet, C., & Jonsson, B. (2001). Ten year probabilities of osteoporotic fractures according to BMD and diagnostic thresholds. Osteoporosis International, 12(12), 989
995. Kim, H. K., Lee, J. S., Kim, J., Seon, J. K., Park, K. S., Jeong, M. H., & Yoon, T. R. (2017). Bone-forming peptide-2 derived from BMP-7 enhances osteoblast differentiation from multipotent bone marrow stromal cells and bone formation. Experimental and Molecular Medicine, 49(5). Komatsu, D. E., Hadjiargyrou, M., Udin, S. M., Trasolini, N. A., & Pentyala, S. (2015). Identification and characterization of a synthetic osteogenic peptide. Calcified Tissue International, 97(6), 611
623. Kousteni, S., & Bilezikian, J. P. (2008). The cell biology of parathyroid hormone in osteoblasts. Current Osteoporosis Reports, 6(2), 72
76. Lee, G., Byun, H., Kim, M., Lee, B., Ko, S., Jung, E., & Jo, H. (2008). The beneficial effect of the sap of Acer mono in an animal with low-calcium diet-induced osteoporosis-like symptoms. British Journal of Nutrition, 100(5), 1011
1018. Lee, J. E., Kim, K. M., Kim, L. K., Kim, K. Y., Oh, T. J., Moon, J. H., & Shin, C. S. (2017). Comparisons of TBS and lumbar spine BMD in the associations with vertebral fractures according to the T-scores: A cross-sectional observation. Bone, 105, 269
275. Lee, J., Choo, J., Park, H. J., Park, J., Lee, S. C., Jo, I., & Park, Y. (2007). Injectable gel with synthetic collagen-binding peptide for enhanced osteogenesis in vitro and in vivo. Biochemical and Biophysical Research Communications, 357(1), 68
74. Lenz, A., Bennett, M., Skelton, W. P., & Vesely, D. L. (2010). Vessel dilator and C-type natriuretic peptide enhance the proliferation of human osteoblasts. Pediatric Research, 68(5), 405
408. Li, G., Ryaby, J. T., Carney, D. H., & Wang, H. (2005). Bone formation is enhanced by thrombin-related peptide TP508 during distraction osteogenesis. Journal of Orthopaedic Research, 23(1), 196
202. Li, J., Hong, J., Zheng, Q., Guo, X., Lan, S., Cui, F., & Chen, C. (2011). Repair of rat cranial bone defects with nHAC/PLLA and BMP-2-related peptide or rhBMP-2. Journal of Orthopaedic Research, 29(11), 1745
1752. Lorget, F., Kaci, N., Peng, J., Benoistlasselin, C., Mugniery, E., Oppeneer, T., & Bunting, S. (2012). Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia. American Journal of Human Genetics, 91(6), 1108
1114. Mada, S. B., Reddi, S., Kumar, N., Kumar, R., & Ahmad, N. (2017). Antioxidative peptide from milk exhibits anti-osteopenic effects through inhibition of oxidative damage and bone-resorbing cytokines in ovariectomized rats. Nutrition, 43, 21. Manolagas, S. C. (2000). Birth and death of bone cells: Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocrine Reviews, 21(2), 115
137. Marcone, S., Belton, O., & Fitzgerald, D. J. (2017). Milk-derived bioactive peptides and their health promoting effects: A potential role in atherosclerosis. British Journal of Clinical Pharmacology, 83(1), 152
162. Matkovic, V., Jelic, T., Wardlaw, G. M., Ilich, J. Z., Goel, P. K., Wright, J. K., & Heaney, R. P. (1994). Timing of peak bone mass in Caucasian females and its implication for the prevention of osteoporosis. Inference from a cross-sectional model. Journal of Clinical Investigation, 93(2), 799
808. Melton, L. J., Chrischilles, E. A., Cooper, C., Lane, A. W., & Riggs, B. L. (2005). How many women have osteoporosis. Journal of Bone and Mineral Research, 20(5), 886
892.

686 Chapter 27 Miller, P. D., & Derman, R. J. (2010). What is the best balance of benefits and risks among anti-resorptive therapies for postmenopausal osteoporosis? Osteoporosis International, 21(11), 1793
1802. Min, J., Yuan, Z., Zhang, Q., Lin, S., Wang, K., & Luo, J. (2018). Analysis of anti-osteoporosis function of chlorogenic acid by gene microarray profiling in ovariectomy rat model. Bioscience Reports, 38(4). Nagel, D. E., Khosla, S., Sanyal, A., Rosen, D. M., Kumagai, Y., & Riggs, B. L. (2004). A fragment of the hypophosphatemic factor, MEPE, requires inducible cyclooxygenase-2 to exert potent anabolic effects on normal human marrow osteoblast precursors. Journal of Cellular Biochemistry, 93(6), 1107
1114. Neerup, T. S. R., Stahlhut, M., Petersen, J. S., Daugaard, J. R., Jensen, J. B., Peng, Z., & Thorkildsen, C. (2011). ZP2307, a novel, cyclic PTH(1-17) analog that augments bone mass in ovariectomized rats. Bone, 48(6), 1319
1327. Neutze, S. A., Gooden, J. M., & Oddy, V. H. (1997). Measurement of protein turnover in the small intestine of lambs. 1. Development of an experimental model. The Journal of Agricultural Science, 128(2), 217
231. Orˇsoli´c, N., Goluˇza, E., Ðiki´c, D., Lisiˇci´c, D., Saˇsilo, K., Roðak, E., & Orct, T. (2014). Role of flavonoids on oxidative stress and mineral contents in the retinoic acid-induced bone loss model of rat. European Journal of Nutrition, 53(5), 1217
1227. Parfitt, A. M. (2002). Targeted and nontargeted bone remodeling: Relationship to basic multicellular unit origination and progression. Bone, 30(1), 5
7. Plotkin, L. I., & Bellido, T. (2013). Beyond gap junctions: Connexin43 and bone cell signaling. Bone, 52(1), 157
166. Reddi, S., Kumar, N., Vij, R., Mada, S. B., Kapila, S., & Kapila, R. (2016). Akt drives buffalo casein-derived novel peptide-mediated osteoblast differentiation. Journal of Nutritional Biochemistry, 38, 134
144. Reginster, J., & Burlet, N. (2006). Osteoporosis: A still increasing prevalence. Bone, 38(2), 4
9. Ren, Z., Yang, L., Xue, F., Meng, Q., Wang, K., Wu, X., & Zhou, L. (2013). Yeast-incorporated gallium attenuates glucocorticoid-induced bone loss in rats by inhibition of bone resorption. Biological Trace Element Research, 152(3), 396
402. Riggs, B. L., & Parfitt, A. M. (2004). Drugs used to treat osteoporosis: The critical need for a uniform nomenclature based on their action on bone remodeling. Journal of Bone and Mineral Research, 20(2), 177
184. Rissanen, J. P., Suominen, M. I., Peng, Z., & Halleen, J. M. (2008). Secreted tartrate-resistant acid phosphatase 5b is a marker of osteoclast number in human osteoclast cultures and the rat ovariectomy model. Calcified Tissue International, 82(2), 108
115. Saini, V., Marengi, D. A., Barry, K. J., Fulzele, K., Heiden, E., Liu, X., & Baron, R. (2013). Parathyroid hormone (PTH)/PTH-related peptide type 1 receptor (PPR) signaling in osteocytes regulates anabolic and catabolic skeletal responses to PTH. Journal of Biological Chemistry, 288(28), 20122
20134. Saito, A., Suzuki, Y., Ogata, S. I., Ohtsuki, C., & Tanihara, M. (2005). Accelerated bone repair with the use of a synthetic BMP-2-derived peptide and bone-marrow stromal cells. Journal of Biomedical Materials Research. Part A, 72(1), 77
82. Sanders, K. M., Nicholson, G. C., Watts, J. J., Pasco, J. A., Henry, M. J., Kotowicz, M. A., & Seeman, E. (2006). Half the burden of fragility fractures in the community occur in women without osteoporosis. When is fracture prevention cost-effective? Bone, 38(5), 0
700. Schuit, S. C. E., Der Klift, M. V., Weel, A. E. A. M., De Laet, C., Burger, H., Seeman, E., & Pols, H. A. P. (2004). Fracture incidence and association with bone mineral density in elderly men and women: The Rotterdam Study. Bone, 34(1), 195
202. Seeman, E. (2006). Osteocytes—Martyrs for integrity of bone strength. Osteoporosis International, 17(10), 1443
1448. Seibel, M. J., Robins, S. P., & Bilezikian, J. P. (1992). Urinary pyridinium crosslinks of collagen: Specific markers of bone resorption in metabolic bone disease. Trends in Endocrinology and Metabolism, 3(7), 263
270. Shapses, S. A., Pop, L. C., & Wang, Y. (2017). Obesity is a concern for bone health with aging. Nutrition Research, 39, 1
13. Shin, H., Zygourakis, K., Farachcarson, M. C., Yaszemski, M. J., & Mikos, A. G. (2004). Attachment, proliferation, and migration of marrow stromal osteoblasts cultured on biomimetic hydrogels modified with an osteopontin-derived peptide. Biomaterials, 25(5), 895
906.

Food-derived osteogenic peptides towards osteoporosis 687 Shiraki, M., & Orimo, H. (1991). The effect of estrogen and, sex-steroids and thyroid hormone preparation on bone mineral density in senile osteoporosis—A comparative study of the effect of 1 alphahydroxycholecalciferol (1 alpha-OHD3) on senile osteoporosis. Nihon Naibunpi Gakkai zasshi, 67(2), 84
95. Sibilia, V., Cocchi, D., Villa, I., Lattuada, N., Soglian, A., Rubinacci, A., & Netti, C. (2002). Bone effects of hexarelin, a GH-releasing peptide, in female rats: Influence of estrogen milieu. European Journal of Endocrinology, 146(6), 855
862. Siddique, N., Odonoghue, M., Casey, M. C., & Walsh, J. B. (2017). Malnutrition in the elderly and its effects on bone health—A review. Clinical Nutrition ESPEN, 21, 31
39. Srivastava, A. K., Mohan, S., Singer, F. R., & Baylink, D. J. (2002). A urine midmolecule osteocalcin assay shows higher discriminatory power than a serum midmolecule osteocalcin assay during short-term alendronate treatment of osteoporotic patients. Bone, 31(1), 62
69. Stevens, J. A., & Rudd, R. A. (2013). The impact of decreasing U.S. hip fracture rates on future hip fracture estimates. Osteoporosis International, 24(10), 2725
2728. Sturmer, A., Mehta, N., Giacchi, J., & Cagatay, T. (2013). Pharmacokinetics of oral recombinant human parathyroid hormone [rhPTH(1
31)NH2] in postmenopausal women with osteoporosis. Clinical Pharmacokinetics, 52(11), 995
1004. Suzuki, Y., Tanihara, M., Suzuki, K., Saitou, A., Sufan, W., & Nishimura, Y. (2000). Alginate hydrogel linked with synthetic oligopeptide derived from BMP-2 allows ectopic osteoinduction in vivo. Journal of Biomedical Materials Research, 50(3), 405
409. Szulc, P., Seeman, E., & Delmas, P. D. (2000). Biochemical measurements of bone turnover in children and adolescents. Osteoporosis International, 11(4), 281
294. Tang, S., Xie, H., Yuan, L., Luo, X., Huang, J., Cui, R., & Liao, E. (2007). Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways. Peptides, 28(3), 708
718. Thorwarth, M., Schultzemosgau, S., Wehrhan, F., Kessler, P., Srour, S., Wiltfang, J., & Schlegel, K. A. (2005). Bioactivation of an anorganic bone matrix by P-15 peptide for the promotion of early bone formation. Biomaterials, 26(28), 5648
5657. Tsuchita, H., Goto, T., Shimizu, T., Yonehara, Y., & Kuwata, T. (1996). Dietary casein phosphopeptides prevent bone loss in aged ovariectomized rats. Journal of Nutrition, 126(1), 86
93. Tu, M., Cheng, S., Lu, W., & Du, M. (2018). Advancement and prospects of bioinformatics analysis for studying bioactive peptides from food-derived protein: Sequence, structure, and functions. Trends in Analytical Chemistry, 105, 7
17. Uehara, K., Takahashi, A., Watanabe, M., & Nomura, Y. (2014). Shark protein improves bone mineral density in ovariectomized rats and inhibits osteoclast differentiation. Nutrition, 30(6), 719
725. Vaananen, H. K., & Laitalaleinonen, T. (2008). Osteoclast lineage and function. Archives of Biochemistry and Biophysics, 473(2), 132
138. Valentijn, K., Gutow, A. P., Troiano, N., Gundberg, C., Gilligan, J. P., & Vignery, A. (1997). Effects of calcitonin gene-related peptide on bone turnover in ovariectomized rats. Bone, 21(3), 0
274. Vestergaard, P. (2007). Discrepancies in bone mineral density and fracture risk in patients with type 1 and type 2 diabetes—A meta-analysis. Osteoporosis International, 18(4), 427
444. Wang, V., Misra, G., & Amsden, B. G. (2008). Immobilization of a bone and cartilage stimulating peptide to a synthetic bone graft. Journal of Materials Science: Materials in Medicine, 19(5), 2145
2155. Weiss, M. J., Ray, K., Henthorn, P. S., Lamb, B. T., Kadesch, T., & Harris, H. (1988). Structure of the human liver/bone/kidney alkaline phosphatase gene. Journal of Biological Chemistry, 263(24), 12002
12010. Wojtowicz, A. M., Shekaran, A., Oest, M. E., Dupont, K. M., Templeman, K. L., Hutmacher, D. W., & Garcia, A. J. (2010). Coating of biomaterial scaffolds with the collagen-mimetic peptide GFOGER for bone defect repair. Biomaterials, 31(9), 2574
2582. Xu, W., Yan, X., Liu, N., & Wu, G. (2018). P1c peptide decorated liposome targeting αvβ3-expressing tumor cells in vitro and in vivo. RSC Advances, 8(45), 25575
25583.

688 Chapter 27 Xu, Z., Chen, H., Fan, F., Shi, P., Tu, M., Cheng, S., & Du, M. (2019). Bone formation activity of an osteogenic dodecapeptide from blue mussels (Mytilus edulis). Food & Function, 10(9), 5616
5625. Xu, Z., Chen, H., Wang, Z., Fan, F., Shi, P., Tu, M., & Du, M. (2019). Isolation and characterization of peptides from Mytilus edulis with osteogenic activity in mouse MC3T3-E1 preosteoblast cells. Journal of Agricultural and Food Chemistry, 67(5), 1572
1584. Xu, Z., Zhao, F., Chen, H., Xu, S., Fan, F., Shi, P., & Du, M. (2019). Nutritional properties and osteogenic activity of enzymatic hydrolysates of proteins from the blue mussel (Mytilus edulis). Food & Function, 10 (12), 7745
7754. Yang, F., Williams, C. G., Wang, D., Lee, H., Manson, P. N., & Elisseeff, J. H. (2005). The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials, 26(30), 5991
5998. Yang, L., Yang, J., Pan, T., & Zhong, X. (2019). Liraglutide increases bone formation and inhibits bone resorption in rats with glucocorticoid-induced osteoporosis. Journal of Endocrinological Investigation, 42 (9), 1125
1131. Yathisha, U. G., Bhat, I., Karunasagar, I., & Mamatha, B. S. (2019). Antihypertensive activity of fish protein hydrolysates and its peptides. Critical Reviews in Food Science and Nutrition, 59(15), 2363
2374. Yoo, S. Y., Kobayashi, M., Lee, P. P., & Lee, S. (2011). Early osteogenic differentiation of mouse preosteoblasts induced by collagen-derived DGEA-peptide on nanofibrous phage tissue matrices. Biomacromolecules, 12(4), 987
996. Zaidi, M., Fuller, K., Bevis, P. J. R., Gainesdas, R. E., & Macintyre, I. (1987). Calcitonin gene-related peptide inhibits osteoclastic bone resorption: A comparative study. Calcified Tissue International, 40(3), 149
154. Zhao, Z., Shao, L., Zhao, H. M., Zhong, Z. H., Liu, J., & Hao, C. (2011). Osteogenic growth peptide accelerates bone healing during distraction osteogenesis in rabbit tibia. Journal of International Medical Research, 39 (2), 456
463. Zvereva, I., Dudko, G., & Dikunets, M. (2017). Strategies of GnRH synthetic analogues determination in doping control: In vitro and in vivo metabolism data; evaluation of LH and steroid profile parameters fluctuations as suitable biomarkers. Drug Testing & Analysis, 10(4), 711
722.

CHAPTER 28

Applications in medicine: mental health Yorick Janssens1, Evelien Wynendaele1, Kurt Audenaert2 and Bart De Spiegeleer1 1

Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium, 2Department of Psychiatry and Medical Psychology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium

28.1 Introduction Until the 1960s, it was believed that information processing and communication between neurons occurs only by the release of amine neurotransmitters such as acetylcholine and serotonin in synapses followed by an appreciation that amino acids such as gammaaminobutyric acid, glutamic acid, aspartic acid, and glycine also might serve as transmitters. Then, due to pioneering work of Nobel prize winners Schally and Guillemin, it was demonstrated that most hypothalamic releasing and inhibiting hormones could be chemically identified as small peptides (Hokfelt et al., 2000). These “neuropeptides” are defined as “small proteinaceous substances produced and released by neurons through the regulated secretory route and acting on neural substrates”. They are thus distinguished from other peptides such as peptide hormones by the fact that they are both synthesized by and act on neurons. Neuropeptides are very diverse and over 100 known neuropeptides have been identified to date. Based on structural homologies, these peptides can be grouped into families such as the opioids, natriuretic factors, gonadotropin-releasing hormones, and many others. Neuropeptides are posttranslationally processed from a precursor protein and are released via secretory vesicles. The difference with classic neurotransmitters is that they do not require presynaptic machinery for their release and can be released at other regions of the neuronal cell as well. Once released, neuropeptides diffuse from their point of release and can act on their targets at a relatively large distance. For this reason, neuropeptides are widely distributed in both the peripheral and central nervous system (CNS) where they can act as brain modulators as well as peripheral signaling molecules (Russo, 2017). After release, the neuropeptides are generally degraded by proteases while classic neurotransmitters are reinternalized and recuperated. This results in lower endogenous concentrations of neuropeptides, but their potency is much higher. Target receptors of these peptides have almost exclusively a G protein-coupled receptor (GPCR) structure. After interaction with its receptor, a conformational change of the transmembrane region takes

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00020-0 © 2021 Elsevier Inc. All rights reserved.

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690 Chapter 28 place, allowing the intracellular domain to activate the heterotrimeric G-protein. Neuropeptides almost always coexist and are thus complementary, with one or more classic neurotransmitters; for example, by modulating their actions. However, contrary to the classic neurotransmitters, these peptides can also exert trophic actions elsewhere in the body; for example, by altering bone mineralization (vasoactive intestinal peptide) or stimulating growth of fibroblasts and smooth muscle cells (substance P) (Hokfelt et al., 2000). In addition to neuropeptides, peripheral peptides, originating from other anatomical locations, also have effects on the CNS. One of the best-known examples is their effects on food intake control. For example, ghrelin, a 28-amino acid acylated peptide, which is mainly produced by the stomach, plays important roles in maintaining growth hormone release, stimulation of appetite, stimulation of prolactin, and others. Other gastrointestinal peptides such as glucagon-like peptides, cholecystokinin, bombesin, and Peptide YY are involved in the control of food intake and have either orexigenic (stimulate food intake) or anorexigenic (attenuate food intake) effects (Tassone et al., 2007). Leptin, a peptide hormone produced by adipose tissue, is also involved in the regulation of food intake and induces satiety and reduces food intake. This peptide also regulates the expression of various other neuropeptides in the brain which are involved in satiety control and food intake (e.g., Neuropeptide Y, POMC,. . .) (Guzman, Hernandez-Coronado, Rosales-Torres, & Hernandez-Medrano, 2019). These systems can be exploited to develop peptide therapeutics against food-related diseases such as obesity and anorexia and are discussed in another chapter in this book. More recently, it has been found that also peptides produced by bacteria, namely quorum sensing peptides, are able to reach the brain and have effects on brain cells in vitro (Janssens et al., 2018; Wynendaele et al., 2015). Food-derived peptides, which are ingested due to proteolytic cleavage of food proteins, also affect the brain. For instance, ingestion of soy-derived peptides led to a significant increase of noradrenaline expression in the brain stem of rats (Imai et al., 2017). Ingestion of different dietary proteins (zein, gluten, casein, lactalbumin, and soy-derived protein) have differential effects on brain tryptophan uptake and serotonin synthesis, probably mediated via cryptic peptides of these proteins (Fernstrom et al., 2013). Casein-derived opioid peptides, derived from bovine milk, show epigenetic effects on neuronal cells in vitro. Bovine β-casomorphin-7 decreased global DNA methylation in differentiating neuronal stem cells, hereby promoting neurogenesis (Trivedi, Zhang, Lopez-Toledano, Clarke, & Deth, 2016). Other opioid peptides, derived mainly from milk (casomorphins, lactorphins, and casoxins) and wheat (gluten exorphins and gliadorphins) and to a lesser extent from soy (soymorphins), spinach (rubiscolins), and rice (oryzatensin) have effects on nociception, spontaneous behavior, and learning and memory in in vivo models (Liu & Udenigwe, 2019).

Applications in medicine: mental health 691 This chapter discusses the broad effects of peptides on the CNS and their applications in medicine. The use of peptides in diagnostics and treatment of psychiatric and neurodegenerative disorders is discussed. For more information concerning neuropeptides and the different neuropeptide families, we refer to the numerous reviews in the literature and the Handbook of Biologically Active Peptides (Hokfelt et al., 2000; Hokfelt, Bartfai, & Bloom, 2003; Kastin, 2013; Russo, 2017).

28.1.1 Peptide transport across the blood
brain barrier and use as shuttles The blood
brain barrier (BBB) is a structural and functional barrier between the blood and the interstitial fluid of the brain. It protects the brain from peripheral toxins and maintains the brain’s homeostatic biochemical environment which is necessary for correct neural functioning and essential for survival. The barrier is mainly formed by the capillary endothelial cells which, in contrast to capillaries outside the CNS, are not fenestrated. These endothelial cells are connected to each other by tight junctions which makes paracellular transport of molecules quasi-impossible and are surrounded by the basal membrane. In addition, the endothelial cells are surrounded by pericytes and astroglial foot processes; and in the space between the endothelial cells and the interstitial fluid, perivascular macrophages play a role in the CNS immune function. Finally, the endothelial cells also form a metabolic barrier as they contain membrane and cytosolic enzymes. The assembly of these endothelial cells, pericytes, astroglia, and macrophages form the neurovascular unit which is the structural and functional unit of the BBB (Lee & Jayant, 2019). Despite the presence of this barrier, molecules are still able to cross the BBB by passive transmembrane diffusion and/or by specific transport systems which transport compounds by carrier-, receptor-, or adsorptive-mediated transfer mechanisms (Sanchez-Navarro, Giralt, & Teixido, 2017) (Fig. 28.1). In contrast, active efflux transporters such as the P-glycoprotein which pump compounds back into the blood exist as well (Van Dorpe et al., 2012). Different neurological disorders such as multiple sclerosis, stroke, Alzheimer’s disease (AD), and traumatic brain injuries, but also normal aging, can affect the integrity of the BBB, thereby increasing its permeability (Daneman & Prat, 2015; Farrall & Wardlaw, 2009). However, certain areas in the brain lack the presence of this barrier. The circumventricular organs (CVOs) are midline structures located around the third and fourth ventricles that permit polypeptide hypothalamic hormones to leave the brain without disrupting the BBB and permit substances that do not cross the BBB to trigger changes in brain function. The pineal gland, neurohypophysis, and subcommisural terminalis are classified as the secretory CVOs, whereas the subfornical organ, area postrema, and organum vasculosum are classified as the sensory CVOs. These organs are important sites for communication with the cerebrospinal fluid (CSF) as well as between the brain and peripheral organs via blood-borne molecules. The lack of the BBB is necessary to enable this communication. For example, the area postrema is involved in the detection of toxins in the blood and acts as a vomit-inducing

692 Chapter 28

Figure 28.1 Schematic representation of the blood
brain barrier (BBB). (A) The BBB is formed by a monolayer of endothelial cells in close contact with astrocytes end-feet and pericytes. The endothelial cells are strongly bound by tight junctions (TJs) proteins and adherens junctions (AJs) proteins. Transport mechanisms at the BBB are classified according to their energy requirements; (B) active transport mechanisms, which include transport mediated by receptor or transporter and adsorptive-mediated transport; (C) passive transport mechanisms that encompass transcellular passive diffusion and paracellular diffusion. Source: Reprinted from Sanchez-Navarro M., Giralt E., Teixido M. (2017). Blood-brain barrier peptide shuttles. Current Opinion in Chemical Biology; 38: 134
140.

center. These organs are lined by an ependymal lining which contain modified ependymal cells called tanycytes, which are involved in transport of molecules (Kaur & Ling, 2017). Whether peripheral peptides are able to cross this BBB remained a matter of great skepticism as it was initially believed that peptides could not pass this barrier. It was due to the pioneering work of Abba J. Kastin and William A. Banks in the early 80s that this skepticism was refuted (Banks, 2015). To date, over 250 peptide structures are included in the Brainpeps database, a database that contains BBB transport information of peptides (Van Dorpe et al., 2012). Based on this data, Stalmans et al. (2015) developed a classification scheme to classify peptides from a very high brain influx to a very low brain influx. Peptides can enter the brain by different transport mechanisms. Small peptides may enter via transcellular diffusion or carrier-mediated transport across the endothelial cells which involves influx and efflux of the peptide via membrane solute carriers (Lee & Jayant, 2019). Six different types of these peptide transport systems have already been identified in which some work from the brain to blood, some from blood to brain, and some in both

Applications in medicine: mental health 693 directions. These transporters are not static and can adapt to various conditions and/or be impacted by disease (Banks, 2015). Larger peptides and proteins cross the BBB by receptor-mediated transport, in which the ligand binds to its receptor on the luminal plasma membrane of the endothelial cell. The ligand
receptor complex is then internalized by endocytosis and an internal vesicle is formed which shuttles to the abluminal membrane where exocytosis occurs in the brain parenchyma. The above-mentioned transport mechanisms can also be exploited to improve or deteriorate peptide-specific transport across the BBB in which different strategies can be applied (Lee & Jayant, 2019). Peptides, which show a high brain influx, can be used as shuttles to circumvent the BBB and transport therapeutics in the CNS. Several compounds have been proposed for this purpose. However, major developments are taking place in the field of peptide shuttles (Sanchez-Navarro et al., 2017). Cargoes that can be coupled to the peptides are small drugs, proteins, nanoparticles, and DNA/RNA/siRNA molecules. In 2007 it was demonstrated for the first time that a peptide is able to transport a cargo molecule into the brain in a selective fashion. In this study, a siRNA molecule was delivered to the brain using the RVG-9R peptide resulting in specific gene silencing in the brain and protection against fatal viral encephalitis (Kumar et al., 2007). Later, other promising peptides such as Angiopep-2 and glutathione (GSH) were identified and formulations using these peptides are already under clinical trial. For example, Angiopep-2 conjugated to paclitaxel (ANG1005) reached phase II for the treatment of recurrent high-grade glioma. The vast majority of studies with Angiopep-2 describes conjugates with small molecules for the diagnosis or treatment of brain tumors; delivery of an Angiopep-2-coated nanoparticle loaded with hGDNF on the other hand boosts the neuroprotective effect of this protein and improves locomotor activity and dopaminergic neuron recovery in a mouse model for Parkinson’s disease (PD) (OllerSalvia, Sanchez-Navarro, Giralt, & Teixido, 2016). Larger molecules such as antibodies can also be coupled to Angiopep-2: systemic administration of a conjugate between Angiopep-2 and an anti-HER2-mAb results in the increased survival of mice with HER2-positive brain tumors (Regina et al., 2015). GSH is mainly applied to target PEGylated nanoliposomes which are loaded with drugs and thereby protected against degradation and clearance, a formulation known as G-Technology. Two conjugates making use of this technology already reached the clinical phase for the treatment of brain tumors and multiple sclerosis. Since then, numerous other peptides such as TAT, g7, and apamine have also been described to be used as peptide BBB shuttles (Oller-Salvia et al., 2016). Various strategies can be applied to identify new peptide sequences which show BBB-penetrating behavior, with phage display being one of the most popular. This technique allows for the screening of wide libraries of different peptides against targets of different complexities. More than 10 BBB peptide shuttles have already been discovered using this technology (Bakhshinejad, Karimi, & Khalaj-Kondori, 2015; Diaz-Perlas et al., 2017). Using this technique, a cyclic peptide is also found that targets the neurovascular unit endothelial cells and reactive

694 Chapter 28 astrocytes in different mouse models of neurological disorders. This peptide may thus potentially be used as a tool to enhance the delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation (Mann et al., 2017). Moreover, peptides can also be used to cross other barriers such as the intestinal wall, thereby increasing the oral bioavailability of drugs (Sanchez-Navarro, Garcia, Giralt, & Teixido, 2016).

28.2 Peptides as diagnostic tools in brain tumors and CNS disorders 28.2.1 Peptide-based imaging tracers Radiolabeled peptides have become an important topic in nuclear oncology. These peptides target GPCR which are overexpressed in tumor cells and can be visualized using positron emission tomography (PET) or single-photon emission computed tomography (SPECT), or can be used to specifically target the tumor cells for radio-induced necrosis. These radiopeptides have thus an important impact not only on diagnosis but also on targeted radionuclide therapy of these tumors (Ambrosini, Fani, Fanti, Forrer, & Maecke, 2011). These peptides can be labeled with different chelating agents and radionuclides, where there is a difference between radionuclides for therapy and radionuclides for PET and SPECT imaging (Tornesello, Buonaguro, Tornesello, & Buonaguro, 2017). In all cases of glioblastoma, the most frequent and malignant type of brain tumor, the neurokinin type 1 receptor is overexpressed, making it a good target for radiopeptides. The natural ligand of this receptor is substance P, an 11-amino acid peptide. Different studies demonstrate that coupling this peptide to different radioisotopes facilitates resectability of the tumor by improving demarcation and radiation-induced antiangiogenic effects (Cordier et al., 2010; 2010; Kneifel et al., 2006, 2007; Krolicki et al., 2019). Novel substance P-based radiopeptides are continuously being developed for the treatment of recurrent glioblastomas (Majkowska-Pilip et al., 2018). Another peptide, TM-601, a 36-amino acid neurotoxin which is derived from scorpion venom, has been coupled to 131I for the visualization of glioblastoma (Hockaday et al., 2005; Mamelak et al., 2006). However, the peptide itself also has antiangiogenic and antitumoral effects. In addition, it can also be used as a cargo for other chemotherapeutics (Cohen-Inbar and Zaaroor, 2013). More recently, radiolabeled RGD peptide motifs and bombesin analogs are being investigated as PET tracers for glioblastoma (Fan et al., 2015; Isal et al., 2018; Karimi, Sadeghzadeh, Abediankenari, Rezazadeh, & Hallajian, 2017; Zhang et al., 2016). Another example is octreotide, an 8-amino acid cyclic somatostatin analog that inhibits growth hormone release and is therefore being used for the treatment of acromegaly which is caused by a neuroendocrine tumor on the pituitary gland. Analogs of the peptide, which are conjugated to different chelators and radiometals, are also used for PET imaging and treatment of other

Applications in medicine: mental health 695 neuroendocrine tumors which overexpress the somatostatin subtype 2 receptor (Ambrosini et al., 2011; Lamberts and Hofland, 2019). The use of PET imaging in psychiatry also has a place and has provided new insights into the neurobiology of major psychiatric disorders. For example, PET studies have provided evidence of dopamine dysregulation in schizophrenia patients and loss of monoamines in patients with depression. However, peptide-based tracers have not been developed yet (Pagani, Carletto, & Ostacoli, 2019; Zipursky, Meyer, & Verhoeff, 2007).

28.2.2 Peptides as biomarkers During disease, expression profiles of peptides can be altered, making them excellent candidates as biomarkers for diagnosis. These peptide biomarkers can also lie on the etiological basis of pathogenesis. An example is the amyloid-β (Aβ) peptide in AD, the main cause of dementia and one of the greatest health-care challenges of the 21st century (Scheltens et al., 2016). The Aβ oligomer, which is thought to be the toxic form of Aβ, is often used as a biomarker as increased levels of this peptide oligomer are observed in both the CSF and plasma of patients. However, a large overlap with healthy controls is seen, making this test insufficient to be used as a diagnostic test on its own (Holtta et al., 2013; Zhou et al., 2012). Only 67% of AD patients had Aβ levels in the CSF above the optimal cutoff (Holtta et al., 2013). To increase the sensitivity and specificity of Aβ-based diagnosis in CSF, the Aβ/tau ratio can be applied, with tau being a protein that is also involved in AD pathogenesis (Adamczuk et al., 2015). Another approach is the visualization of Aβ with PET ligands. To date, three ligands have been approved by the Food and Drug Administration (FDA) and European Medicines Agency: that is, florbetapir, florbetaben, and flutemetamol (Scheltens et al., 2016; Tolboom, Ossenkoppele, & van Berckel, 2019). It is demonstrated that Aβ PET imaging correlates well with oligomeric Aβ levels in the CSF, making both tests complementary (Muller et al., 2019; Schipke et al., 2017). PET imaging has different advantages over Aβ determination in the CSF: the technique is noninvasive and provides localized information of Aβ depositions in the brain which can be useful for longitudinal follow-up of disease progression or to track changes after therapeutic intervention. Different studies also investigated the potential of B-type natriuretic peptide (BNP) plasma levels as a diagnostic marker for cognitive impairment. Results concerning this peptide are discordant since some studies found elevated levels of this peptide and its N-terminal pro-peptide in mild cognitive impairment (MCI) and AD (Daniels et al., 2011; Hiltunen et al., 2013; Kerola et al., 2010), while a more recent study found declined plasma levels in both MCI and AD patients (Begic et al., 2019). Further investigation is thus necessary to evaluate the diagnostic value of this peptide in cognitive impairment.

696 Chapter 28 In depressive disorders, different circulating peptides are able to discriminate between patients and healthy persons. One example is the lower C-peptide levels in depressive patients compared to healthy controls (odds ratio 5 0.466) (Takekawa et al., 2019). Also, lower Aβ-42 levels are observed in the serum of patients with depression, also indicating a potential role of this “Alzheimer peptide” in the pathophysiology of depression (Yasuda et al., 2019). On the other side, different circulating peptides can be elevated in depression as well: higher ghrelin levels may be associated with depressive symptoms in Japanese women, but not in men (Akter et al., 2014), while cathelicidin LL-37, an antibacterial peptide which is produced by immune cells in response to different microbial stimuli, is elevated in both major depressive disorder (MDD) and bipolar disorder (Kozlowska, Wysokinski, & Brzezinska-Blaszczyk, 2017; Kozlowska, Zelechowska, BrzezinskaBlaszczyk, Margulska, & Wysokinski, 2018). This peptide is expressed in both epithelial cells as immune cells and destroys the lipoprotein membranes of bacterial cells, which are enveloped in phagosomes after engulfment, after fusion with the lysosome (Du¨rr, Sudheendra, & Ramamoorthy, 2006). Also in bipolar disorder, lower plasma levels of orexin A and Aβ-42 are observed (Piccinni et al., 2012; Tsuchimine et al., 2019). In the CSF, lower cocaine- and amphetamine-regulated transcript (CART) peptide levels are observed in MDD patients compared to healthy controls, while levels of the calcitonin gene-related peptide are increased (Mathe, Agren, Lindstrom, & Theodorsson, 1994; Yoon, Hattori, Sasayama, & Kunugi, 2018). In schizophrenia, lower oxytocin and higher arginine
vasopressin (AVP) levels are observed in serum, two nonapeptides known to play regulatory roles in social behaviors (Guzel et al., 2018). Increased serum insulin, pancreatic polypeptide, and calcitonin gene-related peptide levels are also observed in schizophrenic patients compared to healthy controls (Guest et al., 2011; Urban-Kowalczyk, Smigielski, & Strzelecki, 2017). In addition, in plasma of children with autism spectrum disorder (ASD), lower oxytocin levels are observed in plasma as well (Zhang et al., 2016). In the urine of ASD patients, an increase in milk- and wheat-derived casomorphin 8 and exorphin C is observed, indicating an increased gut permeability or uptake of these food-derived peptides in ASD patients (Bojovic et al., 2019). In PD patients with comorbid depression, both neuropeptide Y and calcitonin gene-related peptide are elevated in the CSF compared to patients with depression alone (Svenningsson, Palhagen, & Mathe, 2017). On the contrary, the C-type natriuretic peptide levels in plasma are lower in PD patients compared to the reference range (Woodward, Prickett, Espiner, & Anderson, 2017). Finally, in attentiondeficit hyperactivity disorder (ADHD), a tetra-peptide (GSEN) which is found in the C-terminal part of αS1-casein and stimulates the uptake of serotonin in platelets could be isolated and detected from the urine of patients, while it was not found in the urine of healthy controls (Liu and Reichelt, 2001). An overview of the above-discussed peptide biomarkers in mental disorders is given in Table 28.1 as examples of the use of peptides as biomarkers.

Applications in medicine: mental health 697 Table 28.1: Overview of possible peptide biomarkers in mental disorders. Disease ADHD Alzheimer’s disease

Autism spectrum disorder

Bipolar disorder

Cognitive impairment Major depressive disorder

Parkinson’s disease

Schizophrenia

Peptide

Sample

Decreased/increased

GSEN Amyloid-β oligomer

Urine CSF Brain Plasma Plasma Plasma Urine Urine Plasma Plasma Plasma Plasma Plasma Serum Plasma Plasma CSF CSF CSF CSF Plasma Serum Serum Serum Serum Plasma

Increased Increased Increased Increased Both Decreased Increased Increased Increased Decreased Decreased Both Decreased Decreased Increased Increased Decreased Increased Increased Increased Increased Increased Increased Decreased Increased Increased

BNP Oxytocin Casomorphin 8 Exorphin C Cathelicidin LL-37 Orexin A Amyloid-β 42 BNP C-peptide Amyloid-β 42 Cathelicidin LL-37 Ghrelin CART Calcitonin gene-related peptide Neuropeptide Y Calcitonin gene-related peptide C-type natriuretic peptide Insulin Pancreatic polypeptide Oxytocin Arginine
vasopressin Calcitonin gene-related peptide

ADHD, Attention-deficit hyperactivity disorder; CSF, cerebrospinal fluid; CART, cocaine- and amphetamine-regulated transcript.

28.3 Therapeutic applications of peptides for mental health Since disease-related mutations in the brain peptidergic system occur, this system may be an excellent target for novel therapeutic strategies (Hokfelt et al., 2000). One of the first breakthroughs was the disruption of the substance P system by a small molecule neurokinin 1 antagonist in the treatment of major depression in 1998 (Hokfelt et al., 2003). In addition, due to the great potency of peptides in the brain, peptides themselves can be promising to be used as novel therapeutics. For example, compared to the classic neurotransmitters, neuropeptides already exert their effects at very low concentrations (Russo, 2017). Here, we discuss the possible therapeutic applications of bioactive peptides, both in the preclinical as in different clinical phases, in different classes of psychiatric and neurodegenerative diseases. The growing importance of peptides in mental disorders becomes clear in Fig. 28.2. A search on PubMed with the keywords “peptide AND mental disorder” yields about 22,000 publications in 2020, clearly showing a trajectory for continued growth and

698 Chapter 28

Figure 28.2 Number of publications found in PubMed since 1980 for “peptide* AND mental disorder” by year.

interest (Fig. 28.2). The number of publications vastly increased over the past few decades, with a strong increase in the 2000s. A schematic overview of the peptide drug development cycle is given in Fig. 28.3 and an overview of the discussed peptides with therapeutic potential is given in Table 28.2.

28.3.1 Neurodevelopmental disorders The term neurodevelopmental has been applied to a very broad group of disabilities that involve some form of disruption in brain development. This definition groups together a very wide range of neurological and psychiatric conditions that are both clinically and causally disparate (Thapar, Cooper, & Rutter, 2017). A nice overview of different neurodevelopmental disorders (NDD) and their prevalence is given by D’Souza and Karmiloff-Smith (2017). One of the key characteristics of NDD is that they typically onset in childhood before puberty and can be distinguished from other neuropsychiatric disorders due to their clinical course. NDD tend to show a more steady course rather than a remitting and relapsing one. These disorders show also a prominent early onset of neurocognitive deficits, affect more males than females and are highly heritable but are also typically multifactorial in origin; a single major cause for the onset of these diseases is rare. Overall, this group of disorders is highly heterogeneous in terms of clinical characteristics, causes, treatment responses, and outcomes (Thapar et al., 2017). In this chapter, two of the most prevalent NDD are discussed: ASD and ADHD. Autism is a developmental disorder with deficits in social interaction, communication, and language. To date, neither the etiological mechanisms are fully understood nor any treatment is capable of completely reversing the symptoms. Different peptides have been already under clinical trial to improve and/or treat symptoms of ASD. One of the most extensively

Applications in medicine: mental health 699

Figure 28.3 Schematic overview of the peptide drug development cycle. Peptide discovery starts with either the phenotype- or target-based approach in which peptides are selected based on in vitro effects on cell-cultures or having a high probability of binding the target receptor, respectively. Lead peptides are selected for further in vivo studies in rodents and nonrodents. The peptide
protein binding scaffold from peptides, of which the target is known, is investigated and optimized using docking studies. The peptide structure can be optimized to increase proteolytic stability and bioavailability using multiple approaches (e.g., amino acid substitution, terminal protection, backbone modifications. . .). After preclinical investigations, lead peptides are selected for human clinical trials.

investigated peptides is oxytocin, of which the plasma levels are decreased in patients (Zhang et al., 2016). This peptide has been associated with various social behaviors in mammals, including social attachment and pair bonding; in addition, some oxytocin-related genes (e.g., oxytocin receptor genes) are associated with the pathogenesis of ASD as well. Some clinical characteristics of ASD (e.g., social behavior) can be considered at least partially as a phenotype of oxytocin-related genes. In the last decade, many studies have

700 Chapter 28 Table 28.2: Overview of discussed peptides with potential therapeutic properties in mental disorders. Peptide

Effects

Indication(s)

Status

Oxytocin

Reduction repetitive behavior, improved speech, social interactions, task performance and eye gazing Improved social interaction, anxiety reduction, and decreased repetitive behavior Improvement on aberrant behavior checklist and childhood autism rating scale Promotes locomotion, increases dopamine levels, attenuates hyperactivity, and improves spontaneous alternation behavior Improvement of higher-level social cognition, eye gazing Cognition improvement Behavior and monoamine normalization Improvement on performance-based skill assessment score Restores social interaction, dopamine enhancement Improvement on HDRS scale Improvement on Montgomery-Asberg Depression Rating Scale Increasing serotonin Decreasing HPA-axis activation Δ-opioid agonist CRF2 agonist TREK-1 antagonist Suppresses microglia activation Neurogenesis stimulation Prevents LPS-induced depression and anxiety behavior Antianxiety effects Antianxiety effects Antianxiety effects Antianxiety effects Antianxiety effects Improves short-term memory, induction of neuronal activation Stimulates neurogenesis and synaptic efficiency Stimulates neurogenesis and synaptic efficiency Stimulates neurogenesis and synaptic efficiency Stimulates neurogenesis and synaptic efficiency Stimulates neurogenesis and synaptic efficiency Improvement of delayed memory and cognitive function Improves motor and nonmotor symptoms, blocks microglia activation Promotes neuronal cell proliferation and anti-inflammatory

Autism

Clinical

Autism

Clinical

Autism

Clinical

ADHD

Preclinical

Schizophrenia

Clinical

Schizophrenia Schizophrenia Schizophrenia

Clinical Preclinical Clinical

Schizophrenia MDD MDD

Preclinical Clinical Clinical

MDD MDD MDD MDD MDD MDD MDD Anxiety Anxiety Anxiety Anxiety Anxiety Anxiety Dementia

Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Clinical Preclinical Preclinical Preclinical Preclinical Preclinical

Dementia Dementia Dementia Dementia Dementia Dementia

Preclinical Preclinical Preclinical Preclinical Preclinical Clinical

PD

Clinical

PD

Preclinical

AVP GRP TAT-DATNT

Oxytocin POP-inhibitors Lunasin Davunetide CART MIF-1 Nemifitide CART GLP-2 NIH 11082 Urocortin 2/3 Spadin LH FGF2 TLQP-62 Oxytocin Angiotensin 1-7 GHK GLP-2 HLDF-6 Orexin Ghrelin Neuropeptide Y Galanin Obestatin GRP Insulin Exenatide Lumbricusin

(Continued)

Applications in medicine: mental health 701 Table 28.2: (Continued) Peptide WY Liraglutide P2 Peptide T

CART ANP Oxytocin Oxytocin Oxytocin

Effects

Indication(s)

Status

Inhibits monoamine oxidase-B, increases dopamine levels, suppresses microglia activation Promotes neurogenesis Stimulation of neurite outgrowth and promotion of neuronal survival Blocks interaction of gp120 with brain tissue, cognitive improvement

Cognitive decline

Preclinical

Cognitive decline Traumatic brain injury HIV-associated cognitive impairment Drug withdrawal Alcohol withdrawal Heroin withdrawal Drug abuse Nicotine withdrawal

Preclinical Preclinical

Anxiolytic effects Anxiolytic effects Craving reduction Reverse neuro-adaptions Improves somatic signs

Clinical

Preclinical Preclinical Clinical Preclinical Preclinical

ADHD, attention-deficit hyperactivity disorder; MDD, major depressive disorder; PD, Parkinson’s disease.

investigated the effect of oxytocin administration on autistic behavior (Yamasue and Domes, 2017), both via intravenous as intranasal administration routes. Intranasal administration of drugs has the advantage that the BBB is bypassed, resulting in a higher brain exposure and thus a lower dose that needs to be administered. Other advantages are less side effects and a higher systemic bioavailability (Erdo, Bors, Farkas, Bajza, & Gizurarson, 2018). Peptides and proteins enter the brain after intranasal administration after coming in contact with the cribriform plate in the nasal cavity. The peptide is then transported directly to the brain via the olfactory bulb or trigeminal nerve or indirectly by the lymphatic system and the circulation. However, this indirect transport mechanism still requires crossing of the BBB (Meredith, Salameh, & Banks, 2015). Other administration routes may also be applied. Recently, advances are being made in the oral administration of peptides (Drucker, 2020) or by the use of a biodegradable implant after brain surgery for further treatment of brain tumors (Lee et al., 2019). The main results of a single intravenous (10 IU) or intranasal (24 IU) dose of oxytocin administration to ASD patients are a reduction in repetitive behavior, improved speech, improved task performance, increased eye gazing, and increased social interactions. The effects on social reward are mediated by direct activation of dopamine neurons in the ventral tegmental area (Hung et al., 2017; Xiao, Priest, Nasenbeny, Lu, & Kozorovitskiy, 2017). Oxytocin is expected to be a key molecule in developing new treatments for the core symptoms of ASD, despite the fact that some clinical trials failed to yield positive results (Yamasue and Domes, 2017; Young and Barrett, 2015). AVP, an oxytocin-related peptide, shows similar effects in the treatment of ASD. Daily intranasal treatment (24 IU) during 4

702 Chapter 28 weeks resulted in enhanced social abilities in patients compared to the placebo group. Treatment also diminished anxiety symptoms and some repetitive behaviors (Parker et al., 2019). Another peptide that is implicated in the pathogenesis of ASD is the gastrinreleasing peptide, with mutations in its receptor observed in autistic patients (Seidita et al., 2008). In addition, different pharmacological and genetic studies in rodents have demonstrated that this receptor is involved in regulating synaptic plasticity and aspects of behavior (food intake, stereotypy, social behavior, learning, and memory) that might be altered in different psychiatric disorders such as schizophrenia, depression, autism, and dementia (Roesler, Henriques, & Schwartsmann, 2006). Different placebo-controlled trials have investigated the effects of this peptide in autistic patients on different core symptoms, demonstrating that intravenous administration of 160 pmol/kg during 4 consecutive days resulted in significant improvement on the aberrant behavior checklist. This checklist is a scoring system of different core symptoms of autism such as irritability, social withdrawal, stereotypical behavior, hyperactivity, and communication problems. Improvements on the childhood autism rating scale, which has even more evaluation criteria, were also observed (Becker et al., 2016; Marchezan et al., 2017). ADHD is characterized by hyperactivity and inattention and affects around 5% of the children and adolescents worldwide. Current pharmacological agents are stimulants that enhance dopaminergic neurotransmission by directly blocking reuptake dopamine transporters (DAT) or by stimulating dopamine release. Although these are clinically effective, the risk for future substance abuse in patients is present since other illegal drugs such as cocaine and amphetamines also increase dopamine in ventral striatal nuclei and share some similar binding patterns within the dopaminergic system. Therefore other treatments that enhance dopaminergic neurotransmission without directly blocking DAT are necessary. Already in 2006, Kinkead et al. designed an algorithm to computationally design peptides that target the extracellular and paratransmembrane amino acid loops of the ADHD-involved dopamine D2 receptor. Different peptides were found that evoke a positive allosteric and indirect agonistic effect on the receptor and one 15 amino acid-long peptide improved sensor motor gating in the spontaneously hypertensive rat (SHR) model, a frequently used animal model for ADHD, while leaving nonselective attention, impulsive behavior and activity levels unchanged (Kinkead, Selz, Owens, & Mandell, 2006). In addition, some preclinical data suggest that a TAT-DATNT fusion peptide promotes locomotor behavior and elevates extracellular dopamine levels in rats by indirectly blocking DAT. More importantly, the peptide also attenuates hyperactivity and improves spontaneous alternation behavior in SHR rats (Lai, Su, Zhang, & Liu, 2018). This is also an application of the TAT sequence being used as a BBB shuttle fused to an active sequence (Stalmans et al., 2015). These fusion peptides coupled to a BBB shuttle sequence may have, in the future, a place in ADHD treatment in humans. In contrast, peptides can also aggravate the symptoms of these NDD. For example, opioid β-casomorphins, cryptic peptides derived from the milk casein protein, can induce

Applications in medicine: mental health 703 locomotor hyperactivity and stereotypy and disrupt social behavior (Lister, Fletcher, Nobrega, & Remington, 2015).

28.3.2 Psychotic disorders Psychotic disorders are characterized by symptoms, defined as manifestations of cognitive and perceptual dysfunction, delusions, or hallucinations. Three broad groups can be distinguished: idiopathic psychoses (e.g., schizophrenia), psychoses due to medical conditions (e.g., neurodegenerative), and toxic psychoses (e.g,. due to medication or toxins). The most common psychotic disorders are schizophrenia, schizoaffective disorders, bipolar disorders, and MDDs with psychotic features. Different factors are involved in the pathogenesis of these disorders. One of the main factors is an altered neurotransmission in the dopamine and glutamate pathway of the hippocampus, midbrain, corpus striatum, and prefrontal cortex; an excess in synaptic levels of dopamine and glutamate is then observed. Genetic factors also play an important role as epidemiological studies suggest a strong heredity of idiopathic psychotic disorders; specific genetic markers and the mode of heritance are however still not determined. Other factors that may contribute to psychotic disorders are neurodevelopmental factors and autoimmune or inflammatory disorders. Current pharmacological treatments work largely by blocking or mitigating the activity of dopamine D2 receptors (typical antipsychotics) or combined blocking of dopamine D2 and serotonin-2A receptors (atypical antipsychotics). These drugs are considered symptom suppressing rather than disease modifying. Moreover, different side effects such as extrapyramidal neurologic effects, weight gain, and disturbance of glucose and lipid metabolism are then also observed. Other treatments focus on neuromodulation by different brain stimulation techniques such as deep brain stimulation and psychosocial approaches such as cognitive behavioral therapy (Lieberman and First, 2018). Since current pharmacological treatments are only partially effective against the negative symptoms (e.g., apathy, social disruption) in schizophrenia and that these are the greatest contributor to disease burden, an unmet clinical need is present. Just like in ASD, there was a lot of focus in the scientific community towards oxytocin since this peptide is involved in enhancing social interaction and affective functions. However, the obtained findings are inconsistent and inconclusive: different studies investigated the effect of oxytocin administration on different clinical outcomes in patients and found improvements in higherlevel social cognition and social cognition deficits (Davis et al., 2013; De Coster, Lin, Mathalon, & Woolley, 2019), while other studies found no significant improvements over placebo-treated patients (Jarskog et al., 2017; Lee et al., 2019). A meta-analysis performed in 2017 which included eight clinical trials indeed found no evidence for oxytocin to improve any aspect of symptomology in schizophrenic patients using multivariate analysis. Moreover, the Bayes factor indicated a small favor toward the null hypothesis (no effect of

704 Chapter 28 oxytocin) for negative symptoms, while it was inconclusive for positive symptoms and general psychopathology (Williams and Burkner, 2017). A recent study now indicates that oxytocin could improve eye gazing in schizophrenia; further investigation is however needed to conclude whether this impacts social cognition and functional outcomes or not (Bradley et al., 2019). Another approach for the treatment of cognitive deficits can be the selective inhibition of prolyl oligopeptidase, a serine peptidase in the brain which is responsible for neuropeptide degradation; specially proline-containing neuropeptides (e.g., substance P, neurotensin, and AVP) are substrates of this enzyme. Different peptidomimetic inhibitors of this enzyme have already been designed and show cognition-enhancing properties in schizophrenic patients and mouse models due to increased neuropeptide levels (Lopez et al., 2013). Other peptides are also under investigation for the treatment of schizophrenia. Lunasin, a 43 amino acid soybean-derived peptide which is a histone acetylation inhibitor, shows a multitude of biological effects such as antioxidant, anticancer, anti-inflammatory, cholesterol-regulating, and immunomodulating (Fernandez-Tome and Hernandez-Ledesma, 2019; Janssens, Wynendaele, Vanden Berghe, & De Spiegeleer, 2019). Recently, preclinical evidence demonstrated that this intranasal administered peptide normalized behavior and brain monoamine levels in an experimental psychosis mice model by binding to serotonin receptors, thereby indicating the possible antipsychotic effect of the peptide (Dzirkale, Nakurte, Jekabsons, Muceniece, & Klusa, 2019). Davunetide, an intranasally administered peptide under clinical development for AD, also shows promising effects in schizophrenia. It contains an 8-amino acid peptide fragment, called NAP, which interacts with microtubules and promotes neurite outgrowth in different animal models of AD. Moreover, intranasal administration of the peptide shows a significant beneficial effect in a performance-based skill assessment score in schizophrenic patients as well (Javitt et al., 2012). This is explained by increasing N-acetylaspartate, which is considered a useful measure of neuronal integrity, and choline levels, a marker of cell membrane turnover and white matter integrity, in the brain (Jarskog et al., 2013). The protective actions of NAP are mediated by triggering the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI-3K), and cyclic adenosine monophosphate signaling pathways in neuronal cells (Pascual and Guerri, 2007). Other peptides are still in the preclinical research phase, showing promising results against schizophrenia in animal models. For example, the CART peptide restores social interaction in MK-804-induced schizophrenic dementia-like symptoms in rats by positively modulating the actions of dopamine (Borkar et al., 2018). This hypothalamic peptide is an anorectic peptide with similar effects as cocaine; however, when coadministered, it blunts the effects of cocaine. The peptide is released in response to dopamine in the nucleus accumbens and acts as a negative feedback loop. Therefore it can be used to blunt the effects of abusive drugs (Hubert, Jones, Moffett, Rogge, & Kuhar, 2008; Kristensen et al., 1998).

Applications in medicine: mental health 705 Besides direct effects on clinical symptoms of schizophrenia, peptides are also associated with the working mechanisms of small molecule antipsychotic drugs such as clozapine and haloperidol. These dopaminergic drugs are able to modify orexin functions: D1- and D2like dopamine receptor agonists activate orexin cells which may cause the weight gain side effects of some antipsychotic drugs (since orexins are appetite-stimulating peptides). Vice versa, orexin is also able to increase extracellular dopamine levels in the prefrontal cortex which promotes cognitive function. Moreover, orexin antagonists are able to block the effects of these antipsychotic drugs as well, indicating that orexin induction is necessary for the drug’s actions (Deutch and Bubser, 2007).

28.3.3 Depressive, bipolar, and anxiety disorders Depressive, bipolar, and anxiety disorders are three domains in the DSM-5 that include the currently most prevalent disorders in psychiatric practice (Otte et al., 2016). MDD is a debilitating disease that is characterized by a depressed mood, diminished interests, impaired cognitive function, and vegetative symptoms such as a disturbed sleep and appetite. The etiology is multifactorial and besides genetic factors (heritability of approximately 35%), environmental factors such as sexual, physical, and/or emotional abuse during childhood are strongly associated with the risk of MDD development. Different mechanisms such as smaller hippocampal volumes, alterations in the stress response, activation or connectivity of neural networks, and neuroinflammation are known. However, no established mechanism can explain all aspects of the disease. Currently, there are two main treatment options, that is, psychotherapy, which is used in mild depressive episodes, and pharmacotherapy, which is used in more moderate to severe episodes. Current pharmacological treatments consist of drugs that increase the monoamine levels in the synaptic cleft by either blocking the reuptake or degradation or by having effects directly on postsynaptic monoaminergic receptors, resulting in different neuronal responses such as changes in gene expression and neural and synaptic plasticity (Otte et al., 2016). In early clinical trials, the tripeptide MIF-1, a hypothalamic peptide, showed significant improvement on the Hamilton depression rating scale (decrease of 6 23 points) after daily subcutaneous injection of 10 mg during 5 consecutive days in depressed patients compared to placebo-treated patients (Ehrensing and Kastin, 1978; Ehrensing, Kastin, Wurzlow, Michell, & Mebane, 1994). The peptide increases c-Fos expression, a sensitive marker for CNS activation, in brain regions critically involved in the regulation of mood, anxiety, depression, and memory. This increased c-Fos expression is mediated by an increase in the MAPK pERK and a reduction of pSTAT3 in neuronal cells (Khan et al., 2010, 2018). Despite these promising results, the peptide never reached the market for the treatment of depression. An analog of this peptide (nemifitide) is currently in the clinical phase but also did not reach the market yet (Feighner et al., 2008). Since those early days, several other peptides with antidepressant effects have been identified which all

706 Chapter 28 have different working mechanisms, such as glucagon-like peptide 2 (GLP-2), CART, urocortins, TRH-like peptides, spadin, and NIH 11082. The CART peptide exerts its antidepressant effects by increasing serotonin levels in the dorsal raphe nucleus and nucleus accumbens probably by the activation of a CART-specific receptor. GLP-2 works by improving the negative feedback loop of the hypothalamo
pituitary
adrenal axis, hereby decreasing its activation and/or by restoring neurogenesis in the subgranular zone. NIH 11082 is a delta-opioid agonist and the urocortins (2 and 3) bind to the CRF2 receptor which elicits antianxiety and antidepressant actions; the working mechanism of TRH-like peptides remains largely unknown (Khan et al., 2018). Spadin is a 16-amino acid peptide formed during posttranslational maturation of sortilin, a membrane glycoprotein, and is an antagonist of TREK-1, a crucial target in the treatment of depression (Pietri, Djillani, Mazella, Borsotto, & Heurteaux, 2019). TREK-1 is a background K1 channel that is controlled by 5-HT and its deletion in mice results in resistance to depression, indicating that this ion-channel plays a causative role in the pathogenesis of depression (Heurteaux et al., 2006). Spadin increases the firing activity of serotonergic neurons, neurogenesis, and synaptogenesis. Also shortened analogs such as mini-spadin (6 amino acids) have been identified and show better efficacy and in vivo stability compared to spadin. This peptide also shows protective effects against poststroke depression and shows no cardiac side effects which are observed with other TREK-1 inhibitors (Pietri et al., 2019). Taken together, this peptide and its analogs show promising results but are still in the preclinical phase; however, the peptide is considered safe to continue to the clinical phase (Djillani, Pietri, Mazella, Heurteaux, & Borsotto, 2019). Wheat-derived cryptic peptides also show antidepressant actions: pyroglutamyl leucine and pyroglutamyl glutaminyl leucine exhibit antidepressive effects by enhancing neuronal proliferation in the hippocampus (Khan et al., 2018). The LH dipeptide, which is enriched in fermented dairy products (e.g., cheese), suppresses activation of microglia cells and depression-like behavior in a mouse model (Ano, Kita, Kitaoka, & Furuyashiki, 2019). Indeed, daily consumption of these fermented dairy products is inversely correlated with depressive symptoms in humans (Cui et al., 2017). Also, the neuropeptide fibroblast growth factor-2 (FGF2) has been shown to take part in the pathophysiology of depression. The peptide is expressed in both neurons and glial cells and levels are elevated after repeated administration of antidepressants. Exogenous FGF2 administration was shown to have antidepressant effects in mice and the peptide is also involved in anxiolytic actions of benzodiazepines (see further) since endogenous hippocampal levels are elevated after acute diazepam injection. The main working mechanism of this peptide is the stimulation of neurogenesis in the adult brain as its receptor is expressed on proliferating neural stem cells (Karatas, Yemisci, Eren-Kocak, & Dalkara, 2018). Increasing evidence suggests the presence of a gut
brain axis, a bidirectional communication pathway between the gut and the brain (Dinan and Cryan, 2017). Also in depression and

Applications in medicine: mental health 707 anxiety, the link with gut function is well appreciated with changes in colonic motility, physiology and morphology and alterations in the gut microbiota composition (Janssens et al., 2018; Lach, Schellekens, Dinan, & Cryan, 2018); several animal studies already demonstrated that the gut microbiota modulates anxiety and depressive-like behaviors. Gut peptides are peptides that are produced by the enteroendocrine cells (EECs) of the gastrointestinal tract. Several of these peptides and their receptors (ghrelin, GLP-1, peptide YY, oxytocin, cholecystokinin, and corticotropin-releasing factor) are also present in the brain where they play well-established roles in the neurobiology of anxiety and depression [for review: see (Holzer, Reichmann, & Farzi, 2012)]. Since the activity of the EECs is modulated by the gut microbiota, these peptides may be one of the messengers responsible for gut
brain communication and the pathophysiology of anxiety and depression. Targeting these gut peptides by manipulating the gut microbiota composition by so-called “psychobiotics” may represent a promising opportunity to target mental disorders (Lach et al., 2018). Finally, possible pathogenic roles for the soluble Aβ 42 peptide have also been proposed in depression (Pomara and Sidtis, 2010). Bipolar disorders differ from depressive disorders by the fact that recurrent depressive episodes are accompanied by episodes of elevated mood. Bipolar I disorder is characterized by episodes of severe elevated mood (mania), while bipolar II disorders are accompanied by less severe mood elevations (hypomania). These manic and depressive symptoms often co-occur resulting in mixed states. Management of bipolar disorders is complex: treatment with antidepressants has only very limited effect and can exacerbate elevated mood (mood switch) and is therefore not appropriate in treating bipolar depression. Pharmacological treatment of bipolar disorders is based on mood stabilization drugs, such as lithium, antiepileptics, and atypical antipsychotics (Anderson, Haddad, & Scott, 2012). Different peptides such as orexin A, Aβ 42, and LL-37 are discriminative between bipolar patients and healthy individuals in plasma and can be potentially used as biomarkers (decrease of orexin A and Aβ and an increase of LL-37 in patients) (Kozlowska et al., 2018; Piccinni et al., 2012; Tsuchimine et al., 2019). However, no specific peptides have been described in the literature for the potential treatment of bipolar disorders in particular. Anxiety disorders are the largest group of mental disorders and are a leading cause of disability in most western countries with a lifetime prevalence of 20%. This group includes separation anxiety disorder, selective mutism, specific phobias, social anxiety disorder, panic disorder, agoraphobia, and generalized anxiety disorder. The essential features of anxiety disorders are excessive and enduring fear, anxiety, or avoidance of perceived threats and also include panic attacks. The onset of most disorders occurs during childhood, adolescence, or early adulthood and remains often undetected and untreated. In general, treatment is not sought until two decades after onset. Several psychological (cognitive behavior therapy, interpersonal therapy, and psychodynamic approaches) and pharmacological treatments (antidepressants, benzodiazepines, atypical antipsychotics, and β-adrenergic blockers) are available for the management of anxiety disorders

708 Chapter 28 (Craske et al., 2017). Numerous peptides have been described with anxiolytic effects in animals of which the majority are still in the preclinical phase; often, these peptides also have antidepressant effects at the same time. The VGF neuropeptide C-terminal fragment TLQP-62 prevents Lipopolysaccharide (LPS)-induced depression-like and anxiety-like behavior in mice by reinforcing BDNF/TrkB signaling which regulates memory consolidation and antidepressant-like actions (Li et al., 2017). Just like in ASD, a lot of attention was given to oxytocin, playing roles in all kinds of anxiety disorders and alleviating symptoms after administration in patients (Gottschalk and Domschke, 2017). Besides oxytocin, a wide variety of different peptides with different origins show anxiolytic effects in mice or rats, mostly resulting in more time exploring the open arms of an elevated plus maze, the golden standard for assessing anxiety in in vivo models. Examples are peptides isolated from wasp venom (Gomes et al., 2016), foods such as spinach (rubiscolin-6), soy (soymoprhin-5), eggs (ovolin), and milk (multiple cryptic peptides from casein, lactoglobulin, and whey proteins) (Lister et al., 2015; Mizushige, Sawashi, Yamada, Kanamoto, & Ohinata, 2013; Oda et al., 2012; Yamada, Mizushige, Kanamoto, & Ohinata, 2014) or endogenous peptides such as GLP-2, angiotensin 1
7, GHK, urocortin, and HLDF-6 (Almeida-Santos et al., 2016; Bobyntsev, Chernysheva, Dolgintsev, Smakhtin, & Belykh, 2015; Iwai et al., 2015; Zolotarev et al., 2016). The modes of action of these peptides are explained by their effects on the different neurotransmitter systems such as the glutamatergic, serotonergic, and dopaminergic systems (Iwai et al., 2015; Telegdy and Adamik, 2013; Zolotarev et al., 2016).

28.3.4 Neurocognitive and neurodegenerative disorders Neurocognitive disorders are the collection of all disorders which cause cognitive impairment. A distinction is made between MCI, in which the cognitive decline is higher than what is expected for an individual’s age or education but does not affect normal functioning, and major cognitive impairment (or dementia), in which the normal daily function is affected by the cognitive decline (Gauthier et al., 2006). Dementia can be caused by different neurodegenerative pathological conditions such as AD, PD, Huntington’s disease, HIV infection, and Prion disease, and also by a variety of other medical conditions such as schizophrenia and alcohol abuse. Numerous peptides have been shown to improve memory and learning and are promising as alternative therapeutic approaches for the treatment of dementia. The majority of these peptides are still in the preclinical phase. Intranasal administration of orexin peptides may be useful in treating a variety of cognitive disorders. It has been demonstrated in sleep-deprived macaques that the peptide improves the performance on a short-term memory task and induces neuronal activation in different brain regions such as the piriform cortex, agranular insular, and nucleus basalis; also increasing glutamatergic and cholinergic neurotransmission in the prefrontal cortex has been observed (Calva and Fadel, 2018). Other feeding regulatory

Applications in medicine: mental health 709 peptides (ghrelin, Neuropeptide Y, galanin, obestatin, and gastrin-releasing peptide) also ameliorate memory impairment by stimulating neurogenesis and synaptic efficiency in the hippocampus (Beck and Pourie, 2013; Eslami, Sadeghi, & Goshadrou, 2018; Yang et al., 2017). Two other peptides that improve memory and cognition after intranasal administration are insulin and PACAP; for insulin, already a pilot clinical trial has been performed and resulted in the improvement of delayed memory and cognitive function in memory-impaired older adults (Meredith et al., 2015). Peptides can also act as cognitive enhancers by improving synaptic functioning using different mechanisms and pathways (Asua, Bougamra, Calleja-Felipe, Morales, & Knafo, 2018). Food-derived peptides also play a role in keeping the memories alive. A milk-derived tripeptide is able to attenuate Aβ42-induced cognitive decline by suppressing the expression of inflammatory cytokines and oxidative stress and increasing cerebral blood flow (Min et al., 2017). The Trp-Tyr (WY) dipeptide, present in fermented dairy products, helps to prevent age-related cognitive decline via inhibition of monoamine oxidase-B activity, increasing dopamine levels and suppressing microglial activation (Ano et al., 2018; Ano et al., 2019). The soy peptide Tyr-Pro attenuates Aβ25
35 memory impairment via the cholinergic neurotransmission pathway (Tanaka et al., 2020). Other soy-derived di- and tripeptides suppress cognitive decline by increasing the expression of neurotrophic factors such as BDNF, neural growth factor, and NT-3 by CREB-dependent pathways (Katayama, Imai, Sugiyama, & Nakamura, 2014). Fermented rice-derived peptides also prevent memory impairment by similar pathways (Corpuz, Fujii, Nakamura, & Katayama, 2019). AD is the leading cause of major cognitive decline worldwide and is characterized by hyperphosphorylated tau tangles and extracellular aggregated Aβ plaques in the brain followed by brain tissue atrophy. Oligomers of the Aβ peptide are the toxic form of the peptide and cause different pathological effects such as brain capillary constriction and contribute to neuronal dysfunction and activation of microglia (Nortley et al., 2019; Scheltens et al., 2016; Zott et al., 2019). Besides orexin, PACAP and insulin, which are all able to improve neurocognition, numerous other (synthetic) peptides have been suggested as potential therapeutics in the treatment of AD (Cheng et al., 2017; Ribaric, 2018; Ruderisch et al., 2017). Most of these peptides target Aβ aggregation and plaque formation, but also other pathological mechanisms of the disease can be targeted, such as attenuation of brain inflammation and/or Tau filament formation; stimulation of neurogenesis, and improvement of cognition can be observed as well. Just like peptide BBB shuttles, Aβ interacting peptides can be identified using the phage display technology (Wang et al., 2011). Besides insulin, also one other peptide (i.e., NAP) reached Phase II clinical trial for the treatment of MCI. This peptide attenuates Aβ aggregation and Tau filament formation and improves cognitive functioning in AD patients (Ribaric, 2018). PD, the second most neurodegenerative disorder in adults over the age of 65, is characterized by motor symptoms such as tremor, postural and gait impairment,

710 Chapter 28 bradykinesia, and muscular rigidity; nonmotor symptoms, encompassing mainly olfactory dysfunction, cognitive impairment, and other psychiatric symptoms are observed as well. The disease is caused by the loss of dopaminergic neurons in the substantia nigra and by the formation of Lewy bodies, that is, abnormal aggregates of the α-synuclein protein, throughout the brain. Disease management is purely symptomatic with drugs that increase dopamine levels or act directly on dopamine receptors (Kalia and Lang, 2015). Activated microglia, the innate immune cells of the brain, also actively participate in the pathogenesis of PD. Different peptides have already been described which have neuroprotective properties in an in vivo PD mouse model by blocking microglia activation, thereby blocking the expression of proinflammatory cytokines and nitric oxide (Delgado and Ganea, 2003; Kim, Moon, & Park, 2009; Moon et al., 2009). One of these peptides has reached the clinical phase for the treatment of PD. Exenatide, a 39-amino acid GLP-1 agonist, showed long-lasting improvement of both motor and nonmotor symptoms and represents a major new avenue for investigation (Athauda et al., 2017; Athauda et al., 2018). In addition, these peptides are already approved by the FDA for the treatment of type 2 diabetes which can fasten approval for other indications since long-term safety data is already available. Another already approved peptide therapeutic and GLP-1 agonist, Liraglutide, has also come to attention for the treatment of psychiatric disorders. A lot of preclinical evidence suggests that the peptide promotes neurogenesis and could improve cognition and prevent cognitive decline (Camkurt, Lavagnino, Zhang, & Teixeira, 2018). Lumbricusin, a peptide isolated from the earthworm Lumbricus terrestris also promotes neuronal cell proliferation and shows neuroprotective effects in a PD mouse model (Kim et al., 2015). In addition, this peptide also shows antineuroinflammatory effects on microglial cells (Seo et al., 2017). Another approach is, just like in AD pathology, to design peptide inhibitors that attenuate plaque formation, in this case the aggregation of α-synuclein into Lewy bodies (Mason and Fairlie, 2015). Cerebrolysin, a parenteral peptide preparation which is produced by controlled digestion of lipid-free porcine brains, contains cryptic peptides from porcine brain proteins and neuropeptides and can contain up to more than 600 unique peptide fragments (Gevaert et al., 2015). This digest has neurotrophic effects and shows beneficial effects in different neurodegenerative disorders and traumatic brain injury. Some of the beneficial effects are reduction of the pathophysiology of PD, neuroprotection in AD, improvement of learning and memory in senescent rats, and acceleration of neurorehabilitation after acute ischemic stroke and brain injury (Flores-Paez, Pacheco-Rosado, Alva-Sanchez, & Zamudio, 2019; Ozkizilcik et al., 2019; Poon et al., 2019; Sharma et al., 2019; Stan, Birle, Blesneag, & Iancu, 2017). Products like cerebrolysin are freely accessible on the market and can be easily ordered over the internet while these are not approved in most countries worldwide. This issue raises concerns as there can be an insufficiently assured quality. Furthermore, regulations and classifications of these products are not consistent in different regions of the world, further international harmonization concerning these products is thus necessary (Gevaert, Veryser, Verbeke, Wynendaele, & De Spiegeleer, 2016).

Applications in medicine: mental health 711 Finally, also in other neurocognitive disorders, peptides can have potential promising therapeutic effects. Treatment with an NCAM-derived P2 peptide improves postlesion recovery of motor and cognitive functions, reduces neuronal degeneration, protects cells against oxidative stress, and increases reactive astrogliosis and neuronal plasticity after traumatic brain injury; this is performed by stimulation of neurite outgrowth and promotion of neuronal survival (Klementiev, Novikova, Korshunova, Berezin, & Bock, 2008). Peptide T, which blocks the binding of the HIV protein gp120 to brain tissue, is associated with overall cognitive improvement in HIV-associated cognitive impairment. However, the results of this clinical trial were not significant (Heseltine et al., 1998). Next to their neuroprotective properties, peptides can be neurotoxic as well. While it is well established that the Aβ peptide is one of the causal factors for AD, other peptides also have neurotoxic effects and stimulate memory loss or cognitive decline. For example, some studies suggest that β-casomorphins, derived from milk, induce memory loss in animal models (Lister et al., 2015) and venoms from spiders, snakes, snails, scorpions, and wasps contain bioactive peptides which mainly target neuron ion-channels resulting in paralysis of their prey (Kastin, 2013). Peptides can also stimulate neuroinflammation which is involved in all types of psychiatric- and neurodegenerative disorders. For instance, LL-37, which is increased in the plasma of patients of both MDD and bipolar disorder, stimulates glia-mediated neuroinflammation in vivo (Kozlowska et al., 2017; Kozlowska et al., 2018; Lee, Shi, Barron, McGeer, & McGeer, 2015). This proinflammatory property is a potential causal factor of the disease.

28.3.5 Others Different studies suggest that peptides can be used in drug abuse-related disorders and during withdrawal of chronic drug abuse. The CART peptide, for example, has been shown to have anxiolytic functions after drug withdrawal (Meng et al., 2018). There also seems to be a correlation between atrial natriuretic peptide (ANP) plasma levels and anxiety during alcohol abstinence; moreover, administration of ANP during alcohol withdrawal shows anxiolytic activity in mice (von der Goltz, Jahn, Mutschler, Wiedemann, & Kiefer, 2014). Other peptides like neuropeptide Y are able to reduce alcohol intake and can have therapeutical potential against binge drinking (Sparrow et al., 2012). Finally, oxytocin reduces craving but not anxiety in heroin-dependent patients (Moeini, Omidi, Sehat, & Banafshe, 2019). This peptide also improves somatic signs after nicotine withdrawal and may reverse neuroadaptations after drug and alcohol abuse making it a good therapeutic candidate (Lee, Rohn, Tanda, & Leggio, 2016; Manbeck, Shelley, Schmidt, & Harris, 2014).

28.4 Conclusion Peptides play vital roles in the normal functioning and signal transmission in the CNS. In addition to neuropeptides, peripheral peptides are also involved in regulating diverse

712 Chapter 28 functions of the brain since these are able to cross the BBB by different transport mechanisms; in this way, communication between the CNS and the rest of the body is possible. This property can also be exploited by the so-called peptide shuttles, which show a high brain influx, thereby transporting other molecules and therapeutics into the brain. Peptide expression and/or levels were found to be altered in psychiatric disorders, making it therefore possible biomarkers for these diseases. Moreover, peptide-based PET and SPECT tracers can be used for the diagnosis and treatment of glioblastoma and neuroendocrine tumors as well. Finally, numerous peptides are under preclinical and clinical investigation for the treatment of different psychiatric and neurodegenerative disorders to alleviate symptoms or to target underlying causal mechanisms. Bioactive peptides can thus form new and innovative therapeutic possibilities in the field of psychiatry and other mental disorders.

References Adamczuk, K., Schaeverbeke, J., Vanderstichele, H. M., Lilja, J., Nelissen, N., Van Laere, K., . . . Vandenberghe, R. (2015). Diagnostic value of cerebrospinal fluid Abeta ratios in preclinical Alzheimer’s disease. Alzheimer’s Research & Therapy, 7(1), 75. Akter, S., Pham, N. M., Nanri, A., Kurotani, K., Kuwahara, K., Jacka, F. N., . . . Mizoue, T. (2014). Association of serum leptin and ghrelin with depressive symptoms in a Japanese working population: A cross-sectional study. BMC Psychiatry, 14, 203. Almeida-Santos, A. F., Kangussu, L. M., Moreira, F. A., Santos, R. A., Aguiar, D. C., & Campagnole-Santos, M. J. (2016). Anxiolytic- and antidepressant-like effects of angiotensin-(1-7) in hypertensive transgenic (mRen2)27 rats. Clinical Science (London), 130(14), 1247
1255. Ambrosini, V., Fani, M., Fanti, S., Forrer, F., & Maecke, H. R. (2011). Radiopeptide imaging and therapy in Europe. Journal of Nuclear Medicine, 52(Suppl 2), 42S
55S. Anderson, I. M., Haddad, P. M., & Scott, J. (2012). Bipolar disorder. British Medical Journal, 345, e8508. Ano, Y., Ayabe, T., Kutsukake, T., Ohya, R., Takaichi, Y., Uchida, S., . . . Nakayama, H. (2018). Novel lactopeptides in fermented dairy products improve memory function and cognitive decline. Neurobiology of Aging, 72, 23
31. Ano, Y., Kita, M., Kitaoka, S., & Furuyashiki, T. (2019). Leucine-histidine dipeptide attenuates microglial activation and emotional disturbances induced by brain inflammation and repeated social defeat stress. Nutrients, 11(9). Ano, Y., Yoshino, Y., Kutsukake, T., Ohya, R., Fukuda, T., Uchida, K., . . . Nakayama, H. (2019). Tryptophanrelated dipeptides in fermented dairy products suppress microglial activation and prevent cognitive decline. Aging (Albany NY), 11(10), 2949
2967. Asua, D., Bougamra, G., Calleja-Felipe, M., Morales, M., & Knafo, S. (2018). Peptides acting as cognitive enhancers. Neuroscience, 370, 81
87. Athauda, D., Maclagan, K., Budnik, N., Zampedri, L., Hibbert, S., Skene, S. S., . . . Foltynie, T. (2018). What effects might exenatide have on non-motor symptoms in Parkinson’s disease: A post hoc analysis. Journal of Parkinson’s Disease, 8(2), 247
258. Athauda, D., Maclagan, K., Skene, S. S., Bajwa-Joseph, M., Letchford, D., Chowdhury, K., . . . Foltynie, T. (2017). Exenatide once weekly versus placebo in Parkinson’s disease: A randomised, double-blind, placebo-controlled trial. Lancet, 390(10103), 1664
1675. Bakhshinejad, B., Karimi, M., & Khalaj-Kondori, M. (2015). Phage display: Development of nanocarriers for targeted drug delivery to the brain. Neural Regeneration Research, 10(6), 862
865. Banks, W. A. (2015). Peptides and the blood-brain barrier. Peptides, 72, 16
19.

Applications in medicine: mental health 713 Beck, B., & Pourie, G. (2013). Ghrelin, neuropeptide Y, and other feeding-regulatory peptides active in the hippocampus: Role in learning and memory. Nutrition Reviews, 71(8), 541
561. Becker, M. M., Riesgo, R. S., Roesler, R., Bosa, C., Ohlweiler, L., Backes, B., . . . Schwartsmann, G. (2016). Improvement in symptoms of autism spectrum disorder in children with the use of gastrin-releasing peptide: An open trial. Clinical Neuropharmacology, 39(5), 215
219. Begic, E., Hadzidedic, S., Kulaglic, A., Ramic-Brkic, B., Begic, Z., & Causevic, M. (2019). SOMAscan-based proteomic measurements of plasma brain natriuretic peptide are decreased in mild cognitive impairment and in ’Alzheimer’s dementia patients. PLoS One, 14(2), e0212261. Bobyntsev, I. I., Chernysheva, O. I., Dolgintsev, M. E., Smakhtin, M. Y., & Belykh, A. E. (2015). Anxiolytic effects of Gly-His-Lys peptide and its analogs. Bulletin of Experimental Biology and Medicine, 158(6), 726
728. Bojovic, K., Stankovic, B., Kotur, N., Krstic-Milosevic, D., Gasic, V., Pavlovic, S., . . . Ignjatovic, D. (2019). Genetic predictors of celiac disease, lactose intolerance, and vitamin D function and presence of peptide morphins in urine of children with neurodevelopmental disorders. Nutritional Neuroscience, 22(1), 40
50. Borkar, C. D., Bharne, A. P., Nagalakshmi, B., Sakharkar, A. J., Subhedar, N. K., & Kokare, D. M. (2018). Cocaine- and amphetamine-regulated transcript peptide (CART) alleviates MK-801-induced schizophrenic dementia-like symptoms. Neuroscience, 375, 94
107. Bradley, E. R., Seitz, A., Niles, A. N., Rankin, K. P., Mathalon, D. H., O’Donovan, A., & Woolley, J. D. (2019). Oxytocin increases eye gaze in schizophrenia. Schizophrenia Research, 212, 177
185. Calva, C. B., & Fadel, J. R. (2018). Intranasal administration of orexin peptides: Mechanisms and therapeutic potential for age-related cognitive dysfunction. Brain Research, 145921. Camkurt, M. A., Lavagnino, L., Zhang, X. Y., & Teixeira, A. L. (2018). Liraglutide for psychiatric disorders: Clinical evidence and challenges. Hormone Molecular Biology and Clinical Investigation, 36(2). Cheng, Y. S., Chen, Z. T., Liao, T. Y., Lin, C., Shen, H. C., Wang, Y. H., . . . Tu, P. H. (2017). An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice. EMBO Molecular Medicine, 9(5), 703
715. Cohen-Inbar, O., & Zaaroor, M. (2013). Glioblastoma multiforme targeted therapy: The Chlorotoxin story. Journal of Clinical Neuroscience, 33, 52
58. Cordier, D., Forrer, F., Bruchertseifer, F., Morgenstern, A., Apostolidis, C., Good, S., . . . Merlo, A. (2010). Targeted alpha-radionuclide therapy of functionally critically located gliomas with 213Bi-DOTA-[Thi8, Met(O2)11]-substance P: A pilot trial. European Journal of Nuclear Medicine and Molecular Imaging, 37 (7), 1335
1344. Cordier, D., Forrer, F., Kneifel, S., Sailer, M., Mariani, L., Macke, H., . . . Merlo, A. (2010). Neoadjuvant targeting of glioblastoma multiforme with radiolabeled DOTAGA-substance P--results from a phase I study. Journal of Neuro-Oncology, 100(1), 129
136. Corpuz, H. M., Fujii, H., Nakamura, S., & Katayama, S. (2019). Fermented rice peptides attenuate scopolamineinduced memory impairment in mice by regulating neurotrophic signaling pathways in the hippocampus. Brain Research, 1720, 146322. Craske, M. G., Stein, M. B., Eley, T. C., Milad, M. R., Holmes, A., Rapee, R. M., & Wittchen, H. U. (2017). Anxiety disorders. Nature Reviews Disease Primers, 3, 17024. Cui, Y., Huang, C., Momma, H., Ren, Z., Sugiyama, S., Guan, L., . . . Nagatomi, R. (2017). Consumption of low-fat dairy, but not whole-fat dairy, is inversely associated with depressive symptoms in Japanese adults. Social Psychiatry and Psychiatric Epidemiology, 52(7), 847
853. D’Souza, H., & Karmiloff-Smith, A. (2017). Neurodevelopmental disorders. Wiley Interdisciplinary Reviews: Cognitive Science, 8(1-2). Daneman, R., & Prat, A. (2015). The blood-brain barrier. Cold Spring Harbor Perspectives in Biology, 7(1), a020412. Daniels, L. B., Laughlin, G. A., Kritz-Silverstein, D., Clopton, P., Chen, W. C., Maisel, A. S., & BarrettConnor, E. (2011). Elevated natriuretic peptide levels and cognitive function in community-dwelling older adults. The American Journal of Medicine, 124(7), 670 e1-8.

714 Chapter 28 Davis, M. C., Lee, J., Horan, W. P., Clarke, A. D., McGee, M. R., Green, M. F., & Marder, S. R. (2013). Effects of single dose intranasal oxytocin on social cognition in schizophrenia. Schizophrenia Research, 147(2-3), 393
397. De Coster, L., Lin, L., Mathalon, D. H., & Woolley, J. D. (2019). Neural and behavioral effects of oxytocin administration during theory of mind in schizophrenia and controls: A randomized control trial. Neuropsychopharmacology, 44(11), 1925
1931. Delgado, M., & Ganea, D. (2003). Neuroprotective effect of vasoactive intestinal peptide (VIP) in a mouse model of Parkinson’s disease by blocking microglial activation. The FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 17(8), 944
946. Deutch, A. Y., & Bubser, M. (2007). The orexins/hypocretins and schizophrenia. Schizophrenia Bulletin, 33(6), 1277
1283. Diaz-Perlas, C., Sanchez-Navarro, M., Oller-Salvia, B., Moreno, M., Teixido, M., & Giralt, E. (2017). Phage display as a tool to discover blood-brain barrier (BBB)-shuttle peptides: Panning against a human BBB cellular model. Biopolymers, 108(1). Dinan, T. G., & Cryan, J. F. (2017). The microbiome-gut-brain axis in health and disease. Gastroenterology Clinics of North America, 46(1), 77
89. Djillani, A., Pietri, M., Mazella, J., Heurteaux, C., & Borsotto, M. (2019). Fighting against depression with TREK-1 blockers: Past and future. A focus on spadin. Pharmacology & Therapeutics, 194, 185
198. Drucker, D. J. (2020). Advances in oral peptide therapeutics. Nature Reviews Drug Discovery, 19(4), 277
289. Du¨rr, U. H. N., Sudheendra, U. S., & Ramamoorthy, A. (2006). LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1758 (9), 1408
1425. Dzirkale, Z., Nakurte, I., Jekabsons, K., Muceniece, R., & Klusa, V. (2019). Intra-nasally administered oligopeptide lunasin acts as a possible anti-psychotic agent in mice models. Medicina (Kaunas, Lithuania), 55(7). Ehrensing, R. H., & Kastin, A. J. (1978). Dose-related biphasic effect of prolyl-leucyl-glycinamide (MIF-I) in depression. The American Journal of Psychiatry, 135(5), 562
566. Ehrensing, R. H., Kastin, A. J., Wurzlow, G. F., Michell, G. F., & Mebane, A. H. (1994). Improvement in major depression after low subcutaneous doses of MIF-1. Journal of Affective Disorders, 31(4), 227
233. Erdo, F., Bors, L. A., Farkas, D., Bajza, A., & Gizurarson, S. (2018). Evaluation of intranasal delivery route of drug administration for brain targeting. Brain Research Bulletin, 143, 155
170. Eslami, M., Sadeghi, B., & Goshadrou, F. (2018). Chronic ghrelin administration restores hippocampal longterm potentiation and ameliorates memory impairment in rat model of Alzheimer’s disease. Hippocampus, 28(10), 724
734. Fan, D., Zhang, X., Zhong, L., Liu, X., Sun, Y., Zhao, H., . . . Wang, F. (2015). (68)Ga-labeled 3PRGD2 for dual PET and Cerenkov luminescence imaging of orthotopic human glioblastoma. Bioconjugate Chemistry, 26(6), 1054
1060. Farrall, A. J., & Wardlaw, J. M. (2009). Blood-brain barrier: Ageing and microvascular disease--systematic review and meta-analysis. Neurobiology of Aging, 30(3), 337
352. Feighner, J. P., Sverdlov, L., Hlavka, J., Nicolau, G., Cartwright, K., & Freed, J. S. (2008). Clinical effect of nemifitide, a novel pentapeptide antidepressant, in the treatment of severely depressed refractory patients. International Clinical Psychopharmacology, 23(1), 29
35. Fernandez-Tome, S., & Hernandez-Ledesma, B. (2019). Current state of art after twenty years of the discovery of bioactive peptide lunasin. Food Research International, 116, 71
78. Fernstrom, J. D., Langham, K. A., Marcelino, L. M., Irvine, Z. L., Fernstrom, M. H., & Kaye, W. H. (2013). The ingestion of different dietary proteins by humans induces large changes in the plasma tryptophan ratio, a predictor of brain tryptophan uptake and serotonin synthesis. Clinical Nutrition, 32(6), 1073
1076. Flores-Paez, L. A., Pacheco-Rosado, J., Alva-Sanchez, C., & Zamudio, S. R. (2019). Cerebrolysin enhances the expression of the synaptogenic protein LRRTM4 in the hippocampus and improves learning and memory in senescent rats. Behavioural Pharmacology.

Applications in medicine: mental health 715 Gauthier, S., Reisberg, B., Zaudig, M., Petersen, R. C., Ritchie, K., Broich, K., . . . Winblad, B. (2006). Mild cognitive impairment. The Lancet, 367(9518), 1262
1270. Gevaert, B., D’Hondt, M., Bracke, N., Yao, H., Wynendaele, E., Vissers, J. P., . . . De Spiegeleer, B. (2015). Peptide profiling of Internet-obtained Cerebrolysin using high performance liquid chromatography electrospray ionization ion trap and ultra high performance liquid chromatography - ion mobility quadrupole time of flight mass spectrometry. Drug Testing and Analysis, 7(9), 835
842. Gevaert, B., Veryser, L., Verbeke, F., Wynendaele, E., & De Spiegeleer, B. (2016). Fish hydrolysates: A regulatory perspective of bioactive peptides. Protein and Peptide Letters, 23(12), 1052
1060. von der Goltz, C., Jahn, H., Mutschler, J., Wiedemann, K., & Kiefer, F. (2014). Intraperitoneal atrial natriuretic peptide attenuates anxiety-related behaviour during alcohol withdrawal in mice. Pharmacopsychiatry, 47(3), 97
100. Gomes, F. M., Paniago, C. K., Freire, D. O., Souza, A. C., Lima, M. R., Oliveira-Junior, N. G., . . . Mortari, M. R. (2016). Anxiolytic-like effect of a novel peptide isolated from the venom of the social wasp Synoeca surinama. Toxicon, 122, 39
42. Gottschalk, M. G., & Domschke, K. (2017). Oxytocin and anxiety disorders. In R. Hurlemann, & V. Grinevich (Eds.), Behavioral pharmacology of neuropeptides: Oxytocin (pp. 467
498). Cham: Springer International Publishing. Guest, P. C., Schwarz, E., Krishnamurthy, D., Harris, L. W., Leweke, F. M., Rothermundt, M., . . . Bahn, S. (2011). Altered levels of circulating insulin and other neuroendocrine hormones associated with the onset of schizophrenia. Psychoneuroendocrinology, 36(7), 1092
1096. Guzel, D., Yazici, A. B., Pek, T. M., Doganay, S., Simsek, A. B. S., Saglam, K., . . . Yazici, E. (2018). Atrial natriuretic peptide and posterior pituitary neurohormone changes in patients with acute schizophrenia. Neuropsychiatric Disease and Treatment, 14, 1855
1860. Guzman, A., Hernandez-Coronado, C. G., Rosales-Torres, A. M., & Hernandez-Medrano, J. H. (2019). Leptin regulates neuropeptides associated with food intake and GnRH secretion. Annales D’endocrinologie, 80(1), 38
46. Heseltine, P. N., Goodkin, K., Atkinson, J. H., Vitiello, B., Rochon, J., Heaton, R. K., . . . Stover, E. S. (1998). Randomized double-blind placebo-controlled trial of peptide T for HIV-associated cognitive impairment. Archives of Neurology, 55(1), 41
51. Heurteaux, C., Lucas, G., Guy, N., El Yacoubi, M., Thummler, S., Peng, X. D., . . . Lazdunski, M. (2006). Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype. Nature Neuroscience, 9(9), 1134
1141. Hiltunen, M., Nieminen, T., Kettunen, R., Hartikainen, S., Sulkava, R., Vuolteenaho, O., & Kerola, T. (2013). B-type natriuretic peptide and severity of cognitive disorder. European Journal of Clinical Investigation, 43(11), 1171
1177. Hockaday, D. C., Shen, S., Fiveash, J., Raubitschek, A., Colcher, D., Liu, A., . . . Mamelak, A. N. (2005). Imaging glioma extent with 131I-TM-601. Journal of Nuclear Medicine, 46(4), 580
586. Hokfelt, T., Bartfai, T., & Bloom, F. (2003). Neuropeptides: Opportunities for drug discovery. Lancet Neurology, 2(8), 463
472. Hokfelt, T., Broberger, C., Xu, Z. Q., Sergeyev, V., Ubink, R., & Diez, M. (2000). Neuropeptides--An overview. Neuropharmacology, 39(8), 1337
1356. Holtta, M., Hansson, O., Andreasson, U., Hertze, J., Minthon, L., Nagga, K., . . . Blennow, K. (2013). Evaluating amyloid-beta oligomers in cerebrospinal fluid as a biomarker for ’Alzheimer’s disease. PLoS One, 8(6), e66381. Holzer, P., Reichmann, F., & Farzi, A. (2012). Neuropeptide Y., peptide Y.Y. and pancreatic polypeptide in the gut-brain axis. Neuropeptides, 46(6), 261
274. Hubert, G. W., Jones, D. C., Moffett, M. C., Rogge, G., & Kuhar, M. J. (2008). CART peptides as modulators of dopamine and psychostimulants and interactions with the mesolimbic dopaminergic system. Biochemical Pharmacology, 75(1), 57
62. Hung, L. W., Neuner, S., Polepalli, J. S., Beier, K. T., Wright, M., Walsh, J. J., . . . Malenka, R. C. (2017). Gating of social reward by oxytocin in the ventral tegmental area. Science, 357(6358), 1406
1411. Imai, H., Moriyasu, K., Nakahata, A., Maebuchi, M., Ichinose, T., & Furuya, S. (2017). Soy peptide ingestion augments the synthesis and metabolism of noradrenaline in the mouse brain. Bioscience, Biotechnology, and Biochemistry, 81(5), 1007
1013.

716 Chapter 28 Isal, S., Pierson, J., Imbert, L., Clement, A., Collet, C., Pinel, S., . . . Maskali, F. (2018). PET imaging of (68) Ga-NODAGA-RGD, as compared with (18)F-fluorodeoxyglucose, in experimental rodent models of engrafted glioblastoma. EJNMMI Research, 8(1), 51. Iwai, T., Jin, K., Ohnuki, T., Sasaki-Hamada, S., Nakamura, M., Saitoh, A., . . . Oka, J. (2015). Glucagon-like peptide-2-induced memory improvement and anxiolytic effects in mice. Neuropeptides, 49, 7
14. Janssens, Y., Nielandt, J., Bronselaer, A., Debunne, N., Verbeke, F., Wynendaele, E., . . . De Spiegeleer, B. (2018). Disbiome database: Linking the microbiome to disease. BMC Microbiology, 18(1), 50. Janssens, Y., Wynendaele, E., Vanden Berghe, W., & De Spiegeleer, B. (2019). Peptides as epigenetic modulators: Therapeutic implications. Clinical Epigenetics, 11(1), 101. Janssens, Y., Wynendaele, E., Verbeke, F., Debunne, N., Gevaert, B., Audenaert, K., . . . De Spiegeleer, B. (2018). Screening of quorum sensing peptides for biological effects in neuronal cells. Peptides, 101, 150
156. Jarskog, L. F., Dong, Z., Kangarlu, A., Colibazzi, T., Girgis, R. R., Kegeles, L. S., . . . Lieberman, J. A. (2013). Effects of davunetide on N-acetylaspartate and choline in dorsolateral prefrontal cortex in patients with schizophrenia. Neuropsychopharmacology, 38(7), 1245
1252. Jarskog, L. F., Pedersen, C. A., Johnson, J. L., Hamer, R. M., Rau, S. W., Elliott, T., & Penn, D. L. (2017). A 12-week randomized controlled trial of twice-daily intranasal oxytocin for social cognitive deficits in people with schizophrenia. Schizophrenia Research, 185, 88
95. Javitt, D. C., Buchanan, R. W., Keefe, R. S., Kern, R., McMahon, R. P., Green, M. F., . . . Marder, S. R. (2012). Effect of the neuroprotective peptide davunetide (AL-108) on cognition and functional capacity in schizophrenia. Schizophrenia Research, 136(1-3), 25
31. Kalia, L. V., & Lang, A. E. (2015). ’Parkinson’s disease. Lancet, 386(9996), 896
912. Karatas, H., Yemisci, M., Eren-Kocak, E., & Dalkara, T. (2018). Brain peptides for the treatment of neuropsychiatric disorders. Current Pharmaceutical Design, 24(33), 3905
3917. Karimi, H., Sadeghzadeh, N., Abediankenari, S., Rezazadeh, F., & Hallajian, F. (2017). Radiochemical evaluation and in vitro assessment of the targeting ability of a novel 99mTc-HYNIC-RGD for U87MG human brain cancer cells. Current Radiopharmaceuticals, 10(2), 139
144. Kastin, A. J. (2013). In A. J. Kastin (Ed.), Handbook of biologically active peptides (Second edition). Boston: Academic Press. Katayama, S., Imai, R., Sugiyama, H., & Nakamura, S. (2014). Oral administration of soy peptides suppresses cognitive decline by induction of neurotrophic factors in SAMP8 mice. Journal of Agricultural and Food Chemistry, 62(16), 3563
3569. Kaur, C., & Ling, E. A. (2017). The circumventricular organs. Histology and Histopathology, 32(9), 879
892. Kerola, T., Nieminen, T., Hartikainen, S., Sulkava, R., Vuolteenaho, O., & Kettunen, R. (2010). B-type natriuretic peptide as a predictor of declining cognitive function and dementia--A cohort study of an elderly general population with a 5-year follow-up. Annals of Medicine, 42(3), 207
215. Khan, H., Khattak, S., Mubarak, M. S., Bawazeer, S. S., Abu-Izneid, T., & Kamal, M. A. (2018). Antidepressant potential of peptides: New insights as future therapeutic. CNS & Neurological Disorders Drug Targets, 17(1), 9
13. Khan, R. S., Yu, C., Kastin, A. J., He, Y., Ehrensing, R. H., Hsuchou, H., . . . Pan, W. (2010). Brain activation by peptide Pro-Leu-Gly-NH(2) (MIF-1). International Journal of Peptides, 2010. Kim, D. H., Lee, I. H., Nam, S. T., Hong, J., Zhang, P., Lu, L. F., . . . Kim, H. (2015). Antimicrobial peptide, lumbricusin, ameliorates motor dysfunction and dopaminergic neurodegeneration in a mouse model of ’Parkinson’s disease. Journal of Microbiology and Biotechnology, 25(10), 1640
1647. Kim, S., Moon, M., & Park, S. (2009). Exendin-4 protects dopaminergic neurons by inhibition of microglial activation and matrix metalloproteinase-3 expression in an animal model of ’Parkinson’s disease. The Journal of Endocrinology, 202(3), 431
439. Kinkead, B., Selz, K. A., Owens, M. J., & Mandell, A. J. (2006). Algorithmically designed peptides ameliorate behavioral defects in animal model of ADHD by an allosteric mechanism. Journal of Neuroscience Methods, 151(1), 68
81.

Applications in medicine: mental health 717 Klementiev, B., Novikova, T., Korshunova, I., Berezin, V., & Bock, E. (2008). The NCAM-derived P2 peptide facilitates recovery of cognitive and motor function and ameliorates neuropathology following traumatic brain injury. The European Journal of Neuroscience, 27(11), 2885
2896. Kneifel, S., Bernhardt, P., Uusijarvi, H., Good, S., Plasswilm, L., Buitrago-Tellez, C., . . . Merlo, A. (2007). Individual voxelwise dosimetry of targeted 90Y-labelled substance P radiotherapy for malignant gliomas. European Journal of Nuclear Medicine and Molecular Imaging, 34(9), 1388
1395. Kneifel, S., Cordier, D., Good, S., Ionescu, M. C., Ghaffari, A., Hofer, S., . . . Merlo, A. (2006). Local targeting of malignant gliomas by the diffusible peptidic vector 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7, 10-triacetic acid-substance p. Clinical Cancer Research, 12(12), 3843
3850. Kozlowska, E., Wysokinski, A., & Brzezinska-Blaszczyk, E. (2017). Serum levels of peptide cathelicidin LL-37 in elderly patients with depression. Psychiatry Research, 255, 156
160. Kozlowska, E., Zelechowska, P., Brzezinska-Blaszczyk, E., Margulska, A., & Wysokinski, A. (2018). Circulating cathelicidin LL-37 level is increased in euthymic patients with bipolar disorder. Journal of Clinical Neuroscience, 48, 168
172. Kristensen, P., Judge, M. E., Thim, L., Ribel, U., Christjansen, K. N., Wulff, B. S., . . . Hastrup, S. (1998). Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature, 393(6680), 72
76. Krolicki, L., Bruchertseifer, F., Kunikowska, J., Koziara, H., Krolicki, B., Jakucinski, M., . . . Morgenstern, A. (2019). Safety and efficacy of targeted alpha therapy with (213)Bi-DOTA-substance P in recurrent glioblastoma. European Journal of Nuclear Medicine and Molecular Imaging, 46(3), 614
622. Kumar, P., Wu, H., McBride, J. L., Jung, K. E., Kim, M. H., Davidson, B. L., . . . Manjunath, N. (2007). Transvascular delivery of small interfering RNA to the central nervous system. Nature, 448(7149), 39
43. Lach, G., Schellekens, H., Dinan, T. G., & Cryan, J. F. (2018). Anxiety, depression, and the microbiome: A role for gut peptides. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics, 15(1), 36
59. Lai, T. K. Y., Su, P., Zhang, H., & Liu, F. (2018). Development of a peptide targeting dopamine transporter to improve ADHD-like deficits. Molecular Brain, 11(1), 66. Lamberts, S. W. J., & Hofland, L. J. (2019). ANNIVERSARY REVIEW: Octreotide, 40 years later. European Journal of Endocrinology, 181(5), R173
R183. Lee, J., Cho, H. R., Cha, G. D., Seo, H., Lee, S., Park, C. K., . . . Kim, D. H. (2019). Flexible, sticky, and biodegradable wireless device for drug delivery to brain tumors. Nature Communications, 10(1), 5205. Lee, M., Shi, X., Barron, A. E., McGeer, E., & McGeer, P. L. (2015). Human antimicrobial peptide LL-37 induces glial-mediated neuroinflammation. Biochemical Pharmacology, 94(2), 130
141. Lee, M. R., & Jayant, R. D. (2019). Penetration of the blood-brain barrier by peripheral neuropeptides: New approaches to enhancing transport and endogenous expression. Cell and Tissue Research, 375(1), 287
293. Lee, M. R., Rohn, M. C., Tanda, G., & Leggio, L. (2016). Targeting the oxytocin system to treat addictive disorders: Rationale and progress to date. CNS Drugs, 30(2), 109
123. Lee, M. R., Wehring, H. J., McMahon, R. P., Liu, F., Linthicum, J., Buchanan, R. W., . . . Kelly, D. L. (2019). The Effect of intranasal oxytocin on measures of social cognition in schizophrenia: A negative report. Journal of Psychiatry and Brain Science, 4(1). Li, C., Li, M., Yu, H., Shen, X., Wang, J., Sun, X., . . . Wang, C. (2017). Neuropeptide VGF C-terminal peptide TLQP-62 alleviates lipopolysaccharide-induced memory deficits and anxiety-like and depression-like behaviors in mice: The role of BDNF/TrkB signaling. ACS Chemical Neuroscience, 8(9), 2005
2018. Lieberman, J. A., & First, M. B. (2018). Psychotic disorders. The New England Journal of Medicine, 379(3), 270
280. Lister, J., Fletcher, P. J., Nobrega, J. N., & Remington, G. (2015). Behavioral effects of food-derived opioid-like peptides in rodents: Implications for schizophrenia? Pharmacology, Biochemistry, and Behavior, 134, 70
78. Liu, Y., & Reichelt, K. L. (2001). A serotonin uptake-stimulating tetra-peptide found in urines from ADHD children. The World Journal of Biological Psychiatry, 2(3), 144
148. Liu, Z., & Udenigwe, C. C. (2019). Role of food-derived opioid peptides in the central nervous and gastrointestinal systems. Journal of Food Biochemistry, 43(1), e12629.

718 Chapter 28 Lopez, A., Mendieta, L., Prades, R., Royo, S., Tarrago, T., & Giralt, E. (2013). Peptide POP inhibitors for the treatment of the cognitive symptoms of schizophrenia. Future Medicinal Chemistry, 5(13), 1509
1523. Majkowska-Pilip, A., Rius, M., Bruchertseifer, F., Apostolidis, C., Weis, M., Bonelli, M., . . . Morgenstern, A. (2018). In vitro evaluation of (225) Ac-DOTA-substance P for targeted alpha therapy of glioblastoma multiforme. Chemical Biology & Drug Design, 92(1), 1344
1356. Mamelak, A. N., Rosenfeld, S., Bucholz, R., Raubitschek, A., Nabors, L. B., Fiveash, J. B., . . . Gonda, M. A. (2006). Phase I single-dose study of intracavitary-administered iodine-131-TM-601 in adults with recurrent high-grade glioma. Journal of Clinical Oncology, 24(22), 3644
3650. Manbeck, K. E., Shelley, D., Schmidt, C. E., & Harris, A. C. (2014). Effects of oxytocin on nicotine withdrawal in rats. Pharmacology, Biochemistry, and Behavior, 116, 84, -. Mann, A. P., Scodeller, P., Hussain, S., Braun, G. B., Molder, T., Toome, K., . . . Ruoslahti, E. (2017). Identification of a peptide recognizing cerebrovascular changes in mouse models of ’Alzheimer’s disease. Nature Communications, 8(1), 1403. Marchezan, J., Becker, M., Schwartsmann, G., Ohlweiler, L., Roesler, R., Renck, L. B., . . . Riesgo, R. D. S. (2017). A placebo-controlled crossover trial of gastrin-releasing peptide in childhood autism. Clinical Neuropharmacology, 40(3), 108
112. Mason, J. M., & Fairlie, D. P. (2015). Toward peptide-based inhibitors as therapies for ’Parkinson’s disease. Future Medicinal Chemistry, 7(16), 2103
2105. Mathe, A. A., Agren, H., Lindstrom, L., & Theodorsson, E. (1994). Increased concentration of calcitonin generelated peptide in cerebrospinal fluid of depressed patients. A possible trait marker of major depressive disorder. Neuroscience Letters, 182(2), 138
142. Meng, Q., Kim, H. C., Oh, S., Lee, Y. M., Hu, Z., & Oh, K. W. (2018). Cocaine- and amphetamine-regulated transcript (CART) peptide plays critical role in psychostimulant-induced depression. Biomolecules and Therapeutics (Seoul), 26(5), 425
431. Meredith, M. E., Salameh, T. S., & Banks, W. A. (2015). Intranasal delivery of proteins and peptides in the treatment of neurodegenerative diseases. The AAPS journal, 17(4), 780
787. Min, L. J., Kobayashi, Y., Mogi, M., Tsukuda, K., Yamada, A., Yamauchi, K., . . . Horiuchi, M. (2017). Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice. PLoS One, 12(2), e0171515. Mizushige, T., Sawashi, Y., Yamada, A., Kanamoto, R., & Ohinata, K. (2013). Characterization of Tyr-Leu-Gly, a novel anxiolytic-like peptide released from bovine alphaS-casein. The FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 27(7), 2911
2917. Moeini, M., Omidi, A., Sehat, M., & Banafshe, H. R. (2019). The effects of oxytocin on withdrawal, craving and stress response in heroin-dependent patients: A randomized, double-blind clinical trial. European Addiction Research, 25(1), 41
47. Moon, M., Kim, H. G., Hwang, L., Seo, J. H., Kim, S., Hwang, S., . . . Park, S. (2009). Neuroprotective effect of ghrelin in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of ’Parkinson’s disease by blocking microglial activation. Neurotoxicity Research, 15(4), 332
347. Muller, E. G., Edwin, T. H., Stokke, C., Navelsaker, S. S., Babovic, A., Bogdanovic, N., . . . Revheim, M. E. (2019). Amyloid-beta PET-Correlation with cerebrospinal fluid biomarkers and prediction of Alzheimer s disease diagnosis in a memory clinic. PLoS One, 14(8), e0221365. Nortley, R., Korte, N., Izquierdo, P., Hirunpattarasilp, C., Mishra, A., Jaunmuktane, Z., . . . Attwell, D. (2019). Amyloid beta oligomers constrict human capillaries in ’Alzheimer’s disease via signaling to pericytes. Science, 365(6450). Oda, A., Kaneko, K., Mizushige, T., Lazarus, M., Urade, Y., & Ohinata, K. (2012). Characterization of ovolin, an orally active tryptic peptide released from ovalbumin with anxiolytic-like activity. Journal of Neurochemistry, 122(2), 356
362. Oller-Salvia, B., Sanchez-Navarro, M., Giralt, E., & Teixido, M. (2016). Blood-brain barrier shuttle peptides: an emerging paradigm for brain delivery. Chemical Society reviews, 45(17), 4690
4707. Otte, C., Gold, S. M., Penninx, B. W., Pariante, C. M., Etkin, A., Fava, M., . . . Schatzberg, A. F. (2016). Major depressive disorder. Nature Reviews Disease Primers, 2, 16065.

Applications in medicine: mental health 719 Ozkizilcik, A., Sharma, A., Lafuente, J. V., Muresanu, D. F., Castellani, R. J., Nozari, A., . . . Sharma, H. S. (2019). Nanodelivery of cerebrolysin reduces pathophysiology of ’Parkinson’s disease. Progress in Brain Research, 245, 201
246. Pagani, M., Carletto, S., & Ostacoli, L. (2019). PET and SPECT in psychiatry: The past and the future. European Journal of Nuclear Medicine and Molecular Imaging, 46(10), 1985
1987. Parker, K. J., Oztan, O., Libove, R. A., Mohsin, N., Karhson, D. S., Sumiyoshi, R. D., . . . Hardan, A. Y. (2019). A randomized placebo-controlled pilot trial shows that intranasal vasopressin improves social deficits in children with autism. Science Translational Medicine, 11(491). Pascual, M., & Guerri, C. (2007). The peptide NAP promotes neuronal growth and differentiation through extracellular signal-regulated protein kinase and Akt pathways, and protects neurons co-cultured with astrocytes damaged by ethanol. Journal of Neurochemistry, 103(2), 557
568. Piccinni, A., Origlia, N., Veltri, A., Vizzaccaro, C., Marazziti, D., Catena-Dell’Osso, M., . . . Dell’Osso, L. (2012). Plasma beta-amyloid peptides levels: A pilot study in bipolar depressed patients. Journal of Affective Disorders, 138(1), 160
164. Pietri, M., Djillani, A., Mazella, J., Borsotto, M., & Heurteaux, C. (2019). First evidence of protective effects on stroke recovery and post-stroke depression induced by sortilin-derived peptides. Neuropharmacology, 158, 107715. Pomara, N., & Sidtis, J. J. (2010). Brain neurotoxic amyloid-beta peptides: Their potential role in the pathophysiology of depression and as molecular therapeutic targets. British Journal of Pharmacology, 161(4), 768
770. Poon, W., Matula, C., Vos, P. E., Muresanu, D. F., von Steinbuchel, N., von Wild, K., . . . Vester, J. C. (2019). Safety and efficacy of Cerebrolysin in acute brain injury and neurorecovery: CAPTAIN I-a randomized, placebo-controlled, double-blind, Asian-Pacific trial. Neurological ScienceS. Regina, A., Demeule, M., Tripathy, S., Lord-Dufour, S., Currie, J. C., Iddir, M., . . . Lachowicz, J. E. (2015). ANG4043, a novel brain-penetrant peptide-mAb conjugate, is efficacious against HER2-positive intracranial tumors in mice. Molecular Cancer Therapeutics, 14(1), 129
140. Ribaric, S. (2018). Peptides as potential therapeutics for ’Alzheimer’s disease. Molecules, 23(2). Roesler, R., Henriques, J. A., & Schwartsmann, G. (2006). Gastrin-releasing peptide receptor as a molecular target for psychiatric and neurological disorders. CNS & Neurological Disorders Drug Targets, 5(2), 197
204. Ruderisch, N., Schlatter, D., Kuglstatter, A., Guba, W., Huber, S., Cusulin, C., . . . Freskgard, P. O. (2017). Potent and selective BACE-1 peptide inhibitors lower brain abeta levels mediated by brain shuttle transport. EBioMedicine, 24, 76
92. Russo, A. F. (2017). Overview of neuropeptides: Awakening the senses? Headache, 57(Suppl 2), 37
46. Sanchez-Navarro, M., Garcia, J., Giralt, E., & Teixido, M. (2016). Using peptides to increase transport across the intestinal barrier. Advanced Drug Delivery Reviews, 106(Pt B), 355
366. Sanchez-Navarro, M., Giralt, E., & Teixido, M. (2017). Blood-brain barrier peptide shuttles. Current Opinion in Chemical Biology, 38, 134
140. Scheltens, P., Blennow, K., Breteler, M. M., de Strooper, B., Frisoni, G. B., Salloway, S., & Van der Flier, W. M. (2016). ’Alzheimer’s disease. Lancet, 388(10043), 505
517. Schipke, C. G., Koglin, N., Bullich, S., Joachim, L. K., Haas, B., Seibyl, J., . . . Peters, O. (2017). Correlation of florbetaben PET imaging and the amyloid peptide Ass42 in cerebrospinal fluid. Psychiatry Research: Neuroimaging, 265, 98
101. Seidita, G., Mirisola, M., D’Anna, R. P., Gallo, A., Jensen, R. T., Mantey, S. A., . . . Cali, F. (2008). Analysis of the gastrin-releasing peptide receptor gene in Italian patients with autism spectrum disorders. American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics: the Official Publication of the International Society of Psychiatric Genetics, 147B(6), 807
813. Seo, M., Lee, J. H., Baek, M., Kim, M. A., Ahn, M. Y., Kim, S. H., . . . Hwang, J. S. (2017). A novel role for earthworm peptide Lumbricusin as a regulator of neuroinflammation. Biochemical and Biophysical Research Communications, 490(3), 1004
1010.

720 Chapter 28 Sharma, H. S., Muresanu, D. F., Castellani, R. J., Nozari, A., Lafuente, J. V., Tian, Z. R., . . . Sharma, A. (2019). Nanowired delivery of cerebrolysin with neprilysin and p-Tau antibodies induces superior neuroprotection in ’Alzheimer’s disease. Progress in Brain Research, 245, 145
200. Sparrow, A. M., Lowery-Gionta, E. G., Pleil, K. E., Li, C., Sprow, G. M., Cox, B. R., . . . Thiele, T. E. (2012). Central neuropeptide Y modulates binge-like ethanol drinking in C57BL/6J mice via Y1 and Y2 receptors. Neuropsychopharmacology, 37(6), 1409
1421. Stalmans, S., Bracke, N., Wynendaele, E., Gevaert, B., Peremans, K., Burvenich, C., . . . De Spiegeleer, B. (2015). Cell-penetrating peptides selectively cross the blood-brain barrier in vivo. PLoS One, 10(10), e0139652. Stalmans, S., Gevaert, B., Wynendaele, E., Nielandt, J., De Tre, G., Peremans, K., . . . De Spiegeleer, B. (2015). Classification of peptides according to their blood-brain barrier influx. Protein and Peptide Letters, 22(9), 768
775. Stan, A., Birle, C., Blesneag, A., & Iancu, M. (2017). Cerebrolysin and early neurorehabilitation in patients with acute ischemic stroke: A prospective, randomized, placebo-controlled clinical study. Journal of Medicine and Life, 10(4), 216
222. Svenningsson, P., Palhagen, S., & Mathe, A. A. (2017). Neuropeptide Y and calcitonin gene-related peptide in cerebrospinal fluid in ’Parkinson’s disease with comorbid depression versus patients with major depressive disorder. Frontiers in Psychiatry, 8, 102. Takekawa, D., Kudo, T., Saito, J., Kimura, F., Nikaido, Y., Sawada, K., . . . Hirota, K. (2019). Higher plasma leptin and lower C-peptide levels are associated with depression: A cross-sectional study. Journal of Affective Disorders, 243, 70
74. Tanaka, M., Kiyohara, H., Yoshino, A., Nakano, A., Takata, F., Dohgu, S., . . . Matsui, T. (2020). Braintransportable soy dipeptide, Tyr-Pro, attenuates amyloid β peptide(25-35)-induced memory impairment in mice. npj Science of Food, 4, 7. Tassone, F., Broglio, F., Gianotti, L., Arvat, E., Ghigo, E., & Maccario, M. (2007). Ghrelin and other gastrointestinal peptides involved in the control of food intake. Mini Reviews in Medicinal Chemistry, 7(1), 47
53. Telegdy, G., & Adamik, A. (2013). Involvement of transmitters in the anxiolytic action of urocortin 3 in mice. Behavioural Brain Research, 252, 88
91. Thapar, A., Cooper, M., & Rutter, M. (2017). Neurodevelopmental disorders. Lancet Psychiatry, 4(4), 339
346. Tolboom, N., Ossenkoppele, R., & van Berckel, B. N. (2019). Amyloid-beta PET imaging in aging and dementia. In F. Fraioli (Ed.), PET/CT in brain disorders (pp. 119
129). Cham: Springer International Publishing. Tornesello, A. L., Buonaguro, L., Tornesello, M. L., & Buonaguro, F. M. (2017). New insights in the design of bioactive peptides and chelating agents for imaging and therapy in oncology. Molecules, 22(8). Trivedi, M., Zhang, Y., Lopez-Toledano, M., Clarke, A., & Deth, R. (2016). Differential neurogenic effects of casein-derived opioid peptides on neuronal stem cells: Implications for redox-based epigenetic changes. The Journal of Nutritional Biochemistry, 37, 39
46. Tsuchimine, S., Hattori, K., Ota, M., Hidese, S., Teraishi, T., Sasayama, D., . . . Kunugi, H. (2019). Reduced plasma orexin-A levels in patients with bipolar disorder. Neuropsychiatric Disease and Treatment, 15, 2221
2230. Urban-Kowalczyk, M., Smigielski, J., & Strzelecki, D. (2017). Olfactory identification in patients with schizophrenia - the influence of beta-endorphin and calcitonin gene-related peptide concentrations. European Psychiatry, 41, 16
20. Van Dorpe, S., Bronselaer, A., Nielandt, J., Stalmans, S., Wynendaele, E., Audenaert, K., . . . De Spiegeleer, B. (2012). Brainpeps: The blood-brain barrier peptide database. Brain structure & function, 217(3), 687
718. Wang, F., Zhou, X. L., Yang, Q. G., Xu, W. H., Wang, F., Chen, Y. P., & Chen, G. H. (2011). A peptide that binds specifically to the beta-amyloid of ’Alzheimer’s disease: selection and assessment of anti-betaamyloid neurotoxic effects. PLoS One, 6(11), e27649. Williams, D. R., & Burkner, P. C. (2017). Effects of intranasal oxytocin on symptoms of schizophrenia: A multivariate Bayesian meta-analysis. Psychoneuroendocrinology, 75, 141
151.

Applications in medicine: mental health 721 Woodward, Z., Prickett, T. C. R., Espiner, E. A., & Anderson, T. J. (2017). Central and systemic C-type natriuretic peptide are both reduced in Parkinson’s disease. Parkinsonism & Related Disorders, 43, 15
19. Wynendaele, E., Verbeke, F., Stalmans, S., Gevaert, B., Janssens, Y., Van De Wiele, C., . . . De Spiegeleer, B. (2015). Quorum sensing peptides selectively penetrate the blood-brain barrier. PLoS One, 10(11), e0142071. Xiao, L., Priest, M. F., Nasenbeny, J., Lu, T., & Kozorovitskiy, Y. (2017). Biased oxytocinergic modulation of midbrain dopamine systems. Neuron, 95(2), 368
384, e5. Yamada, A., Mizushige, T., Kanamoto, R., & Ohinata, K. (2014). Identification of novel β-lactoglobulin-derived peptides, wheylin-1 and -2, having anxiolytic-like activity in mice. Molecular Nutrition & Food Research, 58(2), 353
358. Yamasue, H., & Domes, G. (2017). Oxytocin and autism spectrum disorders. In R. Hurlemann, & V. Grinevich (Eds.), Behavioral pharmacology of neuropeptides: Oxytocin (pp. 449
465). Cham: Springer International Publishing. Yang, J., Yao, Y., Wang, L., Yang, C., Wang, F., Guo, J., . . . Ming, D. (2017). Gastrin-releasing peptide facilitates glutamatergic transmission in the hippocampus and effectively prevents vascular dementia induced cognitive and synaptic plasticity deficits. Experimental Neurology, 287(Pt 1), 75
83. Yasuda, S., Baba, H., Maeshima, H., Shimano, T., Inoue, M., Ichikawa, T., . . . Arai, H. (2019). Serum levels and mutual correlations of amyloid beta in patients with depression. Geriatrics & Gerontology International. Yoon, H. S., Hattori, K., Sasayama, D., & Kunugi, H. (2018). Low cocaine- and amphetamine-regulated transcript (CART) peptide levels in human cerebrospinal fluid of major depressive disorder (MDD) patients. Journal of Affective Disorders, 232, 134
138. Young, L. J., & Barrett, C. E. (2015). Neuroscience. Can oxytocin treat autism? Science, 347(6224), 825
826. Zhang, H. F., Dai, Y. C., Wu, J., Jia, M. X., Zhang, J. S., Shou, X. J., . . . Han, J. S. (2016). Plasma oxytocin and arginine-vasopressin levels in children with autism spectrum disorder in China: Associations with symptoms. Neuroscience Bulletin, 32(5), 423
432. Zhang, J., Li, D., Lang, L., Zhu, Z., Wang, L., Wu, P., . . . Chen, X. (2016). 68Ga-NOTA-Aca-BBN(7-14) PET/ CT in healthy volunteers and glioma patients. Journal of Nuclear Medicine, 57(1), 9
14. Zhou, L., Chan, K. H., Chu, L. W., Kwan, J. S., Song, Y. Q., Chen, L. H., . . . Lam, K. S. (2012). Plasma amyloid-beta oligomers level is a biomarker for ’Alzheimer’s disease diagnosis. Biochemical and Biophysical Research Communications, 423(4), 697
702. Zipursky, R. B., Meyer, J. H., & Verhoeff, N. P. (2007). PET and SPECT imaging in psychiatric disorders. Canadian Journal of Psychiatry, 52(3), 146
157. Zolotarev, Y. A., Kovalev, G. I., Kost, N. V., Voevodina, M. E., Sokolov, O. Y., Dadayan, A. K., . . . Myasoedov, N. F. (2016). Anxiolytic activity of the neuroprotective peptide HLDF-6 and its effects on brain neurotransmitter systems in BALB/c and C57BL/6 mice. Journal of Psychopharmacology, 30(9), 922
935. Zott, B., Simon, M. M., Hong, W., Unger, F., Chen-Engerer, H. J., Frosch, M. P., . . . Konnerth, A. (2019). A vicious cycle of beta amyloid-dependent neuronal hyperactivation. Science, 365(6453), 559
565.

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CHAPTER 29

Applications in medicine: joint health Ezequiel R. Coscueta, Marı´a Emilia Brassesco, Patrı´cia Batista, Sandra Borges and Manuela Pintado Universidade Cato´lica Portuguesa, CBQF—Centro de Biotecnologia e Quı´mica Fina—Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal

29.1 Introduction Rheumatic and joint diseases, such as osteoarthritis and rheumatoid arthritis, are among the most widespread painful and disabling pathologies across the globe (Fusco, Skaper, Coaccioli, Varrassi, & Paladini, 2017). Osteoarthritis and joint injury are characterized by remodeling and degradation of cartilage, bone, and other joint tissues. Rheumatoid arthritis is a chronic, inflammatory joint disorder with a worldwide prevalence of nearly 5/1000 adults (Aletaha & Smolen, 2018). The pathophysiology of rheumatoid arthritis is related to chronic inflammation of the synovial membrane, which can destroy articular cartilage (Aletaha, Funovits, Smolen, & Editorial, 2011). The therapeutic strategies widely used for joint disorders are the administration of analgesics and nonsteroidal antiinflammatory. Nevertheless, these drugs are inept to stop or slow the evolution of structural injury and commonly have gastrointestinal and digestive adverse effects (Puigdellivol et al., 2019). Alternative treatments with dietary supplements have received much attention due to the higher levels of safety and effectiveness. Natural supplements, such as collagen derivatives, embody bioactive peptides that display physiological activities being beneficial to joint health (Kumar, Sugihara, Suzuki, Inoue, & Venkateswarathirukumara, 2015). Collagen peptides are classified as safe by the European Food Safety Authority (EFSA) (Journal, 2005) and by the Food and Drug Administration (FDA) (Bello & Oesser, 2006). Various studies demonstrated that collagen derivatives are absorbed and allocated to joint tissues. Preclinical studies in the animal model showed that oral administration of collagen derivative was specifically found in cartilage (Oesser, Adam, Babel, & Seifert, 1999). Clinical trials have already been performed to prove the health benefits of dietary supplementation of collagen derivatives in the management of osteoarthritis, reducing the pain, and increasing the mobility of patients (Czajka et al., 2018; Puigdellivol et al., 2019). Prophylaxis and therapy approaches were developed and supported by different kinds of peptides, which will be addressed throughout this chapter.

Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00029-7 © 2021 Elsevier Inc. All rights reserved.

723

724 Chapter 29 A better understanding of the action mechanisms in articular tissue may help to identify the more effective targets to counteract the joint pathologies. Understanding the proliferation and regeneration process of cartilage tissue, clarify the role of antioxidant, antimicrobial, and antiinflammatory agents and relate the neuroactivity with articular and allow to better understand the mechanisms of action and the fundamental role played by peptides. These mechanisms are involved with peptides, and their incorporation in therapies and treatment can be very useful in cartilage regeneration. The interest and research in this area are improving with technological development (purification, mass-spectrometry, peptidomics, and cartilage tissue engineering) and allow the increase of development and performance at the level of prevention, diagnosis by biological markers, treatments, and therapeutics (Hu et al., 2020). In this chapter, we will critically review the state of the art of the emerging field of application of bioactive peptides in the prevention and control of joint diseases and their mechanisms of action to achieve those benefits.

29.2 Overview of joint diseases Joint diseases are those disorders or injuries that affect human joints, of which arthritis is the greatest exponent (Sokoloff, 2019). Joint diseases, common in aging, can be short-lived or extremely chronic, very painful or simply bothersome, and uncomfortable; they may be limited to one joint, or they may affect many parts of the skeleton. Inflammation may or may not be an essential feature of joint disease. Arthritis is a generic term for a set of inflammatory joint diseases. This includes more than 100 rheumatic diseases and conditions, with osteoarthritis being the most common. Other common forms of arthritis are rheumatoid arthritis, lupus, fibromyalgia, and gout. In general, they are characterized by causing pain, stiffness, swelling, and inflammation in and around the joints. The inflammation can be of such a nature and severity that it destroys the articular cartilage and underlying bone and causes irreparable deformities. In such cases, the fusion between the articulated limbs is frequent, with a consequent stiffness and loss of mobility (ankylosis). Some forms of arthritis, such as rheumatoid arthritis and lupus, can affect various organs and cause widespread symptoms (Sokoloff, 2019).

29.2.1 Osteoarthritis Osteoarthritis is the most common type of arthritis. It mainly affects the common cartilage, which is the tissue that cushions the ends of bones within the joint (D. Horowitz, J. Hanrahan, & R.K, Turley). Once this process begins, the cartilage begins to erode and may eventually suffer severe straining. This leads to joint pain and stiffness. The severity of osteoarthritis symptoms can vary greatly from person to person and between different affected joints. In some cases, the symptoms may be mild and may be recurring. In other

Applications in medicine: joint health 725 cases, the problems may be more continuous and serious, to the point of making it difficult to carry out daily activities. Almost any joint can be affected by osteoarthritis, but the most common locations involved include the knees, hips, and small joints of the hands. The exact cause of osteoarthritis remains unknown, but several things are thought to increase the risk of developing this disease. For example, overuse of a joint that has not had enough time to heal after injury or surgery may result in osteoarthritis. Osteoarthritis can also occurs in joints severely damaged by a previous condition (another type of arthritis), such as rheumatoid arthritis or gout. Obesity can also lead to consistent osteoarthritis, since it puts excess stress on the joints, particularly those that support most of the weight, such as the knees and hips. Gender can also be a determining factor since osteoarthritis is more common in women than in men. Also, the risk of developing the condition increases with aging.

29.2.2 Rheumatoid arthritis Rheumatoid arthritis is an inflammatory disease of the joint lining, generally affecting several joints at the same time (Aletaha & Smolen, 2018). The patients experience pain, stiffness, swelling of the joint lining, and eventually joint damage, leading to deformity and pain, with loss of function of the affected joints. It can affect both hands and both feet. Some people with rheumatoid arthritis also experience problems in other parts of the body or more general symptoms, such as tiredness and weight loss. Rheumatoid arthritis is an autoimmune disease. This means that your immune system mistakenly attacks the cells that line your joints, causing the joints to be swollen, stiff, and painful. Over time, this can damage joints, cartilage, and nearby bone. It is not clear what triggers this problem with the immune system, although an increased risk has been seen in women, smokers, or with family histories suffering this condition.

29.3 Peptides activity and characterization Biologically active peptides take an important part in various biological processes of joint health. In recent years, with the technological development of natural peptide identification and their mechanisms of study the peptidome is playing a more and more important role in the study of biological markers (Malmstro¨m, Catrina, & Klareskog, 2017) and therapeutic targets (Park et al., 2018) for the development of new molecular tools for the diagnosis, prevention, and therapy of joint diseases based on bioactive peptides.

29.3.1 Natural bioactive peptide sources Collagen is one of the most abundant proteins in the world that is widely available and inexpensive to recover from animal (Fu, Therkildsen, Aluko, & Lametsch, 2018; Silva et al., 2014)

726 Chapter 29 by-products. Currently, utilization of collagen as high value-added source of ingredients via enzyme technology has been the top trend in the processing industry, leading to a high benefit-tocost ratio. The resultant collagen peptides have enormous commercial potential as food ingredients or nutraceuticals, because they are recognized as safe components of pharmaceuticals and foods by the FDA Center for Food Safety and Nutrition (Bello & Oesser, 2006) and by the EFSA (Journal, 2005). In this respect, scientific evidence suggests that collagen hydrolysates exert a positive therapeutic effect on osteoarthritis (Bello & Oesser, 2006; Kumar et al., 2015). However, the EFSA panel on dietetic products, nutrition, and allergies recently concluded that so far, no causeand-effect relationship between the maintenance of joints and the use of collagen hydrolysates has been shown (EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2011). So, Schadow et al. (Schadow et al., 2013) evaluated for the first time whether different bovine collagen hydrolysate preparations indeed modulate the metabolism of collagen and proteoglycans from human OA cartilage explants and determined the chemical composition of oligopeptides representing collagen fragments. They concluded that collagen hydrolysates from various sources differ significantly with respect to both their chemical composition of oligopeptides representing collagen fragments as well as their effects on human articular cartilage, and metabolized collagen fragments or other collagen hydrolysate preparations might contain therapeutically useful peptides. Thus their biomedical properties must be studied thoroughly both in vitro and in animal as well as clinical trials before being applied as safe and effective nutraceuticals in patients. Also, collagen peptides are biocompatible and safe due to their unique biological and structural characteristics. Regardless of different collagen types, they all share the nearly identical sequence and structure (Fig. 29.1), which guarantee weak in vivo immunogenicity (Banerjee & Shanthi, 2016). Collagen peptides, especially those with C-terminal Pro or Hyp residue, can be transported across the intestinal epithelial monolayer, enter the bloodstream, and exert their bioactive properties. The unique amino acid composition and structure confer collagen peptides with excellent stability and less protease cleavage sites. The high Pro level existent in collagen peptides (10%
30% abundance) may increase their stability toward digestive enzymes and intestinal peptidases in comparison to other therapeutic peptides (Banerjee & Shanthi, 2016). This is due to the presence of a Pro residue adjacent to the cleavage site that prevents proteases from cleaving the normally susceptible peptide bond (Fu et al., 2018). It is important to highlight that the direct intake of collagen supported by simple digestion may not reach peptides with dimensions for absorption. So, it seems probable that the biological activities of ingested collagen are mediated, at least partly, by collagen-derived oligopeptides such as Pro-Hyp and/or Hyp-Gly. Transportation of an oligopeptide into a cell can be mediated by proton-coupled oligopeptide transporters that support low-molecular weight of peptides. Several studies indicate that collagen-derived oligopeptides appear in blood at fairly high concentrations and suggest that the beneficial effects of collagen ingestion are mediated by these oligopeptides. Other studies described the mechanism of absorption and distribution

Applications in medicine: joint health 727

Figure 29.1 Raw collagen molecular structure and collagen peptides.

of collagen peptides in the body. It has been demonstrated that C14-labeled collagen peptides can reach the skin, cartilage, bones, and muscles and remain in these tissues up to 14 days after a single ingestion (Kawaguchi, Nanbu, & Kurokawa, 2012; WatanabeKamiyama et al., 2010). Iwai and colleagues showed that hydrolyzed collagen from porcine skin, chicken cartilage, and chicken feet, which was ingested by healthy subjects after 12 h of fasting was absorbed and detected in the plasma as small peptides (Iwai et al., 2005). Hydroxyproline-containing peptides detected in the plasma peaked 2 h after oral ingestion and decreased to half of the maximum after 4 h. Several in vivo studies have demonstrated the efficacy of collagen peptides on skin and cartilage health and ageing (Czajka et al., 2018).

29.3.2 Peptidome analysis Peptidomics is a new branch of proteomics which is based on investigating endogenous protein fragments in tissues or body fluid (Hu et al., 2020). Biologically active peptides are involved in almost all physiological processes, including cell differentiation, immune regulation, and even tumor formation (Ferro, Rioli, Castro, & Fricker, 2014). So, the study of

728 Chapter 29 bioactive peptides as biological markers and therapeutic targets for the diagnosis, prevention, and therapy of joint diseases is a very important research to be performed in the near future. From a peptidome perspective, the pathophysiology process of joint diseases, as well as the functional mechanism of bioactive peptides, can be disclosed (Hu et al., 2020). For therapeutics targets, there is extensive evidence to support a role for C-type natriuretic peptide (CNP) in maintaining homeostatic function in cartilage and bone. However, the biology of CNP signaling in joint tissues is complex and is influenced by several factors leading to dysfunction and disease. The differences in the mechanism of natriuretic peptide receptor (NPR) signaling affect the ability of the peptide to function normally. This has widespread impact due to the role of CNP in maintaining joint homeostasis and may crosstalk with other mechanisms linking angiogenesis and osteogenesis with function in vascular systems. The differential effects of NPRs in response to signals that influence their expression will determine the CNP signaling system and their effects on tissue function. Therapeutic application of CNP or interventions targeted to NPR to influence the actions of CNP should therefore be considered to speed up repair mechanisms and stabilize tissue homeostasis. Evidence indicates that enhanced CNP signaling may prevent growth retardation and protect cartilage in patients with inflammatory joint disease (Peake et al., 2014). Another example is the vasoactive intestinal peptide (VIP) that can prevent chronic cartilage damage and joint remodeling. Evidence suggests that VIP is downregulated in synovial fluid of osteoarthritis, and VIP downregulation leads to an increase in the production of proinflammatory cytokines that might contribute to the pathogenesis of osteoarthritis (Jiang, Wang, Li, & Luo, 2016).

29.4 Mechanisms of action The function of joint involves several processes, and understanding the mechanisms of action (proliferation, degradation, regeneration, and infection) in the joint disease process is important for the diagnostic, prophylaxis, and treatment. Understanding the processes is also important for the development of biomaterials and bioingredients to promote healthy joint. These mechanisms are associated with active peptides that are involved in almost all physiological processes, including cell differentiation, proliferation, inflammatory process, and immune regulation (see Table 29.1).

29.4.1 Cartilage proliferation Cartilage is a connective tissue composed by a low density of cells, chondrocytes, embedded within an extracellular matrix (ECM). The cartilage ECM is responsible for biomechanical functions, structural support, and resistance to deformation. To ensure these mechanisms of action, various proteins and peptides are present, such as proteoglycans

Applications in medicine: joint health 729 Table 29.1: Endogenous and exogenous peptides with intervention in the function of joint. Protein/peptides TGF-β BMPs NF-Kβ (RANKL) SASP CNP Cytomodulins NLS-TAT E7, L7 HA-binding peptide Chondroitin sulfate-binding peptide RADA 16-1 KLD-12 RGD peptide Collagen Fish gelatine peptides Resveratrol impact in PI3K/AKT signaling pathway KAFAK TNF-α IL-1β, IL-6, IL-10, IL-17A 5-LOX, 15-LOX FPR2 Antimicrobial peptides (AMPs) Lysozyme Lactoferrin Secretory phospholipase A2 RNase 7 CAP37 Cathelicidin LL37 HBD-2/-3 VIP SP CGRP

Function/impact

References

Improve cartilage proliferation/repair

Varela-Eirin et al. (2018) Tuan et al. (2013) Deng et al. (2018) Parmar et al. (2015) Peake et al. (2014) Haque Bhuyan et al. (2017) Faust et al. (2018) Liu et al. (2018) Dar et al. (2017)

Antioxidant peptides and antiinflammatory function

Kim & Mendis (2006) Yu et al. (2018) Lin et al. (2016) McMasters et al. (2017) Al-Madol et al. (2017) Janakiraman et al. (2018)

Antiinflammatory and immune responses

Antimicrobial peptides

Pinto et al. (2019) Elezagic et al. (2019) Varoga et al. (2005)

Neuroactivity

Jiang et al. (2016) Kanemitsu et al. (2020) Zhang et al. (2018) Courties et al. (2017)

tangled in a collagen network, noncollagenous proteins, and glycoproteins (Hu et al., 2020). The abundance and distribution of these molecules are different in each cartilage type (hyaline, fibrous, and elastic), according to their function. The cartilage tissue has a vascular nature and a limited capacity for self-repair, and its repair remains a challenge (Lam, Reuveny, & Oh, 2020; Liu et al., 2018; Tuan, Chen, & Klatt, 2013). Therefore cartilage loss and degeneration are often associated with pathologies, which consequently triggers the appearance of joint pathologies. To fill this gap, researchers have been studying therapies and new techniques for cartilage repair. So, it

730 Chapter 29 is important to understand the role of peptides in this process, because it can be promising in the proliferation and/or inhibition of cartilage degeneration. Currently, there is an interest in using human mesenchymal stem cells (MSC) as cell therapy of cartilage, because they have an extensive proliferation potential and can undergo chondrogenesis (Lam et al., 2020). These cells (MSC) have emerged as a promising cell source, and it can be functionalized with different coating materials, such as ECM proteins and peptides. For example, ECM molecule affinity peptides are frequently used in biomaterials development, because they can help mimic the native environment of chondrocytes and influencing the biological functions of these molecules (Liu et al., 2018). The articular cartilage degradation, synovial inflammation, and joint degeneration are typical processes implicit in joint pathologies. During these processes, chondrocytes can undergo phenotypic changes that increase cell proliferation and cluster formation and enhance the production of matrix-remodeling enzymes. Recent investigations suggest that alterations in different proteins, such as transforming growth factor-β (TGF-β)/bone morphogenetic proteins (BMPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Kß), or senescence-associated secretory phenotype factors involved in signaling pathways in wound healing response, could be directly implicated in these pathologies (Liu et al., 2018; VarelaEirin et al., 2018). Understand these mechanisms are advantageous in identifying novel targets and designing therapies to promote effective cartilage repair and successful joint ageing by preventing functional limitations and disability (Varela-Eirin et al., 2018). Studies report that BMPs (the most widely studied are BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, and BMP-9) play an important role in the protection of cartilage degradation (by inflammation or trauma), bind to different receptor combinations and therefore may be active in different intracellular signaling pathways (Deng, Li, Gao, Lei, & Huard, 2018; Tuan et al., 2013). This loss of function of BMP-related receptors contributes to the decrease in intrinsic repair capacity of damaged cartilage, and so, the multifunctional effects make them attractive. So, the use of BMPs and their combination with stem cells and biomaterials can be a promising new therapeutic modality for cartilage repair. Bhuyan and colleagues (Haque Bhuyan et al., 2017) found that the receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3
4 stimulated the differentiation of both chondrocytes and inhibited cartilage degeneration. Their data suggest that the stimulation of mesenchymal cell proliferation by the RANKL-binding peptides might lead to the inhibition of cartilage degeneration. Therefore the introduction of peptides in cartilage tissue engineering has proven to be advantageous and is increasingly a solution in cartilage repair. Other therapies/treatments have been developed with the aid of peptides, for example, the use of CNP has shown evidence for its ability to regulate cartilage and bone homeostasis. Data results by in vitro studies reported that exogenous CNP influenced chondrocyte differentiation, proliferation, and matrix synthesis. However, the CNP signaling systems are

Applications in medicine: joint health 731 complex and influenced by multiple factors, but the evidence showed that enhanced CNP signaling may prevent growth retardation and protect cartilage in the presence of inflammatory disease (Peake et al., 2014). In addition to the examples presented, other peptides, such as cytomodulins, NLS-TAT, E7, L7, TGFBP, and HA-binding peptide, chondroitin sulfate-binding peptide, RADA 16-1, KLD-12, and RGD peptide may also improve cartilage proliferation/repair (Liu et al., 2018).

29.4.2 Antioxidant, antimicrobial, and antiinflammatory activities The future of cartilage tissue engineering not only lies in cartilage matrix production, preventing matrix and cellular degradation, promoting appropriate cartilage integration but also in the delivery of antimicrobial, antioxidant, and antiinflammatory factors to provide durable cartilage constructs. The antimicrobial, antioxidant, antiinflammatory, and immunomodulatory effects of biologically active peptides in cartilage repair have been reported (see Table 29.1). The biological potential of antioxidant peptides in human clinics has been scarcely reported. One example is the fish gelatine, which showed the potential to maintain normal tendon and bone integrity, treat brittle nails, and improve joint health (Kim & Mendis, 2006). In addition, some natural plant extracts or compounds have been used in combination or isolated. For example, the polyphenol curcumin has shown a promising potential in terms of antiinflammatory and antioxidant effects, being beneficial for joint pathologies and in delaying cell death in the joint tissue (Kim, In Kim, Sim, & Khang, 2017). Kim and collaborators (Kim et al., 2017) developed a scaffold for cartilage regeneration using curcumin/silk composite scaffold, and the results may provide clinical support for the patients with various cartilage diseases. Yu and colleagues (Yu et al., 2018) designed a bioactive resveratrol
PLA
gelatin porous nano-scaffold to repair articular cartilage defects. Resveratrol is a poly-phenolic compound with antiaging, antiinflammatory, and antioxidant functions. This bioactive molecule can alleviate damage to cartilage, as it can regulate inflammation signaling (PI3K/AKT signaling pathway) in human chondrocytes. Other agents involved in the inflammatory process are proinflammatory (TNF-α, IL-1β, and 5-LOX) and antiinflammatory (15-LOX, FPR2, and IL-10) mediators. Al-Madol and colleagues (Al-Madol et al., 2017) suggested that the inflammatory mediators such as TNF-α, IL-1β, and 5-LOX play a key role in driving the inflammation and synovial cell proliferation in rheumatoid arthritis-associated joint destruction. On the other hand, other results showed that the antiinflammatory cytokines IL-10 is a potent immunoregulatory cytokine and plays a role in preventing exaggerated inflammatory and immune responses and protect the patient from

732 Chapter 29 immune-mediated damage (Al-Madol et al., 2017). Cytokines control a wide variety of inflammatory processes in joints, which provide the rationale for current treatments with the use of monoclonal antibodies against TNF-α, IL-1β, IL-6, and IL-17A (Janakiraman, Krishnaswami, Rajendran, Natesan, & Kandasamy, 2018; Krishnan & Grodzinsky, 2018). There is no treatment to recover cartilage, and the antiinflammatory therapies used for joint diseases provide short-term relief but can have adverse side effects. Lin and colleagues (Lin, Poh, & Panitch, 2016) developed a nanoparticle system that delivers therapeutics (in this case an antiinflammatory peptide KAFAK) intracellularly with improved efficacy by triggering degradation and suppressing inflammation in multiple cell types within an inflamed joint. Further, a study by McMasters and collaborators (McMasters, Poh, Lin, & Panitch, 2017), showed that hollow, sulfated poly(N-isopropylacrylamide) nanoparticles are an effective platform for the loading and delivery of antiinflammatory cell-penetrating peptides (KAFAK), with higher loading capacity, and prolonged-release profiles compared to other delivery systems. To enhance cartilage regeneration, various strategies have been developed with protective agents, such as antioxidants, antimicrobial, and antiinflammatory factors. The development of nutraceuticals composed by cartilage matrix elements has been studied in supporting joint health. Food supplements with chondroitin and glucosamine have been tested and presented several positive results. Recently, Dar and colleagues (Dar et al., 2017) reported that the oral consumption of a hydrolyzed type 1 collagen preparation, showed chondroprotective and antiapoptotic effects in articular chondrocytes, promoting antiinflammatory effect, and could represent a strategy for supporting joint health. On the other hand, in addition to the antioxidant and antiinflammatory agents, it is also important to know the potential of antimicrobial agents. The factors controlling the production of joint-associated antimicrobial peptides (AMPs) are unknown; however, it is known that AMPs may act by altering the cellular membrane permeability, protein synthesis inhibition, nucleic acid binding, or inactivation of the toxins that enable microbial infection (Pinto et al., 2019). So, several AMPs mechanisms action may be more advantageous in decreasing the ability of bacteria to acquire resistance and inducing the antiinflammatory response (by T cells stimulation and other cells with immunomodulatory properties) (Pinto et al., 2019). The AMPs may induce the expression of chemokines such as CXCL8 (IL-8), CCL2, and proinflammatory cytokines, and act directly through the binding-receptor interaction between LL37 and formyl peptide receptor-like 1 (FPRL1) (Pinto et al., 2019). So, the bioinspired AMPs have become a promising alternative, namely for cartilage tissue regeneration application. Varoga and colleagues (Varoga et al., 2005) showed a systematic analysis of in vitro and in vivo antimicrobial active proteins in healthy articular joints and disease states such as pyogenic arthritis, rheumatoid arthritis, and osteoarthritis. The review identified the most prominent antimicrobial proteins in articular joints

Applications in medicine: joint health 733 (Table 29.1), as lysozyme, lactoferrin, secretory phospholipase A2, RNase 7, CAP37, the cathelicidin LL37, and especially the human beta-defensin-2 and -3 (HBD-2/-3). For example, during arthroplasty (in arthroplasty-associated septic arthritis) bacteria can adhere to implant surfaces and therefore be introduced into the patient’s joint, as well as when adhesion of bacteria contributes to biofilm formation in surfaces of implants. So, theses AMPs prevent bacterial adhesion to the substrates, exhibit an antimicrobial effect, and coating joint prostheses with CLEC3A-derived AMPs could be an application in cartilage tissue engineering (Elezagic et al., 2019).

29.4.3 Neuroactivity Joint diseases are associated with pain and despite therapeutics to pain control and the use of effective disease-modifying antirheumatic drugs, sometimes the pain persists. Several studies reported the interaction between the nervous and immune systems, by neuropeptides and cytokines. The nervous system can modulate immunological and inflammatory responses, and several neuropeptides show potent ability to induce vasodilatation, edema and pain. Some neuropeptides, such as vasoactive intestinal polypeptide (VIP), substance P, and calcitonin-related peptide have been detected in the synovial fluid from patients with rheumatic disease. Recent studies reported the role of the VIP that is a neuropeptide acting as a neurotransmitter or neuromodulator in many organs and tissues (Jiang et al., 2016). This peptide operates in the stimulation of contractility in the heart, vasodilation, promoting neuroendocrine-immune communication, lowering arterial blood pressure, and possesses antiinflammatory, and immune-modulatory activity. Kanemitsu and colleagues (Kanemitsu et al., 2020) concluded that the inhibition of VIP signaling has the potential to be a therapeutic target to prevent osteoarthritis progression. On the other hand, according to Zhang & Lee (2018), persistent pain in join may not be associated with inflammatory processes but to the dysregulation of central nervous system pain regulatory pathways. Many cells of osteoarticular tissue have receptors for sympathetic and parasympathetic neurotransmitters and thus may respond to their stimuli (Courties, Sellam, & Berenbaum, 2017). Sympathetic nerves also innervate the synovium and maybe induce joint diseases. However, scarce dataare available on parasympathetic innervation of the joint tissue and more research is needed to understand the relation by pain—neuroactive peptides—joint diseases.

29.5 Evidence in joint health benefits Research studies about compounds that modify the progress of joint diseases have received increasing attention. Collagen derivatives have been studied to prevent or

734 Chapter 29 decrease the deterioration of joint tissue. The collagen derivatives are shown in different forms, as undenatured collagen (300 kDa), gelatine (20
90 kDa) and, as mentioned before, collagen hydrolysates (1
10 kDa) (Van Vijven et al., 2012). Collagen and gelatine hydrolysates studies proposed that peptides can be used as building blocks for the cartilage. Collagen hydrolysates showed a stimulatory effect on type II collagen biosynthesis in a cell culture model of chondrocytes, observing a collagen turnover in cartilage tissue (Oesser & Seifert, 2003). In preclinical studies in mice, gelatine hydrolysates demonstrated a cartilage tissue accumulation and intestinal absorption, where 95% of compounds were absorbed in the first 12 h, observing its clinical benefit on degenerative diseases by oral administration (Oesser et al., 1999). Animal trials have proposed that oral ingestion of collagen derivatives might have positive effects on joint health such as osteoarthritis, showing that collagen peptide decreases the morphological alteration related to osteoarthritic cartilage destruction in knee joints (Ohara, Iida, Ito, Takeuchi, & Nomura, 2010). Therefore the potential function of collagen derivatives in repairing damaged cartilage might relate to the accumulation of orally administered collagen derivatives. The connective tissues modify with the natural process of ageing. The cartilage ageing can be associated with the softening of the articular surface, reduction of proteoglycan amount, injury of matrix biomechanical properties, and the decrease of chondrocytes. The aged cartilage is more exposed to deformation throughout joint activities and to produce osteoarthritic alterations. To slow down the evolution of the signs of ageing and to improve the cartilage tissues, collagen hydrolysates have been used as a nutraceutical supplement (Czajka et al., 2018). Collagen peptides can reach and remain in several tissues (cartilage, skin, bones, and muscles) after 14 days of ingestion. Gelatine hydrolysates from porcine skin, chicken cartilage, and chicken feet have demonstrated to be absorbed and detected in the plasma of healthy subjects after oral ingestion (Iwai et al., 2005). The oral supplementation of collagen hydrolysates has also proved that can improve the quality of life of patients with osteoarthritis. It has been already reported that the ingestion of 10 g of hydrolyzed collagen during 6 months by patients with osteoarthritis, reduced the pain, and improved the knee joint comfort (Benito-Ruiz et al., 2009; McAlindon et al., 2011). Moreover, it is suggested that the intake of collagen peptides have a potential protective role and might delay osteoarthritis progression (McAlindon et al., 2011). The collagen peptides were also used to reduce knee joint pain in young athletes during physical activity (Zdzieblik, Oesser, Gollhofer, & Ko¨nig, 2017). Table 29.2 summarizes some of the randomized controlled trials that establish the effectiveness of the ingestion of collagen derivatives for the joint disorders’ prevention/treatment and symptoms relief.

Applications in medicine: joint health 735 Table 29.2: Randomized controlled clinical trials of oral supplementation of collagen derivatives towards joint disorders. Intervention

Participants

Followup

Outcomes

Reference

90 Improvement of clinical Czajka et al. (2018) 122 volunteer Daily consumption of days parameters related to subjects between 50 mL of a test product joint health, such as 21 and 70 years with 8% of hydrolyzed reduction of joint pain by old collagen from fish 43% and improvement of combined with vitamins joint mobility by 39% and other compounds 1 Rapidly reduction of pain Kilinc et al. (2018) 92 subjects Daily consumption of between 40 and 69 month and stiffness in the knee 720 mg of promerim for osteoarthritis the first 15 days and then years old with knee pain 360 mg for the second 15 days. Promerim is a dietary supplement that contains hydrolyzed fish collagen 16 Improvement of 100 subjects Daily consumption of Kanzaki, Ono, between 40 and 74 weeks locomotor functions and Shibata, & Moritani eight tablets containing relieve knee pain years old with knee shark cartilage extract (2015) pain (45 mg of type II collagen peptides and 60 mg chondroitin sulfate), glucosamine hydrochloride, among other ingredients 91 Improvement of the 30 subjects Twice a day consumption Kumar et al. overall physical between 30 and 65 days of 5 g of test product (2015) discomforts resulting from dissolved in 250 mL water years old diagnosed the osteoarthritis, such as with knee or milk containing pain, stiffness, and osteoarthritis collagen peptides isolated physical functions from pork skin or bovine bone 180 Amelioration of knee joint 191 subjects Consumption of a daily Lugo, Saiyed, & symptoms, namely pain, between 40 and 75 days dose of 40 mg of Lane (2016) stiffness and physical years old with undenatured type II functions. Undenatured moderate-to-severe collagen derived from type II collagen presented osteoarthritis chicken sternum better clinical outcomes than glucosamine hydrochloride plus chondroitin sulfate (widely available supplement used for reducing joint pain) (Continued)

736 Chapter 29 Table 29.2: (Continued) Intervention

Participants

Followup

Outcomes

Reference

Oral administration of one tablet three times per day, that comprises mainly hydrolyzed gelatine (500 mg/tablet), chondroitin sulfate, glucosamine sulfate, and devil’s claw and bamboo extracts

130 subjects aged $ 18 years with osteoarthritis

180 days

Nutritional supplement reduced articular pain and improved locomotor function of the knee and/ or hip

Puigdellivol et al. (2019)

A daily dose of 5 g of collagen peptides

160 athletic subjects between 18 and 30 years old

12 weeks

Supplementation of collagen derivative led to an improvement of activity-related joint pain in a young adult with functional knee problems

Zdzieblik et al. (2017)

29.6 Potential applications, production, and commercialization 29.6.1 Diagnostic Patients with rheumatoid arthritis have a decreased quality of life triggered by the pain, fatigue, and damage of some body-functions related with disease evolution. Patients have also an improved risk of lung and cardiovascular disorders and premature mortality. Therefore the early diagnosis of rheumatoid arthritis is imperative to ensure effective treatment, because it has been revealed that the patients who receive early an antirheumatics drug have an improvement of health outcomes (Alm et al., 2018). The clinical criteria to identify this disease is generally based on history and physical exam discoveries, laboratory, and radiographic results. Several laboratory tests are already used, including the detection of rheumatoid factor (RF), antibodies directed against the Fc portion of immunoglobulin G, and also antibodies against peptides containing citrulline (Aggarwal, Liao, Nair, Ringold, & Costenbader, 2009). The anticyclic citrullinated peptide ELISA has been an ideal rheumatoid arthritis indicator because it showed a high specificity (98%) and sensitivity (79%) (Vallbracht & Helmke, 2005). The cyclic citrullinated peptide (CCP) test is based on purified synthetic peptides comprising modified arginine residues (citrulline) acting as antigen. The CCP ELISA can anticipate the clinical manifestation of rheumatoid arthritis and is suitable to evaluate the disease development. Besides, the combination of anti-CCP and RF positive results has been proven to enhance the possibility of a true positive result compared to either of the antibody tests in separated. The high evidence of

Applications in medicine: joint health 737 the value of CCP assays for the diagnosis of rheumatoid arthritis led to the integration of this test in the American College of Rheumatology guidelines (Alm et al., 2018). The biochemical markers have the potential to identify the different joint diseases and measure the pathology progression. So, some research studies focus on the development of strategies addressed to the detection of protein fragments that are generated in joint diseases, such as the degradation products of collagen. Additionally, biomarkers can be essential to appraise the individual patients responses to the administered treatment (Saberi Hosnijeh, Bierma-Zeinstra, & Bay-Jensen, 2019). Rheumatoid arthritis and osteoarthritis have a similar clinical manifestation of abnormal and degraded cartilage in joints. These alterations occur in an early stage of the diseases long before noteworthy damage can be perceived in the radiographic analysis. Herein, it is crucial to recognize the biomarkers of cartilage degradation. The degradation of type II collagen via different enzymatic processes is a very early sign of injury in rheumatoid arthritis and osteoarthritis; so, the detection of type II collagenderived fragments in biological samples has been an area of interesting research. In this context, an ELISA using the 622
632 peptide derived from the sequence of the α1 chain of type II collagen (HELIX-II) as immunogen was developed to measure that fragment in the urine. The urinary levels of HELIX-II in 89 patients with rheumatoid arthritis and 90 patients with osteoarthritis and 162 healthy persons were measured. The HELIX-II ELISA showed to be a useful noninvasive assay to distinguish patients with rheumatoid arthritis or osteoarthritis from healthy people (Charni, Juillet, & Garnero, 2005). The diagnosis of prosthetic joint infections (PJI) is also a challenge because a positive synovial fluid culture can detect a PJI; but, a negative culture does not exclude the hypothesis of PJI. Besides that, the skin commensal bacteria (Staphylococcus spp., Propionibacterium acnes, among others) can confuse the diagnose of PJI; so, other criteria are required to support the diagnosis (Vaishya, Sardana, Butta, & Mendiratta, 2019). A range of new laboratory methods for PJI includes molecular biology tests, antigen and antibody assays, and immune markers in a biological fluid. When the first-line investigations fail to supply a decisive diagnosis, a multidisciplinary discussion for complex PJI is better to define the following diagnostic strategy (Arvieux & Common, 2019). An array of biomarkers has been proposed for PTFI diagnoses, such as inflammatory proteins and AMPs. The AMPs that have been studied comprise alpha-defensins and lactoferrin. Alpha-defensins are released by the neutrophils as a defense mechanism against microorganisms. Alpha-defensin has been the best synovial fluid biomarker for the advance of immunodiagnostic assays (Vaishya et al., 2019). This test is not affected by antibiotherapy or inflammatory conditions. The alpha-defensin PJI assay revealed high specificity (100%) and sensitivity (92.1%) (Hosny & Keenan, 2020).

738 Chapter 29

29.6.2 Prophylaxis/therapeutic The joint diseases are currently considered to be diseases without a cure. For this reason, the available treatments are aimed at reducing the risk of joint damage or reducing the pain caused by the established disease. These disorders are mostly treated through exercise combined with the administration of analgesics or nonsteroidal antiinflammatory drugs. These drugs are for symptoms relief but do not alter the disease and still cause adverse effects. However, as we already mentioned, there is increasing evidence of the beneficial effects of bioactive peptides, given their high potential for the development of new treatments. For example, daily oral supplementation with a liquid nutraceutical containing hydrolyzed fish collagen, vitamins, antioxidants, and other active ingredients may improve the quality of life of subjects suffering from osteoarthritis (Czajka et al., 2018). Intake of collagen peptides (5 g/day for 12 weeks) may lead to the relief of joint pain and stimulate regeneration of type II collagen and the biosynthesis of proteoglycans in cartilage tissue osteoarthritis patients, improving joint mobility (Czajka et al., 2018; McAlindon et al., 2011; Zdzieblik et al., 2017). As for rheumatoid arthritis, a promising way to reduce the autoimmune response is to use peptides target anticitrullinated protein/peptide antibodies (Benham et al., 2015). Besides, one of the putative functional peptides derived from the domain of galectin-1 showed a significant therapeutic effect in TNF-α induced MH7A rheumatoid arthritis model in vitro (Hu et al., 2020). Another rheumatoid arthritis therapy is targeted on the delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide (Wythe et al., 2013). Parmar and colleagues (Parmar et al., 2015) developed a biodegradable hydrogel, by modified a streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate-binding peptides and then cross-linked with a matrix metalloproteinase 7-sensitive peptide. Subsequently, human MSCs were incorporated into hydrogels improving viability and significantly enhancing chondrogenic differentiation. This novel biomaterial showed the potential to act in cell-mediated processes and improve the cartilage repair. Other therapies/treatments have been developed with the aid of peptides, for example, the use of CNP has shown evidence for its ability to regulate cartilage and bone homeostasis. Data results by in vitro studies reported that exogenous CNP influenced chondrocyte differentiation, proliferation, and matrix synthesis. However, the CNP signaling systems are complex and influenced by multiple factors; but, the evidence showed that enhanced CNP signaling may prevent growth retardation and protect cartilage in the presence of inflammatory disease (Peake et al., 2014). Faust and colleges (Faust et al., 2018) developed a peptide-polymer composed of an HA-binding peptide conjugated to a heterobifunctional poly(ethylene glycol) chain and

Applications in medicine: joint health 739 a collagen-binding peptide as a technology that can be implemented after a cartilage defect to slow further degeneration of the cartilage tissue without additional HA supplementation. The results were promising, and this platform could be conjugated to other active molecules or drugs for targeted delivery to damaged areas of cartilage in vivo. However, more studies are needed to validate their therapeutic efficacy. Even some studies have been performed, it is considered that the evidence is not enough and that a larger critical mass of human studies is necessary to get a correct validation of all the findings.

29.6.3 Production and commercialization Biomaterials and ingredients based on collagen and its peptides are very important for biomedical applications, mainly for tissue engineering and regenerative medicine. This is due to its superior biocompatibility and low immunogenicity, always depending on the sources from which the collagen is taken (Silva et al., 2014). In that sense, researchers are focusing on different sources to avoid the use of bovine collagen due to protein misfolding and allergenicity. For example, marine collagen (type I) is considered an excellent alternative of functional ingredients, also because its source is cheap and prevents bovine spongiform encephalopathy, making it an attractive option for product developers (Avila Rodrı´guez, Rodrı´guez Barroso, & Sa´nchez, 2018). Beyond this, many more types of this protein have yet to be discovered, as well as other alternative sources to prevent outbreaks of communicable disease and immune problems. Because of this, research is still underway to identify the various unexplored sources of collagen that may be used in the future. In the last decade, the size of the world market for collagen and its derivatives (hydrolyzed) has grown and is expected to witness significant growth in the coming years, reaching USD 6.63 billion by 2025 (Avila Rodrı´guez et al., 2018). This is mainly due to the growing demand for food and beverages, cosmetics and healthcare. The global market for collagenbased biomaterials and bioingredients for tissue repair and care applications is likely to progress to a solid compound annual growth rate of 10.4% over the next 5 years (Avila Rodrı´guez et al., 2018). It is important to say that due to all the forms of collagen currently on the market (intact, hydrolyzed, gelatine, hydrolyzed gelatine), all cosmetic and pharmaceutical products must indicate what type they are using in their formula and why. Not only to know the role it will have in the body, but also because of the sources from which the used collagen was taken that could interfere with the patient’s homeostasis. In the case of peptides, it is also important to know if the hydrolysis was chemical or enzymatic, as well as the size profile of the peptides since their bioactivity depends on it (Coscueta, Campos, Oso´rio, Nerli, & Pintado, 2019).

740 Chapter 29 All the natural sources of collagen that have been found, as well as the development of synthetic sources, had a major impact on the collagen derivatives industry. It is expected to expand more and more in the coming years, thus opening new strong and interesting opportunities in the field of research applicable to the biomedical industry whose main application is in the prophylaxis and treatment of joints.

29.7 Summary A growing body of evidence provides a rationale for the use of collagen hydrolysate/ peptides from different natural sources in patients with joint diseases. The joint function involves several mechanisms of action, such as cartilage proliferation, degradation, regeneration; and positive impact through the action by antimicrobial, antioxidant, and antiinflammatory agents; and neuroactivity. Understanding these mechanisms is important to develop new molecules or biomaterials for diagnosis, prophylaxis, treatments, and therapies for joint diseases. In that sense, peptidomics is playing a vital role in the study of biological markers and therapeutic targets. So, research studies are crucial for the advancement and validation of clinically applicable biomarkers. It is also imperative to evaluate their specificity and sensitivity for a suitable and early diagnosis. Furthermore, it is hoped that ongoing and future research will develop tools and methodologies for the prevention and therapy of joint diseases based on bioactive peptides. There is evidence that, for example, collagen derivatives are resorbed in the small intestine and are directly transported from the gastrointestinal tract into the bloodstream. In preclinical trials, it has been revealed a significant quantity of biopeptides accumulated in the articular cartilage. In clinical trials, the effectiveness of oral supplementation of collagen derivatives was demonstrated in osteoarthritic patients, and an improvement of clinical parameters related to joint health was reported, that is, reduction of pain and improvement of joint mobility. This therapy was also effective in athletic subjects suffering from joint pain related to excessive physical activity. Therefore although the application of bioactive peptides in joint pathologies has a promising future, there is still a long way to go. Mainly, the critical mass of clinical work should be increased, adopting the new strategies developed in real cases.

Acknowledgments This work was supported by National Funds from FCT—Fundac¸a˜o para a Cieˆncia e a Tecnologia through project UID/Multi/50016/2019.

Applications in medicine: joint health 741

References Aggarwal, R., Liao, K., Nair, R., Ringold, S., & Costenbader, K. H. (2009). Anti-citrullinated peptide antibody assays and their role in the diagnosis of rheumatoid arthritis. Arthritis Care and Research, 61, 1472
1483. Aletaha, D., Funovits, J., Smolen, J. S., & Editorial, S. (2011). Physical disability in rheumatoid arthritis is associated with cartilage damage rather than bone destruction. Annals of the Rheumatic Diseases, 733
739. Available from https://doi.org/10.1136/ard.2010.138693. Aletaha, D., & Smolen, J. S. (2018). Diagnosis and management of rheumatoid arthritis: A review. JAMA: The Journal of the American Medical Association, 320, 1360
1372. Alm, L. M., et al. (2018). The performance of anti-cyclic citrullinated peptide assays in diagnosing rheumatoid arthritis: A systematic review and meta-analysis. Clinical and Experimental Rheumatology, 36, 144
152. Al-Madol, M. A., et al. (2017). Comparative expression analyses of pro- versus anti-inflammatory mediators within synovium of patients with joint trauma, osteoarthritis, and rheumatoid arthritis. Mediators of Inflammation, 2017. Arvieux, C., & Common, H. (2019). New diagnostic tools for prosthetic joint infection. Orthopaedics and Traumatology: Surgery and Research, 105, S23
S30. Avila Rodrı´guez, M. I., Rodrı´guez Barroso, L. G., & Sa´nchez, M. L. (2018). Collagen: A review on its sources and potential cosmetic applications. Journal of Cosmetic Dermatology, 17, 20
26. Banerjee, P., & Shanthi, C. (2016). Cryptic peptides from collagen: A critical review. Protein and Peptide Letters, 23, 664
672. Bello, A. E., & Oesser, S. (2006). Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: A review of the literature. Current Medical Research and Opinion, 22, 2221
2232. Benham, H., et al. (2015). Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype—Positive rheumatoid arthritis patients. Rheumatoid arthritis, 7. Benito-Ruiz, P., et al. (2009). A randomized controlled trial on the efficacy and safety of a food ingredient, collagen hydrolysate, for improving joint comfort. International Journal of Food Sciences and Nutrition, 60, 99
113. Charni, N., Juillet, F., & Garnero, P. (2005). Urinary type II collagen helical peptide (HELIX-II) as a new biochemical marker of cartilage degradation in patients with osteoarthritis and rheumatoid arthritis. Arthritis and Rheumatism, 52, 1081
1090. Coscueta, E. R., Campos, D. A., Oso´rio, H., Nerli, B. B., & Pintado, M. (2019). Enzymatic soy protein hydrolysis: A tool for biofunctional food ingredient production. Food Chemistry: X, 1, 100006. Courties, A., Sellam, J., & Berenbaum, F. (2017). Role of the autonomic nervous system in osteoarthritis. Best Practice and Research: Clinical Rheumatology, 31, 661
675. Czajka, A., et al. (2018). Daily oral supplementation with collagen peptides combined with vitamins and other bioactive compounds improves skin elasticity and has a beneficial effect on joint and general wellbeing. Nutrition Research (New York, N.Y.), 57, 97
108. Dar, Q.-A. A., et al. (2017). Daily oral consumption of hydrolyzed type 1 collagen is chondroprotective and anti-inflammatory in murine posttraumatic osteoarthritis. PLoS One, 12, e0174705. Deng, Z. H., Li, Y. S., Gao, X., Lei, G. H., & Huard, J. (2018). Bone morphogenetic proteins for articular cartilage regeneration. Osteoarthritis and Cartilage, 26, 1153
1161. EFSA Journal. (2005). Undefined. Opinion of the Food Safety Authority on safety of collagen and a processing method for the production of collagen. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA). (2011). Scientific Opinion on the substantiation of a health claim related to collagen hydrolysate and maintenance of joints pursuant to Article 13(5) of Regulation (EC) No 1924/2006. EFSA Journal, 9. Elezagic, D., et al. (2019). Antimicrobial peptides derived from the cartilage-specific C-type lectin domain family 3 member A (CLEC3A)—Potential in the prevention and treatment of septic arthritis. Osteoarthritis and Cartilage, 27, 1564
1573. Faust, H. J., et al. (2018). A hyaluronic acid binding peptide-polymer system for treating osteoarthritis. Biomaterials, 183, 93
101.

742 Chapter 29 Ferro, E. S., Rioli, V., Castro, L. M., & Fricker, L. D. (2014). Intracellular peptides: From discovery to function. EuPA Open Proteomics, 3, 143
151. Fu, Y., Therkildsen, M., Aluko, R. E., & Lametsch, R. (2018). Exploration of collagen recovered from animal by-products as a precursor of bioactive peptides: Successes and challenges. Critical Reviews in Food Science and Nutrition, 8398. Fusco, M., Skaper, S. D., Coaccioli, S., Varrassi, G., & Paladini, A. (2017). Degenerative Joint diseases and neuroinflammation. Pain Practice, 17, 522
532. Haque Bhuyan, M. Z., et al. (2017). The intra-articular injection of RANKL-binding peptides inhibits cartilage degeneration in a murine model of osteoarthritis. Journal of Pharmacological Sciences, 134, 124
130. Hosny, H. A., & Keenan, J. (2020). Management of prosthetic joint infection. Surgery (United Kingdom), 38, 114
120. Hu, J., et al. (2020). Peptidomic analysis on synovial tissue reveals galectin-1 derived peptide as a potential bioactive molecule against rheumatoid arthritis. Cytokine, 131, 155020. Horowitz, D., Hanrahan, J., & Turley, R. K. (n.d.). Osteoarthritis. Health Encyclopedia—University of Rochester Medical Center Available at: https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid 5 85&contentid 5 p00061. Iwai, K., et al. (2005). Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysates. Journal of Agricultural and Food Chemistry, 53, 6531
6536. Janakiraman, K., Krishnaswami, V., Rajendran, V., Natesan, S., & Kandasamy, R. (2018). Novel nano therapeutic materials for the effective treatment of rheumatoid arthritis-recent insights. Materials Today Communications, 17, 200
213. Jiang, W., Wang, H., Li, Y. S., & Luo, W. (2016). Role of vasoactive intestinal peptide in osteoarthritis. Journal of Biomedical Science, 23, 1
6. Kanemitsu, M., et al. (2020). Role of vasoactive intestinal peptide in the progression of osteoarthritis through bone sclerosis and angiogenesis in subchondral bone. Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. Available from https://doi.org/10.1016/j.jos.2019.11.010. Kanzaki, N., Ono, Y., Shibata, H., & Moritani, T. (2015). Glucosamine-containing supplement improves locomotor functions in subjects with knee pain: A randomized, double-blind, placebo-controlled study. Clinical Interventions in Aging, 10, 1743
1753. Kawaguchi, T., Nanbu, P. N., & Kurokawa, M. (2012). Distribution of prolylhydroxyproline and its metabolites after oral administration in rats. Biological & Pharmaceutical Bulletin, 35, 422
427. Kilinc, B. E., et al. (2018). An observational 1-month trial on the efficacy and safety of promerim for improving knee joint. Clinical Medicine Insights: Arthritis and Musculoskeletal Disorders, 11. Kim, D. K., In Kim, J., Sim, B. R., & Khang, G. (2017). Bioengineered porous composite curcumin/silk scaffolds for cartilage regeneration. Materials Science and Engineering C, 78, 571
578. Kim, S. K., & Mendis, E. (2006). Bioactive compounds from marine processing byproducts—A review. Food Research International, 39, 383
393. Krishnan, Y., & Grodzinsky, A. J. (2018). Cartilage diseases. Matrix Biology, 71
72, 51
69. Kumar, S., Sugihara, F., Suzuki, K., Inoue, N., & Venkateswarathirukumara, S. (2015). A double-blind, placebo-controlled, randomised, clinical study on the effectiveness of collagen peptide on osteoarthritis. Journal of the Science of Food and Agriculture, 95, 702
707. Lam, A. T. L., Reuveny, S., & Oh, S. K. W. (2020). Human mesenchymal stem cell therapy for cartilage repair: Review on isolation, expansion, and constructs. Stem Cell Research, 44, 101738. Lin, J. B., Poh, S., & Panitch, A. (2016). Controlled release of anti-inflammatory peptides from reducible thermosensitive nanoparticles suppresses cartilage inflammation. Nanomedicine: Nanotechnology, Biology and Medicine, 12, 2095
2100. Liu, Q., et al. (2018). Functional peptides for cartilage repair and regeneration. American Journal of Translational Research, 10, 501
510. Lugo, J. P., Saiyed, Z. M., & Lane, N. E. (2016). Efficacy and tolerability of an undenatured type II collagen supplement in modulating knee osteoarthritis symptoms: A multicenter randomized, double-blind, placebocontrolled study. Nutrition Journal, 15, 1
15.

Applications in medicine: joint health 743 Malmstro¨m, V., Catrina, A. I., & Klareskog, L. (2017). The immunopathogenesis of from triggering to targeting. Nature Reviews. Immunology, 17, 60
75. McAlindon, T. E., et al. (2011). Change in knee osteoarthritis cartilage detected by delayed gadolinium enhanced magnetic resonance imaging following treatment with collagen hydrolysate: A pilot randomized controlled trial. Osteoarthritis and Cartilage, 19, 399
405. McMasters, J., Poh, S., Lin, J. B., & Panitch, A. (2017). Delivery of anti-inflammatory peptides from hollow PEGylated poly(NIPAM) nanoparticles reduces inflammation in an ex vivo osteoarthritis model. Journal of Controlled Release: Official Journal of the Controlled Release Society, 258, 161
170. Oesser, S., Adam, M., Babel, W., & Seifert, J. (1999). Oral administration of 14C labeled gelatin hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/BL). The Journal of Nutrition, 129, 1891
1895. Oesser, S., & Seifert, J. (2003). Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen. Cell and Tissue Research, 311, 393
399. Ohara, H., Iida, H., Ito, K., Takeuchi, Y., & Nomura, Y. (2010). Effects of Pro-Hyp, a collagen hydrolysate-derived peptide, on hyaluronic acid synthesis using in vitro cultured synovium cells and oral ingestion of collagen hydrolysates in a guinea pig model of osteoarthritis. Bioscience, Biotechnology, and Biochemistry, 74, 2096
2099. Park, Y. J., et al. (2018). A novel antimicrobial peptide acting via formyl peptide receptor 2 shows therapeutic effects against rheumatoid arthritis. Scientific Reports, 8, 4
13. Available from https://doi.org/10.1038/ s41598-018-32963-5. Parmar, P. A., et al. (2015). Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration. Biomaterials, 54, 213
225. Peake, N. J. J., et al. (2014). Role of C-type natriuretic peptide signalling in maintaining cartilage and bone function. Osteoarthritis and Cartilage, 22, 1800
1807. Pinto, I. B., et al. (2019). Utilization of antimicrobial peptides, analogues and mimics in creating antimicrobial surfaces and bio-materials. Biochemical Engineering Journal, 150, 107237. Puigdellivol, J., et al. (2019). Effectiveness of a dietary Supplement containing hydrolyzed collagen, chondroitin sulfate, and glucosamine in pain reduction and functional capacity in osteoarthritis patients. Journal of Diet, 16, 379
389. Saberi Hosnijeh, F., Bierma-Zeinstra, S. M., & Bay-Jensen, A. C. (2019). Osteoarthritis year in review 2018: Biomarkers (biochemical markers). Osteoarthritis and Cartilage, 27, 412
423. Schadow, S., et al. (2013). Collagen metabolism of human osteoarthritic articular cartilage as modulated by bovine collagen hydrolysates. PLoS One, 8. Silva, T. H., et al. (2014). Marine origin collagens and its potential applications. Marine Drugs, 12, 5881
5901. Sokoloff, L. (2019). Joint disease. Encyclopædia Britannica. Available at. https://www.britannica.com/science/ joint-disease. Tuan, R. S., Chen, A. F., & Klatt, B. A. (2013). Cartilage regeneration. Journal of the American Academy of Orthopaedic Surgeons, 21, 303
311. Vaishya, R., Sardana, R., Butta, H., & Mendiratta, L. (2019). Laboratory diagnosis of prosthetic joint infections: Current concepts and present status. Journal of Clinical Orthopaedics and Trauma, 10, 560
565. Vallbracht, I., & Helmke, K. (2005). Additional diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis. Autoimmunity Reviews, 4, 389
394. Varela-Eirin, M., et al. (2018). Cartilage regeneration and ageing: Targeting cellular plasticity in osteoarthritis. Ageing Research Reviews, 42, 56
71. Varoga, D., et al. (2005). Expression and regulation of antimicrobial peptides in articular joints. Annals of Anatomy (Anatomischer Anzeiger: Official Organ of the Anatomische Gesellschaft), 187, 499
508. Van Vijven, J. P. J., et al. (2012). Symptomatic and chondroprotective treatment with collagen derivatives in osteoarthritis: A systematic review. Osteoarthritis and Cartilage, 20, 809
821. Watanabe-Kamiyama, M., et al. (2010). Absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats. Journal of Agricultural and Food Chemistry, 58, 835
841. Wythe, S. E., et al. (2013). Targeted delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide. Annals of the Rheumatic Diseases, 129
135. Available from https://doi.org/10.1136/annrheumdis2012-201457.

744 Chapter 29 Yu, F., et al. (2018). Mechanism research on a bioactive resveratrol
PLA
gelatin porous nano-scaffold in promoting the repair of cartilage defect. International Journal of Nanomedicine, 13, 7845
7858. Zdzieblik, D., Oesser, S., Gollhofer, A., & Ko¨nig, D. (2017). Improvement of activity-related knee joint discomfort following supplementation of specific collagen peptides. Applied Physiology, Nutrition, and Metabolism (Physiologie Appliquee, Nutrition et Metabolisme), 42, 588
595. Zhang, A., & Lee, Y. C. (2018). Mechanisms for joint pain in rheumatoid arthritis (RA): From cytokines to central sensitization. Current Osteoporosis Reports, 16, 603
610. Zhang, Y., et al. (2018). Soy peptide nanoparticles by ultrasound-induced self-assembly of large peptide aggregates and their role on emulsion stability. Food Hydrocolloids, 74, 62
71.

CHAPTER 30

Applications in food technology: antimicrobial peptides En Huang1, Walaa E. Hussein2,3, Emily P. Campbell4 and Ahmed E. Yousef4,5 1

Department of Environmental and Occupational Health, College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR, United States, 2Department of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, OH, United States, 3Department of Microbiology and Immunology, National Research Center, Giza, Egypt, 4Department of Food Science and Technology, The Ohio State University, Columbus, OH, United States, 5Department of Microbiology, The Ohio State University, Columbus, OH, United States

30.1 Introduction Finding the appropriate antimicrobial additives is an important undertaking for any food product developer. These additives are needed to improve the safety and shelf life of the new product, hence, improving the product’s commercial feasibility. The challenge facing these product developers is the limited choices of antimicrobials available. The shortage of antimicrobials is particularly severe in the case of products subject to spoilage by fungi or contamination by Gram-negative bacteria. It is, therefore, critical that researchers spend extra efforts and funding agencies provide the needed resources to discover new antimicrobial agents and to test the feasibility of using these agents in upcoming food products. Currently used antimicrobial food additives include synthetic compounds (e.g., sorbic and benzoic acids) or potentially hazardous chemicals (e.g., nitrites); these have been used for decades in food preservation, but interest in alternative agents from natural sources is rising. This shift is driven, in part, by changing societal needs. Antimicrobial products of microbial origin are promising alternatives to the commonly used preservatives, particularly if the antimicrobial producer is familiar to the food processor. Propionic acid bacteria, for example, are familiar starter cultures in the Swiss cheese industry; these bacteria are used in fermentations resulting in food ingredients rich in propionic acid, which serves as an antifungal agent. The propionic acid bacteria fermentate may be used as an ingredient capable of extending food’s shelf life. Many microbial peptides are more potent, as antimicrobial agents, than organic acids or other metabolites; these are collectively called antimicrobial peptides (AMPs). Being from Biologically Active Peptides. DOI: https://doi.org/10.1016/B978-0-12-821389-6.00006-6 © 2021 Elsevier Inc. All rights reserved.

745

746 Chapter 30 natural sources, AMPs have great potential in food applications. These peptides can be produced by all forms of life, but bacteria have been sought heavily as the most desirable source. AMPs currently used by food processors are products of lactic acid bacteria (LAB), even though equivalently usable peptides are produced by non-LAB. Within the LAB group, preferable antimicrobial-producing species are those used often in the production of fermented food (e.g., Lactococcus and Pediococcus spp.). Unfortunately, bacteria that fit this description are not prolific producers of AMPs. Limiting food applications to AMPs produced by specific species is a response to the difficulties that food processors face while trying to obtain regulatory approval for new agents or antimicrobial producer strains. Researchers have been actively discovering new potent AMPs or characterizing previously discovered agents; many of these are promising candidates for food application. However, the path for the regulatory approval for many of these agents is very treacherous, as explained in a later section of this chapter. While a limited number of AMPs are available for use in food, processors are encountering new strains of foodborne spoilage and pathogenic microorganisms that cannot be controlled by existing antimicrobial agents or processes. It is therefore critical that researchers, processors, and regulators cooperate in securing a steady supply of new antimicrobials for ensuring the safety and quality of future foods.

30.2 Classification AMPs are host defense peptides produced by all living organisms. They can be grouped into categories based on the producers (e.g., LAB vs non-LAB AMPs), structural features (e.g., peptides vs lipopeptides), the biosynthetic pathway used by the producer to synthesize these agents (e.g., ribosomal vs nonribosomal synthesis), and other factors. Fig. 30.1 shows examples of AMPs representing different categories. In terms of food applications, a highly sought-after AMP category is bacteriocins. These are bacterially produced, ribosomally synthesized, AMPs with or without posttranslational modifications. Posttranslational modifications of the ribosomally synthesized bacteriocin include N-terminal acetylation, dehydration, thioether bridge formation, and others. Many well-known bacteriocins are produced by LAB. The bacteriocin subcategory of AMPs has been traditionally classified into four classes, where classes I and II being the most applicable to food. These two classes have small molecular masses (,10 kDa) and relative heat stability. Class I includes posttranslationally modified peptides such as lantibiotics and sactibiotics. Class II includes posttranslationally unmodified bacteriocins such as the pediocin family, some two-peptide bacteriocins, and some circular bacteriocins. Pediocins, produced by Pediococcus spp., and similar unmodified peptides are class II bacteriocins. Coagulin, an anti-Listeria bacteriocin, is a member of the pediocin family and

Applications in food technology: antimicrobial peptides 747

Produced by: Non-LAB LAB

Primary Structure

Modificaon

Synthesis

Anmicrobial Pepdes

Ribosomal

Post Translaon

Non-ribosomal

Post Translaon Unmodified

Modified

Pepde

Lanbiocs

Sacbiocs

Pediocin-Like

Two-Pepde

Circular

Nisin

N/A

Pediocin

Plantaricin

Enterocin AS-48

Linear

N/A

Lipopepde

Cyclic

Linear

Cyclic

Cyclodipepdes

N/A

Mutanobacn D

E/F Paenibacillin

Sublosin A

Coagulin

Brochocin-C

Amylocyclin

ε-Polylysine

Mycobacillin

Paenipepn

Daptomycin

Brevibacillin

Paenibacterin

Figure 30.1 Examples of different categories of antimicrobial peptides produced by LAB and non-LAB. LAB, Lactic acid bacteria.

produced by Bacillus coagulans I4 (Le Marrec, Hyronimus, Bressollier, Verneuil, & Urdaci, 2000). There are at least 15 unmodified two-peptide bacteriocins, which require the presence of both peptides for optimal activities; most of them are produced by LAB. For example, plantaricin E/F and plantaricin J/K are produced by Lactobacillus plantarum C11 (Collins et al., 2017; Moll et al., 1999; Nissen-Meyer, Oppega˚rd, Rogne, Haugen, & Kristiansen, 2010). Brochocin-C, a two-peptide bacteriocin with anti-Clostridium activity is produced by Brochothrix campestris ATCC 43754 (Mccormick et al., 1998). Circular peptides, such as enterocin AS-48 (Ananou et al., 2005) and amylocyclin (Scholz et al., 2014), possess a unique head-to-tail circulation. It is less common to find bacteriocins produced by Gram-negative, compared to Gram-positive, bacteria. Colicins are high molecular mass (30
80 kDa) unmodified bacteriocins produced by Gram-negative bacteria under stress conditions (Cascales et al., 2007). Class I bacteriocins include posttranslationally modified peptides such as lantibiotics and sactibiotics. Structurally, lantibiotics contain dehydrated amino acids and lanthionine residues that form intramolecular thioether rings. The most well-known lantibiotic in the food industry is nisin, which is produced by Lactococcus lactis. Paenibacillin also is a lantibiotic produced by Paenibacillus polymyxa OSY-DF (He et al., 2007; He, Yuan, Zhang, & Yousef, 2008). Subtilosin A, produced by Bacillus subtilis 168, belongs to the sactibiotic subclass of bacteriocins. Sactibiotics are ribosomally synthesized AMPs with a linkage between Cys sulfur and the α-carbon of neighboring amino acid residue (Acedo, Chiorean, Vederas, & van Belkum, 2018; Mathur, Rea, Cotter, Hill, & Ross, 2015). Subtilosin A is active against the foodborne pathogen, Listeria monocytogenes (van Kuijk,

748 Chapter 30 Noll, & Chikindas, 2012). Other sactibiotics include thuricin CD, thurincin H, and propionicin F. To the best of our knowledge, no sactibiotic bacteriocin has been isolated and identified in LAB. Microcins are a class of low molecular mass peptides (1
10 kDa) produced by Gram-negative bacteria under environmental stress (Duquesne, DestoumieuxGarzo´n, Peduzzi, & Rebuffat, 2007). Some microcins undergo posttranslational modifications to form the active molecules. Nonribosomal peptides are synthesized by nonribosomal peptide synthetases (NRPSs) which contribute to the structural diversity of peptides. ε-Polylysine is a linear AMP approved for food applications. Cyclo (L-Phe-L-Pro) is an example of antifungal cyclic dipeptide from LAB (Stro¨m, Sjo¨gren, Broberg, & Schnu¨rer, 2002) and mycobacillin is an antifungal peptide produced by B. subtilis (Majumdar & Bose, 1958). In addition, some nonribosomal peptides possess a lipid chain in the structure; these are known as lipopeptides. For example, brevibacillin (Yang, Huang, Yuan, Zhang, & Yousef, 2016) and paenipeptin (Huang, Yang, Zhang, Moon, & Yousef, 2017) are linear lipopeptides. In contrast, paenibacterin (Guo, Huang, Yuan, Zhang, & Yousef, 2012) and daptomycin (Tally & DeBruin, 2000) are examples of cyclic lipopeptides. Genes encoding NRPS are rarely found in bacteria with a genome smaller than 3 Mb (Lin et al., 2015). It was argued that bacteriocin production may supplant the need for nonribosomal peptides in LAB given the large size of the NRPS enzymes (Collins et al., 2017).

30.3 Current and potential food applications Viability of any food application is rooted in finding the right AMP. Considering the large number of bacterial producers and the variety of AMP products, choice of producer-product combination is dictated by industry’s urgent needs. For example, food processors have been searching for safer alternatives to nitrites, which are used in many meat products to control Clostridium botulinum. It is therefore logical that the AMP capable of combating this pathogen will be well-suited candidate as a meat additive, and obviously, it would be given application priority. AMPs application in food can be achieved typically by three approaches: addition of purified AMPs, inoculation of AMP-producer bacteria, and use as ingredient that has been fermented with the antimicrobial-producing bacteria (Fig. 30.2). The ultimate goal in the application of AMPs in food is their use as antimicrobial additives. This application requires producing the AMP in high enough concentration so that when a small amount is added to food, it demonstrates the desired effect. However, the use of AMPs as additives requires rigorous regulatory approvals (US FDA, 2006). If the AMP-producing species or strain has the generally-recognized-as-safe (GRAS) status, implementation is possible in the form of protective food ingredients. For example, if the food formulation can benefit from the addition of cheese whey solids, fermentation of liquid whey with an AMP producer may

Applications in food technology: antimicrobial peptides 749

Figure 30.2 Various approaches for the application of AMPs in food. AMPs, Antimicrobial peptides.

impart this ingredient with a protective effect against pathogenic and spoilage contaminants in the resulting food. If the AMP-producing species has the GRAS status such as certain LAB and is suitable for use as a starter culture in a fermented food, it may be feasible to use the producer as a member of the multistrain starter culture for that food. In this case, the AMP-producing strain should not be inhibitory to other members of the starter culture cocktail. The path from AMP-producing isolate to the point of application involves scale-up to mass produce the culture or the AMP. Industrial-scale fermentors or bioreactors are often needed to accomplish this task. Subsequently, downstream processing of fermentation or bioreaction product is needed to prepare the AMP in a form acceptable for application in the product. Processes such as concentration, formulation into a commercial product, and preservation (e.g., pasteurization, drying, or freezing) are often implemented. Considering the large number of AMPs known, it is possible to find peptides that are active against most undesirable microorganisms in food. Some AMPs are active against bacteria (Table 30.1) whereas others have antifungal action (Table 30.2). Unlike antibiotics that target many bacterial functions and metabolic pathways, most AMPs act by binding and disrupting the cytoplasmic membrane of the sensitive organism (Lee, Hofferek, Separovic, Reid, & Aguilar, 2019). Many AMPs have a net positive charge to allow for binding to the negatively charged bacterial membrane. AMPs are generally small, ranging from 6 to 100 amino acid residues. They are often highly charged and amphipathic, containing hydrophobic and hydrophilic residues.

750 Chapter 30 Table 30.1: Examples of antibacterial peptides active against foodborne microorganisms. Antibacterial peptide

Producer

Typical application

References

Approved peptide preparations Nisin

Lactococcus lactis

Clostridium botulinum in meat products

Pediocin

Pediococcus spp.

Listeria monocytogenes and other Grampositive pathogens in meat and cheese Broad spectrum and intended for use in beverages, baked goods, cheeses, egg products, poultry, and meat L. monocytogenes in smoked fish

ε-Polylysine

Divergicin Colicins

Streptomyces albulus

Carnobacterium divergens M35 Escherichia coli or plant-based production from plant leaves

E. coli in fruits, vegetables, or meat products

GRAS Notice No. (GRN) 65 Hugo and Hugo (2015) GRN 135 GRN 336 GRN 762 GRN 775

Promising peptide preparations Enterocin AS-48 Paenibacillin

Fermentate with Paraplantaricin TC318

Enterococcus faecalis

Gram-positive bacteria

Ananou et al. (2005)

Paenibacillus polymyxin OSY-DF

Listeria and other Gram-positive bacteria

Lactobacillus paraplantarum TC318

Gram-positive and Gram-negative bacteria

Gerst, Huang, Zhang, and Yousef (2015), He et al. (2007, 2008) Hussein et al. (2020)

Most known AMPs have not been applied commercially. According to the AMP database, there have been at least 2700 natural AMPs discovered and cataloged as of 2016. Many studies have investigated the potential of applying bacteriocins in foods such as dairy products (O’Sullivan, Ryan, Ross, & Hill, 2003), bakery products (Martı´nez Viedma, Abriouel, Ben Omar, Lo´pez, & Ga´lvez, 2011), fermented vegetables (Settanni & Corsetti, 2008), fish (Bali, Panesar, Bera, & Kennedy, 2016), and meat (Valenzuela, Benomar, Abriouel, Can˜amero, & Ga´lvez, 2010). The AMPs’ activity in food products is affected by environmental factors, such as ionic strength and the presence of solutes or ions. At high salt concentrations, antimicrobial activity of the peptide is decreased. Divalent cations stabilize bacterial membranes and compete with cationic AMPs for the binding to anionic lipids (Sa´nchez-Go´mez et al., 2008). A wide range of AMPs has been investigated for potential application in food to limit the growth of pathogenic and spoilage microorganisms. AMPs have been proposed to inhibit Gram-positive and Gram-negative bacteria including but not limited to C. botulinum, L. monocytogenes, and Escherichia coli (Table 30.1). Although AMPs that target bacteria are widely investigated, some AMPs acting against fungal contaminants have been also explored (Table 30.2).

Applications in food technology: antimicrobial peptides 751 Table 30.2: Antifungal peptides produced by lactic acid bacteria. Antifungal peptidesa

Producer

Targeted species

References

Peptide sequences identified Cyclodipeptide Lactobacillus plantarum

Durancins YML007

LR/14

IS10 DU15

Aspergillus sp. Fusarium sp. Enterococcus durans Debaryomyces A5-11 hansenii L. plantarum YML007 Aspergillus parasiticus Penicillium expansum L. plantarum LR/14 Aspergillus niger Rhizopus stolonifer Mucor racemosus Penicillium chrysogenum L. plantarum IS10 Molds Leuconostoc A. niger mesenteroides DU15

¨m, Roos, Dal Bello et al. (2007), Magnusson, Stro ¨gren, and Schnu ¨m et al. (2002) Sjo ¨rer (2003), Stro

Belguesmia et al. (2013) ˜es, and Meca (2017) Luz, Saladino, Luciano, Man

Gupta and Srivastava (2014)

Muhialdin, Hassan, Bakar, and Saari (2016) Muhialdin, Hassan, Abu Bakar, Algboory, and Saari (2015)

Peptide partially characterized Bacteriocin F1 Lactobacillus paracasei subsp. tolerans FX-6

Bacteriocinlike peptide Proteinaceous peptide Proteinaceous peptide a

Lactobacillus coryniformis Lactobacillus brevis NCDC 02 Lactobacillus fermentum CRL 251

Miao et al. (2014, 2015)

Aspergillus flavus A. niger Rhizopus nigricans Penicillium glaucum Molds and yeasts Molds

Falguni, Shilpa, and Mann (2010)

A. niger

Gerez, Torres, Font de Valdez, and Rolla´n (2013)

Magnusson and Schnu ¨rer (2001)

If the antifungal compound was not named by the authors, the producer strain was used to indicate the active peptide.

30.3.1 Commercial application of nisin Nisin is a commercially utilized lantibiotic produced by L. lactis that is active against Gram-positive bacteria (Kim, 1997; Tai, McGuire, & Neff, 2008). Nisin inhibits Bacillus, Clostridium, Listeria, and Staphylococcus spp. (Sobrino-Lo´pez & Martı´n-Belloso, 2008).

752 Chapter 30 It is currently approved in over 50 countries and has been used as a natural biopreservative in a variety of food products including cooked meats, liquid eggs, and dairy products (de Arauz, Jozala, Mazzola, & Vessoni Penna, 2009). In the United States, nisin was approved by the Food and Drug Administration (FDA) in 1988 and was designated as a GRAS additive (Cotter, Hill, & Ross, 2005). It is utilized in processed cheese and in canned products to prevent the growth of C. botulinum (Jones, Salin, & Williams, 2005). Recommendations and regulations on nisin levels vary by country. The FDA has set a maximum limit of 10,000 IU/g in the United States (Jones et al., 2005). The use of starter cultures with nisin producers is unregulated due to L. lactis GRAS status. There is no maximum level of nisin in process cheeses in Australia, France, or Great Britain. However, the Joint FAO (Food and Agriculture Organization)/WHO (World Health Organization) Expert Committee on Food Additives recommended a daily limit of 60 mg of pure nisin for a 70 kg person. Nisin is not often utilized in a highly concentrated form commercially. The level of active nisin in commercially available additives ranges from 0.5% to 5%. The typical dose of nisin in canned, dairy, and meat products ranges from 100 to 200 mg/kg (Jones et al., 2005). Applications of nisin outside the food preservation arena have also been investigated due to the recognition of its potential clinical use (Shin et al., 2016). Researchers have investigated this AMP for potential application to combat or prevent biofilm-associated infections in humans (Fauci & Morens, 2012) and control oral microbiota (Johnson, Hayday, & Colman, 1978).

30.3.2 Commercial application of pediocin Pediocins are class II bacteriocins produced by Pediococcus spp.; these producers are utilized as protective cultures against pathogens and spoilage bacteria in commercial food products. Application of pediocin, produced by Pediococcus spp., may improve food quality and sensory attributes through control of microbial contaminants. Similar to the approval of nisin, pediocin produced by Pediococcus acidilactici and Pediococcus pentosaceus are considered GRAS in certain food applications (Hugo & Hugo, 2015). Pediocin has been found to be active against foodborne pathogens including Staphylococcus aureus and L. monocytogenes (Cintas, Casaus, Ferna´ndez, & Herna´ndez, 1998; Eijsink, Skeie, Middelhoven, Brurberg, & Nes, 1998). The bacteriocin was tested in meat products to control L. monocytogenes (Porto, Kuniyoshi, Azevedo, Vitolo, & Oliveira, 2017). Pediocin effectiveness in different foods is altered by numerous factors including pH, proteolytic enzymes, and heat treatment (Papagianni & Anastasiadou, 2009). Application of pediocins in food products may be accomplished through addition of a producer organism or a previously fermented ingredient. Alta 2341 is a pediocin-containing commercial preparation that is used in the United States and Europe as a preservative. Cultures of P. acidilactici, marketed as CHOOZIT Lyo. Flav 43, are suggested for use in semihard cheese manufacturing to accelerate ripening and enhance product flavor, but it is likely that it also has antimicrobial effect.

Applications in food technology: antimicrobial peptides 753

30.3.3 Commercial application of MicroGARD Fermented ingredients such as MicroGARD are collections of natural antimicrobial ingredients designed to assist in production of minimally processed foods with acceptable shelf life. MicroGARD is developed through Propionibacterium fermentation and the fementate is used as a protective ingredient; the product has been approved by the FDA for use in food [GRAS Notice No (GRN). 128]. Currently, a variety of commercial formulations of MicroGARD fermentates are available for different food applications (Dupont Nutrition and Biosciences, 2021). The preservation is believed to be achieved by a heat-stable protease-sensitive polypeptide, organic acids, and diacetyl (Al-Zoreky, Ayres, & Sandine, 1991; Salih, Sandine, & Ayres, 1990). It is an effective inhibitor of Gram-negative bacteria, such as Pseudomonas, Salmonella, and Yersinia (Al-Zoreky et al., 1991) and some fungi (Weber & Broich, 1986). It is typically added to dairy products but can also be added to prepared meals and cooked, raw, or cured meat.

30.3.4 Commercial application of ε-polylysine ε-Polylysine is a natural homopolymer of approximately 30 L-lysine residues produced by Streptomyces albulus. This compound has broad spectrum antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens and fungi (GRN 336). ε-Polylysine is biosynthesized by S. albulus through the membrane-bound NRPSs, which catalyze L-lysine polymerization (Yamanaka, Maruyama, Takagi, & Hamano, 2008). ε-Polylysine industrial production is carried out through aerobic bioreaction using food-grade raw materials. The producer cells are removed from the bioreaction product using filter sterilization with 0.1 μm filter, followed by a series of ion-exchange chromatographic purification, activated charcoal clarification, and evaporation to form a final fine powder product (GRN 336). ε-Polylysine has been approved for food use in Japan for a variety of foods, including sliced fish, fish sushi, boiled rice, soups, noodles, and cooked vegetables. Under the self-affirmation GRAS process in 2004, ε-polylysine is permitted to be used in the United States as an antimicrobial preservative in cooked rice and sushi rice at 5
50 ppm (GRN 135). In 2011 the application of ε-polylysine was expanded to other products, including beverages, baked goods, cheeses, egg products (up to 0.025%), and poultry and meat (0.06%) (GRN 336).

30.3.5 Other antimicrobial peptide preparations received regulatory approval Colicins are bacteriocins produced by E. coli or other enteric bacteria. Colicins are active against Gram-negative microorganisms. Plant-produced recombinant colicins (colicin preparations) from leaves of spinach, red beet, or lettuce received GRAS status (GRN 593) in the United States in 2015. Recombinant colicins are expressed from tobacco mosaic virus

754 Chapter 30 or potato virus X containing the target gene in the plant hosts. Colicins harvested from plants are concentrated by pH-assisted precipitation and filtration steps (GRN 593). Colicin preparations are intended for use as an antimicrobial on vegetables, fruits, or meat products at levels of 0.5
5 mg/lb (GRN 775). Divergicin M35, produced by Carnobacterium divergens M35, is a bacteriocin preparation that received regulatory approval in 2016 in Canada for use as a live culture antimicrobial preservative against L. monocytogenes in ready-to-eat cold smoked salmon and trout (Canada Health Department approval reference number: NOM/ADM-0079). After spraying the fermentate on fish products, the live C. divergens M35 culture can survive the cold-smoking process and grow at refrigeration temperatures, allowing the production of bacteriocin in situ (NOM/ADM-0079).

30.4 Hurdle approach The multihurdle approach combines several food preservation methods, including physical, chemical, and biological factors, to control foodborne spoilage and pathogenic microorganisms while maintaining food in a minimally processed state. Synergistic hurdles may be achieved by controlling food’s pH, water activity, oxidation-reduction potential, antimicrobial additives, and temperature. The application of AMPs as the main preservation factor in food can be limited due to insufficient antimicrobial characteristics and high cost. One way to improve the preservation effect of AMPs is to combine them with other hurdles such as chemical additives [EDTA (ethylenediaminetetraacetic acid), potassium diacetate, sodium lactate, and others], heating, and high-pressure treatments (Egan et al., 2016). Research groups have observed Gram-negative bacteria sensitization to AMPs with the addition of chelating agents capable of damaging cell’s outer membrane (Chen & Hoover, 2003). Synergistic effects of AMPs after heat treatments have also been observed. Bacteriocin-resistant bacteria exposed to sublethal stresses such as heat, and treated with bacteriocins were found to have reduced viability (Kalchayanand, Hanlin, & Ray, 1992). Studies have shown the synergistic effect of AMPs and high-pressure processing for the inactivation of foodborne microorganisms (Zhao et al., 2013).

30.5 Application of antimicrobial peptides for improving human health Many studies suggested expanding AMPs application from ensuring safety and extending shelf life of food to the arena of improving human health. In addition, bacteriocin production is sometimes used as one of the selection criteria for novel probiotic strains.

30.5.1 Antimicrobial peptides production by probiotic strains Dairy products such as yogurts contain probiotic strains. Probiotics are sometimes prescribed by physicians to patients who are being treated by antibiotics. Self-induction

Applications in food technology: antimicrobial peptides 755 phenomenon, which is observed in nisin producers, leads to increased biosynthesis of the AMPs in response to its presence in small quantities in the growth medium (Chatterjee, Paul, Xie, & van der Donk, 2005; Gonza´lez-Toledo, Domı´nguezDomı´nguez, Garcı´a-Almenda´rez, Prado-Barraga´n, & Regalado-Gonza´lez, 2010). It was hypothesized that bacteriocin induction could occur in the human intestine by gut microbiota (Dicks, Dreyer, Smith, & van Staden, 2018). The capabilities of gut commensals for producing AMPs are thought to give these bacteria the superiority to compete with harmful pathogens in the gastrointestinal tract (GIT) (Schuijt, van der Poll, de Vos, & Wiersinga, 2013). Thus AMP production is one of the criteria for selecting new probiotic strain as it could protect the treated hosts from bacterial infections (Hussein, Abdelhamid, Rocha-Mendoza, Garcı´a-Cano, & Yousef, 2020). Probiotic strains are characterized by their capability to survive the GIT environment. After colonizing the GIT, AMP-producing probiotics may continuously produce AMPs, so these strains were proposed as an AMP delivery vehicle (Marteau & Shanahan, 2003). There are many commercially available probiotic strains that have the capability of producing AMPs even though they are not labeled and marketed as such. L. plantarum 423 is a probiotic strain that was found to adhere to the small intestine, preventing L. monocytogenes from attaching to the GIT and causing systemic infection (van Zyl, Deane, & Dicks, 2016). L. plantarum 423 produces plantaricin 423, a class II bacteriocin, which is active against several Gram-positive bacteria such as L. monocytogenes and Enterococcus faecalis (Van Reenen, Dicks, & Chikindas, 1998). Another example is the probiotic strain Enterococcus mundtii ST4SA, which was found to colonize the lower part of the GIT and prevent L. monocytogenes EGDe infection (van Zyl et al., 2016). E. mundtii ST4SA produces a class II bacteriocin (ST4SA) that is active against L. monocytogenes, S. aureus, and E. faecalis (Granger, van Reenen, & Dicks, 2008). AMP-producing probiotic strains are thought to have superiority in promoting host health over non-AMP producers. For example, in an in vivo study, oral administration of Lactobacillus salivarius UCC 118, a salivaricin producer strain, resulted in protecting mice against L. monocytogenes infection, while the oral administration of a nonbacteriocin-producer variant of L. salivarius UCC 118 did not result in this protective effect (Corr et al., 2007). In another example, bacteriocinproducing strains have been used in livestock to decrease shedding of bacterial pathogens (Diez-Gonzalez, 2007).

30.5.2 Antiinfective activity of antimicrobial peptides Considering the threatening rate at which bacteria have been developing antibiotic resistance, investigators were prompted to search for effective substitutes to treat infections caused by multidrug-resistant bacteria. Some foodborne pathogens also develop resistance to antibiotics. AMPs, particularly the lantibiotic group, are believed to be good candidates

756 Chapter 30 against infections considering these AMPs are (1) effective against some strains of antibiotic-resistant bacteria such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) and Clostridioides difficile (formerly, Clostridium difficile); (2) safe to use, especially if produced by LAB; and (3) active against targets through mechanisms different than those of many antibiotics (Meade, Slattery, & Garvey, 2020). AMPs are therefore of great interest as promising alternatives to antibiotics or to be coupled with antibiotics to decrease their concentrations during use. Recently, many researchers investigated the efficacy of lantibiotics as therapeutic agents for skin, respiratory, and GIT bacterial infections in animals (Field et al., 2015). Nisin was the most prominent bacteriocin and the only one approved for use in topical preparations. Despite the wide range of AMPs proven in vivo as effective for treating bacterial infections (Yang, Lin, Sung, & Fang, 2014), approving AMPs for clinical application faces many challenges including their solubility and stability, production cost, and any potential cytotoxicity. Some of these challenges can be addressed through different approaches. For cost-effective production, many approaches have been developed to facilitate industrial-scale production such as fermentation optimization and heterologous gene expression.

30.5.3 Antiviral effect of antimicrobial peptides Researchers have reported antiviral activities for several bioactive peptides. Among these, enterocins AAR-71 and AAR-74 were tested against the dsDNA bacterial virus, coliphage HAS, at plaque-forming units (PFU) of 4.2 3 103 per mL using plaque assay. Enterocin AAR-71 completely prevented plaque formation, whereas enterocin AAR-74 decreased plaque formation by 10-fold (Zhou, Bao, Zhang, & Wang, 2015). In another report, enterocin CRL35 showed antiviral activity on herpes simplex virus (HSV) type 1 and 2 in Vero and BHK-21 cell lines. In both cell lines, enterocin CRL35 inhibited, in a dose-dependent manner, the intracellular viral multiplication (Wachsman et al., 1999). A bacteriocin produced by Lactobacillus delbrueckii showed antiviral effect against influenza virus A strains H7N7 and H7N1 in chicken embryo fibroblasts (CFE) cell line. The crude extract containing the AMP reduced the infectious virus yield, hemagglutinin production, virus-induced cytopathic effect, and expression of viral neuraminidase, hemagglutinin, and nucleoprotein on the surface of infected cells (Serkedjieva, Danova, & Ivanova, 2000).

30.5.4 Bioavailability and metabolism There are no adequate scientific data on the bioavailability and health impact of AMPs consumption. Due to their proteinaceous nature, most AMPs are believed to be digested by GIT proteases (Bruno, Miller, & Lim, 2013; Cavera, Arthur, Kashtanov, & Chikindas, 2015). However, posttranslationally modified bacteriocins might be more stable to digestive enzymes and other factors due to cyclization and presence of sulfur bridges

Applications in food technology: antimicrobial peptides 757 (Hols, Ledesma-Garcı´a, Gabant, & Mignolet, 2019). The survival and stability of AMPs in the GIT are also affected by many factors in the surrounding environment such as digested food matrix, pH, and interactions with gut microbiota. Furthermore, molecular size, physiochemical characteristics, charge, and hydrophobicity of AMPs affect their interactions in the GIT (Spadoni, Zagato, & Bertocchi, 2015). The gut
blood barrier (GBB) in the intestine consists of multiple layers (mucous layer, epithelial layer, basement membrane, and vascular endothelium) that selectively permeate the GIT content (Farhadi, Banan, Fields, & Keshavarzian, 2003). Besides its role in regulating the absorption of nutrients, electrolytes, and water from the gut lumen, the GBB works as a defensive barrier against pathogens and harmful substances (Kelly et al., 2015). Hydrophobic and membrane-active peptides (e.g., AMPs) favor nonspecific binding to macromolecules, which decrease their absorption from the GIT (Knoetze, Todorov, & Dicks, 2008). However, studies on microbiome
host interaction showed that intestinal epithelial cells interact with microbial products including AMPs (Iacob & Iacob, 2019). Limited studies addressed AMPs migration across the human or animal cells. Therefore more in vivo studies are required for better understanding of the destiny of AMPs after dietary intake. Any orally applied compound must survive the harsh GIT environment and cross the selective GBB in the GIT before reaching blood. After crossing the GBB, stability in the proteolytic blood plasma can also affect the compound’s bioavailability and bioactivity (Dicks et al., 2018). Among the limited reports on the migration of these compounds across intestinal epithelial and vascular endothelial cells, the migration of fluorescently labeled nisin, plantaricin 423, and bacST4SA across colonic adenocarcinoma (Caco-2) cells and human umbilical vein endothelial cells (HUVECs) was studied in vitro (Dreyer, Smith, Deane, Dicks, & van Staden, 2019). After 3 hours of treatment, nisin, plantaricin 423, and bacST4SA migrated across Caco-2 monolayer at 75%, 85%, and 82%, respectively, and across the HUVEC monolayer at 88%, 93%, and 91%, respectively. This finding represents the first indirect evidence of bacteriocin capability to cross the GBB. The authors suggested that the bacteriocins diffused through the GBB using a paracellular pathway without resulting in pores in the cellular membrane or causing cell death. They also found that the investigated bacteriocins had different stabilities after incubation with human plasma for 3 days, supporting the notion that bacteriocins size, hydrophobicity, and chemical structure affect their stability during contact with blood components (Boo¨ttger, Hoffmann, & Knappe, 2017). Viewing the possibility of introducing AMPs as potent antimicrobials to treat intestinal infection, the bioavailability of thuricin CD, a two-peptide bacteriocin effective against C. difficile, was assessed (Rea et al., 2014). The two peptides, Trn-α and Trn-β showed varied stability to proteolytic gastric enzymes, both in vitro and in vivo. Trn-α was stable to digestion by α-chymotrypsin and pepsin and thus was detected in pig intestinal digesta after its oral administration. However, Trn-β was digested by these enzymes. The researchers also found that the rectal administration of thuricin CD to mice resulted in

758 Chapter 30 .1.5 log reduction of C. difficile 027 in the colon content after 1 hour and an additional 1.5 log reduction after 6 hours.

30.6 Mechanisms of action The mechanism of AMPs’ action is largely diverse and is based on their structure, physicochemical characteristics, and presence of posttranslational modifications (Cotter, Ross, & Hill, 2013). The principal traits that contribute to their mechanism of action are the cationic charge and the hydrophobic/amphiphilic nature; the latter depends on the ratio of the hydrophobic residues in the AMP amino acid sequence.

30.6.1 Mechanisms of action against bacteria and fungi The wall teichoic acids and membrane phospholipids of Gram-positive bacteria and the lipopolysaccharide of the outer membrane of Gram-negative bacteria are all negatively charged. These anionic components are targets for the cationic AMPs through an electrostatic interaction that initiates the antimicrobial activity of AMPs against sensitive bacteria (He´chard & Sahl, 2002). The insertion of AMPs in the bacterial cellular membrane leads to the formation of ion-permeable channels resulting in passive efflux and leakage of the intracellular magnesium, potassium, ATP, amino acids, and other vital intracellular components; this leakage in turn leads to cell death (Todorov, 2009). The activity of nisin, a class I bacteriocin, comes from its electrostatic interaction with the anionic cell wall components and its binding to lipid II, forming nisin-lipid II complex. Subsequently, nisin prevents the incorporation of peptidoglycan monomer in the cell wall synthesis and forms pores in the cytoplasmic membrane, resulting in subsequent depolarization, cell components efflux, and cell death (Wiedemann et al., 2001). The majority of class II bacteriocins disrupt the proton motive force of the sensitive target cell through pore formation (He´chard & Sahl, 2002). Although the main mechanism of antimicrobial action is by disrupting the cell membranes of sensitive bacteria, some AMPs interfere with vital cellular pathways such as protein and DNA synthesis, as well as cell wall synthesis. The antimicrobial activity of Ruminococcin C, isolated from Ruminococcus gnavus, was found to result from interrupting the nucleic acids biosynthesis in target bacteria (Chiumento, Roblin, & Kieffer-Jaquinod, 2019). The antimicrobial activity of microcin produced by Gramnegative bacteria occurs through inhibiting Asp-tRNA synthetase, DNA gyrase, and RNA polymerase (Metlitskaya et al., 2006). The antimicrobial mechanism may also occur through the specific cleavage of 16S rRNA, or through the disruption and degradation of DNA (Heu et al., 2001; Wessels et al., 2004). Antifungal peptides may affect fungi membrane permeabilization. For example, treatment with the antifungal bacteriocin

Applications in food technology: antimicrobial peptides 759 durancins from Enterococcus durans A5
11, resulted in the loss of cellular membrane integrity but also led to other structural changes such as nucleus contraction and vacuole fragmentation in yeast cells (Belguesmia et al., 2013).

30.6.2 Mechanisms of action against viruses Compared to their antibacterial action, little is known about AMPs antiviral activity. In contrast to the bacterial cellular membrane, the formation of ion channels in the viral envelope has little effect on the virus infectivity because the envelope function is not related to maintaining osmotic pressure. Thus antiviral peptides may exhibit different membrane-disruptive activities (Egal et al., 1999). The hydrophobicity of antiviral peptides has been proposed to favor the binding with lipid membranes thus interrupting the cellular-viral fusion (Badani, Garry, & Wimley, 2014; Soltani et al., 2020). Other studies have shown that the antiviral activity could be due to the interference with viral multiplication. The antiviral effect of the amphipathic peptides, melittin and cecropin, was studied on human immunodeficiency virus 1 (HIV-1) and it was reported that the exhibited activity occurred through inhibiting the cell-associated production by suppressing HIV-1 gene expression (Wachinger et al., 1998). Polylysine and polyarginine were reported to interact competitively with BHK cellular receptors specific for HSV-1, blocking the viral binding to the receptors (Langeland, Moore, Holmsen, & Haarr, 1988). Enterocin CRL35, produced by Enterococcus faecium CRL35, was found to inhibit HSV-1 and HSV-2 in Vero cells by preventing late glycoprotein synthesis, thus preventing viral multiplication (Wachsman et al., 2003).

30.7 Safety considerations and regulations It is critical to screen candidate AMPs for safety to eukaryotic cells and model animals before proposing their use in food. These tests are often required for regulatory approval. The structure of AMPs may offer clues on their potential cytotoxic effect, but rigorous testing is needed to provide the evidence for their safety.

30.7.1 Safety of antimicrobial peptides Safety evaluation studies showed that some AMPs are not toxic in vitro, particularly when applied at the minimum inhibitory concentration levels. Among these, nisin A, bacST4SA, and plantaricin 423, at concentrations up to 50 μM, were reported not to result in significant decreases in the viability of Caco-2 cell and HUVEC cell lines using neutral red and XTT assays (Dreyer et al., 2019). Using the LDH release assay, nisin A, plantaricin 423, and bacST4SA, at concentrations up to 100 μM, were found to have minimal cytotoxic effect on Caco-2 and HUVECs. Among the AMPs investigated, nisin showed the highest

760 Chapter 30 cytotoxicity, which could be associated with its higher hydrophobicity. However, the reported cytotoxic concentration, 100 μM, was much higher than that required to inhibit sensitive bacterial strains. In another report, nisin A, at concentration of 80 μM, did not result in any significant cytotoxicity against Caco-2 cell lines (Maher & McClean, 2006). In vivo studies showed that lantibiotics were used successfully to treat skin infections without showing any toxicity in mice (Heunis, Smith, & Dicks, 2013; Van Staden, Heunis, Smith, Deane, & Dicks, 2016). In another report, nisin and pediocin showed different cytotoxic effect levels on colonic cells and kidney cells (Murinda, Rashid, & Roberts, 2003). Considering the variability in AMPs’ structure and mode of action, more in vivo studies are required to evaluate potential toxic effect of promising AMPs at the maximum concentrations to be used either as food preservatives or therapeutic agents.

30.7.2 Regulatory aspects of using AMPs or AMP producers in food There are various criteria that regulatory agencies consider for approving the use of AMP-producing microorganisms or AMPs in foods. Beneficial microorganisms such as yeast used for making bread or LAB used in making fermented dairy, meat, or vegetable products have been well known for safe use and consumption for decades. Therefore many LAB strains have the GRAS status when used as important components in the fermented food product. However, safety documentation must be prepared if these bacteria are intended for a different usage (Wessels et al., 2004). Approving AMP producers that have been genetically engineered is managed by applying the already-in-use regulations on a case-specific basis (Harlander, 1993). Preservatives are a class of food additives used to protect food against pathogenic and spoilage bacteria. The regulations that allow application of AMPs as food preservatives depend on several criteria. Among these, the application mode (AMP-producer culture vs AMP preparations), the targeted food category, and the country’s laws that regulate packaging, labeling, and export. Nisin is an AMP approved to be used as a food preservative (Adams & Smid, 2003) but the levels of nisin permitted in different food products are regulated by different national legislations. In 1969, nisin was first evaluated as a safe ingredient by the FAO and the WHO; this evaluation was followed by adding nisin to the European food additive list in 1983. Fermented products are regulated by general food laws and the resulting fermentates, in the form of concentrates or lyophilized powders, may be used as food ingredients. AMP-containing preparation can be applied as processing aids for preserving food. Processing aids, according to Regulation 1333/2008/ EC, could be any substance that is (1) added during food processing for a certain technological function, (2) not considered as food by itself, and (3) may be present as residues in the final food product as long as it does not cause any adverse health effects (Wessels et al., 2004).

Applications in food technology: antimicrobial peptides 761 Bacteriocin producer strains could be applied in food as bioprotective or starter cultures to ensure food microbial safety and extend its shelf life. The use of these bioprotective cultures is subject to the regulations of the country where it is intended to be used. For the United States a new strain intended for application in food may be considered as an additive, which requires premarket FDA approval, or as a substance with GRAS status (Wessels et al., 2004). The premarket FDA approval of additives requires efficacy and toxicological data, while GRAS status is evaluated by qualified experts and approved by the FDA. Toxicological evaluation for a new food additive involves assigning the compound to a concern level (low, intermediate, or high) based on the compound’s toxicological potential as predicted from its chemical structure and the estimation of cumulative human exposure. Genetic toxicity tests and short-term toxicity tests with rodents are recommended for compounds with low concern level. Additional studies on subchronic toxicity, reproduction, developmental toxicity, metabolism, and pharmacokinetic are recommended for compounds with intermediate and high concern levels. In addition, 1-year toxicity studies with nonrodents, chronic toxicity, and carcinogenicity studies with rodents, and human studies are needed for high concern level compounds (US FDA, 2006). GRAS status can be considered if the substance is “generally recognized, among experts qualified by scientific training and experience to evaluate its safety, as having been adequately shown through scientific procedures (or, in the case as a substance used in food prior to January 1, 1958, through either scientific procedures or experience based on common use in food) to be safe under the conditions of its intended use” (US FDA, 2018). Thus the intended use is an important factor to be considered for GRAS status. For example, the GRAS status for a specific strain intended to be used in a yogurt is not valid for this strain in infant formula (Wessels et al., 2004).

30.8 Limitations Search for new AMPs to serve as natural food preservatives or as clinical therapeutic agents has been promoted by advances in genome mining and further characterization (Abdelhamid, Hussein, Gerst, & Yousef, 2019; Garcı´a-Cano, Hussein, Rocha-Mendoza, Yousef, & Jime´nez-Flores, 2020; Hussein, Huang, Ozturk, & Yousef, 2019; Hussein, Xiaoli, & Yousef, 2019). However, there are several limitations that face the wide application and commercialization of AMPs for various industrial applications. The main challenges are the long-term stability, narrow spectrum, cost of large-scale production and purification, and lack of adequate studies on safety, bioavailability, and adverse effects on beneficial bacteria either in form of food starter cultures or gut microbiome. Several strategies have been developed to overcome these limitations such as using advanced biotechnological approaches for AMP production, utilizing different delivery systems, and coupling with other preservation techniques. Some of these limitations and the corresponding strategies are discussed here.

762 Chapter 30 Different delivery systems have been developed to improve AMP stability and efficiency in various environments such as a food matrix or the body. Although nisin is active and stable at acidic and neutral pH, alkaline pH decreases its solubility and makes it ineffective (Shin et al., 2016). The effectiveness of antimicrobials added directly into food can be diminished due to diffusion and interaction with the food matrix. Incorporation of AMPs into edible coatings and films is another way to control pathogenic microorganisms in food products (Valde´s, Ramos, Beltra´n, Jime´nez, & Garrigo´s, 2017). Through interacting with the food product surface, the AMPs contained in the packaging can improve product safety and extend its shelf life. Currently, there are several AMPs that are approved for application commercially as food preservatives (Table 30.1); it is hopeful that the usage of other AMPs will be approved in the future. To overcome the shortcoming of AMP usability in food due to proteolytic enzymes activity, interaction with food matrix components, and uneven incorporation in the food matrix, researchers proposed encapsulating, adding to the food surface, or incorporating AMPs in the food packaging materials. Using liposomes for nisin Z encapsulation was reported to improve the stability and antimicrobial activity of the bacteriocin in cheddar cheese (Benech, Kheadr, Lacroix, & Fliss, 2002). Applying plantaricin BM1 to ham surface has been found to be an efficient approach to inhibit L. monocytogenes compared to mixing with the cooked ham followed by homogenization. Uneven distribution in the food matrix, slow diffusion, and adsorption of AMPs to the food components have been proposed to be the reasons for the inefficiency of AMPs mixed in the meat of the cooked ham (Zhou et al., 2015). Gram-negative bacteria generally exhibit less sensitivity to cationic AMPs produced by Gram-positive bacteria due to the presence of an outer membrane that constitutes a barrier against these AMPs. Peptides that are most active against Gram-positive bacteria may show efficacy against Gram-negative bacteria if combined with other treatments. Combining nisin with chelating agents such as EDTA sensitized the Gram-negative bacterium, Pseudomonas fluorescens, to nisin (Delves-Broughton, 1993). Subjecting bacteriocin resistant E. coli O157:H7, Salmonella enterica serovar Typhimurium, and Yersinia enterocolitica to sublethal stresses such as heating or freezing and thawing followed by treatment with pediocin AcH and nisin was found to decrease the survival of these bacteria (Kalchayanand et al., 1992). In a further study, the combination of nisin and pediocin AcH with ultra-high hydrostatic pressure (UHP) and electroporation (EP) techniques was reported to have higher antibacterial effect than using UHP or EP alone (Kalchayanand, Sikes, Dunne, & Ray, 1994). Despite many limitations, a number of AMPs were proven effective in vivo for treating various bacterial infections (Yang et al., 2014). To overcome the limited stability and activity of many promising AMPs, researchers attempted to improve their physicochemical characteristics and enhance their biological activity through bioengineering. A number of bioengineered AMPs showed better pharmacological characteristics and increased

Applications in food technology: antimicrobial peptides 763 production yield than their native counterparts (Field, Cotter, Hill, & Ross, 2015). Changing as little as one amino acid residue in different sites of the nisin peptide sequence has improved the product activity against clinically relevant pathogens such as MRSA, VRE, and C. difficile and showed a wider spectrum against Gram-negative bacteria. Site-saturation mutagenesis of lysine 12 was used to generate nisin A derivative producers; among these, nisin A K12A producer showed enhanced antimicrobial activity against selected Bacillus, Enterococcus, Lactococcus, Staphylococcus, and Streptococcus strains (Molloy et al., 2013). Site-saturation mutagenesis for altering residue 29, serine, of nisin A was also used to create nisin S29 derivatives that exhibited a superior antimicrobial activity to nisin A against Gram-negative pathogens, such as E. coli, Cronobacter sakazakii, and S. enterica serovar Typhimurium (Field et al., 2012). Two nisin Z derivatives with lysine residues replacing Asn-27 and His-31 showed increased solubilities at pH 7 by factors of 4 and 7, respectively, compared to unmodified nisin Z. Another nisin Z derivative with a dehydrobutyrine (Dhb) at position 5 replacing dehydroalanine (Dha) showed a lower activity but significantly higher resistance to acid-catalyzed chemical degradation (Rollema, Kuipers, Both, De Vos, & Siezen, 1995). In addition, bioengineering approaches could be used to improve the stability of AMPs to proteolytic digestion by altering the amino acids that serve as digestive enzyme recognition sites in these peptides (Field, Cotter, et al., 2015). For cost-effective production of AMPs, many approaches have been developed to facilitate industrial-scale production, such as fermentation optimization, utilization of low-cost production media, heterologous gene expression, and bioengineering. Advances in bioengineering technologies can be successfully implemented not only for developing novel AMP analogs with improved biological and physicochemical characteristics but also for synthesizing AMPs for a cost-effective production (Ongey & Neubauer, 2016).

30.9 Summary Increased investigations into applying AMPs in food is a desirable trend. AMPs are generally viewed as viable and natural alternatives to the synthetic or potentially hazardous chemicals that are currently used. There are many AMPs that can be used effectively in controlling spoilage and pathogenic organisms in food. Some AMPs have been successfully used in food preservation. AMPs can be used as purified or semipurified compounds, in the form of food additives, or simply allowed to be produced in food (or food ingredients) by adding the antimicrobial-producing bacteria. The ability to produce AMPs is a desired trait in bacteria selected for use as commercial probiotics. It is advantageous to use AMPs in combination with other preservation factors in a multihurdle approach. When compared to other antimicrobial agents, AMPs have many advantages. They are active at micromolar levels against target microorganisms. These peptides act against target microorganisms by

764 Chapter 30 compromising the permeability of cytoplasmic membranes, inhibiting the synthesis of cell wall, or reacting with important components imbedded in the membranes. These mechanisms of action make target microorganisms less capable of developing resistance, when comparing AMPs with antibiotics. There are many factors that limit the application of AMPs in food. These include AMPs narrow antimicrobial spectrum, high production costs, and regulatory hurdles that make commercial approval a tedious, lengthy, and costly task.

References Abdelhamid, A. G., Hussein, W. E., Gerst, M. M., & Yousef, A. E. (2019). Draft genome sequence of Bacillus velezensis OSY-GA1, which encodes multiple antimicrobial metabolites and expresses antimicrobial activity against foodborne pathogens. Microbiology Resource Announcements, 8(7). Available from https:// doi.org/10.1128/MRA.01725-18, e01725-18. Acedo, J. Z., Chiorean, S., Vederas, J. C., & van Belkum, M. J. (2018). The expanding structural variety among bacteriocins from Gram-positive bacteria. FEMS Microbiology Reviews, 42, 805
828. Adams, M., & Smid, E. (2003). Nisin in multifactorial food preservation. Natural antimicrobials for the minimal processing of foods (pp. 11
33). Elsevier. Al-Zoreky, N., Ayres, J. W., & Sandine, W. E. (1991). Antimicrobial activity of Microgardt against food spoilage and pathogenic microorganisms. Journal of Dairy Science, 74, 758
763. Ananou, S., Garriga, M., Hugas, M., Maqueda, M., Martı´nez-Bueno, M., Ga´lvez, A., & Valdivia, E. (2005). Control of Listeria monocytogenes in model sausages by enterocin AS-48. International Journal of Food Microbiology, 103, 170
190. Badani, H., Garry, R. F., & Wimley, W. C. (2014). Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity. Biochimica et Biophysica Acta—Biomembranes, 1838, 2180
2197. Bali, V., Panesar, P. S., Bera, M. B., & Kennedy, J. F. (2016). Bacteriocins: Recent trends and potential applications. Critical Reviews in Food Science and Nutrition, 56, 817
834. Belguesmia, Y., Choiset, Y., Rabesona, H., Baudy-Floc’h, M., Le Blay, G., Haertle´, T., & Chobert, J. M. (2013). Antifungal properties of durancins isolated from Enterococcus durans A5
11 and of its synthetic fragments. Letters in Applied Microbiology, 56, 237
244. Benech, R. O., Kheadr, E. E., Lacroix, C., & Fliss, I. (2002). Antibacterial activities of nisin Z encapsulated in liposomes or produced in situ by mixed culture during Cheddar cheese ripening. Applied and Environmental Microbiology, 68, 5607
5619. Boo¨ttger, R., Hoffmann, R., & Knappe, D. (2017). Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum. PLoS One, 12, e0178943. Available from https://doi. org/10.1371/journal.pone.017. Bruno, B. J., Miller, G. D., & Lim, C. S. (2013). Basics and recent advances in peptide and protein drug delivery. Therapeutic Delivery, 4, 1443
1467. Cascales, E., Buchanan, S. K., Duche´, D., Kleanthous, C., Lloube`s, R., Postle, K., . . . Cavard, D. (2007). Colicin biology. Microbiology and Molecular Biology Reviews, 71, 158
229. Cavera, V. L., Arthur, T. D., Kashtanov, D., & Chikindas, M. L. (2015). Bacteriocins and their position in the next wave of conventional antibiotics. International Journal of Antimicrobial Agents, 46, 494
501. Chatterjee, C., Paul, M., Xie, L., & van der Donk, W. A. (2005). Biosynthesis and mode of action of lantibiotics. Chemical Reviews, 105, 633
683. Chen, H., & Hoover, D. G. (2003). Bacteriocins and their food applications. Comprehensive Reviews in Food Science and Food Safety, 2, 82
100. Chiumento, S., Roblin, C., Kieffer-Jaquinod, S., et al. (2019). Ruminococcin C, a promising antibiotic produced by a human gut symbiont. Science Advances, 5, 9969
9994.

Applications in food technology: antimicrobial peptides 765 Cintas, L. M., Casaus, P., Ferna´ndez, M. F., & Herna´ndez, P. E. (1998). Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria. Food Microbiology, 15, 289
298. Collins, F. W. J., O’Connor, P. M., O’Sullivan, O., Go´mez-Sala, B., Rea, M. C., Hill, C., & Ross, R. P. (2017). Bacteriocin gene-trait matching across the complete Lactobacillus Pan-genome. Scientific Reports, 7, article number: 3481. Available from https://doi.org/10.1038/s415. Corr, S. C., Li, Y., Riedel, C. U., O’Toole, P. W., Hill, C., & Gahan, C. G. M. (2007). Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118. Proceedings of the National Academy of Sciences of the United States of America, 104, 7617
7621. Cotter, P. D., Hill, C., & Ross, R. P. (2005). Food microbiology: Bacteriocins: Developing innate immunity for food. Nature Reviews. Microbiology, 3, 777
788. Cotter, P. D., Ross, R. P., & Hill, C. (2013). Bacteriocins—A viable alternative to antibiotics? Nature Reviews. Microbiology, 11, 95
105. Dal Bello, F., Clarke, C. I., Ryan, L. A. M., Ulmer, H., Schober, T. J., Stro¨m, K., . . . Arendt, E. K. (2007). Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7. Journal of Cereal Science, 45, 309
318. de Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Vessoni Penna, T. C. (2009). Nisin biotechnological production and application: A review. Trends in Food Science and Technology, 20, 146
154. Delves-Broughton, J. (1993). The use of EDTA to enhance the efficacy of nisin towards Gram-negative bacteria. International Biodeterioration and Biodegradation, 32, 87
97. Dicks, L. M. T., Dreyer, L., Smith, C., & van Staden, A. D. (2018). A review: The fate of bacteriocins in the human gastro-intestinal tract: Do they cross the gut
blood barrier? Frontiers in Microbiology, 9, 2297. Available from https://doi.org/10.3389/fmicb.2018.02297. Diez-Gonzalez, F. (2007). Applications of bacteriocins in livestock. Current Issues in Intestinal Microbiology, 8, 15
24. Dreyer, L., Smith, C., Deane, S. M., Dicks, L. M. T., & van Staden, A. D. (2019). Migration of bacteriocins across gastrointestinal epithelial and vascular endothelial cells, as determined using in vitro simulations. Scientific Reports, 9, 11481. Available from https://doi.org/10.1038/s41598-019-47843-9. Dupont Nutrition and Biosciences. (2021). MicroGARD® fermentates. , https://www. dupontnutritionandbiosciences.com/products/microgard.html . . (Accessed 23 March 2021). Duquesne, S., Destoumieux-Garzo´n, D., Peduzzi, J., & Rebuffat, S. (2007). Microcins, gene-encoded antibacterial peptides from enterobacteria. Natural Product Reports, 24, 708
734. Egal, M., Conrad, M., MacDonald, D. L., Maloy, W. L., Motley, M., & Genco, C. A. (1999). Antiviral effects of synthetic membrane-active peptides on herpes simplex virus, type 1. International Journal of Antimicrobial Agents, 13, 57
60. Egan, K., Field, D., Rea, M. C., Ross, R. P., Hill, C., & Cotter, P. D. (2016). Bacteriocins: Novel solutions to age old spore-related problems? Frontiers in Microbiology, 7, 461. Available from https://doi.org/10.3389/ fmicb.2016.00461. Eijsink, V. G. H., Skeie, M., Middelhoven, P. H., Brurberg, M. B., & Nes, I. F. (1998). Comparative studies of class IIa bacteriocins of lactic acid bacteria. Applied and Environmental Microbiology, 64, 3275
3281. Falguni, P., Shilpa, V., & Mann, B. (2010). Production of proteinaceous antifungal substances from Lactobacillus brevis NCDC 02. International Journal of Dairy Technology, 63, 70
76. Farhadi, A., Banan, A., Fields, J., & Keshavarzian, A. (2003). Intestinal barrier: An interface between health and disease. Journal of Gastroenterology and Hepatology (Australia), 18, 479
497. Fauci, A. S., & Morens, D. M. (2012). The perpetual challenge of infectious diseases. New England Journal of Medicine, 366, 454
461. Field, D., Begley, M., O’Connor, P. M., Daly, K. M., Hugenholtz, F., Cotter, P. D., . . . Ross, R. P. (2012). Bioengineered nisin A derivatives with enhanced activity against both Gram positive and Gram negative pathogens. PLoS One. Available from https://doi.org/10.1371/journal.pone.0046884.

766 Chapter 30 Field, D., Cotter, P. D., Hill, C., & Ross, R. P. (2015). Bioengineering lantibiotics for therapeutic success. Frontiers in Microbiology, 6, 1363. Available from https://doi.org/10.3389/fmicb.2015.01363. Field, D., Daly, K., O’Connor, P. M., Cotter, P. D., Hill, C., & Paul Ross, R. (2015). Efficacies of nisin A and nisin V semipurified preparations alone and in combination with plant essential oils for controlling Listeria monocytogenes. Applied and Environmental Microbiology, 81, 2762
2769. Garcı´a-Cano, I., Hussein, W. E., Rocha-Mendoza, D., Yousef, A. E., & Jime´nez-Flores, R. (2020). Draft genome sequence of Lactobacillus rhamnosus OSU-PECh-69, a cheese isolate with antibacterial activity. Microbiology Resource Announcements, 9(37), e00803
e00820. Available from https://doi.org/10.1128/ MRA.00803-20. Gerez, C. L., Torres, M. J., Font de Valdez, G., & Rolla´n, G. (2013). Control of spoilage fungi by lactic acid bacteria. Biological Control, 64, 231
237. Gerst, M. M., Huang, E., Zhang, L., & Yousef, A. E. (2015). Development of a new paenibacillin-producing strain and testing its usability in improving food safety. Journal of Food Science, 80, M1538
M1543. Gonza´lez-Toledo, S. Y., Domı´nguez-Domı´nguez, J., Garcı´a-Almenda´rez, B. E., Prado-Barraga´n, L. A., & Regalado-Gonza´lez, C. (2010). Optimization of nisin production by Lactococcus lactis UQ2 using supplemented whey as alternative culture medium. Journal of Food Science, 75, M347
M353. Granger, M., van Reenen, C. A., & Dicks, L. M. T. (2008). Effect of gastro-intestinal conditions on the growth of Enterococcus mundtii ST4SA, and production of bacteriocin ST4SA recorded by real-time PCR. International Journal of Food Microbiology, 123, 277
280. Guo, Y., Huang, E., Yuan, C., Zhang, L., & Yousef, A. E. (2012). Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic. Applied and Environmental Microbiology, 78, 3156
3165. Gupta, R., & Srivastava, S. (2014). Antifungal effect of antimicrobial peptides (AMPs LR14) derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage. Food Microbiology, 42, 1
7. Harlander, S. K. (1993). Regulatory aspects of bacteriocin use. Bacteriocins of lactic acid bacteria (pp. 233
247). Elsevier. He, Z., Kisla, D., Zhang, L., Yuan, C., Green-Church, K. B., & Yousef, A. E. (2007). Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Applied and Environmental Microbiology, 73, 168
178. He, Z., Yuan, C., Zhang, L., & Yousef, A. E. (2008). N-terminal acetylation in paenibacillin, a novel lantibiotic. FEBS Letters, 582, 2787
2792. He´chard, Y., & Sahl, H. G. (2002). Mode of action of modified and unmodified bacteriocins from Grampositive bacteria. Biochimie, 84, 545
557. Heu, S., Oh, J., Kang, Y., Ryu, S., Cho, S. K., Cho, Y., & Cho, M. (2001). gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra. Applied and Environmental Microbiology, 67, 4105
4110. Heunis, T. D. J., Smith, C., & Dicks, L. M. T. (2013). Evaluation of a nisin-eluting nanofiber scaffold to treat Staphylococcus aureus-induced skin infections in mice. Antimicrobial Agents and Chemotherapy, 57, 3928
3935. Hols, P., Ledesma-Garcı´a, L., Gabant, P., & Mignolet, J. (2019). Mobilization of microbiota commensals and their bacteriocins for therapeutics. Trends in Microbiology, 27, 690
702. Huang, E., Yang, X., Zhang, L., Moon, S. H., & Yousef, A. E. (2017). New Paenibacillus strain produces a family of linear and cyclic antimicrobial lipopeptides: Cyclization is not essential for their antimicrobial activity. FEMS Microbiology Letters, 364. Available from https://doi.org/10.1093/femsle/fnx049. Hugo, C. J., & Hugo, A. (2015). Current trends in natural preservatives for fresh sausage products. Trends in Food Science and Technology, 45, 12
23. Hussein, W. E., Abdelhamid, A. G., Rocha-Mendoza, D., Garcı´a-Cano, I., & Yousef, A. E. (2020). Assessment of safety and probiotic traits of Enterococcus durans OSY-EGY, isolated from Egyptian artisanal cheese, using comparative genomics and phenotypic analyses. Frontiers in Microbiology, 11, 608314. Available from https://doi.org/10.3389/fmicb.2020.608314.

Applications in food technology: antimicrobial peptides 767 ´ ., Yang, X., Liu, B., & Yousef, A. E. (2020). Genome-guided Hussein, W. E., Huang, E., Ozturk, I., Somogyi, A mass spectrometry expedited the discovery of paraplantaricin TC318, a lantibiotic produced by Lactobacillus paraplantarum strain isolated from cheese. Frontiers in Microbiology, 11, 1381. Available from https://doi.org/10.3389/fmicb.2020.01381. Hussein, W. E., Huang, E., Ozturk, I., & Yousef, A. E. (2019). Draft genome sequence of Lactobacillus paraplantarum OSY-TC318, a producer of the novel lantibiotic paraplantaracin TC318. Microbiology Resource Announcements, 8(19). Available from https://doi.org/10.1128/MRA.00274-19, e00274-19. Hussein, W. E., Xiaoli, L., & Yousef, A. E. (2019). Draft genome sequence of Enterococcus durans OSY-EGY, a multiple-antimicrobial-peptide producer isolated from Egyptian hard cheese. Microbiology Resource Announcements, 8(28), e00303
e00319. Available from https://doi.org/10.1128/MRA.00303-19. Iacob, S., & Iacob, D. G. (2019). Infectious threats, the intestinal barrier, and its trojan horse: Dysbiosis. Frontiers in Microbiology, 10, 1676. Available from https://doi.org/10.3389/fmicb.2019.01676. Johnson, I. H., Hayday, H., & Colman, G. (1978). The effects of nisin on the microbial flora of the dental plaque of monkeys (Macaca fascicularis). Journal of Applied Bacteriology, 45, 99
109. Jones, E., Salin, V., & Williams, G. (2005). NISIN and the market for commercial bacteriocins. https:// ageconsearch.umn.edu/record/90779. Kalchayanand, N., Hanlin, M. B., & Ray, B. (1992). Sublethal injury makes Gram-negative and resistant Grampositive bacteria sensitive to the bacteriocins, pediocin AcH and nisin. Letters in Applied Microbiology, 15, 239
243. Kalchayanand, N., Sikes, T., Dunne, C. P., & Ray, B. (1994). Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Applied and Environmental Microbiology, 60, 4174
4177. Kelly, J. R., Kennedy, P. J., Cryan, J. F., Dinan, T. G., Clarke, G., & Hyland, N. P. (2015). Breaking down the barriers: The gut microbiome, intestinal permeability and stress-related psychiatric disorders. Frontiers in Cellular Neuroscience, 9, 392. Available from https://doi.org/10.3389/fncel.2015.00392. Kim, W. S. (1997). Nisin production by Lactococcus lactis using two-phase batch culture. Letters in Applied Microbiology, 25, 169
171. Knoetze, H., Todorov, S. D., & Dicks, L. M. T. (2008). A class IIa peptide from Enterococcus mundtii inhibits bacteria associated with otitis media. International Journal of Antimicrobial Agents, 31, 228
234. Langeland, N., Moore, L. J., Holmsen, H., & Haarr, L. (1988). Interaction of polylysine with the cellular receptor for herpes simplex virus type 1. Journal of General Virology, 69, 1137
1145. Le Marrec, C., Hyronimus, B., Bressollier, P., Verneuil, B., & Urdaci, M. C. (2000). Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I4. Applied and Environmental Microbiology, 66, 5213
5220. Lee, T. H., Hofferek, V., Separovic, F., Reid, G. E., & Aguilar, M. I. (2019). The role of bacterial lipid diversity and membrane properties in modulating antimicrobial peptide activity and drug resistance. Current Opinion in Chemical Biology, 52, 85
92. Lin, X. B., Lohans, C. T., Duar, R., Zheng, J., Vederas, J. C., Walter, J., & Ga¨nzle, M. (2015). Genetic determinants of reutericyclin biosynthesis in Lactobacillus reuteri. Applied and Environmental Microbiology, 81, 2032
2041. Luz, C., Saladino, F., Luciano, F. B., Man˜es, J., & Meca, G. (2017). In vitro antifungal activity of bioactive peptides produced by Lactobacillus plantarum against Aspergillus parasiticus and Penicillium expansum. LWT—Food Science and Technology, 81, 128
135. Magnusson, J., & Schnu¨rer, J. (2001). Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Applied and Environmental Microbiology, 67, 1
5. Magnusson, J., Stro¨m, K., Roos, S., Sjo¨gren, J., & Schnu¨rer, J. (2003). Broad and complex antifungal activity among environmental isolates of lactic acid bacteria. FEMS Microbiology Letters, 219, 129
135. Maher, S., & McClean, S. (2006). Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro. Biochemical Pharmacology, 71, 1289
1298. Majumdar, S. K., & Bose, S. K. (1958). Mycobacillin, a new antifungal antibiotic produced by B. subtilis. Nature, 181, 134
135.

768 Chapter 30 Marteau, P., & Shanahan, F. (2003). Basic aspects and pharmacology of probiotics: An overview of pharmacokinetics, mechanisms of action and side-effects. Bailliere’s Best Practice and Research in Clinical Gastroenterology, 17, 725
740. Martı´nez Viedma, P., Abriouel, H., Ben Omar, N., Lo´pez, R. L., & Ga´lvez, A. (2011). Inhibition of spoilage and toxigenic Bacillus species in dough from wheat flour by the cyclic peptide enterocin AS-48. Food Control, 22, 756
761. Mathur, H., Rea, M., Cotter, P., Hill, C., & Ross, R. (2015). The sactibiotic subclass of bacteriocins: An update. Current Protein & Peptide Science, 16, 549
558. Mccormick, J. K., Poon, A., Sailer, M., Gao, Y., Roy, K. L., Mcmullen, L. M., . . . Van Belkum, M. J. (1998). Genetic characterization and heterologous expression of brochocin-C, an antibotulinal, two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754. Applied and Environmental Microbiology, 64, 4757
4766. Meade, E., Slattery, M. A., & Garvey, M. (2020). Bacteriocins, potent antimicrobial peptides and the fight against multi drug resistant species: Resistance is futile? Antibiotics, 9, 32. Available from https://doi.org/ 10.3390/antibiotics9010032. Metlitskaya, A., Kazakov, T., Kommer, A., Pavlova, O., Praetorius-Ibba, M., Ibba, M., . . . Severinov, K. (2006). Aspartyl-tRNA synthetase is the target of peptide nucleotide antibiotic microcin C. Journal of Biological Chemistry, 281, 18033
18042. Miao, J., Guo, H., Ou, Y., Liu, G., Fang, X., Liao, Z., . . . Cao, Y. (2014). Purification and characterization of bacteriocin F1, a novel bacteriocin produced by Lactobacillus paracasei subsp. tolerans FX-6 from Tibetan kefir, a traditional fermented milk from Tibet, China. Food Control, 42, 48
53. Miao, J., Peng, W., Liu, G., Chen, Y., Chen, F., & Cao, Y. (2015). Biopreservative effect of the natural antimicrobial substance from Lactobacillus paracasei subsp. tolerans FX-6 on fresh pork during chilled storage. Food Control, 56, 53
56. Moll, G. N., Van Den Akker, E., Hauge, H. H., Nissen-Meyer, J., Nes, I. F., Konings, W. N., & Driessen, A. J. M. (1999). Complementary and overlapping selectivity of the two-peptide bacteriocins plantaricin EF and JK. Journal of Bacteriology, 181, 4848
4852. Molloy, E. M., Field, D., O’Connor, P. M., Cotter, P. D., Hill, C., & Ross, R. P. (2013). Saturation mutagenesis of lysine 12 leads to the identification of derivatives of nisin A with enhanced antimicrobial activity. PLoS One, 8(3), e58530. Available from https://doi.org/10.1371/journal.pone.0058. Muhialdin, B. J., Hassan, Z., Abu Bakar, F., Algboory, H. L., & Saari, N. (2015). Novel antifungal peptides produced by Leuconostoc mesenteroides DU15 effectively inhibit growth of Aspergillus niger. Journal of Food Science, 80, M1026
M1030. Muhialdin, B. J., Hassan, Z., Bakar, F. A., & Saari, N. (2016). Identification of antifungal peptides produced by Lactobacillus plantarum IS10 grown in the MRS broth. Food Control, 59, 27
30. Murinda, S. E., Rashid, K. A., & Roberts, R. F. (2003). In vitro assessment of the cytotoxicity of nisin, pediocin, and selected colicins on simian virus 40-transfected human colon and Vero monkey kidney cells with trypan blue staining viability assays. Journal of Food Protection, 66, 847
853. Nissen-Meyer, J., Oppega˚rd, C., Rogne, P., Haugen, H. S., & Kristiansen, P. E. (2010). Structure and mode-ofaction of the two-peptide (class-IIb) bacteriocins. Probiotics and Antimicrobial Proteins, 2, 52
60. O’Sullivan, L., Ryan, M. P., Ross, R. P., & Hill, C. (2003). Generation of food-grade lactococcal starters which produce the lantibiotics lacticin 3147 and lacticin 481. Applied and Environmental Microbiology, 69, 3681
3685. Ongey, E. L., & Neubauer, P. (2016). Lanthipeptides: Chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production. Microbial Cell Factories, 15, 1
16. Papagianni, M., & Anastasiadou, S. (2009). Pediocins: The bacteriocins of Pediococci. Sources, production, properties and applications. Microbial Cell Factories, 8. Available from https://doi.org/10.1186/1475-28598-3. Porto, M. C. W., Kuniyoshi, T. M., Azevedo, P. O. S., Vitolo, M., & Oliveira, R. P. S. (2017). Pediococcus spp.: An important genus of lactic acid bacteria and pediocin producers. Biotechnology Advances, 35, 361
374.

Applications in food technology: antimicrobial peptides 769 Rea, M. C., Alemayehu, D., Casey, P. G., O’Connor, P. M., Lawlor, P. G., Walsh, M., . . . Hill, C. (2014). Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract. Microbiology (United Kingdom), 160, 439
445. Rollema, H. S., Kuipers, O. P., Both, P., De Vos, W. M., & Siezen, R. J. (1995). Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering. Applied and Environmental Microbiology, 61, 2873
2878. Salih, M. A., Sandine, W. E., & Ayres, J. W. (1990). Inhibitory effects of Microgardt on yogurt and cottage cheese spoilage organisms. Journal of Dairy Science, 73, 887
893. Sa´nchez-Go´mez, S., Lamata, M., Leiva, J., Blondelle, S. E., Jerala, R., Andra¨, J., . . . Martı´nez-De-Tejada, G. (2008). Comparative analysis of selected methods for the assessment of antimicrobial and membranepermeabilizing activity: A case study for lactoferricin derived peptides. BMC Microbiology, 8. Available from https://doi.org/10.1186/1471-2180-8-196, article number: 196. Scholz, R., Vater, J., Budiharjo, A., Wang, Z., He, Y., Dietel, K., . . . Borriss, R. (2014). Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42. Journal of Bacteriology, 196, 1842
1852. Schuijt, T. J., van der Poll, T., de Vos, W. M., & Wiersinga, W. J. (2013). The intestinal microbiota and host immune interactions in the critically ill. Trends in Microbiology, 21, 221
229. Serkedjieva, J., Danova, S., & Ivanova, I. (2000). Antiinfluenza virus activity of a bacteriocin produced by Lactobacillus delbrueckii. Applied Biochemistry and Biotechnology: Part A, Enzyme Engineering and Biotechnology, 285
298. Settanni, L., & Corsetti, A. (2008). Application of bacteriocins in vegetable food biopreservation. International Journal of Food Microbiology, 121, 123
138. Shin, J. M., Gwak, J. W., Kamarajan, P., Fenno, J. C., Rickard, A. H., & Kapila, Y. L. (2016). Biomedical applications of nisin. Journal of Applied Microbiology, 120, 1449
1465. Sobrino-Lo´pez, A., & Martı´n-Belloso, O. (2008). Use of nisin and other bacteriocins for preservation of dairy products. International Dairy Journal, 18, 329
343. Soltani, S., Hammami, R., Cotter, P. D., Rebuffat, S., Said, L. B., Gaudreau, H., . . . Fliss, I. (2020). Bacteriocins as a new generation of antimicrobials: Toxicity aspects and regulations. FEMS Microbiology Reviews, 39. Available from https://doi.org/10.1093/femsre/fuaa039. Spadoni, I., Zagato, E., Bertocchi, A., et al. (2015). A gut-vascular barrier controls the systemic dissemination of bacteria. Science (New York, N.Y.), 350, 830
834. Stro¨m, K., Sjo¨gren, J., Broberg, A., & Schnu¨rer, J. (2002). Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Applied and Environmental Microbiology, 68, 4322
4327. Tai, Y.-C., McGuire, J., & Neff, J. A. (2008). Nisin antimicrobial activity and structural characteristics at hydrophobic surfaces coated with the PEO-PPO-PEO triblock surfactant Pluronic F108. Journal of Colloid and Interface Science, 322, 104
111. Tally, F. P., & DeBruin, M. F. (2000). Development of daptomycin for Gram-positive infections. Journal of Antimicrobial Chemotherapy, 46, 523
526. Todorov, S. D. (2009). Bacteriocins from Lactobacillus plantarum—Production, genetic organization and mode of action. Brazilian Journal of Microbiology, 40, 209
221. US FDA. (2006). Guidance for industry: Summary table of recommended toxicological testing for additives used in food. , https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidanceindustry-summary-table-recommended-toxicological-testing-additives-used-food . . (Accessed 29 November 2020). US FDA. (2018). History of the GRAS list and SCOGS reviews. , https://www.fda.gov/food/gras-substancesscogs-database/history-gras-list-and-scogs-reviews . . (Accessed 23 March 2021). Valde´s, A., Ramos, M., Beltra´n, A., Jime´nez, A., & Garrigo´s, M. (2017). State of the art of antimicrobial edible coatings for food packaging applications. Coatings, 7, 56. Available from https://doi.org/10.3390/ coatings7040056.

770 Chapter 30 Valenzuela, A. S., Benomar, N., Abriouel, H., Can˜amero, M. M., & Ga´lvez, A. (2010). Isolation and identification of Enterococcus faecium from seafoods: Antimicrobial resistance and production of bacteriocin-like substances. Food Microbiology, 27, 955
961. van Kuijk, S., Noll, K. S., & Chikindas, M. L. (2012). The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A. Letters in Applied Microbiology, 54, 52
58. Van Reenen, C. A., Dicks, L. M. T., & Chikindas, M. L. (1998). Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum. Journal of Applied Microbiology, 84, 1131
1137. Van Staden, A. D. P., Heunis, T., Smith, C., Deane, S., & Dicks, L. M. T. (2016). Efficacy of lantibiotic treatment of Staphylococcus aureus-induced skin infections, monitored by in vivo bioluminescent imaging. Antimicrobial Agents and Chemotherapy, 60, 3948
3955. van Zyl, W. F., Deane, S. M., & Dicks, L. M. T. (2016). Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 excludes Listeria monocytogenes from the GIT, as shown by bioluminescent studies in mice. Beneficial Microbes, 7, 227
235. Wachinger, M., Kleinschmidt, A., Winder, D., Von Pechmann, N., Ludvigsen, A., Neumann, M., . . . BrackWerner, R. (1998). Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression. Journal of General Virology, 79, 731
740. Wachsman, M. B., Castilla, V., De Ruiz Holgado, A. P., De Torres, R. A., Sesma, F., & Coto, C. E. (2003). Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro. Antiviral Research, 58, 17
24. ´ . B., Farı´as, M. E., Takeda, E., Sesma, F., De Ruiz Holgado, A. P., De Torres, R. A., & Coto, Wachsman, M. O C. E. (1999). Antiviral activity of enterocin CRL35 against herpesviruses. International Journal of Antimicrobial Agents, 12, 293
299. Weber, G. H., & Broich, W. A. (1986). Shelf life extension of cultured dairy foods. Cultured Dairy Products Journal, 21, 19
23. Wessels, S., Axelsson, L., Bech Hansen, E., De Vuyst, L., Laulund, S., La¨hteenma¨ki, L., . . . Von Wright, A. (2004). The lactic acid bacteria, the food chain, and their regulation. Trends in Food Science and Technology, 15, 498
505. Wiedemann, I., Breukink, E., Van Kraaij, C., Kuipers, O. P., Bierbaum, G., De Kruijff, B., & Sahl, H. G. (2001). Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. Journal of Biological Chemistry, 276, 1772
1779. Yamanaka, K., Maruyama, C., Takagi, H., & Hamano, Y. (2008). ε-Poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase. Nature Chemical Biology, 4, 766
772. Yang, S. C., Lin, C. H., Sung, C. T., & Fang, J. Y. (2014). Antibacterial activities of bacteriocins: Application in foods and pharmaceuticals. Frontiers in Microbiology, 5, 241. Available from https://doi.org/10.3389/ fmicb.2014.00241. Yang, X., Huang, E., Yuan, C., Zhang, L., & Yousef, A. E. (2016). Isolation and structural elucidation of brevibacillin, an antimicrobial lipopeptide from Brevibacillus laterosporus that combats drug-resistant Gram-positive bacteria. Applied and Environmental Microbiology, 82, 2763
2772. Zhao, L., Wang, S., Liu, F., Dong, P., Huang, W., Xiong, L., & Liao, X. (2013). Comparing the effects of high hydrostatic pressure and thermal pasteurization combined with nisin on the quality of cucumber juice drinks. Innovative Food Science and Emerging Technologies, 17, 27
36. Zhou, H., Xie, Y., Liu, H., Jin, J., Duan, H., & Zhang, H. (2015). Effects of two application methods of plantaricin BM-1 on control of Listeria monocytogenes and background spoilage bacteria in sliced vacuumpackaged cooked ham stored at 4 C. Journal of Food Protection, 78, 1835
1841. Zhou, Y., Bao, H., Zhang, H., & Wang, R. (2015). Isolation and characterization of lytic phage vB-EcoM-JS09 against clinically isolated antibiotic-resistant avian pathogenic Escherichia coli and enterotoxigenic Escherichia coli. Intervirology, 58, 218
231.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Absorption enhancers, 6 Absorption of peptides, 555
556 approaches for enhancing, 6
8 in large intestine (colon), 5 in small intestine, 3
9 approaches for enhancing the absorption of peptides, 6
8 paracellular transport, 3
4 structure-activity relationship of bioactive peptides, 8
9 transcellular transport, 4
5 N-Acetylaspartate, 704 Acetylcholinesterase inhibitory activity, assay of, 135
136 alternative methods/procedures, 136 analysis and statistics, 136 definition, 135 materials, equipment, and reagents, 135 precursor and related techniques, 136 protocols, 135
136 Acetylthiocholine iodide (ATCI), 135 Active transport, 5 Adherent cultures, subculturing, 165
166 Adherent vs. suspension culture, 162t Adipotide, 639
640 Advanced glycation end-products (AGEs), 573 AEINMPDT, 15
16 AEKTK, 13
15 AFKAWAVAR, 646
647

AGFAGDDAPR, 648
649 Aging, generation of peptides during, 408 AHTPDB, 317
319 Albigutide, 638
639 Alcalase-digested canola protein hydrolysate, 598 Alcalase hydrolysates, 376 Alcohol, 158 Aldehydes, 158 Aliskiren, 581
582 Alkaline phosphatase (ALP), 667
669 α-amylase, 608 α-amylase inhibitory activity, assay of, 121
124 alternative methods/procedures, 123
124 analysis and statistics, 123 definition, 122 materials, equipment, and reagents, 122
123 precursor and related techniques, 123 protocols, 123 α-glucosidase, 608 α-glucosidase inhibitory activity, assay of, 124
126 alternative methods/procedures, 126 analysis and statistics, 125 definition, 124 materials, equipment, and reagents, 124
125 precursor and related techniques, 125 protocols, 125

771

ALPMH, 558 Alzheimer’s disease (AD), 135, 709 Amaranth, 557 American College of Gastroenterology (ACG), 496 7-Amino-4-methylcoumarin (AMC), 38
39, 119
120 Amino acids (AAs), 163, 275 6-Amino quinolyl-Nhydroxysuccinimidyl carbamate (AccQ), 221
223, 228 AMPGLSRLFTALK, 12
13 Angiogenesis, 59 Angiotensin-I-converting enzyme (ACE) inhibition, 54, 114
117, 115f, 434
435, 497
498, 500, 562 alternative methods/procedures, 117 analysis and statistics, 116 definition, 115 materials, equipment, and reagents, 115 protocol, 116 and renin-inhibitory peptides, 584
598 animal protein-derived hydrolysates and peptides, 584
592 plant protein-derived hydrolysates and peptides, 592
598 Animal models, 191
193 analysis and statistics, 209
210 multivariate analysis of animal studies, 209
210

772 Index Animal models (Continued) normal and nonnormal distributed data, handling of, 209 sample size, 209 to evaluate hypertension, 199
204 classical animal models, 200
203 newfangled animal models, 203
204 food peptides and animal safety, administration of, 193
199 development of oral and injectable peptides derived from food, 199 gender and age, distribution of, 195
199 meal feeding information, 194
195 safety and toxicological evaluation of peptides, 193
194 metabolic dysfunction, animal models to evaluate, 204
209 knockout mice models, 208
209 safety considerations and standards during the development of, 210
211 bioethics considerations, 210
211 sick animals, clinical evaluation of, 211 Animal protein-derived hydrolysates and peptides, 584
592 Animal sources, generation of bioactivities from proteins of, 403
405, 558
561 animal by-products, 410
411 blood, 410 collagen, 410
411 egg peptides, 560
561 marine sources, 411
413 commercial development of marine-derived peptides, 412
413 seafood, 411
412 meat, 407
409, 559
560

aging, generation of peptides during, 408 fermentation, generation of peptides during, 408
409 gastrointestinal digestion, generation of peptides by, 407
408 protease treatments, generation of peptides by, 409 milk, 405
407, 558
559 cheese whey for producing peptides, 406 milk proteins evaluation for bioactive peptides, 407 royal jelly peptides, 561 Antiangiogenic peptides, 58
59 Anticancer activities of bioactive peptides, 9
11, 10t Anticancer effect of plant-derived peptides, 379
381 Antidiabetic effect, 16
17 Antidiabetic properties of protein hydrolysates/peptides clinical studies, 612
623 in vivo studies, 612 Antihypertensive/ACE inhibitory activity, 406 Antihypertensive activities of bioactive peptides, 14t Antihypertensive cassette design, 59
60 Antihypertensive effect, 13
15 Antihypertensive peptides, 57
58 Antihypertensive protein hydrolysates and peptides, foods formulated with, 598
601 Antiinfective activity of antimicrobial peptides, 755
756 Anti-inflammatory peptides, 11
12, 509, 509t, 533 Antimetabolic dysfunction mechanisms, 207f Antimicrobial peptides (AMPs), 12
13, 745
746 application for improving human health, 754
758 antiinfective activity of antimicrobial peptides, 755
756

antiviral effect of antimicrobial peptides, 756 bioavailability and metabolism, 756
758 probiotic strains, antimicrobial peptides production by, 754
755 classification, 746
748 ε-polylysine, commercial application of, 753 hurdle approach, 754 limitations, 761
763 mechanisms of action, 758
759 against bacteria and fungi, 758
759 against viruses, 759 MicroGARD, commercial application of, 753 nisin, commercial application of, 751
752 pediocin, commercial application of, 752 regulatory approval, 753
754 regulatory aspects of using AMPs or AMP producers in food, 760
761 safety of, 759
760 Antiobesity potential, commercial dietary protein hydrolyzates with, 652
655 Antioxidant activity, 534 Antioxidant activity assays, 104
114 Antiviral effect of antimicrobial peptides, 756 Anxiety disorders, 707
708 Arabidopsis thaliana, 474 Arthritis, 724 Aspergillus oryzae, 373
375 Atherogenic index, 559 Attention-deficit hyperactivity disorder (ADHD), 696, 702 Autism, 698
701 Autism spectrum disorder (ASD), 696 2,2’-Azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS•1), 110
111 2,2’-Azo-bis (2-amidinopropane) dihydrochloride (AAPH), 108

Index B Bacteria, recombinant protein expression in, 52f Bacteriocin, 761 β-casomorphin-7, 193
194 β-casomorphins (β-CMs), 645
646 β-cell dysfunction, 609
610 β-conglycinin, 553
554 Bioactive food-derived peptides, 1 Bioactive peptide, 344 Bioactive peptide-loaded microgels, fabrication of, 339
342 emulsion templating, 342 injection
gelation method, 341
342 Bioactivity assay, methodologies for, 103
104 antioxidant activity assays, 104
114 ferric-reducing antioxidant power (FRAP) assay, 105
107, 107t oxygen radical absorbance capacity (ORAC) assay, 108
110, 110t Trolox-equivalent antioxidant capacity (TEAC) assay, 110
112, 112t enzyme inhibitory assays, 114
138 acetylcholinesterase inhibitory activity, assay of, 135
136 α-amylase inhibitory activity, assay of, 121
124 α-glucosidase inhibitory activity, assay of, 124
126 angiotensin-I-converting enzyme inhibition, assay of, 114
117, 115f chymotrypsin inhibitory activity, assay of, 133
134 dipeptidyl peptidase IV inhibitory activity, assay of, 119
121 lipase inhibitory activity, assay of, 126
129 pros and cons, 137
138

renin inhibition, assay of, 117
119 troubleshooting and optimization, 138
139 trypsin inhibitory activity, assay of, 131
133 tyrosinase inhibitory activity, assay of, 129
131 Bioactivity prediction of peptides, 240
241 BIOPEP-UWM database, 277 Biopharmaceutics and bioactive peptides, 55t Biosafety levels (BSL) 1 through 4, 157 Bipolar disorders, 696, 707 Bitter taste, 336
337, 572 Bitter taste inhibitory peptides, 573 Bitter taste receptor, 572
573 Blood, generation of peptides from, 410 Blood
brain barrier (BBB), peptide transport across, 691
694, 692f Blood pressure, 581. See also Hypertension Bone, 666
667 Bone formation cells, 169
172 in vitro osteoblasts culturing, 169
171 materials, equipment, and reagents, 169
170 MC3T3-E1 cell line, 169 method, 170
171 mineralization assay, 171
172 materials, equipment, and reagents, 171 method, 171
172 Bone marrow monocytes (BMMs), 172 Bone mineral density (BMD), 667 Bone resorption cells, 172
177 in vitro macrophage RAW 264.7 cell culture, 172
173 materials, equipment, and reagents, 173 method, 173 RAW 264.7 cell line, 172

773

osteoclast generation from macrophage RAW 264.7, 173
175 materials, equipment, and reagents, 173
174 method, 174
175 osteoclastic resorption assay, 176
177 materials, equipment, and reagents, 176 method, 176
177 tartrate resistant acid phosphatase (TRAP) staining, 175
176 materials, equipment, and reagents, 175 method, 175
176 Bovine lactoferricin (LfcinB), 8
9 Bowman
Birk inhibitor (BBI), 357
358, 364
367 Brain tumors and CNS disorders, peptides as diagnostic tools in, 694
696 peptide-based imaging tracers, 694
695 peptides as biomarkers, 695
696 Brassica carinata, 37 Buforin IIb, 8
9

C Caco-2 cell model, 681 Caco-2 monolayer, peptides passing through, 231
232 Calcium, 459, 461 Calcium-chelating peptides, 462 Calcium-sensing receptor (CaSR), 206
207, 279
280 Cancer, 58
59 application of biologically active peptides in the clinical treatment of, 501
503 Captopril, 581
582 Carbohydrate digestion and glucose homeostasis, 608
609 Carbohydrates, 163 Carcinogenesis, 379
380 Cardiovascular benefits of food protein-derived bioactive peptides, 581
583

774 Index Cardiovascular benefits of food protein-derived bioactive peptides (Continued) angiotensin-converting enzyme (ACE)- and renin-inhibitory peptides, 584
598 animal protein-derived hydrolysates and peptides, 584
592 plant protein-derived hydrolysates and peptides, 592
598 antihypertensive protein hydrolysates and peptides, foods formulated with, 598
601 Cardiovascular disease (CVD), 551 application of biologically active peptides in the clinical treatment of, 500
501 newfangled animal models to evaluate, 203
204 Cartilage and functional joint pain, 537
538 Casein hydrolysate, 335, 337
338 Casein phosphopeptides (CPPs), 461 Cassia leiandra, 364
367 Cassio obtusifolia, 592
593 Cathelicidin LL-37, 696 Cell culture, 156
165. See also Bone formation cells; Bone resorption cells aseptic technique and contamination control, 159
160 personal hygiene, 159 sterile reagent and media, 160 sterile work area, 159
160 basic equipment for, 156 cell types and sourcing of cell lines, 160
162 continuous cultures, 161 primary cultures, 160 selecting the appropriate cell line, 161 sourcing cell lines, 162 culture media, 163
164 amino acids, 163 carbohydrates, 163

fatty acids and lipids, 164 inorganic salts, 163 protein and peptides, 164 serum, 163
164 vitamins, 164 safety aspects of, 156
159 biohazards, 158 disinfection, 158 risk assessment, 156
158 waste disposal, 158
159 subculturing, 165 temperature, pH, CO2 and O2 levels, 164
165 Cell culture laboratory, recommended equipment/supply for, 157t Cell culture protocols, 165
169 adherent cultures, subculturing, 165
166 cryopreserved cells, thawing, 168
169 freezing cells, 167
168 suspension cultures, subculturing, 167 total cell number and cell viability, quantification of, 167 Cell study, biochemical and molecular analysis of, 177
186 quantitative reverse transcription polymerase chain reaction (RT-qPCR), 182
186 analysis of, 184
186 design primers for SYBR Green qPCR assay, 183
184 materials, equipment, and reagents, 182 method, 182 performing, 184 reverse transcription, 183 RNA extraction by TRIzol reagent, 182
183 Western blotting, 177
181 electrophoresis, 179 electrophoretic transfer from gel to membrane, 179
180 materials, equipment, and reagents, 177
178 method, 178

preparation of cell lysate, 178
179 preparation of SDS polyacrylamide gel, 179 protein detection, 180
181 Cheese whey for producing peptides, 406 Chem2Bio2RDF, 316 Chemical synthesis, 2
3 Cheminformatics, 312 Chemoprevention, 9
11 Chicken foot protein hydrolysate (CFPH), 590
591 Chicken skin protein hydrolysates (CSPH), 590
591 Chickpea peptide, 208 Chinese Society for Parenteral and Enteral Nutrition (CSPEN), 496 Cholesterol 7a-hydroxylase (CYP7A1) transactivation, 558, 561 Cholesterol-lowering activity, 551
552 animal sources, peptides from, 558
561 egg peptides, 560
561 fish peptides, 560 meat peptides, 559
560 milk peptides, 558
559 royal jelly peptides, 561 hypocholesterolemic peptides, 557 structure
activity relationship of, 562
563 peptides activity and characterization, 552 plant proteins, peptides from, 552
557 hempseed peptides, 556
557 lupin peptides, 555
556 soybean peptides, 552
555 Chromatographic separation, 80, 83 Chymotrypsin inhibitory activity, assay of, 133
134 alternative methods/procedures, 134 analysis and statistics, 134 definition, 133

Index materials, equipment, and reagents, 133 precursor and related techniques, 134 protocols, 134 Chymotrypsin inhibitory units (CIU), 364
367 CKGGRAKDC, 639
640 Clinical nutrition, 495
498 biologically active peptides, application of, 498
509 clinical biologically active peptide products, design requirements for, 511 clinical nitrogen supplementation products, characteristics of, 510 in clinical nutritional support and therapy, 497
498 in the clinical treatment of cancer, 501
503 in the clinical treatment of cardiovascular diseases, 500
501 in the clinical treatment of diabetes mellitus, 505
507 in the clinical treatment of liver injury, 503
505 in the clinical treatment of other diseases, 507
509 nitrogen intake requirements for different patients, 510
511 product forms, 514
516 source selection of biologically active peptides in products for patients with specific health needs, 511
514 clinical nutritional support and clinical nutrition therapy, 495
497 Clinical nutritional foods, application of biologically active peptides in, 510
516 clinical biologically active peptide products, design requirements for, 511

clinical nitrogen supplementation products, characteristics of, 510 nitrogen intake requirements for different patients, 510
511 product forms, 514
516 in products for patients with specific health needs, 511
514 Coacervation, 336 Colicins, 753
754 Collagen, 725
726 generation of peptides from, 410
411 Collagen peptides, 726 Colloidal carriers, 7
8 Colonic adenocarcinoma (Caco-2) cells, 756
758 Competent cells preparation, 63
64 Complementary DNA (cDNA), 182 Connective tissue, bioactive peptides and, 535
538 cartilage and functional joint pain, 537
538 tendon, 535
537 Copper, 460 Copper deficiency, 457
459 Cotadutide, 637
638 Cowpea, 557 CRISPR (clustered regularly interspaced short palindromic repeats)
Cas (CRISPRassociated protein) systems, 285
286, 290f advancing biological research, 289
292 analysis and quality control, 295
296 beyond Cas9, 288
289 bioactive peptides and, 292
293 generating CRISPR-guided targets for peptide-based studies in mammalian cells, 292
293 ethical reflections, 296
297 future directions, 297
300 materials, equipment, and reagents, 293
294

775

protocols, 294
295 timeline and development of, 286
288 Cryopreserved cells, thawing, 168
169 Cryptides, 581
582 CSPHP (C-fraction soy protein hydrolyzate with bound phospholipids), 652
655 C-type natriuretic peptide (CNP), 728 Cupric ion
reducing antioxidant capacity (CUPRAC), 113 Cyclic adenosine monophosphate signaling pathways, 704

D Dairy proteins, 645 Databases of bioactive peptides, 309
310 bioinformatic databases for the analysis of food proteins and peptides, 320
324 biological and chemical information on peptides, 312
316 sequences, 316
320 and their classification, 310
312 Data mining (DM), 316 Degree of hydrolysis (DH), 37
38 definition, 37
38 precursor techniques and alternative methods/ procedures, 38 DEHQKIHRFRQGDV, 649
652 Delivery systems, 7 Delonix regia carboxymethylated gum/sodium alginate microgels, 341 Depressive, bipolar, and anxiety disorders, 705
708 Derivatization techniques, 228, 233
234 Dextran sulfate sodium (DSS), 279
280 D-Glucose, 608 Diabetes, clinical diagnosis of, 610
611 Diabetes mellitus, 607

776 Index Diabetes mellitus (Continued) application of biologically active peptides in the clinical treatment of, 505
507 Dianthus superbus, 502
503 Dietary iron, 460 Dietary peptides in cellular signaling events, regulatory properties of, 276
282 exploration of molecular basis of dietary peptide modulating cellular signaling transduction, 279
282 in silico approach for characterizing bioactive peptides, 277 for investigation of interaction between bioactive peptides and molecular target, 277
278 Dietary proteins, 612 Dietary Supplement Health and Education Act (DSHEA) system, 413 2,5-Dihydroxybenzoicacid, 243
244 Diketopiperazines, 225
226 2,5-Dimethyl-4-hydroxy-3 (2H)furanone (DMHF), 416
418 Dimethylsulfoxide (DMSO), 167
168 Dioscorin, 368 Dipeptidyl peptidase-IV (DPP-IV), 114, 119
120, 277
278, 434
435, 505
506, 562, 630
631, 648
649 Dipeptidyl peptidase IV inhibitory activity, assay of, 119
121 alternative methods/procedures, 121 analysis and statistics, 121 definition, 119
120 materials, equipment, and reagents, 120 precursor and related techniques, 121 protocols, 120 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, 113
114

DNA cloning into a suitable vector, 61
63 fragment amplification by PCR and purification of PCR product, 61
62 ligation of amplified fragments by PCR into transient vectors, 63 DPYKLRP, 13
15 Dulaglutide, 636

E Edible insects, 432
435 bioactivity of peptides derived from insects, 433
435 extraction of bioactive peptides from insects, 432
433 Efpeglenatide, 637 Egg peptides, 560
561 Egg protein hydrolysates, 588
589 EKERERQ, 591
592 Elanapril, 581
582 Electrically switchable nanolever technology, 476
478 application of switchSENSE for mineral-binding peptide screening, 476
478 principle of the switchSENSE technology, 476 Electrodialysis with ultrafiltration membrane (EDUF), 439 Electron paramagnetic resonance (EPR), 471 Electron spin resonance, 471 Electrospray ionization-mass spectrometry (ESI-MS), 478
480 principle of, 478
479 use of ESI-MS for MBP screening, 479
480 ELLLNPTHQIYPVTQPLAPV, 12
13 Embryonic stem cells (ESCs) technology, 203 Emulsion templating, 342 Encapsulation technology, 331
332 future perspectives, 351

hydrogel delivery systems, 339
344 encapsulation efficiency of bioactive peptides in microgels, 342
343 fabrication of bioactive peptide-loaded microgels, 339
342 release behavior and bioactive properties of encapsulated peptides in microgels, 343
344 microparticulate delivery systems, 332
339 bitter taste and hygroscopicity of microencapsulated peptides, 336
338 food-grade microparticulate carrier materials, 333
335 release characteristics, gastric stability, and bioavailability of microencapsulated peptides, 338
339 techniques for fabricating microparticles, 335
336 nanoparticulate delivery systems for bioactive peptides, 344
350 liposome-based nanoencapsulation system, 345
347 nanoemulsion-based delivery system, 349
350 polyelectrolyte-based nanoencapsulation system, 347
349 solid lipid nanoparticles, 350 Encrypted vasoinhibin peptide, amplification of, 60 Endopeptidase activity, assay of, 38
40 alternative methods/procedures, 40 analysis, 39 definition, 38
40 materials, equipment, and reagents, 39 protocol, 39 Endoproteinases, 221
223

Index Endothelium-independent vasorelaxation, 255
257 Enteral nutrition (EN), 496 Enteroendocrine cells (EECs), 706
707 Enzymatic hydrolysis, 2
3, 225
226 plant-derived bioactive peptides through, 369
373 Enzymatic mechanisms for bioactive peptides generation, 27
37 degree of hydrolysis (DH), 37
38 definition, 37
38 precursor techniques and alternative methods/ procedures, 38 endopeptidase activity, assay of, 38
40 alternative methods/ procedures, 40 analysis, 39 definition, 38
40 materials, equipment, and reagents, 39 protocol, 39 exopeptidase activity, assay of, 40
42 definition, 40 materials, equipment, and reagents, 40 pros and cons, 41
42 protocol, 41 food processing, bioactive peptides generated during, 30 in the hydrolysis of food proteins, 27
30 proteins hydrolysis with commercial peptidases, bioactive peptides generated through, 30
37 Enzymatic membrane reactor system (EMR), 595
597 Enzyme inhibitory assays, 114
138 Epithelium sodium channels (ENaCs), 569
570 ε-polylysine, commercial application of, 753

Escherichia coli, production of recombinant bioactive peptides in, 57
59 antiangiogenic peptides, 58
59 antihypertensive peptides, 57
58 Escherichia coli expression vectors and strains for recombinant protein production, 50
52, 53t Essential amino acids (EAAs), 529 European Society for Clinical Nutrition and Metabolism (ESPEN), 496 Euthanasia, protocol for, 269 Exenatide, 506
507 Exercise-induced muscle damage (EIMD), 531
532 Exopeptidase activity, assay of, 40
42 definition, 40 materials, equipment, and reagents, 40 pros and cons, 41
42 protocol, 41 alternative methods/ procedures, 41 analysis, 41 Exopeptidase-resistant peptides detection in blood, 229
231 Extended X-ray absorption fine structure (EXAFS), 471 Extracellular matrix (ECM), 728
729 Extracted ion chromatogram (EIC), 91

F Fatty acids and lipids, 164 Fermentation generation of peptides during, 408
409 plant-derived bioactive peptides through, 373
375 Ferric-reducing antioxidant power (FRAP) assay, 105
107 analysis and statistics, 106
107 definition, 105 materials, equipment, and reagents, 106

777

precursor techniques and related techniques, 107 pros and cons, 107, 107t protocols, 106 safety considerations and standards, 107 Fibroblast growth factor-2 (FGF2), 705
706 Fick’s law of diffusion, 4
5 Fish peptides, 560 Flavourzyme 1000L, 31
37 Fluorescein (FL), 108 Fluorescein isothiocyanate (FITC), 38
39 Fluorescence spectroscopy (FS), 471
472 Food, defined, 309 Food-based angiotensin-converting enzyme inhibitors, 240
241 Foodborne pathogens, 752 Food-derived peptide, 9
17, 221
225, 640
652 anticancer activity, 9
11 antidiabetic effect, 16
17 antihypertensive effect, 13
15 anti-inflammatory effect, 11
12 antimicrobial activity (AMP), 12
13 biological activity in body, 232
233 Caco-2 monolayer, peptides passing through, 231
232 direct identification in the body, 227
229 exopeptidase-resistant peptides detection in blood, 229
231 future prospects, 233
234 immunomodulatory peptides, 15
16 inhibiting protease dipeptidyl peptidase-4, 648
649 in vitro evidence of foodderived peptides, 649 with modified amino acid residues in blood, 226
227 structure of peptides in foods, 225
226 survival of food-derived peptides during gut transit, 649
652

778 Index Food-derived peptide (Continued) targeting CCK and GI enzymes with proven in vivo efficacy, 644
646 targeting ghrelin, opioid receptor, and GI transit with proven in vivo efficacy, 646
647 targeting lipid metabolism with proven in vivo efficacy, 647
648 Food-grade microparticulate carrier materials, 333
335 lipid-based carriers, 335 polysaccharide-based carriers, 333
334 protein-based carriers, 334 Food peptides administration of food peptides and animal safety, 193
199 development of oral and injectable peptides derived from food, 199 gender and age, distribution of, 195
199 meal feeding information, 194
195 safety and toxicological evaluation of peptides, 193
194 animal experiments in the study of, 192f hypotensive mechanisms of, 201f that have been tested in vivo, 196t Food processing, bioactive peptides generated during, 30 Food proteins bioinformatic databases for the analysis of food proteins and peptides, 320
324 enzymatic hydrolysis of, 2 enzymatic mechanisms in the hydrolysis of, 27
30 Foods for specified health uses (FOSHU), 412 Formic acid, 80
81 FQINMCILR, 592
593 Freezing cells, 167
168

Fungi, 427
431 bioactive properties of peptides derived from, 429
431 major fungi protein and mechanisms of extraction, 428
429 FVVNATSN, 553
554, 647

G γ-glutamyl transferase (GGT)catalyzed reaction, 575
576 Ganoderma lucidum, 504 Gastric inhibitory peptide, 16
17 Gastrointestinal digestion, generation of peptides by, 407
408 GEGSGA, 9
11 Genome editing, 289
290 Gibbs free energy, 472
474 GLDIQK, 558 GLTSK, 9
11 Glucagon-like peptide-1 (GLP-1), 16
17, 193, 505, 631
637, 632t Glucose-dependent insulinotropic peptide (GIP), 505 Glucose homeostasis, 608
609 Glycomacropeptide (GMP), 645 GPETAFLR, 647 G protein-coupled receptors (GPCRs), 569
570, 689
690 GQEQSHQDEGVIVR, 556 Gram-negative bacteria, 762 Gram-positive bacteria, 762 Gut
blood barrier (GBB), 756
758

H HCQRPR, 15
16 Hedyotis diffusa, 380 Hempseed peptides, 556
557 Hemp seed protein hydrolysates (HPH), 593
595 Hepatic injury, 503 Hepatocyte nuclear factor-1α (HNF-1α), 554
555 High blood pressure. See Hypertension

High-performance liquid chromatography (HPLC), 221
223 Hill-plot analysis, 268, 269f HIRL, 646
647 Home enteral nutrition (HEN), 497 Homology-directed repair (HDR), 289
290 Host cells, transformation of, 63
66 competent cells preparation, 63
64 fragment restriction and ligation into expression vector, 65
66 plasmid DNA, preparation of, 65 transformation, 64 Human immunodeficiency virus (HIV), 364
367 Human umbilical vein endothelial cells (HUVECs), 756
758 Hurdle approach, 754 HVLSRAPR, 9
11 Hydrogel delivery systems, 339
344 bioactive peptide-loaded microgels, fabrication of, 339
342 emulsion templating, 342 injection
gelation method, 341
342 encapsulation efficiency of bioactive peptides in microgels, 342
343 release behavior and bioactive properties of encapsulated peptides in microgels, 343
344 Hydrogen atom transfer (HAT), 104 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity, 647 Hydroxyl radical absorbance capacity (HORAC), 113 Hygroscopicity, 337
338 Hyperlipidemia, 501 Hypertension, 13, 57
58 animal models to evaluate, 199
204 classical animal models, 200
203

Index newfangled animal models, 203
204 Hypochlorites, 158 Hypocholesterolemic peptide, 557 structure
activity relationship of, 562
563 Hypoglycemic peptides, 607 antidiabetic properties of protein hydrolysates/peptides, 613t clinical studies, 612
623 in vivo studies, 612 carbohydrate digestion and glucose homeostasis, 608
609 diabetes, clinical diagnosis of, 610
611 protein hydrolysates and bioactive peptides, diverse physiological properties of, 611 type 2 diabetes, pathophysiology of, 609
610 Hypotensive mechanisms of food peptides, 201f

I IAVPGEVA, 553 IIAEK, 558 IKHQGLPQE, 12
13 Immobilized metal-ion affinity chromatography (IMAC), 476, 480
482, 481f immobilized metal-ion affinity chromatography, principle of, 480
482 use of IMAC for MBP screening, 482 Immunomodulatory peptides, 15
16, 507 InChI (The IUPAC International Chemical Identifier), 315 InChIKey, 315 Injection
gelation method, 341
342 Inorganic salts, 163 In silico methods predicting bioactivity in food-derived peptides, 242
243 Insulin resistance, 607, 609
610 Insulin secretion, 607, 609
610

Intestinal drug transport mechanisms, 4f In vitro macrophage RAW 264.7 cell culture, 172
173 materials, equipment, and reagents, 173 method, 173 RAW 264.7 cell line, 172 In vitro osteoblasts culturing, 169
171 materials, equipment, and reagents, 169
170 MC3T3-E1 cell line, 169 method, 170
171 In vitro protein digestibility (IVPD), 373
375 IPP (Ile-Pro-Pro), 13
15, 584 IQQGN, 15
16 IRLIIVLMPILMA, 13
15 Isoleucine, 8 Isopropyl-β-D-thiogalactoside (IPTG), 51 Isothermal titration calorimetry (ITC), 472
474 for MBP screening, 474 principle of, 472
474

J Joint health, 723
724 evidence in joint health benefits, 733
735 mechanisms of action, 728
733 antioxidant, antimicrobial, and antiinflammatory activities, 731
733 cartilage proliferation, 728
731 neuroactivity, 733 osteoarthritis, 724
725 peptides activity and characterization, 725
728 natural bioactive peptide sources, 725
727 peptidome analysis, 727
728 potential applications, production, and commercialization, 736
740 diagnostic, 736
737

779

production and commercialization, 739
740 prophylaxis/therapeutic, 738
739 rheumatoid arthritis, 725

K Kafirin, 357
364 Kappaphycus alvarezii, 437
438 KLAKLAKKLAKLAK peptide, 639
640 KLPPLLLAKLLMSGKLLAEPCTGR, 9
11 Kluyveromyces marxianus Z17, 30 Knockout (KO) cell types, 289
290 Knowledge Discovery from Databases (KDD), 312 KNQDK, 500
501 Kokumi peptides, characteristics of, 575
576 Kokumi taste, 575 Kokumi taste receptors, 575 KPEGMDPPLSEPEDRRDGAAGPK, 9
11 KRQKYDI, 591
592 Kunitz-type inhibitor (KTI), 357
358 KWFKIQMQIRRWKNKR, 349

L Laccase, 341 lac promoter, 51 Lactic acid bacteria (LAB), 30, 373
375 Lactobacillus brevis, 13
15 Lactobacillus helveticus, 584 Lactobacillus pentosus, 30 Lactobacillus plantarum, 373
375 Lacto-ghrestatin, 646 Lactotripeptides, 223
225 LANAK, 9
11 Large intestine (colon), absorption of peptides in, 5 LC
electron spray ionization tandem mass spectrometry (LC
ESI
MS/MS), 221
223 Leucine, 8 L-glutamic acid, 570
571

780 Index Lipase inhibitory activity, assay of, 126
129 Assay A, 127
128 analysis and statistics, 127
128 materials, equipment, and reagents, 127 protocols, 127 Assay B, 128
129 analysis and statistics, 129 materials, equipment, and reagents, 128 protocols, 128 definition, 126 Lipid-based carriers, 335 Lipoprotein receptor-related protein 1 (LRP1), 292
293 Liposome-based nanoencapsulation system for bioactive peptides, 345
347 Lipospheres, 335 Liraglutide, 638
639 Lisinopril, 581
582 Liver injury, application of biologically active peptides in the clinical treatment of, 503
505 LIVTQTMKG, 646 LKPTPEGDL, 505
506 LKPTPEGDLE, 505
506 LKPTPEGDLEIL, 505
506 Low-density lipoprotein cholesterol (LDL-C), 551 LPQNIPPL, 505
506 LPYPR, 554 LRVPAGTTFYVVNPDNDENLRMIA, 553
554 LSGNK, 9
11 LSGYGP, 586 Luetzelburgia auriculate, 364
367 LunaRich X, 652
655 Lunasin, 9
11, 367
368 Lupin peptides, 555
556

M Machine learning tools, 246 Macroalgae, 435
438 Macrophage colony stimulating factor (M-CSF), 172 Magnesium, 459
460

Maillard reaction, bioactive peptides and, 413
418 bioactivities of Maillard reaction products from peptides, 415
416 bioactivities of volatile Maillard reaction products from peptides, 416
418 and meat, 414
415 Maillard reaction products (MRPs), 404, 415
416, 570
571, 573 MAIPPKKNQDK, 500
501 Major depressive disorder (MDD), 696, 705
706 Major royal jelly protein 1 (MRJP1), 561 MALDI SYNAPT Q-TOF mass spectrometer instrument, 90
91 MALDI-TOF-MS analysis, 243
244 Marine macroalgae, 435
438 bioactive properties of peptides from macroalgae proteins, 437
438 mechanisms of extraction of bioactive peptides from, 436
437 Marine sources, bioactive peptides from, 411
413 commercial development of marine-derived peptides, 412
413 seafood, generation of peptides from, 411
412 Mass spectrometers, 88 Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS), 221
223 Meat, bioactive peptides from, 407
409 aging, generation of peptides during, 408 fermentation, generation of peptides during, 408
409 gastrointestinal digestion, generation of peptides by, 407
408

protease treatments, generation of peptides by, 409 Meat, Maillard reaction and, 414
415 Meat peptides, 559
560 Mechanistic insights on the biological activities of bioactive peptides from plants, 377
383 anticancer effect of plantderived peptides, 379
381 metabolic syndrome, role of plant-derived peptides in, 381
383 role of plant-derived peptides in inflammation and immunomodulation, 377
379 Mechanistic target of rapamycin (mTOR) signaling pathway, 527
528 Mental health, 689
694 blood
brain barrier (BBB), peptide transport across, 691
694, 692f brain tumors and CNS disorders, peptides as diagnostic tools in, 694
696 peptide-based imaging tracers, 694
695 peptides as biomarkers, 695
696 therapeutic applications of peptides for, 697
711 depressive, bipolar, and anxiety disorders, 705
708 neurocognitive and neurodegenerative disorders, 708
711 neurodevelopmental disorders (NDD), 698
703 psychotic disorders, 703
705 Messenger RNA (mRNA), 182 Metabolic dysfunction, animal models to evaluate, 204
209 knockout mice models, 208
209 Metabolic syndrome, role of plantderived peptides in, 381
383 Metabotropic glutamate 4 (mGluR4), 571

Index 4-Methylumbelliferyl oleate (4MUO), 126 5-Methylpyrazine-2-methanol (MPM), 416
418 Microbial fermentation, 2
3, 373
375, 384 MicroGARD, commercial application of, 753 Microgels encapsulation efficiency of bioactive peptides in, 342
343 release behavior and bioactive properties of encapsulated peptides in, 343
344 Microparticles, techniques for fabricating, 335
336 coacervation, 336 spray drying, 335
336 Microparticulate delivery systems, 332
339 bitter taste and hygroscopicity of microencapsulated peptides, 336
338 bitter taste, 336
337 hygroscopicity, 337
338 food-grade microparticulate carrier materials, 333
335 lipid-based carriers, 335 polysaccharide-based carriers, 333
334 protein-based carriers, 334 microencapsulated peptides, 338
339 techniques for fabricating microparticles, 335
336 coacervation, 336 spray drying, 335
336 Milk, 15
16 bioactive peptides from, 405
407 evaluation of milk proteins for bioactive peptides, 407 generation of peptides from milk, 405
406 utilization of cheese whey for producing peptides, 406 -derived peptides, 239
240 Milk peptides, 558
559

Mineral-binding peptides, 455
456 application of, 462
464 mineral deficiency, 462
463 oxidation phenomena, 463
464 electrically switchable nanolever technology, 476
478 application of switchSENSE for mineral-binding peptide screening, 476
478 switchSENSE technology, principle of, 476 electrospray ionization-mass spectrometry (ESI-MS), 478
480 principle of, 478
479 use of ESI-MS for MBP screening, 479
480 evidence of health effects of, 460
462 immobilized metal-ion affinity chromatography separation, 480
482 principle of immobilized metal-ion affinity chromatography, 480
482 use of IMAC for MBP screening, 482 isothermal titration calorimetry, 472
474 for MBP screening, 474 principle of, 472
474 in natural resources, 464 peptides
metal ion interactions, 468
469 production of, 465
468 chemical peptide synthesis, 466
468 proteolysis, 465
466 spectroscopic techniques, 469
472 principle of, 469
472 to understand metal
peptide interactions, 472 surface plasmon resonance (SPR), 475
476 for MBP screening, 476 principle of, 475, 475f Mineralization assay, 171
172

781

materials, equipment, and reagents, 171 method, 171
172 Minerals’ importance for nutrition, 456
460 bioavailability and metabolism of minerals, 459
460 main mineral involved in nutrition and their needs in human, 456
457 safety considerations and standards/regulation, 457
459 Mitogen-activated protein kinase (MAPK), 704 Momordica charantia, 364
367 Monosodium glutamate (MSG), 570
571 MPACGSS, 9
11 MTEEY, 9
11 Mucoadhesive systems, 6
7 Multiomics and molecular biology approaches, 279f Multiple actions mimetics, 637
638 Multiple reaction monitoring (MRM), 88, 96
97 Mung bean protein hydrolysate (MPH), 597
598 Muscle damage, bioactive peptides and, 531
535 antiinflammatory effect, 533 antioxidant activity, 534 protein synthesis, effects on, 533 Muscle foods, 30 Muscle recovery, 525, 532
533

N Nanoemulsion-based delivery system for bioactive peptides delivery, 349
350 Nanoencapsulation-based systems, 351 Nanoparticles, 7
8 Nanoparticulate delivery systems for bioactive peptides, 344
350 liposome-based nanoencapsulation system, 345
347

782 Index Nanoparticulate delivery systems for bioactive peptides (Continued) nanoemulsion-based delivery system, 349
350 polyelectrolyte-based nanoencapsulation system, 347
349 solid lipid nanoparticles, 350 Natriuretic peptide receptor (NPR) signaling, 728 Naturally generated peptides absolute quantitation of, 96
99 alternative methods/ procedures, 99 analysis and statistics, 97
99 materials, equipment, and reagents, 97 pros and cons, 99 protocols, 97 troubleshooting and optimization, 99 identification of, 88
89 alternative methods/ procedures, 90
91 analysis and statistics, 90 materials, equipment, and reagents, 89
91 pros and cons, 90 protocol, 89 troubleshooting and optimization, 91 label-free relative quantitation of, 91
96 alternative methods/ procedures, 96 analysis and statistics, 93
95 materials, equipment, and reagents, 92 pros and cons, 95 protocols, 92 troubleshooting and optimization, 96 Naturally occurring bioactive peptides in plants, 364
369 dioscorin, 368 lunasin, 367
368 plant protease inhibitors (PPIs), 364
367

sporamins and patatins, 368
369 Natural peptides, 87
88 Neurocognitive and neurodegenerative disorders, 708
711 Neurodevelopmental disorders (NDD), 698
703 Neutral endopeptidases (NEPs), 635
636 Niemann-Pick C1-like 1 (NPC1L1) protein levels, 558
559 Nisin, commercial application of, 751
752 Nitrilotriacetic acid (NTA), 476
478 3-Nitrobenzyl alcohol (3-NBA), 80
81 Nitrocellulose, 177 Nitrogen intake requirements for different patients, 510
511 Nonobese diabetic (NOD) mice model, 206
207 Nonribosomal peptides, 748 Novel technologies in bioactive peptides production and stability, 47
48 proteins and peptides, stability of, 52
57 protocol, 59
70 antihypertensive cassette design, 59
60 DNA cloning into a suitable vector, 61
63 encrypted vasoinhibin peptide, amplification of, 60 host cells, transformation of, 63
66 induction of the expression of the desired protein under controlled conditions, 66
68 recombinant peptides, preparation and encapsulation of, 69
70 recombinant product, recovery and purification of, 68 recombinant bioactive peptides production in Escherichia coli, 57
59

antiangiogenic peptides, 58
59 antihypertensive peptides, 57
58 recombinant peptides, expression of, 48
52 Nuclear magnetic resonance (NMR), 471

O Obesity, 208 Obesity and satiety control, 629 commercial dietary protein hydrolyzates with antiobesity potential, 652
655 food-derived peptides, 640
652 inhibiting protease dipeptidyl peptidase-4, 648
649 in vitro evidence of foodderived peptides, 649 survival of food-derived peptides during gut transit, 649
652 targeting CCK and GI enzymes with proven in vivo efficacy, 644
646 targeting ghrelin, opioid receptor, and GI transit with proven in vivo efficacy, 646
647 targeting lipid metabolism with proven in vivo efficacy, 647
648 synthetic peptides, 631
640 glucagon-like peptide-1 (GLP-1) mimetics, 631
637, 632t multiple actions mimetics, 637
638 other synthetic peptides in preclinical trials and in vitro development, 639
640 safety considerations and limitations for, 638
639 Osteoarthritis, 724
725, 737 Osteoclast generation from macrophage RAW 264.7, 173
175

Index materials, equipment, and reagents, 173
174 method, 174
175 Osteoclastic resorption assay, 176
177 materials, equipment, and reagents, 176 method, 176
177 Osteogenic agents, 670
679 osteogenic peptides, 672
679 osteoporosis, drugs for, 670
672 Osteogenic peptides, 665
667, 673t bioavailability of, 681
682 absorption analysis, 681 pharmacokinetic analysis, 681
682 characterization of, 679
681 identification, 679
681 preparation, 679 Osteoporosis, 513, 667 drugs for, 670
672 evaluation and diagnosis of, 667
670 bone formation and resorption biomarkers, 667
669 computed tomography diagnosis, 669
670 Oswald ripening, 349
350 Oxygen radical absorbance capacity (ORAC) assay, 108
110 analysis and statistics, 109 definition, 108 materials, equipment, and reagents, 108
109 precursor techniques and related techniques, 110 pros and cons, 109, 110t protocols, 109 safety considerations and standards, 109 Oxytocin, 698
702

P Paenibacillin, 747
748 Paracellular transport, 3
4 Parenteral nutrition (PN), 496

Parkinson’s disease (PD), 709
710 Passaging. See Subculturing Patatins, 368
369 Pea protein hydrolysate (PPH), 593 Pediocin, 746
747 commercial application of, 752 Penicillium roquefortii, 576 Pepsin-pancreatin-hydrolyzed pea protein (PPHPp), 13
15 Peptide-form Hyp, 226 Peptide-induced vasorelaxation, 255
257, 262 Peptide-loaded solid lipid nanoparticles, 350 Peptide transporter 1 (PEPT1), 5 Peptide transporter 2 (PEPT2), 5 Peptidomics, methodologies for, 87
88 naturally generated peptides, absolute quantitation of, 96
99 alternative methods/ procedures, 99 analysis and statistics, 97
99 materials, equipment, and reagents, 97 pros and cons, 99 protocols, 97 troubleshooting and optimization, 99 naturally generated peptides, identification of, 88
89 alternative methods/ procedures, 90
91 analysis and statistics, 90 materials, equipment, and reagents, 89
91 pros and cons, 90 protocol, 89 troubleshooting and optimization, 91 naturally generated peptides, label-free relative quantitation of, 91
96 alternative methods/ procedures, 96 analysis and statistics, 93
95 materials, equipment, and reagents, 92

783

pros and cons, 95 protocols, 92 troubleshooting and optimization, 96 Peptidomics methods, 88 Personal protective equipment (PPE), 158 PGSGCAGTDL, 597
598 Phaseolus lunatus protein hydrolysate, 341 Phenol antioxidant index (PAOXI), 113 Phenyl thiocarbamyl (PTC)peptides, 227 Phosphoinositide 3-kinase (PI-3K), 704 Physicochemical feature of bioactive peptide, 243
244 Plant-derived bioactive peptides through enzymatic hydrolysis, 369
373 through fermentation, 373
375 Plant protease inhibitors (PPIs), 364
367 Plant protein-derived hydrolysates and peptides, 592
598 Plant proteins, peptides from, 552
557 hempseed peptides, 556
557 lupin peptides, 555
556 soybean peptides, 552
555 Plant sources of bioactive peptides, 357
358 challenges and opportunities in studying the health benefits of plant-derived peptides, 383
386 mechanistic insights on the biological activities of bioactive peptides from plants, 377
383 anticancer effect of plantderived peptides, 379
381 role of plant-derived peptides in inflammation and immunomodulation, 377
379 role of plant-derived peptides in metabolic syndrome, 381
383

784 Index Plant sources of bioactive peptides (Continued) naturally occurring bioactive peptides in plants, 364
369 dioscorin, 368 lunasin, 367
368 plant protease inhibitors (PPIs), 364
367 sporamins and patatins, 368
369 plant proteins classification and isolation and extraction methods, 358
364 unique aspects of plant proteins and preparing bioactive peptides from plant sources, 375
377 Plasmid DNA, preparation of, 65 p-nitroanilide (pNA), 38
39 Polyelectrolyte-based nanoencapsulation system for bioactive peptide delivery, 347
349 Polyelectrolyte nanocomplexes, 348 Polymerase chain reaction (PCR), 60, 182 Polymeric microgels, 69
70 Polyphosphoric acid (PPA), 341 Polysaccharide-based carriers, 333
334 Polyvinylidene fluoride (PVDF) membrane, 177 Porcine gelatin hydrolysate (PGH), 586
587 Porous graphitic carbon (PGC) cartridges, 76
77, 79 Porphyra columbina, 437
438 Preparation of bioactive peptides, 2
3 Preservatives, 760 Probiotic strains, antimicrobial peptides production by, 754
755 Pro-Gly, 233 Prolactin (PRL) gene, 60 Proline, 8 Prolyl peptide, 233 Prosthetic joint infections (PJI), 737

Protease AN hydrolyzed lupine, 31
37 Protease treatments, generation of peptides by, 409 Protein and peptides, 164 Proteinase Inhibitor II, 652
655 Protein-based carriers, 334 Protein glycation, 52
54 Protein hydrolysate, 9
12, 194
195, 582
583 alcalase-digested canola protein hydrolysate, 598 chicken foot protein hydrolysate (CFPH), 590
591 chicken skin protein hydrolysates (CSPH), 590
591 diverse physiological properties of, 611 hemp seed protein hydrolysates (HPH), 593
595 mung bean protein hydrolysate (MPH), 597
598 pea protein hydrolysate (PPH), 593 rapeseed enzymatic protein hydrolysates, 593
595 Protein metabolism, 510 Protein
protein interaction (PPI), 556 Proteins, 331 Proteins and peptides, stability of, 52
57 Proteins glycation, 52
54 Proteins hydrolysis with commercial peptidases, bioactive peptides generated through, 30
37 Proteins in foods, 27 Protein synthesis, effects on, 533 Proteolysis, 27
30, 38
39 Proton-coupled oligopeptide transporter 1 (PepT1), 559
560 Protospacer adjacent motif (PAM), 285
286 Psychotic disorders, 703
705 PVNFKFLSH, 646 Pyroglutamyl peptide, 233

Q QAGLSPVR, 586
587 QCQQAVQSAV, 11
12 QSAR (quantitative structure
activity relationships) approach, 320
321 Quantitative reverse transcription polymerase chain reaction, 182
186 analysis of, 184
186 design primers for SYBR Green qPCR assay, 183
184 materials, equipment, and reagents, 182 method, 182 performing, 184 reverse transcription, 183 RNA extraction by TRIzol reagent, 182
183 Quantitative structure
activity relationship (QSAR) methods, 244
245 artificial neural networking and, 246
247

R Ramipril, 581
582 Rapeseed enzymatic protein hydrolysates, 593
595 Reactive oxygen species (ROS), 104, 463 Receptor activator of NF-κB ligand (RANKL), 172 Recombinant bioactive peptides production in Escherichia coli, 57
59 antiangiogenic peptides, 58
59 antihypertensive peptides, 57
58 Recombinant peptides expression of, 48
52 preparation and encapsulation of, 69
70 Recombinant product, recovery and purification of, 68 Recombinant protein expression in bacteria, 52f Renin
angiotensin
aldosterone system (RAAS), 200, 583
601

Index angiotensin-converting enzyme (ACE)- and renin-inhibitory peptides, 584
598 animal protein-derived hydrolysates and peptides, 584
592 plant protein-derived hydrolysates and peptides, 592
598 antihypertensive protein hydrolysates and peptides, foods formulated with, 598
601 Renin inhibition, assay of, 117
119 alternative methods/procedures, 119 analysis and statistics, 118
119 definition, 117
118 materials, equipment, and reagents, 118 protocols, 118 Rheumatic and joint diseases, 723 Rheumatoid arthritis, 725, 737 Rhizopus oligosporus, 373
375 Rice, 557 RKQLQGVN, 9
11 Royal jelly peptides, 561 RQIKIWFQNRRMKWKK, 349 RQSHFANAQP, 9
11 Rubia akane, 502
503

S Salt taste, 573
574 Salty taste receptors, 574 Seafood, generation of peptides from, 411
412 Seaweed-derived peptides, bioactive properties from, 437t Seaweed. See Macroalgae Serum, 163
164 SGFAP, 15
16 Short-chain bioactive peptides, 75
77 alternative methods/procedures, 79, 82
83 definition, 77 materials, equipment and reagents, 77
78, 80
81 pros and cons, 78
79, 82

protocols, 78, 81
82 troubleshooting and optimization, 79
80, 83 Sick animals, clinical evaluation of, 211 SIFIQRFTT, 12
13 Simplified molecular input line entry system (SMILES) code, 277
278 Sinapinic acid, 243
244 Single electron transfer (SET), 104 Size exclusion chromatography (SEC), 227 Slendesta, 652
655 Small intestine, absorption of peptides in, 3
9 approaches for enhancing the absorption of peptides, 6
8 paracellular transport, 3
4 structure-activity relationship of bioactive peptides, 8
9 transcellular transport, 4
5 SMILES (Simplified Molecular Input-Line Entry System), 315 SNVVPLY, 474 Solid lipid nanoparticles for bioactive peptide delivery, 350 Solid-phase peptide synthesis (SPPS), 466
468 Soybean, 554 Soybean-conglycinin peptide, 649
652 Soybean peptides, 552
555 Soymorphin-5, 649
652 Spadin, 705
706 Specific pathogen-free (SPF) pellets, 200
201 Spectroscopic techniques, 469
472 principle of, 469
472 to understand metal
peptide interactions, 472 SpirPep, 322 Spoilage microorganisms, 750 Spontaneously hypertensive rats (SHRs), 195
201, 204, 583
584, 586
587, 702 Sporamins, 368
369 Sport nutrition, 525
526

785

bioactive peptides, body composition, and muscular performance, 527
531 bioactive peptides and connective tissue, 535
538 cartilage and functional joint pain, 537
538 tendon, 535
537 bioactive peptides and muscle damage, 531
535 antiinflammatory effect, 533 antioxidant activity, 534 protein synthesis, effects on, 533 limitations, 538
539 practical applications, 539
541 Spray drying, 335
336 Square wave voltammetry (SWV), 474 Staphylococcus aureus, 437
438 Staphylococcus carnosus, 30 Storage proteins (SPs), 358
359 Streptococcus thermophilus, 286
288 Structural characteristics of salty taste-enhancing peptides, 574
575 Structure-activity relationship of bioactive peptides, 8
9 Structure
function of bioactive peptides, mapping methods to predict, 241
242 Structure
function relationship of food-derived peptides, 239
240 bioactivity prediction of peptides, 240
241 classical bioinformatics and computational biology approach, limitations of, 247
248 future directions, 248 in silico methods predicting bioactivity in food-derived peptides, 242
243 mapping methods to predict structure
function of bioactive peptides, 241
242 physicochemical feature of bioactive peptide, 243
244

786 Index Structure
function relationship of food-derived peptides (Continued) quantitative structure
activity relationship (QSAR) methods, 244
245 artificial neural networking and, 246
247 Subculturing, 165 Subtiliosin A, 747
748 N-Succinyl-leucine-tyrosine-7amido-4-methylcoumarin, 40 Surface plasmon resonance (SPR), 475
476 for MBP screening, 476 principle of, 475, 475f Suspension cultures, subculturing, 167 Sustained release, 338 SwitchSENSE technology application of, for mineralbinding peptide screening, 476
478 principle of, 476, 477f Synthetic peptides, 631
640 glucagon-like peptide-1 (GLP-1) mimetics, 631
637, 632t multiple actions mimetics, 637
638 other synthetic peptides in preclinical trials and in vitro development, 639
640 safety considerations and limitations for, 638
639

T T9 (GQEQSHQDEGVIVR), 556 Tachyplesin I, 8
9 Tartrate resistant acid phosphatase (TRAP) staining, 175
176 materials, equipment, and reagents, 175 method, 175
176 TRACP5b, 669 Taspoglutide, 635 Taste attributes and their corresponding taste receptors, 570t Taste enhancers, peptides as, 569
570

bitter taste, 572 bitter taste inhibitory peptides, 573 bitter taste receptor, 572
573 kokumi peptides, characteristics of, 575
576 Kokumi taste, 575 Kokumi taste receptors, 575 salt taste, 573
574 salty taste receptors, 574 structural characteristics of salty taste-enhancing peptides, 574
575 umami and umami-enhancing peptides, structural characteristics of, 571
572 umami taste, 570
571 umami taste receptors, 571 Tendon, 535
537 Tetradentate nitriloacetic acid (NTA) sensor chips, 476 Tetrahydrofuran (THF), 80
81 TGAPCR, 592
593 Therapeutic peptides, 240
241 Therapeutic recombinant proteins/ peptides, production of, 57 Thyroid C cells, 242
243 Tilapia skin gelatin, 586
587 Tirzepatide, 637
638 Total antioxidant capacity (TAC), 107 Total radical-trapping antioxidant parameter (TRAP) assays, 113 Transcellular transport, 4
5 Transglutaminase, 341 Transient receptor potential vanilloid 1 (TRPV1) channels, 569
570 Trifluoroacetic acid (TFA), 80
81 Trinitrobenzenesulfonic acid (TNBS) method, 38 2,4,6-Trinitrobenzensulfonate, 221
223 Tripeptidylpeptidases, 27
28 Triticum dicoccum, 373
375 Trolox-equivalent antioxidant capacity (TEAC) assay, 110
112 analysis and statistics, 111
112 definition, 110
111

materials, equipment, and reagents, 111 precursor and related techniques, 112 pros and cons, 112, 112t protocol, 111 safety considerations and standards, 112 Trypsin inhibitory activity, assay of, 131
133 alternative methods/procedures, 133 analysis and statistics, 132 definition, 131 materials, equipment, and reagents, 131
132 precursor and related techniques, 132 protocols, 132 TTAGLLE, 376
377 Type 1 diabetes (T1D), 206
207 Type 2 diabetes (T2D), 206
207, 505 pathophysiology of, 609
610 Tyrosinase inhibitory activity, assay of, 129
131 alternative methods/procedures, 131 analysis and statistics, 130 definition, 129 materials, equipment, and reagents, 130 precursor and related techniques, 130
131 protocols, 130

U Umami and umami-enhancing peptides, structural characteristics of, 571
572 Umami taste, 570
571 Umami taste receptors, 571 Underutilized agricultural byproducts, 438
444 bioactivity of peptides derived from, 440
444 mechanisms for extraction of bioactive peptides from, 439
440

Index V Valine, 8 Vascular smooth muscle cells (VSMCs), 255
257 Vasoactive intestinal peptide (VIP), 728 Vasoprotective mechanisms of bioactive peptides, 255
257 alternative methods/procedures, 270
271 measurement of vascular tension using rat mesenteric arteries, 270
271 patch clamp test, 271 analysis and statistics, 266
268 Hill-plot analysis, 268 measurement of [Ca21]i, 267 measurement of vascular tension, 266
267 percentage of Ca21
CaM complex formation, 267
268 materials, equipments, and reagents, 258
261 Ca21
CaM complex formation, assay for, 260
261 intracellular Ca21 concentration [Ca21]i, measurement of, 260 vascular tension, measurement of, 259
260 principles, 257
258 [Ca21]i, measurement of, 258 Ca21
CaM complex formation, assay for, 258

vascular tension, measurement of, 257 pros and cons, 270 [Ca21]i, measurement of, 270 Ca21
CaM complex formation, assay for, 270 vascular tension, measurement of, 270 protocols, 261
266 [Ca21]i, measurement of, 263
266 Ca21
CaM complex formation, assay for, 266 vascular tension, measurement of, 261
263 safety considerations and standards, 268
269 ethical statement, 269 euthanasia, protocol for, 269 troubleshooting and optimization, 271
272 [Ca21]i, measurement of, 272 vascular tension, measurement of, 271
272 VAWWMY, 554, 647 VFVRN, 648 VHVV, 648
652 Vibrational spectroscopies, 470
471 Viola ignobilis, 380 Vitamins, 164 VKKVLGNP, 591
592 VLPVPQK, 474 VPP (Val-Pro-Pro), 13
15, 584 VRIRLLQRFNKRS peptide, 644 VVYP, 652
655 VVYPWTQRF, 500, 591
592

787

VYVEELKPTPEGDLEILLQK, 558

W Western blotting, 177
181 electrophoresis, 179 electrophoretic transfer from gel to membrane, 179
180 materials, equipment, and reagents, 177
178 method, 178 preparation of cell lysate, 178
179 preparation of SDS polyacrylamide gel, 179 protein detection, 180
181 WGAPSL, 554, 647 Wheat-derived cryptic peptides, 705
706 Whey-derived peptides, 406 Whey protein, 529 Wilson’s disease, 457
459 WVYY, 593
595 WYT, 593
595

X X-ray absorption near edge structure (XANES), 471 X-ray absorption spectroscopy, 471

Y YADLVE, 597
598 YPFVV, 646 YPFVVN, 646 YPFVVNA, 646 YVVNPDNDEN, 553
554, 647 YVVNPDNNEN, 553
554, 647