Biopolymer-based Formulations: Biomedical and Food Applications [1 ed.] 0128168978, 9780128168974

Table of contents :
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Biopolymer-Based FormulationsBiomedical and Food ApplicationsEdited byKunal PalIndranil BanerjeePreetam SarkarDoman KimWin- ...
Copyright
Contributors
1 . Introduction of biopolymers: food and biomedical applications
1. Introduction
2. Cross-linking methods employed to design biopolymer-based polymeric architectures
2.1 Physically cross-linked gels
2.1.1 Cross-linking by ionic interactions
2.1.2 Self-assembly of hydrophobized polysaccharides
2.1.3 Cross-linking by the crystallization of the polysaccharides
2.1.4 Cross-linking by induction of hydrogen bonds among the polysaccharide molecules
2.1.5 Cross-linking using protein molecules
2.2 Chemical cross-linking
2.2.1 Glutaraldehyde
2.2.2 Polycarboxylic acids
2.2.3 EDC coupling
2.2.4 Epichlorohydrin
2.2.5 Trisodium metaphosphite
2.2.6 Polysaccharide dialdehydes
2.2.7 Genipin
2.2.8 Proanthocyanidin
2.2.9 Glyoxal
3. Biopolymers and their applications
3.1 Chitosan
3.1.1 Food industry
3.1.2 Biomedical applications
3.2 Cellulose
3.2.1 Food industry
3.2.2 Biomedical applications
3.3 Pullulan
3.3.1 Food industry
3.3.2 Biomedical applications
3.4 Hyaluronic acid
3.4.1 Biomedical applications
3.5 Alginate
3.5.1 Food applications
3.5.2 Biomedical applications
4. Conclusion
Acknowledgments
References
2 . Enzymatic synthesis of flavonoid glucosides and their biochemical characterization
1. Introduction
2. Enzymatic synthesis of glycosylated flavonoids
2.1 Flavonol
2.2 Flavanones
2.3 Flavanonol
2.4 Flavone
2.5 Isoflavones
2.6 Physical and biological characterization of glycosylated flavonoids
2.6.1 Solubility
2.6.2 Biological characterization
2.6.3 Sensory of α-glucosylated flavonoids
3. Conclusion
Acknowledgment
References
3 . Fish gelatin: molecular interactions and applications
1. Introduction
2. Gelatin
3. Fish gelatin
4. Protein–polysaccharide interactions
5. Molecular interactions of fish gelatin with polysaccharides
5.1 Formation of insoluble complexes
5.2 Formation of soluble complexes
5.3 State diagram
5.3.1 Effects of pH and mixing ratio
5.3.2 Effects of NaCl
6. Applications
7. Conclusions
References
4 . Peptides as biopolymers—past, present, and future
1. Introduction
2. Peptides with biomedical applications
2.1 Therapeutic peptides and biopolymers
2.2 Biopolymers and peptides targeting the cardiovascular system
3. Food industry applications
3.1 Biopolymer nanoparticles as delivery systems
3.2 Peptides as value enhancers
3.3 Biopolymers in food processing and packaging
4. Conclusion
References
5 . Microbial production of biopolymers with potential biotechnological applications
1. Introduction
2. Microbial biopolymer production
2.1 Xanthan
2.2 Dextran
2.3 Pullulan
2.4 Glucans
2.5 Gellan
2.6 Alginate
2.7 Cyanophycin
2.8 Poly-γ-glutamic acid
2.9 Levan
2.10 Hyaluronic acid
2.11 Bacterial cellulose
2.12 Organic acid fermentation for polymer synthesis
2.13 Microbial exopolysaccharides
2.14 Polyhydroxyalkanoates
3. Biosynthesis of microbial polyhydroxyalkanoates
4. Conclusion
References
Further reading
6 . Animal-derived biopolymers in food and biomedical technology
1. Introduction
2. General classifications of biopolymers
3. What are animal and natural biopolymers?
4. Types of animal biopolymers
5. Silk
6. General applications of biopolymer
7. Formulation of different biopolymers
8. Applications of biopolymers in food industries
9. Biopolymers and its application in biomedical sciences
10. Some environmental benefits of use of biopolymer
11. Conclusion
References
7 . Application of CRISPR technology to the high production of biopolymers
1. Introduction
2. Application of CRISPR/Cas9 in various research
3. CRISPR/Cas9-based metabolic engineering
4. CRISPR/Cas9-based genome editing in biopolymer production from prokaryotes
5. CRISPR/Cas9-based genome editing in biopolymer production from eukaryotes
6. Conclusions and perspectives
Acknowledgments
References
8 - Biomedical and food applications of biopolymer-based liposome
1. Introduction
2. Starch
3. Zein
4. Xanthan gum
5. Collagen
6. Chitin
7. Gelatin
8. Methacrylate
9. Calcium alginate
10. Chitosan
11. Future prospects
12. Conclusion
References
9 . Nanosized magnetic particles for cancer theranostics
1. Introduction
2. Synthesis techniques of magnetic nanoparticles
3. Physiochemical properties of MNPs
3.1 Size of MNPs
3.2 Shape of MNPs
3.3 Surface properties and biopolymer-functionalized MNPs
3.4 Biocompatibility and safety
4. Magnetic nanoparticles for cancer imaging
5. Magnetic nanoparticles for cancer therapy
5.1 Chemotherapy
5.2 Hyperthermia therapy
6. Smart magnetic nanoparticles for cancer theranostics
7. Conclusion
References
10 . Core–shell biopolymer nanoparticles
1. Introduction
2. Materials used for core–shell nanoparticles
3. Methods involved in the formation of core–shell nanoparticles
3.1 Antisolvent precipitation
3.2 Electrostatic deposition
3.3 Self-assembly
3.4 Thermal treatment
3.5 Covalent cross-linking
3.6 Combinations and others
4. Applications
4.1 Delivery of small molecular drugs and nutraceuticals
4.2 Delivery of peptides and proteins
4.3 Delivery of nucleic acids
5. Current issues and future development
References
11 . Nanotechnology-based sensors
1. Introduction
2. Types of nanomaterials for biosensor
2.1 Gold nanomaterials
2.2 Silver nanomaterials
2.3 Carbon nanostructures
2.4 Silicon nanomaterial
2.5 Magnetic nanoparticles
2.6 Biopolymers-based nanosensors
3. Properties of nanomaterials used for detection in biosensors
4. Principle and classification of biosensors
4.1 Working principle of biosensor
4.2 Classification of biosensor
5. Application of Nanobiosensors
6. Pros and cons of biosensors and strategy to overcome
7. Conclusion
References
Further reading
12 . Functional amyloids
1. Introduction
2. Protein misfolding and aggregation
3. Amyloid and its pathogenesis
4. Mechanism of amyloid formation
5. Functional amyloids found in nature
5.1 As structural components
5.2 As regulatory factors in several biochemical pathways
5.3 As scaffolds for storage and release of proteins
5.4 In sexual reproduction
5.5 As molecular chaperones
6. How toxicity is prevented with functional amyloids?
7. Potential of amyloids in biomedical applications
7.1 As scaffolds for tissue engineering
7.2 As drug delivery vehicles
7.3 In biosensors and bioimaging
8. Conclusion and future perspectives
References
Further reading
13 . Lipid-derived renewable amphiphilic nanocarriers for drug delivery, biopolymer-based formulations: biomedical and food appl ...
1. Introduction
2. Classification of drug delivery systems
2.1 Polymeric nanoparticles
2.2 Micelles
2.3 Dendrimers
3. Emulsions-based drug delivery systems
3.1 Microemulsions
3.2 Nanoemulsions
3.3 Self-emulsifying delivery systems
3.4 Pickering emulsions
4. Vesicular drug delivery systems
4.1 Liposomes
4.2 Phytosomes
4.3 Pharmacosomes
4.4 Ethosomes
4.5 Vesosomes
5. Lipid particulate drug delivery systems
5.1 Solid lipid nanoparticles
5.2 Nanostructured lipid carriers
5.3 Lipid–drug conjugates
6. Types of lipid-based formulation
6.1 Processing techniques for lipid formulations
7. Biomedical applications of lipid-derived nanocarriers
7.1 Gene therapy systems
7.2 Drug delivery vehicles
7.3 Theranostics nanocarriers
8. Applications of lipid nanocarriers in food industry
9. Summary
References
14 . Nanoencapsulation of nutraceutical ingredients
1. Introduction
2. Excipient selection
3. Encapsulation techniques
3.1 Coacervation
3.1.1 Procedure for formation of coacervates
3.2 Electrospraying/spinning
3.2.1 Advantages of electrospraying/spinning
3.2.2 Disadvantages of electrospraying
3.3 Nanolaminated system
3.3.1 Methods of preparation
3.3.2 Advantages
3.4 Nanospray drying
3.4.1 Advantages
3.4.2 Disadvantages
3.5 Nanogels
3.5.1 Classifications of nanogels
3.5.2 Synthesis of nanogels
3.5.3 Advantages of nanogels
3.5.4 Disadvantages
3.6 Nanoemulsions
3.6.1 Types of nanoemulsion
3.6.2 Nanoemulsion formulation techniques
3.6.3 Advantages of nanoemulsion
3.6.4 Limitation of nanoemulsion
3.7 Liposomes
3.7.1 Mechanism of liposome formation
3.7.2 Classification of liposomes
3.7.3 Stability of the liposomes
3.7.4 Advantages of liposomes
3.7.5 Disadvantages of liposomes
3.8 Solid lipid nanoparticles
3.8.1 Choice of lipid and surfactants in the production of SLN
3.8.2 Advantages of solid lipid nanoparticles
3.8.3 Disadvantages of solid lipid molecules
3.9 Nanostructured lipid carrier
3.9.1 Classification of NLC
3.9.1.1 Imperfect NLC
3.9.1.2 Amorphous NLC
3.9.1.3 Multiple NLC
4. Impact of encapsulation on characteristics of nutraceutical compound
4.1 UV stability
4.2 pH stability
4.3 Temperature stability
4.4 Solubility
4.5 Crystallinity
4.6 Oxidative stability
5. Mechanism of release of bioactive compounds from encapsulated matrix
5.1 pH stimuli response delivery
5.2 Temperature stimuli response delivery
5.3 Redox stimuli response delivery
5.4 Magnetic stimuli response delivery
6. Nutraceutical bioavailability improvement
6.1 Gastroretentive delivery systems
6.2 Intestinal targeted delivery
6.3 Colon-specific delivery
7. Conclusion
References
15 . Nutraceutical encapsulation and delivery system for type 2 diabetes mellitus
1. Introduction
2. Encapsulation of nutraceuticals and their characterization
2.1 Spray drying
2.2 Spray chilling
2.3 Fluidized bed
3. Potential of nutraceuticals and their delivery for treating T2DM
3.1 Polyphenolic compounds
3.1.1 Quercetin
3.1.2 Silymarin
3.1.3 Resveratrol
3.1.4 Lipoic acid
4. Conclusion and future prospective
References
16 - Pickering emulsions stabilized by nanoparticles
1. Introduction
2. History of pickering emulsion
3. Physical chemistry of pickering emulsion
3.1 Globule size and formation criteria
3.2 Stabilizing particles
3.3 Stable emulsion formation by adsorbed solid particle
3.4 Parameters of different types of emulsions
3.5 Unbound particles and consequence of rheology
4. Applications
4.1 Potential application to the life science and drug delivery
4.2 Application in material science using polymerization template
5. Conclusion
References
Further Reading
17 . Microencapsulation of bioactive compounds and enzymes for therapeutic applications
1. Introduction
2. Types of microencapsulation
3. Microencapsulation of bioactive compounds and bioactive extracts
4. Microencapsulation of therapeutic enzymes
5. Challenges and future outlooks
Acknowledgments
References
18 . Rice husk silica for the stabilization of food-grade oil-in-water (O/W) emulsions
1. Introduction
2. Silica for stabilization of O/W emulsion
3. Effect of emulsifier addition combined with rice husk silica in stabilizing O/W emulsion
3.1 Addition of lecithin in the oil phase
3.2 Addition of Tween-20 in the aqueous phase
4. Effect of pH of outer continuous phase on the stability of O/W emulsion stabilized with rice husk silica
5. Effect of storage temperature on the stability of O/W emulsion stabilized with rice husk silica
6. Kinetics study on the stability of O/W emulsion stabilized with rice husk silica
Acknowledgments
References
19 . Oil-entrapped films
1. Introduction
2. Hydrocolloid-based films incorporated with essential oils
2.1 Polysaccharide-based edible films
2.1.1 Starch-based edible films
2.1.1.1 Cassava starch
2.1.1.2 Corn starch
2.1.1.3 Banana starch
2.1.2 Chitosan-based edible films
2.1.3 Carrageenan-based edible films
2.1.4 Pectin-based edible films
2.1.5 Gum-based
2.1.5.1 Guar gum
2.1.5.2 Gum tragacanth
2.1.5.3 Gum Arabic
2.2 Protein-based edible films
2.2.1 Soy protein isolate
2.2.2 Whey protein isolate
2.2.3 Zein proteins
3. Composite films incorporated with essential oils
4. Conclusion
References
20 . Tamarind seed polysaccharide: unique profile of properties and applications
1. Introduction
2. Fundamental properties
2.1 Molecular weight and structure
2.2 Rheological properties
2.3 Gelling properties
2.4 Emulsion stabilizing and emulsification property
3. Food applications
3.1 Functions
3.1.1 Thickening
3.1.2 Gelling and water-retaining
3.1.3 Starch modification
3.1.4 Emulsification and emulsion stabilization
3.2 Applications
3.2.1 Bakery products
3.2.2 Dressing and mayonnaise
3.2.3 Frozen desserts
4. Conclusion
Acknowledgments
References
21 . Thermomechanical and surface morphology of biopolymer–nanoparticle composite films
1. Introduction
2. Nanoparticles
3. Biopolymers
4. Biopolymer–nanoparticle fabrication techniques
5. In situ polymerization
6. Melt intercalation
7. Solution cast intercalation
8. Polylactic acid–based nanocomposites
9. Rheology of polylactic acid–based nanocomposites
10. Mechanical properties
11. Thermal analysis
12. Barrier property
13. Microstructure
14. Chitosan-based nanocomposite films
15. Gelatin-based films
16. Starch-based nanocomposite films
17. Hydrocolloid-based films
18. Conclusions
Acknowledgment
References
22 . Natural and bioderived molecular gelator–based oleogels and their applications
1. Introduction
2. Oleogelators
Outline placeholder
Solid matrix
Fluid matrix
2.1 Lipid-based oleogelators
2.1.1 Mono-, di-, and triacylglycerols
2.1.1.1 Monoacylglycerols
2.1.1.2 Triacylglycerols
2.1.2 Fatty acids and fatty alcohols
2.1.2.1 Fatty acids
2.1.2.2 Fatty alcohols
2.1.2.3 Fatty acids+fatty alcohols
2.1.3 γ-Oryzanol+phytosterols
2.1.4 Ceramides
2.1.5 Cocoa butter
2.2 Nonlipid-based oleogelators
2.2.1 Sorbitan derivatives
2.2.2 Lecithin
2.2.3 Carbohydrate-based gelators
2.2.4 Waxes
2.2.4.1 Candelilla wax oleogels
2.2.4 2 Rice bran wax oleogels
2.2.4 3 Beeswax oleogels
2.3 Polymeric gelators
2.3.1 Cellulose derivatives
2.3.1.1 Ethyl cellulose
2.3.1 2 Hydroxypropyl methylcellulose
2.3.2 Gums and resins
2.3.3 Proteins
3. Applications of oleogels
3.1 Oleogels in food
3.1 1 Oleogels in shortenings, spreads, and margarines
3.1 2 Oleogels in creams and pastes
3.1 3 Oleogels in meat products
3.2 Oleogels in pharmaceutics
3.2 1 Oleogels in topical and transdermal drug delivery
3.2 2 Oleogels in the delivery of nutraceuticals
3.3 Oleogels in cosmetics
3.4 Oleogels in lubricants
4. Conclusion
Acknowledgment
References
23 . Hydrogels as biodegradable biopolymer formulations
1. Introduction
1.1 Hydrogels as biopolymer-based formulations
1.2 Hydrogel classification based on structure and biomaterial classification
2. Polysaccharide-based hybrid biopolymer hydrogels
2.1 Hyaluronic acid–containing hydrogels
2.2 Alginate-containing hydrogels
2.3 Xanthan gum–containing hydrogels
2.4 Chitosan-containing hydrogels
2.5 Protein extracellular matrix–containing hydrogels
2.5.1 Elastin biopolymer–based formulations
2.5.2 Collagen biopolymer–based formulations
2.5.3 Fibrin biopolymer–based formulations
3. Conclusion
References
Further reading
24 . Biopolymer-based oleocolloids
1. Introduction
2. Biopolymer-based oleogels
2.1 Chitin-based oleogels
2.2 Oleogels prepared from surface-active biopolymers through colloid-templated approaches
2.2.1 Oleogels from foam-templated approach
2.2.2 Oleogels from emulsion-templated approach
2.3 Gels prepared from protein hydrogels via stepwise solvent exchange route
2.4 Gel prepared from aerogel-templated approach
2.5 Gel prepared from emulsion-encapsulation approach
3. Biopolymer-based O/W/O emulsions
4. Biopolymer-based oleofilms
5. Conclusion and future trends
References
25 . Gum-based hydrogels in drug delivery
1. Introduction
2. Gums and their classifications
3. Chitosan-based hydrogels used in drug delivery
4. Alginate-based hydrogels used in drug delivery
5. Pectin-based hydrogels used in drug delivery
6. Gellan gum-based hydrogels used in drug delivery
7. Tamarind gum-based hydrogels used in drug delivery
8. Sterculia gum-based hydrogels used in drug delivery
9. Guar gum-based hydrogels used in drug delivery
10. Locust bean gum-based hydrogels used in drug delivery
11. Miscellaneous
12. Conclusion
References
Further reading
26 . Implant surface modification strategies through antibacterial and bioactive components
1. Introduction
2. Response of cells and tissues to implant materials
2.1 Bone tissue: the most fundamental unit supporting implant
2.2 Effect of environmental factors to implant performance
3. Implant materials and their surface modification
3.1 Classification of implant materials
3.2 Properties of implant biomaterials
3.3 Technique and methods for surface modification of implant materials
3.4 Implant surface coating techniques
3.4.1 Hydroxyapatite
3.4.2 Growth factors
3.4.3 Extracellular matrix protein
3.4.4 Peptides
3.4.5 Drug coating
3.4.6 Fluoride coating
3.5 Biopolymer materials
3.6 Antimicrobial activity of biopolymer implant coating
3.6.1 Alginate
3.6.2 Hyaluronic acid
3.6.3 Silk fibroin
3.6.4 Albumin
3.6.5 Tannic acid
3.6.6 Collagen
3.6.7 Gelatin
3.6.8 Poly(lactic acid)
3.6.9 Polyhydroxyalkanoates
3.6.10 Chitosan
3.7 Methods for biopolymer processing as surface modification and implant coating
4. Conclusion and future prospects
References
Further reading
27 . Edible films and coatings: an update on recent advances
1. Introduction
2. Novel patterns in basic structural matrices
2.1 New trends in polysaccharides-based edible films
2.1.1 Cellulose nanocrystal and hemicellulose
2.1.2 Pectin–pectin
2.1.3 Carrageenan
2.1.4 Chitosan
2.1.5 Polysaccharide gums
2.2 Protein films
2.2.1 Collagen
2.2.2 Zein
2.2.3 Gelatin
2.2.4 Soy protein films
2.2.5 Milk films
2.3 Lipid films
2.4 Nanobiocomposite edible films
3. Recent advances in the applications of edible films
3.1 Flavor encapsulation
3.2 Carrier of probiotics
3.3 Carriers of antioxidant and antimicrobial compounds
4. Conclusion
References
28 . Rheology and tribology assessment of foods: a food oral processing perspective
1. Introduction
2. Rheology: basic understanding of the tests used in the food industry
2.1 Flow properties of fluid foods using rotational tests
2.1.1 Zero shear viscosity and yield stress point determination
2.2 Small amplitude oscillatory tests
3. Tribology fundamentals
3.1 Background and principle
3.2 Tongue movements and role of saliva
3.3 Tribology configurations
3.3.1 Mount tribological device (a ball-on-three-plates principle)
3.3.2 Friction tester
3.3.3 Optical tribological configuration
3.3.3 The mini-traction machine
3.3.4 The double-ball-on-plate tribological apparatus
3.3.5 Three-ball-on-plate configuration attached to a texture analyzer
4. Conclusions
References
29 . Biopolymer-based scaffolds: development and biomedical applications
1. Introduction
2. Properties of scaffolds
2.1 Biocompatibility
2.2 Biodegradability
2.3 Mechanical properties
2.4 Scaffold architecture and assessment
2.5 Manufacturing technology
3. Types of scaffolds
3.1 Porous scaffold
3.1.1 Materials and methods
3.1.2 Application
3.2 Microsphere scaffold
3.2.1 Materials and methods
3.2.2 Application
3.3 Hydrogel scaffold
3.3.1 Materials and methods
3.3.2 Application
3.4 Fibrous scaffold
3.4.1 Materials and methods
3.4.2 Applications
3.5 Polymer–bioceramic composite scaffold
3.5.1 Materials and methods
3.5.2 Applications
4. Scaffold processing and fabrication techniques
4.1 Fiber felts or mesh
4.2 Fiber bonding
4.2.1 Phase separation
4.3 Solvent casting and particulate leaching
4.4 Membrane lamination
4.5 Melt molding
4.6 Polymer–ceramic fiber composite foam
4.7 High-pressure processing
4.8 Hydrocarbon templating
5. Biopolymer-based scaffolds
5.1 Collagen
5.2 Hyaluronic acid
5.3 Silk fibroin
5.4 Chitosan
6. Application of scaffolds
6.1 Tissue engineering
6.1 1 Bone tissue engineering
6.1 2 Cartilage tissue engineering
6.1 3 Skin tissue engineering
6.1 4 Vascular tissue engineering
6.2 Drug delivery
7. Conclusion
References
Further reading
30 . Phenolic nanoconjugates and its application in food
1. Introduction
2. Phenolic nanoconjugates
2.1 Phenols–polysaccharide nanoconjugates
2.2 Phenols–protein nanoconjugates
2.3 Phenols–protein–polysaccharides nanoconjugates
2.4 Phenols–metal nanoconjugates
3. Production techniques of phenolic nanoconjugates
3.1 Noncovalent methods for phenolic nanoconjugates
3.2 Covalent methods for phenolic nanoconjugates
3.3 Physical methods for phenolic nanoconjugates
4. Identification and characterization of phenolic nanoconjugates
4.1 Particle size
4.2 Zeta potential
4.3 Scanning electron microscope
4.4 Transmission electron microscope
4.5 Atomic force microscopy
4.6 Differential scanning calorimeter
5. Bioavailability and bioaccessibility study of phenolic nanoconjugates
6. Cytotoxicity study of phenolic nanoconjugates
7. Application of phenolic nanoconjugates in the food industry
7.1 Food processing
7.2 Food packaging
8. Conclusion and future perspective
References
Further reading
31 . Oleogels for food applications
1. Introduction
2. Meat products
3. Bakery products
4. Dairy products
5. Other applications
5.1 Spreadable products
5.2 Chocolate and fillings
5.3 Usage of the oleogels as delivering material
6. Conclusion
Acknowledgments
References
Further reading
32 . CNT-tamarind gum–based solid-textured composite hydrogels for drug delivery applications
1. Introduction
2. Review literature
3. Materials and methods
3.1 Materials
3.2 Methods
3.2.1 Preparation of the hydrogel
3.2.2 Microscopy
3.2.3 FTIR analysis
3.2.4 Impedance analysis
3.2.5 Mechanical analysis
3.2.6 Drug release
3.2.7 Antimicrobial analysis
4. Results and discussion
4.1 Preparation of hydrogels
4.2 Microscopy
4.3 FTIR analysis
4.4 Impedance analysis
4.5 Mechanical analysis
4.6 Drug release
4.7 Antimicrobial analysis
5. Conclusion
Acknowledgments
References
33 . Testicular tissue engineering: an emerging solution for in vitro spermatogenesis
1. Introduction
2. Cosmetic prosthesis
3. Androgen-producing implants
4. Fertility restoration through tissue engineering
4.1 Types of cells for testicular tissue engineering
4.1.1 Testis-derived male germline stem cells and sperm progenitor cells
4.1.2 Male germ cells derived from nontesticular pluripotent stem cells
4.1.3 Male germ cells derived from nontesticular multipotent stem cells
4.2 Approaches for in vitro spermatogenesis
4.2.1 Ectopic grafting of testicular tissue or isolated testicular cells
4.2.2 Organ culture of testicular tissue
4.2.3 Engineered testicular tissue
4.2.4 “Classical” tissue engineering
4.2.4.1 Hydrogels and porous scaffolds
4.2.4.2 Fibrous scaffolds
5. Conclusion
References
34 . Enrichment of edible coatings and films with plant extracts or essential oils for the preservation of fruits and vegetables
1. Introduction
2. Development of edible films or coatings from various sources
2.1 Protein based films and coatings
2.2 Polysaccharide based edible films and coatings
2.3 Lipid based edible films coatings
2.4 Composite coatings from protein and polysaccharides
3. Effect of edible coatings on various horticultural commodities
4. Use of plant extract, essential oils and antimicrobial agents in coatings and films formulation
5. Effect of edible coatings with natural plant extracts, essential oil and antimicrobial agents
6. Future trends
References
35 . Collagen-based 3D structures—versatile, efficient materials for biomedical applications
1. Background
1.1 Collagen structure
1.2 Properties and envisaged applications
1.3 Collagen sources
1.4 Collagen cross-linking
2. Collagen for drug/gene delivery
2.1 Solution, gels, films, inserts, and shields
2.2 Collagen-based micro/nanoparticles
2.3 Hybrid and smart collagen–based drug delivery systems
3. Collagen-based formulations for scaffolding—From simple to complex, multifunctional systems—Processing advances
3.1 Introduction
3.2 Collagen-based scaffold preparation methods—basic conventional techniques
3.3 Scaffolds by additive manufacturing
3.4 Applications
4. Collagen dressings
5. Conclusions
References
Index
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X
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Biopolymer-Based Formulations Biomedical and Food Applications Edited by

Kunal Pal Indranil Banerjee Preetam Sarkar Doman Kim Win-Ping Deng Navneet Kumar Dubey Kaustav Majumder

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Contributors Saumya Agarwal Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India Jasim Ahmed Food and Nutrition Program, Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Safat, Kuwait B. Amulyasai Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India C. Anandharamakrishnan Computational Modeling and Nano Scale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, India Arfat Anis Department of Chemical Engineering, King Saud University, Riyadh, Saudi Arabia Muhammad Arshad Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Indranil Banerjee Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Manav Bandhu Bera Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, India Rakesh Bhaskar Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Pravin Bhattarai Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, Haidian, China Nisarani Bishoyi Department of Chemistry, NIT Rourkela, Rourkela, Odisha, India Prasanta Kumar Biswas Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata, West Bengal, India Ragini G Bodade Department of Microbiology, Savitribai Phule Pune University, Pune, Maharashtra, India Anand G Bodade Department of Transfusion Medicine, Seth G S Medical College and KEM Hospital, Mumbai, Maharashtra, India

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Contributors

Subhadeep Bose Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata, West Bengal, India Muhammed Yusuf C¸a
glar Istanbul Sabahattin Zaim University, Faculty of Engineering and Natural Sciences, Department _ of Food Engineering, Istanbul, Turkey Mustafa C¸avu¸s Igdır University, Engineering Faculty, Department of Food Engineering, I
gdır, Turkey Jianshe Chen School of Food Science, University of Idaho, Moscow, ID, United States Harish Kumar Chopra Department of Chemistry, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, India Donghwa Chung Food Technology Major, Graduate School of International Agricultural Technology, Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang, Gangwon, Republic of Korea Geta David Gh. Asachi Technical University of Iasi, Iasi, Romania Raj Deb Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Mehmet Demirci Istanbul Sabahattin Zaim University, Faculty of Engineering and Natural Sciences, Department _ of Food Engineering, Istanbul, Turkey Win-Ping Deng School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; Graduate Institute of Basic Science, Fu Jen Catholic University, New Taipei City, Taiwan Chanda Vilas Dhumal Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India Navneet Kumar Dubey School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan Rajni Dubey Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan Sayantani Dutta Computational Modeling and Nano Scale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, India

Contributors

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Rimpi Foujdar Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, India Advaita Ganguly Comprehensive Tissue Centre, UAH Transplant Services, Alberta Health Services, Edmonton, AB, Canada; Health Sciences Education and Research Commons, University of Alberta, Edmonton, AB, Canada Ann Mary George Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Mukesh Kumar Gupta Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Sadaf Hameed Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, Haidian, China Thi Thanh Hanh Nguyen The Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea; Department of International Agricultural Technology & Institute of Green BioScience and Technology, Seoul National University, Pyeongchang, Gangwon-do, Republic of Korea Md Saquib Hasnain Department of Pharmacy, Shri Venkateshwara University, Amroha, Uttar Pradesh, India Monjurul Hoque Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India Margaret O. Ilomuanya Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Lagos, Surulere, Lagos, Nigeria John Jeslin Department of Biotechnology, St. Joseph’s College of Engineering, Chennai, Tamil Nadu, India Juhui Jin Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea Padmaja Kar Department of Chemistry, NIT Rourkela, Rourkela, Odisha, India Hyo Jin Kim Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea; Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea

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Contributors

Doman Kim Department of International Agricultural Technology & Institutes of Green BioScience and Technology, Seoul National University, Pyeongchang, Gangwon-do, Republic of Korea; The Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea; Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwondo, Republic of Korea Sanjeev Kumar Department of Biotechnology, Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India Chi-Ching Lee Istanbul Sabahattin Zaim University, Faculty of Engineering and Natural Sciences, Department _ of Food Engineering, Istanbul, Turkey Timothy Lee Turner Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States L. Mahalakshmi Computational Modeling and Nanoscale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India Tanushree Maity Defence Research and Development Organization, DRDO Bhawan, Rajaji Marg, New Delhi, India Samrendra Maji SRM Research Institute, SRM Institute of Science and Technology, Kanchipuram, Tamil Nadu, India Kaustav Majumder Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, United States M. Maria Leena Computational Modeling and Nanoscale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India Nupur Mohapatra Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India Jeyan A. Moses Computational Modeling and Nano Scale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, India

Contributors

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Soma Mukherjee Department of Veterinary Medicine School, Mississippi State University, Mississippi State, MS, United States Amit Kumar Nayak Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj, Odisha, India Suraj Kumar Nayak Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Bhagyashree Padhan Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Dilipkumar Pal Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur, Chhattisgarh, India Kunal Pal Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Ashok R. Patel Guangdong Technion Israel Institute of Technology, Shantou, China Rehan Ali Pradhan Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Sushant Prajapati Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Dilshad Qureshi Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India K.J. Rao Department of Biotechnology, Veltech University, Chennai, Tamil Nadu, India Sirsendu S. Ray Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Sai Preetham Reddy Peddireddy Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India

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Contributors

Fiona Concy Rodrigues Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Sai S. Sagiri Department of Chemistry and Biochemistry, The City College of New York, New York, NY, United States Lanny Sapei Department of Chemical Engineering, University of Surabaya, Raya Kalirungkut, Surabaya, East Java, Indonesia Nandini Sarkar Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela, Odisha, India Preetam Sarkar Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India Angana Sarkar Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India Alok Saxena Amity Institute of Food Technology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India Iis Septiana Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea Kumakshi Sharma Health, Safety and Environment Branch, National Research Council Canada, Edmonton, AB, Canada Loveleen Sharma Amity Institute of Food Technology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India Abhinay Kumar Singh School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan Vinay K. Singh Research and Development, Intas Pharmaceuticals Limited, Ahmedabad-Gujarat, India Agustin Wulan Suci Dharmayanti University of Jember, Jember, East Java, Indonesia Yumewo Suzuki DSP Gokyo Food & Chemical Co., Ltd., Osaka, Japan Irshaan Syed Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India

Contributors

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Akira Tabuchi DSP Gokyo Food & Chemical Co., Ltd., Osaka, Japan Goutam Thakur Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India Aman Ullah Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada Rituja Upadhyay School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China; School of Food Science, University of Idaho, Moscow, ID, United States Madan L. Verma Centre for Chemistry and Biotechnology, Deakin University, Geelong, VIC, Australia Lei Wang Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, United States Varsha Wankhade Department of Zoology, Savitribai Phule Pune University, Pune, Maharashtra, India Hiroyuki Yamada DSP Gokyo Food & Chemical Co., Ltd., Osaka, Japan Kazuhiko Yamatoya DSP Gokyo Food & Chemical Co., Ltd., Osaka, Japan Yue Zhang College of Food & Biology Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang, China; Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, United States Muhammad Zubair Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada

CHAPTER

Introduction of biopolymers: food and biomedical applications

1

Dilshad Qureshi1, Suraj Kumar Nayak1, Arfat Anis2, Sirsendu S. Ray1, Doman Kim3, Thi Thanh Hanh Nguyen3, Kunal Pal1 1

Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India; 2 Department of Chemical Engineering, King Saud University, Riyadh, Saudi Arabia; 3Department of International Agricultural Technology & Institute of Green BioScience and Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea

1. Introduction Polymers are macromolecules which usually have monomeric units (same or different) that combine in different ways (Mills and White, 2012). The polymers may be either of synthetic or natural origin (Deb et al., 2019). Nowadays, humankind has become more dependent on the use of synthetic polymers (Buggy, 2016). Unfortunately, the use of these synthetic polymers is associated with a large number of environmental and health issues (Kaushik et al., 2016). This can explain the search of natural polymers by the researchers, which can efficiently be used to replace the synthetic polymers for different applications. The natural origin polymers are also regarded as “biopolymers” (Numata and Kaplan, 2011). Hence, biopolymers can be described as naturally occurring macromolecules which are usually produced by living systems including plants, animals, and microorganisms (Yadav et al., 2015). In recent years, there is a growing tendency to use more of natural polymers for developing various food and biomedical products (Babu et al., 2013; Davidenko et al., 2014; Verbeek, 2012). It is noteworthy to mention over here that the mankind has been using biopolymers not only for food and biomedical applications, where it has found numerous applications, but also in textile, cosmetics, pharmaceuticals, and paper industries (Anwunobi and Emeje, 2011). As previously mentioned biopolymers contain repeating units of monomers and are usually derived from plants, animals, and microorganisms. In general, the repeating units of a biopolymer may either be sugars, amino acids, or fermentative products like aliphatic polyesters (Prameela et al., 2018). These biopolymers may have different functional groups like hydroxyl, amino, amide, carboxyl, phosphate, phenolic, etc. (Li et al., 2012), which impart to their different biological activities (Kim, 2016). The biopolymers are usually broadly classified into three groups, namely, polysaccharides, proteins, and polynucleotides (Mohan et al., 2016). Polysaccharides are generally made up of sugar moieties which are covalently bonded with each other via glycosidic linkages (Garcı´a, 2018). Removal of one water molecule occurs with the formation of each glycosidic bond. On the basis of charge, polysaccharides can be neutral (dextran, pullulan), polycationic (chitosan), and polyanionic (alginate) Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00001-1 Copyright © 2020 Elsevier Inc. All rights reserved.

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Chapter 1 Introduction of biopolymers

(Harding et al., 2015). Polysaccharides are homogenous (containing one type of monomers such as glycogen) or heterogeneous (containing different sugar units, e.g., xanthan gum, gellan gum) (Johnson-Green, 2002). On the other hand, proteins may be defined as the polymers that consist of amino acid moieties as the monomeric unit. These amino acids are joined together by amide linkages resulting in the formation of three-dimensional (3D) structure (Wool and Sun, 2011). This polyamide chain is the basic level in the hierarchy of the protein structure. Various molecular interactions such as hydrogen bonding, salt and disulfide bridges, and hydrophobic and hydrophilic interactions are responsible for the folding of the chain into secondary structures (e.g., a-helix, b-pleated sheets). The structure is further packed closely through the aforementioned interactions into tertiary structures. Interactions among different protein subunits result in the formation of a quaternary structure (Pollock, 2007). Interestingly, the other class of biopolymers, i.e., polynucleotides (such as DNA, RNA), comprise of as many as 13 or more nucleotide monomers (Davidenko et al., 2014). These two heteropolymeric molecules exhibit significance in the living nature. They have a distinct biological function regarding the storage, replication, and discerning of the genetic information. The backbone framework of the two nucleic acids comprises of a phosphate group, sugar moiety, and four nitrogenous bases with few differences (Frank-Kamenetskii, 2005). Due to the availability of the biopolymers with different chemistries, it is possible to formulate various food and biomedical products that have varied structural and physicochemical properties. This is possible because of the presence of different functional groups on the polymeric chains of the biopolymer (SenGupta, 2007). These functional groups help the biopolymers to interact with the different components which are present in the products (Maleki, 2008; Shishir et al., 2018). Despite the aforementioned advantages, the biopolymeric materials have been reported to have inadequate mechanical properties, which make them unsuitable for use in designing specific products (Vieira et al., 2011). To circumvent this problem, many authors have proposed the use of cross-linkers, which are multifunctional chemical agents, and have the ability to form covalent bonds with the polymer chains (Reddy et al., 2015). Further, the employed cross-linking technique may also result in the formation of products with varied properties as the chemical reaction may take place in different ways when environmental conditions of the cross-linking reaction are changed (Akakuru and Isiuku, 2017; Thakur et al., 2017). Considering the above discussion in mind, in this chapter, we will be discussing about the various cross-linking strategies for the biopolymers, the properties of some of the commonly used polysaccharide and protein-based biopolymers, and finally their food and biomedical applications.

2. Cross-linking methods employed to design biopolymer-based polymeric architectures Biopolymers are being investigated thoroughly to design matrices for food and biomedical applications. Some of the common applications include controlled release of bioactive molecules (e.g., flavors, proteins, probiotics, vitamins, drugs, mammalian cells including stem cells etc.), scaffold fabrication for regenerative medicine, gels for food, pharmaceuticals, and biomedical applications (Shit and Shah, 2014; Udenni Gunathilake et al., 2017). In many of the aforesaid applications, designed polymeric architectures are expected to undergo degradation under certain conditions (Jain et al., 2017). It is important that the products generated from the degradation of these polymeric

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

3

architectures are harmless to the human body (Ulery et al., 2011). Such a requirement is necessary for ensuring good biocompatibility of the polymeric architectures. In this section, we will discuss about the different physical and chemical cross-linking strategies that are used for designing polymeric architectures for different applications. It is important to note that the chemical cross-linking strategies help in designing polymeric architectures with very good mechanical stability (Ozbolat, 2016). Unfortunately, the cross-linking agents (the chemical compounds used for cross-linking) have mostly been reported to be toxic (Ozbolat, 2016) and can also react with the bioactive agents which are loaded in the polymeric architectures, thereby rendering them inactive. Such disadvantage can be easily avoided if the physical cross-linking strategies are applied (Puoci, 2014). The common cross-linking strategies employed have been discussed in this section.

2.1 Physically cross-linked gels Recent years has seen an increased interest in employing physical cross-linking strategies to design polymeric architectures (de Oliveira et al., 2019; Ebara et al., 2014; Montaser et al., 2019). This is mainly because the physical cross-linking strategies help in eliminating the use of chemical crosslinking agents. In this section, various physical cross-linking strategies for biopolymers will be discussed.

2.1.1 Cross-linking by ionic interactions Alginate is a commonly used biopolymer that can be easily cross-linked by ionic interactions. The biopolymer consists of b-D-mannuronic acid and a-L-guluronic acid residues. These acidic residues can ionically interact with multivalent cations like calcium (Ca2þ) ions. Guluronate acid residues play a prominent role in the cross-linking of alginate by interacting with Ca2þ ions and forming a characteristic “egg-box” structure (Bruchet and Melman, 2015) (Fig. 1.1). In many cases, alginate beads are being prepared by adding aqueous solutions of sodium alginate (SA) or potassium alginate into an aqueous solution of Ca2þ ions prepared using calcium chloride salt. The cross-linking of the alginate polymer occurs very fast resulting in the formation of globular gels. Unfortunately, this method results in the formation of gel beads with varying cross-linking density and a polymer concentration gradient, thereby resulting in the formation of gel beads with different physical stabilities (Kuo and Ma, 2001) (Fig. 1.2). In an alternative method, calcium carbonate (CaCO3) powder (having very low solubility in water) is homogeneously dispersed in the alginate solution. The gelation of the alginate phase is induced by releasing Ca2þ with the addition of acids (e.g., acetic acid, Glucono-a-lactone) (Connon and Hamley, 2014; Thakur et al., 2017). Since the CaCO3 is homogeneously dispersed within the alginate solution, the cross-linking of the alginate matrices occurs in a near homogeneous manner (Rehm, 2009). Hence, the gels produced by this method are usually uniform gels. The use of calcium sulfate (CaSO4) has been explored for the cross-linking of the alginate polymer by many authors. Unfortunately, it is very difficult to control the gelation kinetics of alginate phase when CaSO4 is used as the ionically crosslinking material. In the year 2001, Kuo and Ma reported that the mixtures of CaCO3 and D-glucono-dlactone, and CaCO3, D-glucono-d-lactone, and CaSO4 were able to reduce the rate of gelation significantly which allowed them to prepare alginate matrices having structurally uniform properties (Kuo and Ma, 2001) (Fig. 1.3).

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Chapter 1 Introduction of biopolymers

FIGURE 1.1 Structural framework of the alginate backbone.

Alginate has been recognized as an important ingredient in food industries as a thickening agent, gelling agent, film forming, stabilizing, and emulsifying agent (Qin et al., 2018). Along with this, alginate has wide applications in pharmaceutics for controlled drug delivery, and in biomedical sciences in cell culture, tissue regeneration, and wound healing (Lee and Mooney, 2012).

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

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FIGURE 1.2 Schematic diagram of the external gelation of the alginate solution using calcium chloride salt solution.

In a similar manner, polycationic polymers like chitosan can be ionically cross-linked using polyanions (Wu et al., 2014). One such example is chitosan. It consists of b-(1 / 4)-linked glucosamine units. The solutions of hydrated glycerol phosphate disodium salt and sodium triphosphate are two of the commonly used physical cross-linkers of the chitosan (Patil, 2008; Saharan and Pal, 2016). Further, it is important to note that when the solution of polymeric anions (e.g., alginate) and polymeric cations (e.g., chitosan) are mixed together, the oppositely charged ionic groups present in the polymers

FIGURE 1.3 Structural diagram of the internal gelation of the alginate solution using calcium carbonate and glucono-dlactone.

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Chapter 1 Introduction of biopolymers

ionically interact with each other to form a gelled network (Kulig et al., 2016). Unfortunately, these gels usually form structurally inhomogeneous matrices (Kuo and Ma, 2008). Due to this reason, matrices formed as per this methodology is not commonly used. Preparation of matrices using this methodology is carried out very carefully. This polymer-polymer complexation process has mainly been restricted to the coating of one of the polymer with the other. As for example, Gandomi et al. (2016) coated porous alginate matrices with a chitosan layer, which resulted in the formation of a semipermeable membrane across the alginate matrix (Gandomi et al., 2016). It is surprising to note that many neutral biopolymers (which do not have any ionic groups) can also result in the formation of gelled structures in the presence of ions. One such example is dextran biopolymer. This polymer can form gelled structure through crystallization by forming hydrogen bonds or in the presence of potassium (Kþ) ions. Although being neutral, the glucose residues in the polymer chains of the dextran molecules form a cage-like structure. The Kþ ions are suitably placed within the cage. This phenomenon results in the formation of a complex cross-linked structure (Mishra, 2017). Though ionic cross-linking allows us to avoid the use of generally toxic chemical cross-linkers (Grumezescu, 2018b), these structures are usually unstable in nature in aqueous/physiological environment (Kuo and Ma, 2008). They get destabilized and undergo structural breakdown when placed in the aforesaid conditions (Grumezescu, 2018a). This phenomenon appears to be useful in some food applications but in many biomedical applications such phenomenon is undesirable. Hence, for biomedical applications usually a second-stage cross-linking is carried out after the initial ionic crosslinking process (Peppas, 2010; Thakur and Thakur, 2015).

2.1.2 Self-assembly of hydrophobized polysaccharides Gelation in water-based systems using amphiphilic molecules has been widely studied. In many of these systems, the amphiphilic molecules undergo self-assembly to form a network-like structure, which, in turn, induces the gelation of the aqueous phase (Malo de Molina and Gradzielski, 2017). Many authors have explored this technique for designing polysaccharide-based physical hydrogels (Fig. 1.4). Hydrophobic modifications of the polysaccharide chains can help in achieving this. The

FIGURE 1.4 Schematic diagram of the cross-linking of hydrophobized polysaccharides through self-assembly.

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

7

hydrophobic modification renders the polysaccharide as amphiphiles. In other words, the biopolymers become polymer amphiphiles (Alhaique et al., 2015). The self-assembly or the aggregation of these polymer amphiphiles to form gelled structures has been greatly explored for pharmaceutical and biotechnological applications (Hassani et al., 2012; Thomas et al., 2018). Some of the common polysaccharides which have been successfully modified for designing hydrogels include carboxymethylcellulose (CMC), hyaluronic acid, guar gum, chitosan (Camponeschi et al., 2015), starch, alginate, agarose (Ahmed, 2015), dextran, pullulan, and carboxymethyl curdlan (Maitra and Shukla, 2014). The hydrophobization of the polysaccharides are usually achieved by preparing palmitoyl, cholesterol, and polyester side-chain-substituted derivatives of the previously mentioned polysaccharides (Liu et al., 2016). Modification of the chitosan using polyacrylic acid and poly-Nisopropylacrylamide polymers has also been proposed for designing pH- and temperature-sensitive hydrogels, respectively (Kim et al., 2000; Yazdani-Pedram et al., 2000). Further, the modification of carboxymethyl dextran with poly-N-isopropylacrylamide to design thermosensitive hydrogels has also been proposed (Huh et al., 2000).

2.1.3 Cross-linking by the crystallization of the polysaccharides The crystallization of the polysaccharides, which results in the formation of a network structure, is prepared by freeze-thaw technique. The hydrogels prepared by this method are also reported as “physical hydrogels.” In these gels, a specific type of gelation is induced by cryogenic treatment of the polysaccharide solution. It is reported that during the freezing and subsequent storage of the polysaccharide, solution in the frozen state results in the formation of side-by-side associations of the polysaccharide chains during the crystallization (freezing) of the solvent (in this case water) (Fig. 1.5). When this frozen structure is thawed the polysaccharide network which has been formed during the crystallization step maintains its structural integrity (Zhang et al., 2013). This methodology has been used to design hydrogels using various polysaccharide including maltodextrins, locus bean gum, starch, agarose, hyaluronan, and xanthan (Bhatia, 2016).

FIGURE 1.5 Schematic diagram of the cross-linking of polysaccharides using freeze-thaw method.

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Chapter 1 Introduction of biopolymers

2.1.4 Cross-linking by induction of hydrogen bonds among the polysaccharide molecules Hydrogen bonding among the polysaccharide molecules is one of the major mechanisms that is employed for the gelation of the aqueous phase. The gelation of the agarose solution in water is one such example. Agarose remains insoluble in cold water. The agarose gels are formed by dissolving the agarose powder in near boiling water, which is subsequently cooled down to room temperature to induce gelation (Zucca et al., 2016). The gelation is induced by the formation of inter- and intramolecular hydrogen bonds, which are responsible for maintaining the 3D structure of the agarose gels (Tako and Nakamura, 1988) (Fig. 1.6). This structure can be destabilized by disrupting the hydrogen bonds by supplying external thermal energy. This results in the conversion of the gelled structure into a liquid state (Bansal and Boccaccini, 2012). Another example of cross-linking by induction of hydrogen bonds is the gelation of the chitosan molecules in a pH-dependent fashion. Chenite et al. (2000) reported the formation of such hydrogels by adding glycerol phosphate salts to aqueous solutions of chitosan (Chenite et al., 2000). The gels were produced by heating the solution to body temperature. They reported that the thermal reversibility of gelation is dependent on the pH of the chitosan and glycerol phosphate solutions. If the final mixture was at a pH value in the range of 6.9e7.2, then the gels formed were partially thermoreversible. On the contrary, completely thermoreversible hydrogels could be produced when the pH values were in the range of 6.5 and 6.9. The complete thermoreversibility of the gels whose solution was having pH in the range of 6.5 and 6.9 was attributed to the inhibition of the hydrogen bond formation with the consequent higher electronic repulsion within the polymeric chains of the chitosan molecules (Chenite et al., 2000). Hydrogen bonding also plays an important role in the preparation of the protein-based gels including gelatin gels (Lean, 2006). It is worthy to note that in the gels prepared by hydrogen bonding, hydrophobic interactions also play an important role to a certain extent, and vice versa (Chang et al., 2018b; Karino et al., 2002).

FIGURE 1.6 Schematic diagram of hydrogen bondingeinduced cross-linking of polysaccharide chains.

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

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2.1.5 Cross-linking using protein molecules The interactions between the specific peptide domains have been reported to synthesize gels. In such a method, two peptide domains that show positive interactions are incorporated separately into noninteracting polymeric chains. When these two protein-engineered polymeric solutions are mixed together, the positive interactions between the peptide domains incorporated within the polymer chains serve as the cross-linking points. This results in the formation of a 3D network structure with a capability to immobilize water molecules (Fig. 1.7). The main advantage in this type of hydrogels is that there is no need to alter the pH, temperature, and ionic strength of the polymeric solution during the formation of the hydrogels. This is beneficial when various living biological entities, e.g., microbial and mammalian cells, and biologically active agents are incorporated within the hydrogel matrix (Foo et al., 2009).

2.2 Chemical cross-linking Physical cross-linking methods provide a good opportunity to improve the properties of the biopolymeric structures. Unfortunately, in many cases where an alteration in the environmental conditions (e.g., pH, temperature, and ionic strength) occurs during the application stage, the polymeric architectures may lose their structural integrity (Saini, 2017). Though in many applications, this property may be useful, but in many applications this property is undesirable. Hence, it has been found that even

FIGURE 1.7 Schematic diagram of the cross-linking of polymers using proteins as cross-linking agents.

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Chapter 1 Introduction of biopolymers

after carrying out the physical cross-linking, the polymeric architectures are further being chemically cross-linked so as to consolidate the structural properties of the physically cross-linked polymeric architectures (Parhi, 2017). Also, many biopolymers/biopolymer mixtures cannot be physically crosslinked. In such cases, chemical cross-linking remains the only available strategy for improving the structural properties of the biopolymeric architectures. The chemical cross-linkers are basically multifunctional chemically reactive molecules which can form covalent bonds with the functional groups present in the biopolymers (Shen et al., 2016). In this way, chemical cross-linkers form interconnecting bridges among the biopolymeric molecules. During the process of chemical crosslinking, there is an increase in the molecular weight with the corresponding increase in the mechanical and the structural ability of the polymeric architectures (Maitra and Shukla, 2014). Though the chemical cross-linkers can provide the aforesaid advantages, they are not devoid of the disadvantages. The primary concern with the use of cross-linkers is the toxicity due to the presence of unreactive monomers within the polymeric architectures (Patel and Mequanint, 2011). Hence, proper care should be taken to eliminate the unreactive monomers after the cross-linking process is over. Further, the sites for the degradation of the polymeric structures can be significantly altered due to the chemical reaction occurring during the cross-linking process. This may significantly reduce the biodegradability of the biopolymeric architectures (Azeredo and Waldron, 2016). Also, there is a decrease in the number of functional groups within the biopolymeric architectures. This may alter the environment-sensitive properties of some of the biopolymers, which, in turn, will compromise the environment-sensitive properties of the final product (He et al., 2012). The researchers have explored various chemical cross-linkers and cross-linking strategies. Some of the common cross-linkers used for the cross-linking of biopolymers include glutaraldehyde, polycarboxylic acids like citric acid and maleic acid, EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), epichlorohydrin, STMP (trisodium metaphosphite), polysaccharide dialdehydes like dextran dialdehyde and alginate dialdehyde, genipin, proanthocyanidin (PA), and glyoxal. In this section, we will be discussing in brief the cross-linking mechanism of the aforesaid cross-linking agents.

2.2.1 Glutaraldehyde Glutaraldehyde is a dialdehyde and is very commonly used for its ability to react with both protein and polysaccharides. It can stabilize protein and polysaccharide-based biopolymeric architectures with a very high efficiency. This cross-linking agent is a nonspecific cross-linker and the cross-linking can happen both between inter- and intramolecular functional groups. The aldehydic group present in the glutaraldehyde molecules has the capability to react with the free amino groups present in the polypeptide chain (Fan et al., 2018). Further, glutaraldehyde can also react with the hydroxyl groups which are present in both proteins and polysaccharide molecules (W. Wang et al., 2016). The mechanism of the chemical reaction has been provided in Fig. 1.8. It is noteworthy to mention that all the glutaraldehyde-based cross-linking occurs at low pH. Hence, the addition of acids in the reaction mixture is an essential step in glutaraldehyde-based cross-linking. Apart from mixing the glutaraldehyde reagent in the solution form to the polymeric solutions to induce cross-linking, many authors have reported vapor-phase cross-linking using glutaraldehyde (Destaye et al., 2013; Lu et al., 2015). In this process, glutaraldehyde solutions are made in different concentrations. The gels/matrices that need to be cross-linked are placed at the top of the container, which contains glutaraldehyde solutions of definite concentration using suitable attachments.

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

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FIGURE 1.8 Schematic representation for the cross-linking of the polymer using glutaraldehyde.

Thereafter, the container is completely sealed. This arrangement is incubated at a particular temperature for a definite time period. During the incubation process, the glutaraldehyde vapors are released from the solution, which then interacts with the samples to be cross-linked. It has been found that an alteration in the concentration of the glutaraldehyde solutions results in the differential cross-linking density (Lu et al., 2015).

2.2.2 Polycarboxylic acids Polycarboxylic acids like citric acid, maleic acid, succinic acid, and tricarballylic acid, which have more than three carboxylic groups, have been used to cross-link cellulose and starch molecules. These polycarboxylic acids are reported to react with the hydroxyl groups present in the cellulose and the starch molecules. During the cross-linking process, an intermediate polycarboxylic acid anhydride is reported to be formed (Fahmy and Fouda, 2008). A plausible mechanism for the reaction process using polycarboxylic acids has been provided in Fig. 1.9.

2.2.3 EDC coupling EDC, chemically known as carbodiimide, has been used to couple carboxyl groups to primary amines. The coupling reaction results in the formation of an intermediate compound, which is known as aminereactive O-acylisourea. This intermediate product is stabilized using NHS or sulfo-NHS to form an amine-reactive NHS or sulfo-NHS ester. Additionally, the O-acylisourea intermediate may also chemically react with another amine group which is present in a separate biopolymeric chain. This leads to the cross-linking of biopolymers having carboxyl and amine groups. It has been reported that the removal of the excess EDC may be done easily using dialysis and gel filtration techniques. This is possible due to the water-soluble nature of EDC (Conde et al., 2014). The probable mechanism of EDC/NHS cross-linking mechanism has been provided in Fig. 1.10.

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FIGURE 1.9 Schematic representation for the cross-linking of cellulose with tricarballylic acid.

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

FIGURE 1.10 Schematic representation for the cross-linking of polymer chains using EDC.

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2.2.4 Epichlorohydrin Epichlorohydrin is a chlorinated cyclic ether and has been used for cross-linking biopolymers like starch and chitosan. It covalently reacts with the hydroxyl groups present in the biopolymer backbone. The main advantage of the epichlorohydrin cross-linking in comparison to the glutaraldehyde crosslinking (which also reacts with the hydroxyl groups) is its ability to selectively react with the hydroxyl groups to form glycerol diether bridges (Motawie et al., 2014; Zhou et al., 2016). This is of importance while cross-linking biopolymers like chitosan that have additional functional groups like amines. Since epichlorohydrin spares the amine groups of chitosan biopolymer, the cross-linked chitosan matrix remains as cationic matrix and accordingly exhibits pH-sensitive character. Unlike glutaraldehyde cross-linking, the epichlorohydrin cross-linking is usually carried out under alkaline conditions (Kuniak and Marchessault, 1972). The schematic diagram of the epichlorohydrin-based cross-linking mechanism has been shown in Fig. 1.11.

2.2.5 Trisodium metaphosphite STMP is a nontoxic cross-linking agent. The Food and Drug Administration (FDA) has approved this cross-linking agent. STMP reacts with the primary alcoholic groups of the polysaccharides under strong alkaline conditions, which helps in achieving the cross-linking of the polysaccharide molecules. Usually, the cross-linking of the biopolymers using STMP is carried out at pH > 9.5. It is reported that the cross-linking of the biopolymers occurs as a two-step reaction. In the first step, the polysaccharide alkoxides, which are formed by the dissolution of the biopolymers in sodium hydroxide solution (alkaline media), undergo reaction with STMP. Due to this reaction, the cyclic structure of the STMP is compromised, and forms tripolyphosphated polymer. In the second step, the tripolyphosphated polymer so-formed reacts with another biopolymer alkoxide molecule, thereby resulting in the formation of a cross-linked matrix (Dulong et al., 2011). The cross-linking mechanism of the STMPbased cross-linking has been shown in Fig. 1.12.

2.2.6 Polysaccharide dialdehydes The mechanism of the reaction of the polysaccharide dialdehydes is similar to that of glutaraldehyde. The polysaccharide dialdehydes are formed by oxidizing the polysaccharides using periodate reaction. The oxidation process results in the cleavage of the C2eC3 bond of glucose residues. During the oxidation process two aldehyde units per monosaccharide units are formed, thereby resulting in the formation of 2,3-dialdehyde polysaccharides (Azeredo and Waldron, 2016). The mechanism of the formation of the oxidized polysaccharide via periodate oxidation has been provided in Fig. 1.13.

2.2.7 Genipin Genipin is a naturally occurring cross-linking agent. It is an iridoid glycoside that is extracted from the fruits of Gardenia jasminoids. The said cross-linker exhibits very low cytotoxicity and hence, has been proposed for cross-linking of biopolymers. The genipin molecules interact with the primary amine groups. The cross-linking reaction occurs via a two-step reaction mechanism. In the first step, the primary amine groups react with the genipin molecules via a nucleophilic reaction. This results in the formation of an unstable intermediate compound, which forms a tautomeric aldehyde. The aldehydic group thereafter forms a covalent bond with another amine group. This results in the cross-linking of

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

FIGURE 1.11 Schematic representation for the cross-linking of chitosan using epichlorohydrin.

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FIGURE 1.12 Schematic representation for the cross-linking of polysaccharide using trisodium metaphosphite (STMP).

2. Cross-linking methods employed to design biopolymer-based polymeric architectures

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FIGURE 1.13 Schematic representation for the cross-linking of polysaccharide and protein molecule through polysaccharide dialdehyde.

the two biopolymeric chains (Pal et al., 2009). The cross-linking mechanism of the genipin molecules has been shown in Fig. 1.14.

2.2.8 Proanthocyanidin PA is a naturally occurring cross-linking agent. It is produced as a side-product of the plant metabolism. This cross-linker has been reported to be available in seeds, flowers, nuts, barks, vegetables, and fruits (Pal et al., 2009). The use of PA has been reported to promote the conversion of the soluble collagen into insoluble collagen. Due to this property, synthetic PA is added to the culture medium to reduce the solubility of the collagen molecules (Pal et al., 2009). PAs are typically condensed tannins. Due to this reason, they consist of a large number of phenolic hydroxyl groups. The presence of such

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FIGURE 1.14 Schematic representation for the cross-linking of chitosan using genipin.

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FIGURE 1.15 Schematic representation for the cross-linking of polymer using proanthocyanidin.

hydroxyl groups allows interaction with the carboxylic groups present in the biopolymers and consequently cross-links them through the formation of ester linkages (Kim et al., 2005). The plausible cross-linking reaction scheme adopted by PA is represented in Fig. 1.15.

2.2.9 Glyoxal It is a dialdehyde with a chemical formula of C2H2O2 and is normally used as disinfectant in dentistry and medical healthcare (Aalto-Korte et al., 2005). Glyoxal, in comparison to glutaraldehyde, presents very little cytotoxicity (Wang and Stegemann, 2011) as its production and metabolism occur through normal cellular pathways (Rojas and Azevedo, 2011). Glyoxal mediates the cross-linking of polymers containing amine functional groups by forming a Schiff’s base (Parhi, 2017). Yang and coworkers (2005) studied the cross-linking of the chitosan fibers using glyoxal in order to enhance their mechanical properties. Glyoxal improves the mechanical strength of the polymer network by increasing the crystalline nature. This is due to the steric effect exerted by it on the cross-linked polymer network (Yang et al., 2005). The plausible mechanism adopted by the cross-linking of the chitosan fibers using glyoxal is presented in Fig. 1.16.

3. Biopolymers and their applications The exploitation of biopolymers in various fields of science is not a new concept. Naturally occurring polymers are now widely utilized in the food industry as the biodegradable and environmental friendly alternatives of their plastic counterparts. They are mainly utilized in the food industry as packaging materials, edible coatings, films, and delivery carriers for bioactive molecules (Grujic et al., 2017). Many different applications of the biopolymers in the medical field can be roughly categorized as: (i) drug delivery systems, (ii) implantable devices, (iii) wound healing products, and (iv) tissue engineering scaffolds (Davidenko et al., 2014). In this section, a comprehensive review of some of the

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FIGURE 1.16 Schematic representation for the cross-linking of chitosan using glyoxal.

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recent applications of the naturally derived polymers in food and biomedical industry will be discussed.

3.1 Chitosan Chitosan is a linear cationic polysaccharide obtained from the deacetylation of chitin. Chitin is considered as an important structural component of the exoskeleton of crustaceans, insects, and fungal cell walls (Cheung et al., 2015). Chemically, the polymer is aminoglucopyran and its structural framework consists of randomly arranged monomers, namely, b-(1 / 4)-linked D-glucosamine and N-acetyl-D-glucosamine (Nimesh, 2013). Structurally, the polymer contains three different kinds of chemical moieties: an amino group at C-2 and primary as well as secondary hydroxyl groups at C-3 and C-6, respectively (Manigandan et al., 2018). Deacetylated chitin, i.e., chitosan, is a cationic polymer and hence demonstrates solubility in an acidic environment. Due to this reason, it can interact with other natural or synthetic negatively charged polymers (Cheung et al., 2015). The degree of deacetylation and molecular weight of chitosan have a prominent effect on its characteristics (S.-K. Kim, 2010). Type of deacetylation procedure involved in the production of chitosan considerably influences its solubility and microstructural properties (Davidenko et al., 2014). Low-molecularweight chitosan (22 kDa) demonstrates solubility in physiological conditions as compared to its highmolecular-weight counterpart which is soluble in dilute acids (Misra, 2010). Chitosan has been reported to possess important biological properties such as antitumor, antimicrobial, antiviral, and hemostatic activities. It is also widely known in the biomedical arena for its biodegradable, biocompatible, nontoxic, and wound healing characteristics (Davidenko et al., 2014). This polymer has gained considerable attention of the researchers for its applications in food, pharmaceuticals, tissue engineering, and other biomedical pursuits due to its appealing features.

3.1.1 Food industry Owing to its inherent antimicrobial activity against fungi, bacteria, and yeast, this cationic polymer holds promising potential to be utilized as a food preservative in the food industry (No et al., 2007). The most hypothesized mechanism responsible for the antimicrobial action of chitosan exploits its strong positive character. This cationic polymer interacts with the negative microbial cell membrane and results in the release of the intracellular and proteinaceous components (Shahidi et al., 1999). Some other mechanisms through which chitosan demonstrates its antimicrobial action are by: (i) acting as a chelating agent, (ii) activating host defense mechanisms, (iii) working as a water-binding agent, and (iv) inhibiting mRNA and protein synthesis (Shahidi et al., 1999). Chitosan is also utilized in food industries as: (i) edible films (food wraps) for extending the shelf life of the vegetables and fruits, (ii) for encapsulation of enzymes, (iii) food additives such as dye-binding, emulsifying, antioxidant, gelling, and thickening agent, and (iv) dietary supplements (Agullo´ et al., 2003). Saral et al. (2019) reported the preparation of biodegradable packaging composite films from mahua oil a polyolbased polyurethane and chitosan. The films were incorporated with zinc oxide (ZnO) nanoparticles. Antimicrobial, mechanical, and thermal characteristics of the films were found to be improved after the incorporation of ZnO particles. The films also demonstrated noncytotoxic nature toward L929 fibroblast cells which suggested their use as a potential food packaging material (Saral, Indumathi and Rajarajeswari, 2019). Lin et al. (2018) reported the preparation of ε-polylysine/chitosan nanofibers as food packaging material exhibiting antimicrobial activity against Salmonella typhimurium and

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Salmonella enteritidis on chicken. The results suggested that the nanofibers maintained the sensory quality of the chicken and also demonstrated the expected antimicrobial activity (Lin et al., 2018). Salama et al. (2018) reported the preparation of chitosan biguanidine hydrochloride (CG) and SAbased edible films. The films were characterized through various techniques and also demonstrated significant antibacterial activity against B. subtilis, S. pneumoniae, and E. coli suggesting its potential applications in food packaging (Salama et al., 2018). Rattanaburi et al. (2018) reported the preparation of a novel emulsion using a combination of bacteriocin-producing lactic acid bacteria (Lactococcus lactis IO-1), chitosan, and hydroxypropyl methylcellulose (HPMC). Studies reported that HPMC/ chitosan/bacteria-based emulsions were highly stable and exhibited antimicrobial activity against gram-positive food pathogens, suggesting their promising potential to be used in the food industry (Rattanaburi et al., 2018). Kaya et al. (2018) proposed the development of chitosan-based edible films supplemented with Berberis crataegina fruit extract and seed oil. The physicochemical and biological characterization of the chitosan fruit extract films revealed the enhancement in their thermal stability, antimicrobial, quorum sensing, and antioxidant activity. The hydrophobicity of the films demonstrated much improved characteristics in comparison to the chitosan control films (Kaya et al., 2018).

3.1.2 Biomedical applications Owing to the excellent biocompatible, biodegradable, and nontoxic features, considerable attention has been paid to its biomedical, tissue engineering, biosensing, and drug delivery applications (Vunain et al., 2017). As a cationic polysaccharide, chitosan can interact electrostatically with negatively charged materials to form polyelectrolyte complexes (PECs) (Aminabhavi and Dharupaneedi, 2017). In a particular study conducted by Kilicarslan and coworkers, chitosan and alginate comprising adhesive polyelectrolyte complex films were prepared. The films were loaded with the drug clindamycin phosphate (CDP) for periodontal therapy (Kilicarslan et al., 2018). Its importance as a drug delivery vehicle is also enhanced due to its mucoadhesive nature and ability to loosen the tight epithelial junctions which aid in the delivery of the therapeutic agents (Ahsan et al., 2018). Chitosan and its derivatives demonstrate promising potential to be used as scaffolds for tissue engineering applications due to its physicochemical properties (Ahsan et al., 2018). Along with this, they also possess bacteriostatic property that offers several advantages for wound healing applications. Khorasani et al. (2018) reported the heparinized poly(vinyl alcohol) (PVA)/chitosan hydrogels incorporated with zinc oxide (ZnO) nanoparticles for the development of wound dressings (Khorasani et al., 2018). Azizian et al. (2018) reported the fabrication of composite scaffolds composed of chitosan and gelatin. Chitosan nanoparticles loaded with basic fibroblast growth factor (bFGF) and bovine serum albumin (BSA) were incorporated within the scaffolds. Characterization of the scaffolds revealed that the loading of the nanoparticles affected their physical properties and provided sustained release of the growth factor. This promoted fibroblast cell proliferation (Azizian et al., 2018). Shamekhi et al. (2019) reported the preparation of chitosan-based scaffolds incorporated with graphene oxide (GO) nanoparticles. The prepared scaffolds were comprehensively characterized through various techniques. It was revealed that with an increasing GO content, the scaffolds were physically and mechanically more stable. The nanocomposite scaffolds stimulated the proliferation and improved the morphology of human articular chondrocytes (Shamekhi et al., 2019). Li et al. (2019b) reported the development of collagen/chitosan gels supplemented with cell-penetrating peptide (CPP) (oligoarginine, R8) for cutaneous wound healing applications. The gels prepared were characterized through different techniques and it was revealed that the supplementation of the gels with CPP increased the inflammatory

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response and antibacterial activity. The authors suggested that the films possess potential wound healing activity (Li et al., 2019b). Ge et al. (2019) reported the fabrication of magnetic scaffolds based on Fe3O4/chitosan by freeze-drying method. The characterization revealed that the prepared scaffolds were highly biocompatible. The authors suggested the utilization of these scaffolds as the promising magnetic implants for tissue engineering applications (Ge et al., 2019).

3.2 Cellulose Cellulose is a natural linear polysaccharide that comprises of b-(1 / 4)-linked glucose monomeric units. It works as a major structural constituent in the cell walls of algae, bacteria, green plants, and vegetables (Mudgil, 2017). It is the most abundant polymer in nature and is well known for its high strength, fibrous, and water-insoluble nature (Zhang et al., 2014). Due to the presence of the three reactive hydroxyl groups per anhydroglucose unit in the polymeric structure of cellulose, this polymer contains large number of hydroxyl groups (Klemm et al., 2005). These functional and reactive hydroxyl groups are mainly responsible for the hydrophilic nature of the cellulose molecules (Bhat et al., 2017). The presence of extensive inter- and intramolecular hydrogen bonds imparts crystallinity to the cellulose which ultimately makes it insoluble in water (Mudgil, 2017). Various types of modified celluloses can be developed using different techniques such as oxidation, etherification, esterification, and micronization. Cellulose derivatives like HPMC, hydroxypropyl cellulose (HPC), microcrystalline cellulose (MCC), silicified microcrystalline cellulose (SMCC), hydroxyethyl cellulose (HEC), sodium carboxymethylcellulose (SCMC), ethyl cellulose (EC), methylcellulose (MC), and oxycellulose (OC) have found tremendous applications in food and biomedical industries (Lavanya et al., 2011). These cellulose derivatives, also known as cellulosic’s, can be modified in accordance with the required industrial applications (Lavanya et al., 2011). The recent advances in nanotechnology have modified the native cellulose fibers into different kinds of materials such as cellulose nanofibers (CNFs), cellulose nanocrystals (CNCs) or nanowhiskers, cellulose acetate (CA), and bacterial nanocellulose (BNC) (Gopinath et al., 2018). Cellulose is utilized in different fields such as pharmaceuticals, food, biomedical, and tissue engineering due to its attractive characteristics.

3.2.1 Food industry Different available forms of cellulose have been approved by FDA for utilization in food products as texturizing, bulking, anticaking, and emulsifying agent, extender, fat substitute, and filter aid in the filtration of juices (Lavanya et al., 2011). The most widely available derivative of cellulose, i.e., microcrystalline cellulose (MCC), has been tremendously exploited as a functional food ingredient in meat and dairy products, beverages, emulsions, bakery, and confectionery (Nsor-Atindana et al., 2017). Feng et al. (2018a) reported the exploitation of MCC along with the carboxymethylcellulose sodium (CMC-Na) for the microencapsulation of the astaxanthin (a carotenoid pigment). The stability, solubility, and antioxidant activity of the pigment was found to be improved after encapsulation. Astaxanthin yogurt demonstrated characteristic orange-red color with significantly improved stability and antioxidant ability in comparison to plain yogurt (Feng et al., 2018a). EC is an ether derivative of cellulose (Gravelle and Marangoni, 2018) and has been used in a range of applications in food, pharmaceutical, and biomedical industries owing to its gelation properties (Davidovich-Pinhas et al., 2018). It possesses excellent film-forming and moisture barrier ability (Gravelle and Marangoni, 2018), considerable mechanical strength, flexible nature, and transparency (Davidovich-Pinhas et al.,

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2018). Go´mez-Estaca et al. (2019) reported the preparation of EC and beeswax oleogels based on olive, linseed, and fish oils. The oleogels were checked for their suitability as potent animal fat replacers for pork liver paˆte´s. As compared to beeswax oleogels, the EC-based oleogels were softer, thermally stable, and flexible (Go´mez-Estaca et al., 2019). MC, another cellulose derivative, is linear polysaccharide with substituent methyl groups. It has promising applications in food packaging owing to its properties like film-forming ability, easy availability, etc. de Dicastillo et al. (2016a, b) reported the preparation of active MC and murta fruit (MU) extractebased films. Films were further crosslinked with glutaraldehyde for improved water resistance (de Dicastillo et al., 2016a). High consumption of solid fat creates serious health issues such as obesity, diabetes, and heart-related diseases due to its high saturated fat content. Lee et al. (2018) reported the preparation of HPMC and sunflower oil based oleogels. Foam-structured HPMC was employed to formulate sunflower oil oleogels. The oleogels were analyzed for their suitability as shortening replacer in muffins. The analysis demonstrated positive indications for the use of HPMC oleogels as a replacer of shortening in muffins without degrading more than 50% of its properties (Lee, 2018). Singh et al. (2019) reported the preparation of edible films based on CMC-Na and HEC cross-linked with citric acid for food packaging applications. The matrices were loaded with the model probiotic bacteria, Lactobacillus rhamnosus GG (LGG). The matrices demonstrated efficient entrapment and good viability of the probiotics (Singh et al., 2019).

3.2.2 Biomedical applications A study conducted by Zulkifli and coworkers (2014) reported the fabrication of fibrous membranes based on HEC and PVA by electrospinning method. The mats were chemically cross-linked with glutaraldehyde and then characterized through different techniques. The nanofibrous scaffolds were assessed for their cytotoxic nature using human melanoma cells by the MTT assay. The results revealed that the mats were highly biocompatible and enhanced the viability of the cells suggesting their potential for skin tissue engineering applications (Zulkifli et al., 2014). El-Naggar et al. (2017) reported the preparation of blend hydrogels using synthetic polymer PVA and different ratios of MC using gamma irradiation. The characterization of the hydrogels revealed their pH-sensitive swelling nature. PVA/MC hydrogels also demonstrated pH-sensitive and controlled release behavior of the model drug named doxorubicin (El-Naggar et al., 2017). The use of CNCs to improve the performance of the polymeric scaffolds has been reported. CNC is used due to its unique properties such as small size, high surface area, and high mechanical strength. Huang et al. (2018) reported the synthesis of poly(butylene succinate) (PBS) and CNC-based biocomposite scaffolds by electrospinning technique for tissue engineering applications. Studies suggested that incorporation of CNC increased the thermostability and crystallinity of the PBS matrix. Cell culture results demonstrated that the scaffolds were suitable for tissue engineering applications (Huang et al., 2018). Ao et al. (2017) fabricated nanofiber scaffolds utilizing cotton cellulose and nano-hydroxyapatite by electrospinning for bone tissue engineering. The nanofibers were characterized and found to be biocompatible with human dental follicle cells (Ao et al., 2017). Zulkifli et al. (2018) reported the preparation of 3D scaffolds based on PVA and HEC by freeze-drying technique. The prepared scaffolds possessed higher porosity. Human fibroblast (hFB) cells, cultured on the polymer matrices, revealed improved proliferation which indicated their applicability in skin tissue engineering (Zulkifli et al., 2018).

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3.3 Pullulan Pullulan (pul) is an important biopolymer that is obtained from the yeast Aureobasidium pullulans. Maltotriose serves as the building block of pullulan. Three glucose units of maltotriose are linked together through a-(1 / 4) glycosidic bond and the consecutive maltotriose units are joined to each other by a-(1 / 6) bonds. It demonstrates activity against some enzymes such as invertase, amylases, and glucosidases (Kumar et al., 2012). It has been reported that the pullulan structure sometimes consist of maltotetrose residues. Pullulan obtained from certain strains contains some other types of linkages such as a-(1 / 3), b-(1 / 3), and b-(1 / 6) in the backbone structure. Interestingly, the dimeric segments of this polymer structure comprise of [/x)-a-D-glucopyranosyl-(1 / 4)-a-Dglucopyranosyl-(1/] and [/4)-a-D-glucopyranosyl-(1 / 6)-a-D-glucopyranosyl-(1/], where x may be either 4 or 6 for the (1 / 4)-linked segment. Its structure is considered to be an intermediate between amylase and dextran structures and is attributed to the presence of both a-(1 / 4) and a-(1 / 6) glycosidic bonds (Singh et al., 2015). However, the differences between them are mainly due to the proportion of these linkages. Pullulan mainly contains 30% of a-(1 / 6) glycosidic linkages, while maltodextrin contains approximately 20% of it (Park and Khan, 2009). The polymer is water-soluble. However, its solubility can be controlled through chemical modifications (Singh et al., 2015). It does not demonstrate solubility in organic solvents except in dimethylformamide and dimethylsulfoxide solvents (Park and Khan, 2009). Its molecular formula is C6H10O5 and molecular weight varies between 45 and 600 kDa. Pullulan is odorless, biodegradable, and tasteless polysaccharide due to which it is used as a nonpolluting film to wrap food supplements (Singh et al., 2015). This biopolymer is nonimmunogenic, nontoxic, noncarcinogenic, edible, nonhygroscopic (Trinetta and Cutter, 2016), anticoagulant, antithrombotic, and antiinflammatory in nature (Tabasum et al., 2018).

3.3.1 Food industry Pullulan is used in various forms such as edible films and coatings, noncalorific food ingredient, and protective coating for food products (Tabasum et al., 2018). Shao et al. (2018) reported the preparation of Pul and CMC-Na-based nanofibers. The nanofibers were incorporated tea polyphenols (TP) by electrospinning technique. The nanofibers were evaluated for their potential use as fruit packaging material. Studies demonstrated that the Pul-CMC-based nanofibers reduced weight loss and maintained the firmness of the strawberries (Shao et al., 2018). Silva et al. (2018) developed nanocomposite films based on Pul and lysozyme nanofibers (LNFs) using a simple solvent casting technique for active packaging of food materials. The antibacterial effect of these nanocomposite films against Staphylococcus aureus was investigated. The results suggested the use of the films in active food packaging (Silva et al., 2018). Zhang et al. (2018c) reported the preparation of SA- and Pul-based composite films incorporated with capsaicin (an active ingredient of red pepper). The films containing different proportion of the capsaicin were prepared and then characterized. Studies revealed that the films demonstrated good antibacterial activity against E. coli and S. aureus. Also, the capsaicin-incorporated films improved the shelf life of apples. This suggested the potential of the films to be used in food packaging (Zhang et al., 2018c). Pattanayaiying et al. (2015) reported the development of Pul films containing antimicrobial agents, namely, lauric arginate (LAE) and nisin Z (isolated form L. lactis), either alone or in combination. The films were investigated to have control over the growth of foodborne pathogens on fresh or ready-to-eat muscle foods. Raw turkey breast

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Chapter 1 Introduction of biopolymers

slices covered with these films demonstrated gradually decreasing population of the pathogens including Salmonella typhimurium and Salmonella enteritidis. The overall studies revealed that these films exhibited good activity against foodborne pathogens on fresh and processed muscle foods (Pattanayaiying et al., 2015).

3.3.2 Biomedical applications The pyranose rings per maltotriose subunit in the structure of pullulan contains nine hydroxyl groups which impart characteristic properties to this biopolymer. For its utilization in the biomedical field, these functional groups can be easily derivatized in different forms (Singh et al., 2017b). Tian et al. (2018) reported the preparation of orodispersible films (ODFs) of trehalose and Pul by air- and freezedrying techniques for the delivery of therapeutic proteins. Characterization of trehalose/Pul-based ODFs revealed their suitability for their application in protein delivery via the oral cavity (Tian et al., 2018). (Li et al. 2018a) reported the fabrication of injectable hydrogel scaffolds based on adipic hydrazide functionalized chondroitin sulfate and oxidized pullulan (oxPL) for cartilage tissue engineering. The hydrogels were prepared through covalent hydrazone cross-linking of these two polysaccharides without the involvement of any chemical cross-linking agents. Rabbit articular chondrocytes encapsulated in this self-cross-linked and biodegradable CS-ADH/oxPL hydrogels demonstrated good viability (Li et al., 2018a). Atila et al. (2016) reported the preparation of 3D electrospun fibrous scaffolds based on Pul and CA for bone tissue engineering. These fibrous matrices were cross-linked with chemical cross-linker STMP in order to provide mechanical stability. To investigate the structural and mechanical properties, the P50/CA50 scaffolds were seeded with human osteogenic sarcoma cell line (Saos-2). Cross-linked P50/CA50 scaffolds demonstrated positive results for bone tissue engineering applications (Atila et al., 2016). Liang et al. (2018) reported the preparation of pH-responsive and mucoadhesive injectable hydrogels based on chitosan-grafted dihydrocaffeic acid (CS-DA) and oxPL. Sustained and pH-sensitive in vitro drug release behavior of the CS-DA/oxPL hydrogels was demonstrated using doxorubicin (DOX) as a model drug. The hydrogels showed good mucoadhesive and pH-responsive properties and their potential for localized drug delivery applications (Liang et al., 2018). Soni and Ghosh (2017) also reported the preparation of interpenetrating network (IPN) microspheres based on Pul and PVA. The microspheres were prepared by employing glutaraldehyde-assisted water-in-oil emulsion cross-linking method. IPN microspheres were investigated as a delivery system for the drug pirfenidone. Studies revealed that the microspheres were noncytotoxic and were promising candidate for the controlled delivery of pirfenidone (Soni and Ghosh, 2017).

3.4 Hyaluronic acid Hyaluronic acid (HA), also referred as “hyaluronan,” is a linear polysaccharide. It is framed from alternating repeating units of b-(1 / 4)-linked D-glucuronic acid and b-(1 / 3)-linked N-acetyl-Dglucosamine (Kogan et al., 2007). It belongs to the family of mammalian glycosaminoglycans. Unlike the other members of the family, HA is nonsulfated and is structurally simpler polysaccharide which is not covalently linked to a core protein (Kogan et al., 2007). It serves as a crucial component of synovial fluid (SF). The molecular weight of HA ranges between 1.6  106 and 10.9  106 g mol1 (Herzog et al. 2019). HA can be extracted from different sources such as animal tissues (tissue joints, brain cartilage, cockscombs), and by fermentation utilizing various bacteria such as Pseudomonas

3. Biopolymers and their applications

27

aeruginosa and Streptococci. On considering the structure, HA molecule contains a large number of hydroxyl and carboxyl groups that can lead to the formation of numerous inter- and intramolecular hydrogen bonds in aqueous solutions. These hydrogen bonds contribute to the rigidity of the HA structure and result in the formation of single or double helical conformation. Due to the absence of noncovalent linkages in its structure, HA exhibits viscoelastic properties. Due to this reason, HA is widely used as biomaterial in tissue engineering and pharmaceutical applications (Huang and Chen, 2018). Interaction with water heavily hydrates the HA structure and results in the formation of viscous gels. This property contributes to its ability to serve as: (i) scaffold for extracellular matrix (ECM) organization, (ii) regulator of viscoelasticity of physiological fluids, and (iii) controller of tissue hydration (Tiwari and Bahadur, 2018). HA interacts with cell-specific glycoproteins such as CD44 and intercellular adhesion molecule-1 (ICAM-1) thereby stimulating signaling pathways (Tiwari and Bahadur, 2018) (Tiwari and Bahadur, 2018). HA exhibits various pharmacological activities, including, but not limited to, anticancer, antiinflammatory, antidiabetic, antiaging, immunomodulatory, skin repairing, and wound healing (Bukhari et al., 2018). Although HA has found numerous applications in the biomedical field, no significant literature could be found for its use in the food industry.

3.4.1 Biomedical applications HA has been used in biomedical applications owing to its peculiar properties such as hydrophilic nature and rheological behavior. Hydrogels prepared from HA can serve as scaffold materials which imitate the natural tissue conditions and provide a 3D elastic and compressive network to the cultured cells (Tiwari and Bahadur, 2018). Larran˜eta et al. (2018) reported the preparation of HA-based hydrogels cross-linked with poly(methyl vinyl ether-alt-maleic acid) using environment-friendly thermal and microwave processes. The hydrogels were then evaluated as a delivery system for the model drug (methylene blue). Additionally, the materials were also used to prepare microneedle arrays to be used as transdermal drug delivery systems. The antimicrobial studies of the hydrogels suggested their potential application as medicated wound dressings or coatings for noninfective catheter (Larran˜eta et al., 2018). Xie et al. (2018) reported the preparation of HA-containing ethosomes (HA-ES) as a potential transdermal drug delivery carrier using rhodamine-B (RB) as the model drug. Characterization of HA-ES-RB carriers through various techniques revealed their small particle size, stable nature, and biocompatibility. Skin permeation tests demonstrated that the penetration effect of HA-ESRB was more prominent in comparison to ES-RB (Xie et al., 2018). Chanda et al. (2018) fabricated bilayered polymeric scaffolds of chitosan (CS)/polycaprolactone (PCL) and HA by electrospinning technique. The CS/PCL mesh network was laid over by electrospun HA fibers for increasing the mechanical stability, cytocompatibility, and hydration of the wound bed. The bilayered scaffolds demonstrated increased swelling, degradation, hydrophilic nature, and water vapor transmission rate. Moreover, antimicrobial studies showed decreased adhesion of bacteria on the bilayered scaffolds (Chanda et al., 2018). Wang et al. (2019) reported the preparation of the composite scaffolds based on silk fibroin, collagen, and HA (SF/COL/HA) incorporated with pilose antler polypeptides (PAPs)PLGA microspheres. The prepared scaffolds were then implanted into rabbit cartilage defect model and studied for their cartilage repair effect. The results demonstrated that the PAP-SF/COL/HA scaffolds had a positive effect on the articular cartilage repair (Wang et al., 2019).

28

Chapter 1 Introduction of biopolymers

3.5 Alginate It is a natural anionic polysaccharide which is obtained from brown seaweeds, such as Laminaria hyperborea, Laminaria digitata, Laminaria japonica, and Macrocystis pyrifera (Lee and Mooney, 2012). It is mainly present in the cell wall and intracellular spaces of brown seaweeds and provides flexibility and strength to the plant (Venkatesan et al., 2014). Structurally, alginate is a linear copolymer of b-(1 / 4)-linked mannuronic acid and a-(1 / 4)-linked guluronic acid. The monomers can be arranged in the form of blocks composed of consecutive G-residues (GGGG), consecutive Mresidues (MMMM), and alternating M or G residues (GMGM) (Venkatesan et al., 2014). Based on the source of extraction, alginates show significant differences in the M or G content and the length of each block. Interestingly, G-blocks are considered to play an important role in the interaction of alginate with divalent metal ions through intermolecular cross-linking to form hydrogels (Lee and Mooney, 2012). Calcium (Ca2þ) ion is generally used for inducing gelation of alginate due to its clinical safety and availability. The mechanism of gelation is based on the substitution of monovalent ions of G-blocks with the Ca2þ ions and consequent formation of “egg-box structure.” High G content results in the formation of strong, brittle, stiff, and porous gels, while higher M content forms more weak and elastic gels (Abedini et al., 2018).

3.5.1 Food applications Alginate and its derivatives have an excellent functionality in the food industry as thickening, gelling, film-forming, and stabilizing agent (Qin et al., 2018). It has also been recently utilized for the encapsulation of bioactives owing to its low toxic and immunogenic nature, simple structure, and high availability (Khaksar et al., 2014). Khaksar et al. (2014) reported the development of alginatehigh methoxy pectin (HMP) microparticles as a delivery system for a natural preservative, nisin. The microparticles were spherical in shape and housed the bacteriocin within the particles. The in vitro release studies demonstrated that the release from the particles was via Fickian diffusion mechanism (Khaksar et al., 2014). Zhang et al. (2016) reported the synthesis of calcium alginate beads for encapsulating whey protein using an extrusion device with a vibrating nozzle. The synthesis was carried out at three different pH values. High retention and encapsulation of the protein were obtained when the pH of the synthesis medium was low while the protein release was highest when the pH of the synthesis medium was high. The results suggested that the beads were suitable for protein delivery applications (Zhang et al., 2016). The disposing problems associated with the use of conventional packaging materials can be eradicated by exploiting biodegradable polymers. Aziz et al. (2018) reported the preparation of edible films comprising of SA and castor oil (CO). Physicochemical characterization of the films suggested that the addition of CO enhanced their thermal and mechanical stability. Incorporation of CO also resulted in a significant increase in the antimicrobial activity of the edible films against gram-positive bacteria. However, no activity was observed against gram-negative bacteria (Aziz et al., 2018). Falco´ et al. (2019) reported the development of SA-oleic acid (OA)-based coatings. The coatings contained green tea extract (GTE) (as an antiviral agent) for preserving fresh strawberries and raspberries. The studies suggested that the antioxidant characteristics of the SA-OA-GTE coatings were not pH dependent. However, their antiviral activity was most effective at pH 5.5. The applied film-forming dispersion on the fresh berries demonstrated effective control over the infectivity of murine norovirus (MNV) and hepatitis A virus (HAV) (Falco´ et al., 2019).

4. Conclusion

29

3.5.2 Biomedical applications Supramaniam et al. (2018) reported the preparation of pH-sensitive hydrogel beads composed of alginate. The beads were incorporated with magnetic-CNCs (m-CNCs). The effect of the m-CNCs on the integrity, swelling, and in vitro drug release behavior of the beads was evaluated. The studies revealed that the incorporation of m-CNCs enhanced the mechanical strength and swelling degree of the hydrogel beads with controlled drug release of ibuprofen (Supramaniam et al., 2018). Montalbano et al. (2018) reported the formation of the thermoresponsive tricomponent hydrogel of collagen, alginate, and fibrin (CAF). The hydrogels showed similar stiffness as that of the native soft tissue. Cytocompatibility studies of the hydrogels were assessed using three different cell lines: L929 murine fibroblasts, pancreatic MIN6 b-cells, and human mesenchymal stem cells (hMSCs). The hydrogels were found to support the cell survival and proliferation by providing favorable microenvironment to the cells (Montalbano et al., 2018). Kamoun et al. (2015) proposed the development of hydrogel membranes comprising of PVA and SA as a potential wound dressing material. The hydrogel matrices were prepared by freeze-thaw method and were loaded with a model antibiotic, sodium ampicillin. The films with increased content of SA exhibited higher protein adsorption, swelling index, surface roughness, and hydrolytic degradation. The PVA-SA films demonstrated good hemocompatibility and in vitro antibacterial activity suggesting their use for wound dressing applications (Kamoun et al., 2015). Diaz-Rodriguez et al. (2018) proposed the preparation of novel mineralized alginate hydrogels incorporated with marine-derived calcium carbonate (biomineral) particles for bone tissue engineering applications. The study suggested that the calcium carbonate microparticles promoted the ECM calcification and osteoblastic differentiation of hMSCs (Diaz-Rodriguez et al., 2018). Park et al. (2015) reported the preparation of cellulosealginate composite hydrogels for cell encapsulation. The hydrogel composite was prepared by incorporating TEMPO-mediated oxidized bacterial cellulose (TOBC) into SA solution. The hydrogels were subsequently cross-linked in the presence of Ca2þ solution. TOBC/SA composites were found to be mechanically and chemically more robust in comparison to SA hydrogels. The cells which were encapsulated in the composite beads demonstrated good viability and proliferation (Park et al., 2015). A concise set of recent examples in the last 5 years of naturally derived biopolymers in the field of food and biomedical applications has been tabulated in Tables 1.1 and 1.2, respectively.

4. Conclusion Natural polymers are obtained from different living organisms such as plants, animals, microbes, and fungi, where they perform particular functions necessary for survival. Biopolymers can be categorized broadly as proteins, polynucleotides, and polysaccharides. In spite of belonging to different backgrounds, these versatile materials are nowadays extensively utilized in food and biomedical industries. Major advancements have been performed in the area of cross-linking techniques to impart adequate mechanical strength to the biopolymer products. However, concerns about the impact of the chemical cross-linking methods on the safety of the products and new nontoxic cross-linking techniques have also been reviewed in this chapter. Along with this, new and sophisticated developments in the use of biopolymer-based products in the field of food and biomedical sciences have been comprehensively summarized.

30

Chapter 1 Introduction of biopolymers

Table 1.1 Examples of cross-linking agents utilized in food applications. S.No.

Components

Cross-linking agents

Application

Reference

1.

Cush-cush yam and cassava starch Gelatin, aminofunctionalized montmorillonite Feather keratin (FK)

Trisodium metaphosphite (STMP) Dialdehyde xanthan gum (DXG)

Food packaging

Gutie´rrez et al. (2015) Ge et al. (2015)

Dialdehyde starch (DAS)

Food packaging

Transglutaminase (TGase)

Food packaging

CaCl2

Enteric microprobioticcarrier for potential probiotic delivery Entrapment matrix for probiotic lactic acid bacteria and their protection from simulated gastrointestinal conditions _

2.

3. 4. 5.

6.

Gelatin and calcium carbonate Chitosan hydrochloride and alginate Gelatin and maltodextrin

TGase

7.

Xanthan gum

STMP

8.

Alginate, hylon starch, chitosan, and poly-L-lysine

Genipin

9.

TGase

10.

Gelatin, curcuminloaded zein nanoparticles, and calcium propionate CMC and chitosan

Genipin

11.

Pectin and starch

CaCl2

12.

CMC and chitosan

Genipin and physical cross-linking

13.

Chitosan and dextran sulfate

Genipin and physical cross-linking

14.

Arabinoxylan

Laccase

15.

Mechanically deboned chicken meat protein (MDCM-P)

TGase

Edible films

Encapsulation and oral delivery of probiotics Lactococcus lactis and Bifidobacterium longum Edible coatings for the storage of Benitaka grapes

Encapsulation and delivery of probiotic bacteria (Lactobacillus rhamnosus GG) Encapsulation and delivery of L. plantarum ATCC: 13643 cells Encapsulation of probiotic bacteria (Lactobacillus rhamnosus GG) Encapsulation of probiotic bacteria (Lactobacillus acidophilus GG) Extension of shelf life of Persian lime Edible packaging film

Dou et al. (2015) Wang et al. (2015) Wu et al. (2016) Nawong et al. (2016)

Tao et al. (2016) Yeung (2016)

Lemes et al. (2017)

Singh et al. (2017a)

Dafe et al. (2017) Singh et al. (2017a) Falco et al. (2017) Gonza´lezEstrada et al. (2017) Yayli et al. (2017)

4. Conclusion

31

Table 1.1 Examples of cross-linking agents utilized in food applications.dcont’d S.No.

Components

Cross-linking agents

Application

Reference

16.

PVA, alginate, and borate-stabilized silver nanoparticles (AgNPs) Zein

CaCl2 and physical crosslinking by freeze-thaw cycles

Active food packaging

Narayanan and Han (2017)

Tannic acid

18.

Sesame protein isolate and guar gum

Citric acid, maleic acid, succinic acid

Santos et al. (2018) Sharma et al. (2018)

19. 20.

Gelatin, rosmarinic acid (RosA) Casein

EDC/NHS and dialdehyde xanthan gum Tannic acid

Enhancing the shelf life of guava Bilayer coatings used for the shelf life extension of fresh-cut pineapple Food and pharmaceutical packaging Food packaging

21.

Whey protein isolate

Self-assembly

22.

Starch and gelatin

23.

Sodium alginate (SA), gum acacia, and silver nanoparticles Gelatin, poly(acrylic acid-co-acrylamide), and montmorillonite clay (MMT) Gelatin

Starchbutanetetracarboxylic acid dianhydride-Nhydroxysuccinimide (starch-BTCAD-NHS) Glutaraldehyde

17.

24.

25. 26.

27. 28. 29. 30.

Peach gum polysaccharide (PGP) and Auricularia polytricha b-glucans (APP) Alginate, pectin, and immortelle extract Alginate Gelatin, zein, and curcumin Chitosan and alginate

Retarding the oxidation and browning of food products Food packaging and extending the shelf life of peeled apples

Ge et al. (2018) Picchio et al. (2018) Feng et al. (2018b) Tao et al. (2018)

Extension of shelf life of black grapes (Vitis vinifera)

Kanikireddy et al. (2019)

Citric acid

Controlled release of vitamin B12

Nath et al. (2019)

Genipin

Vitamin B6 delivery

Fe3þ

Carrier for probiotic strainsdL. plantarum CICC20264 and L. salivarius CICC23174

Teimouri et al. (2019) Zhu et al. (2019)

CaCl2

Active food packaging

CuSO4

Delivery of folic acid

Green tea extract (GTE)

Active food packaging

Layer-by-layer assembly and ferulic acid crosslinking

Food packaging

Karaca et al. (2019) Camacho et al. (2019) Alehosseini et al. (2019) Li et al. (2019)

32

Chapter 1 Introduction of biopolymers

Table 1.2 Examples of cross-linking agents utilized in biomedical applications. S.No.

Components

Cross-linking agent

Application

Reference

1.

Chitosan, poly(vinyl alcohol) (PVA), zinc oxide nanoparticles Poly g-glutamic acid (g-PGA) and ε-polylysine (ε-PL)

Boric acid

e

Nanfang (2015)

Carbodiimide (EDC) and Nhydroxysuccinimide (NHS) Proanthocyanidin

Wound dressing, drug delivery, and contact lens

Hua et al. (2016)

Choi et al. (2016)

Glutaraldehyde

Periodontal tissue engineering for delayed replantation Active food packaging

Ca(NO3)2

Wound healing

Basu et al. (2017)

Polyethylene glycol

Central nervous system tissue engineering Musculoskeletal tissue engineering

Zhuo et al. (2017)

2.

3.

Collagen

4.

6.

Methylcellulose (MC) and maqui (Aristotelia chilensis) berry fruit extract Nanofibrillated cellulose (NFC) Hyaluronan and MC

7.

Silk fibroin, pul

8.

Chitosan and alginate

9.

Gelatin, sodium alginate (SA), and carboxymethylcellulose (CMC) Carboxymethyl chitosan (CMCS)

Glutaraldehyde, CaCl2

Genipin

13.

Carboxymethyl chitosan (CMCS) and poloxamer 407 (F127) CMCdpolyethylene glycol (PEG) Chitosan and gelatin

14.

Portulaca mucilage

15.

Oxidized hyaluronic acid (HA), type I collagen, and betatricalcium phosphate (b-TCP)

5.

10.

11.

12.

Horseradish peroxidase (HRP), hydrogen peroxide (H2O2) Glutaraldehyde, CaCl2

Genipin

Citric acid Oxidized sucrose Alginate, epichlorohydrin Oligomeric proanthocyanidins (OPCs)

De Dicastillo et al. (2016b)

Li et al. (2018b)

Temporomandibular joint disc regeneration e

Bousnaki et al. (2018) Dai et al. (2018)

Modulation of osteogenic and hemostatic activities Ocular drug delivery

Zhang et al. (2018a)

Controlled release of poorly soluble drugs Ocular drug delivery

Ghorpade et al. (2018) El-Feky et al. (2018) Asnani et al. (2018) Wei et al. (2018)

Colon-targeted drug delivery Treatment of advanced chronic periodontitis

Yu et al. (2018)

4. Conclusion

33

Table 1.2 Examples of cross-linking agents utilized in biomedical applications.dcont’d S.No.

Components

Cross-linking agent

Application

Reference

16.

Starch, k-carrageenan

Glyoxal

Drug delivery

17.

Hyperbranched polysaccharide (TM3a), xanthan gum Psyllium seed husk polysaccharide (PSH), and human hair keratins (KER) Hydrolyzed collagen, acrylic acid, and methacrylic acid

Trisodium metaphosphite (STMP)

Drug delivery

Sonawane and Patil (2018) Zhang et al. (2018b)

STMP

Diabetic wound healing

Ponrasu et al. (2018)

N,N0 -methylene bisacrylamide (NMBA), ammonium persulfate (APS), and N,N,N0 ,N0 -tetramethyl ethylenediamine (TEMED) Citric acid

Drug delivery

Noppakundilograt et al. (2018)

Wound dressing material Surgical glue and delivery system for tissue engineering and regenerative medicine Controlled drug release system Faster regeneration of skin in diabetic wounds

El Fawal et al. (2018) Kim et al. (2018)

Shape memory gels

Chang et al. (2018a)

Colon-specific delivery of 5fluorouracil Dynamic tissue engineering Doxorubicin delivery in breast cancer

Asnani et al. (2018)

Gastroretentive drug delivery system Corneal tissue engineering e

Patil-Vibhute and Hajare (2019) Goodarzi et al. (2019) Zhu et al. (2019)

18.

19.

20. 21.

22. 23.

24.

25.

Hydroxyethylcellulose (HEC) Tyramine-conjugated hyaluronic acid and gelatin Chitosan, gelatin, b-cyclodextrin (CD) Curcumin, cytomodulincoupled porous PLGA microparticles (cPMS), and gelatin Poly(ureidopyrimidone methacrylate-co-stearyl acrylate-co-acrylic acid) Portulaca plant mucilage and SA

Novel tyrosinase derived from Streptomyces avermitilis Glutaraldehyde Glutaraldehyde

Hydrophobic interactions and hydrogen bonds Borax and calcium chloride

26.

Gelatin

mTransglutaminase

27.

Chitosan, Nisopropylacrylamide (NiPAAm), itaconic acid (IA) Chitosan and PVA

Glycerophosphate (GP)

28. 29. 30.

Type I collagen (COL) and gelatin Chondroitin sulfate and dopamine

Glyoxal EDC/NHS EDC/NHS

Kaur et al. (2018) Bulbake et al. (2018)

Jepsen et al. (2019) Fathi et al. (2019)

34

Chapter 1 Introduction of biopolymers

Acknowledgments The present study has been conducted under Indo-Korea joint research program of Department of Science and Technology, Government of India (Sanction order # INT/Korea/P-37, June 15, 2017), and under the framework of International Cooperation Program managed by the NRF (2016K1A3A1A19945059), Republic of Korea.

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Chapter 1 Introduction of biopolymers

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CHAPTER

Enzymatic synthesis of flavonoid glucosides and their biochemical characterization

2

Thi Thanh Hanh Nguyen1, Juhui Jin2, Iis Septiana2, Dilshad Quereshi3, Kunal Pal3, Doman Kim1, 2 The Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea; 2Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea; 3Department of Biotechnology & Medical Engineering, National Institute of Technology, Rourkela, Odisha, India 1

1. Introduction Flavonoids are the natural products, mostly extracted from vegetable, fruits, nuts, seeds, stems, and flowers as well as tea and alcohol beverage of plant origin and belong to a class of plant secondary metabolites having a polyphenolic structure (Panche et al., 2016). Currently, approximately 6000 flavonoids have been reported (Panche et al., 2016). The common chemical structure of flavonoids is a 15-carbon skeleton, comprising of two phenyl rings (A and B) and one heterocyclic ring (C). Based on the numbers and positions of the hydroxyl group and the C-ring structures, flavonoids are classified into six major subclasses as flavones (apigenin, luteolin, tangeritin), flavonols (fisetin, kaempferol, myricetin, quercetin), flavanones (hesperetin, naringenin), flavanols (catechin, gallocatechin, epicatechin, epigallocatechin 3-gallate), anthocyanins (cyaniding, delphinidin, malvidin), and isoflavones (genistein, daidzein, glycitein) (Ross and Kasum, 2002). Flavonoids have many biological functions such as protection of the plant against fungal infection and UV irradiation or oxidation (Treutter, 2005), enzyme inhibition (Hua et al., 2018; Walker et al., 2000), antioxidant (Gomes et al., 2016), antiinflammation (Middleton, 1998), antiaging (Lin et al., 2015), neuroprotective (Prakash and Sudhandiran, 2015), antimicrobial (Nguyen et al., 2017), and antitumor activities (Kanadaswami et al., 2005). Based on the National Health and Nutrition Examination Survey (NHANES), the dietary flavonoid consumption of US adults was likely as 1.6
0.2 mg/day of flavone, 12.9
0.4 mg/day of flavonol, 156.5
11.3 mg/day of flavanol, 14.4
0.6 mg/day of flavanone, 3.1
0.5 mg/day of anthocyanin, and 1.2
0.2 mg/day of isoflavone (Chun et al., 2007), and based on the consumption of vegetables and fruits, the flavonoid intake can range between 50e800 mg/day (Pietta, 2000). Despite numerous medicinal properties of flavonoids, their low water solubility leads to slow absorption, inconstant bioavailability, and intestinal mucosal toxicity (Savjani et al., 2012). Previous studies reported that these carbohydrate parts have role in the absorption (Makino et al., 2009), bioavailability (Makino et al., 2009), bioactivity, and water solubility (Lepak et al., 2015) and protect flavonoids from oxygen and/or light degradation (Yoshino et al., 2016). Consequently, the flavonoid Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00002-3 Copyright © 2020 Elsevier Inc. All rights reserved.

47

48

Chapter 2 Enzymatic synthesis of flavonoid glucosides

glucosides have gained attention due to their enhanced solubility, stability, and functionality. The normal flavonoids almost exist in the form of b-glycosides in flavone/flavonol subclass (Plaza et al., 2014). Glycosylation of flavonoid using enzymatic methods is an effective approach to rise their solubility and stability in water (Plaza et al., 2014), which also results to increase in their bioavailability and decrease in the toxicity and harmful effects (Xu et al., 2016). For example, the kaempferol-3-O-b-Dglucopyranosyl-(1-4)-O-a-glucopyranoside synthesized using cyclodextrin glucanotransferase (CGTase) exhibited a 65-fold increase in the water solubility compared to kaempferol-3-O-b-D-glucopyranosyl (Choung et al., 2017). Astragalin 6-glucosides synthesized using glucansucrase from Leuconostoc mesenteroides showed improvement in the inhibitory activity against fructose transporters (GLUT5) (George Thompson et al., 2015). In this review, we provide an overview of enzymatic synthesis of a-glycosylated and b-glycosylate flavonoids and the outcome of glycosylation on the solubility, stability, and biological functions of flavonoids.

2. Enzymatic synthesis of glycosylated flavonoids 2.1 Flavonol The backbone structure of flavonol subclass comprises of the 3-hydroxy-2-phenylchromen-4-one. This subclass includes quercetin, myricetin, kaempferol, isorhamnetin, and rutin. Onions, kale, apples, buckwheat, teas, and broccoli are the major food sources of flavonols (Jacques et al., 2013). The transfer ratio as well as reaction condition of enzymatic synthesis of glycosylated flavonols are shown in Table 2.1. Moon et al. (2007) studied the transglucosylation of quercetin using L. mesenteroides B1299CB4 dextransucrase (2.4 U/mL) in the presence of 150 mM sucrose in 20 mM sodium acetate buffer (pH 5.2) at 28 C for 5 h. The structure of two quercetin-glucosides (quercetin-30 -O-a-D-glucopyranoside and quercetin-40 -O-a-D-glucopyranoside) was determined by NMR with 20.2% and 2.9% transfer ratio, respectively (Table 2.1 and Fig. 2.1). In another study, Chen et al. (2011) used maltose as the substrate; quercetin, kaempferol, and isorhamnetin as acceptors; and cellulase from Penicillium decumbens for the synthesis of quercetin glycoside, kaempferol glycoside, and isorhamnetin glycoside with 45%, 50%, and 41% transfer ratio, respectively (Table 2.1). Quercetin glycoside and kaempferol glycosides were synthesized with a transfer ratio of 64.1% and 78.9% using 7-O-glucosyltransferase from Arabidopsis thaliana (Kim et al., 2006). Quercetin 3-O-glycoside was synthesized using recombinant glucosyltransferase UGT71G1 from Medicago truncatula. Ko et al. (2008) described cloning and expression of four flavonoid glycosyltransferase (UGT706C1, UGT706D1, UGT707A3, and UGT709A4) from rice in Escherichia coli. UGT706C1 and UGT707A3 have been reported to transfer a glucose moiety to the 3-hydroxyl group of quercetin and kaempferol while UGT706D1 has been stated to produce a minimum of two products with glycosylation at different hydroxyl groups (Ko et al., 2008). The sugar-sugar glucosyltransferase from Catharanthus roseus that catalyzes 1,6-glucosylation of flavonol glucosides can synthesize quercetin 3-O-gentibioside, quercetin 3-O-gentiotrioside, and quercetin 3-O-gentiotetroside in the presence of recombinant enzymes and increased level of UPD-glucose (Masada et al., 2009) (Fig. 2.1). Kim et al. (2012) synthesized different astragalin glucosides (DP 1-8) using dextransucrase from L. mesenteroides 512FMCM. Among the synthesized astragalin glucosides, the yield of astragalin G1 (kaempferol-3O-b-D-glucopyranosyl-(1 / 3)-O-a-D-glucopyranoside or kaempferol-3-O-b-D-nigeroside) and

Table 2.1 Summary of the transglycosylation results in flavonoids prepared using enzymatic methods. Acceptor

Enzyme

Enzyme source

Reaction conditions

Glucansucrase

Leuconostoc mesenteroides B1299CB

Alternansucrase

Leuconostoc mesenteroides B23192

Cellulase

Penicillium decumbens

Quercetin Kaempferol

7-Oglucosyltransferase

Arabidopsis thaliana

Myricetin

Dextransucrase

Quercetin

Recombinant glycosyltransferase UGT71G1

Leuconostoc mesenteroides NRRL B-512F Medicago truncatula

Quercetin/ kaempferol

Recombinant glavonoid 3-Oglucosyltransferase UGT706C1/ UGT707A3/ UGT706D1

20.5 mM quercetin, 150 mM sucrose, 2400 U enzyme in 20 mM sodium acetate buffer (pH 5.2) at 28 C for 5 h 9 mM quercetin, 3 U/mL enzyme, 120 mM sucrose, 30% MEE (Diethylene glycol ethyl methyl ether) for 24 h 25 mg flavonoid, 0.5 g/L maltose, 2 mL cellulose (2 mg/mL), 50% ethanol, pH 6.0 at 60 C for 30 h 75 mM flavonoid in phosphate buffer (pH 7.0), 10 mg enzyme, 250 mM MgCl2, 250 mM UDPglucose at 37 C for 3 h 9 mM myricetin, 3 U/mL enzyme, 120 mM sucrose, 30% MEE for 24 h 250 mM quercetin, 4 mg enzyme, 500 mM UDPG, UDP-galactose, or UDPglucuronic acid, 14 mM mercaptoethanol in 50 mM Tris-HCl (pH 7.0) for 1 h at 30 C 100 mM of substrate, 5 mM MgCl2, 500 mM of UDP-glucose, 25e30 mg protein in 10 mM KH2PO4 (pH 6.8) at 37 C for 1 h

Transfer ratio (%)

References

23.1

Moon et al. (2007)

4

Bertrand et al. (2006)

45 50 41

Chen et al. (2011)

64.1 78.9

Kim et al. (2006)

49

Bertrand et al. (2006)

ND

He et al. (2006)

ND

Ko et al. (2008)

Flavonol Quercetin

Rice

2. Enzymatic synthesis of glycosylated flavonoids

Kaempferol Isorhamnetin

49

Continued

Table 2.1 Summary of the transglycosylation results in flavonoids prepared using enzymatic methods.dcont’d Enzyme source

Reaction conditions

Quercetin 3-Oglucoside/ Kaempferol 3-Oglucoside/ myricetin 3-Oglucoside/quercetin 3-O-gentibioside Astragalin

Flavonoid glucoside 1,6glucosyltransferase

Catharanthus roseus

500 mM acceptor substrate, 2 mM UDPglucose, 1e5 mg enzyme in 50 mM Tris-HCl (pH 5.2) at 30 C for 4 h

ND

Masada et al. (2009)

Cyclodextrin glucanotransferase

Bacillus licheniformis

7.7

Choung et al. (2017)

Dextransucrase

Leuconostoc mesenteroides B512FMCM

24.5

Kim et al. (2012)

b-galactosidase

Bacillus circulans

73.8

Han et al. (2017)

Cyclodextrin glucanotransferase “amano”

Bacillus macerans

72.35

Rha et al. (2019)

Cyclodextrin glucanotransferase Toruzyme 3.0 L

Bacillus licheniformis

a-Amylase G “amano” L

Geobacillus sp.

11 mM astragalin, 29 mM maltose, 5 U enzyme in 50 mM sodium acetate buffer (pH 6.0) at 60 C for 3h 10 mM astragalin, 200 mM sucrose, 1.8 U/ mL enzyme in 20 mM sodium acetate buffer pH (5.2) at 28 C for 5 h 50 mM astragalin, 400 mM lactose, 3 U/mL b-galactosidase in 20 mM sodium phosphate buffer (pH 6.0) at 60 C for 12 h 50 mg/mL soluble starch, 1 mg/mL of quercetin 3O-glucoside, 1 U/mL enzyme in 50 mM citrate phosphate buffer (pH 6.0) at 60 C for 24 h 50 mg/mL soluble starch, 1 mg/mL of quercetin 3O-glucoside, 1 U/mL enzyme in 50 mM citrate phosphate buffer (pH 5.0) at 70 C for 24 h 50 mg/mL soluble starch, 1 mg/mL of quercetin 3O-glucoside, 1 U/mL enzyme in 50 mM citrate phosphate buffer (pH 5.0) at 70 C for 24 h

Quercetin 3-Oglucoside

74.66

76.77

References

Chapter 2 Enzymatic synthesis of flavonoid glucosides

Enzyme

50

Transfer ratio (%)

Acceptor

Amylosucrase

Deinococcus geothermalis

50 mg/mL sucrose, 1 mg/ mL of quercetin 3-Oglucoside, 1 U/mL enzyme in 50 mM Tris HCl (pH 7.0) at 45 C for 24 h

97.64

Cyclodextrin glucanotransferase “amano”

Bacillus macerans

Acceptor, 1% (w/v) a-CD, CGTase enzyme at 55 C for 0.5e1 h

46

Lee et al. (2017)

Cyclodextrin glucanotransferase

Bacillus sp.

44 ND ND

Kometani et al. (1996)

Hesperetin

Cyclodextrin glucanotransferase

Thermoanaerobacter sp.

Eriodictyol Naringenin

7-Oglucosyltransferase

Arabidopsis thaliana

Liquiritin

Flavonoid glucoside 1,6glucosyltransferase

Catharanthus roseus

Naringenin

Recombinant flavonoid 3-Oglucosyltransferase UGT706D1

Rice

0.5% w/v neohesperidin/ naringin, 5% soluble starch, 2 U/mL CGTase at 40 C (pH 10) 15 mg/mL hesperetin, 180 mg/mL soluble starch, 10% CGTase, 30% bis(2-methoxyethyl) ether in 10 mM sodium citrate buffer (pH 5.0) at 60 C for 18 h 75 mM flavonoid in phosphate buffer (pH 7.0), 10 mg enzyme, 250 mM MgCl2, 250 mM UDPglucose at 37 C for 3 h 500 mM acceptor substrate, 2 mM UDPglucose, 1e5 mg enzyme in 50 mM Tris-HCl (pH 5.2) at 30 C for 4 h 100 mM of substrate, 5 mM MgCl2, 500 mM of UDP-glucose, 25e30 mg protein in 10 mM KH2PO4 (pH 6.8) at 37 C for 1 h

Flavanone Hesperidin 7-Oglucoside Prunin Neohesperidin Naringin

Gonzalez-Alfonso et al. (2018)

100 71.6

Kim et al. (2006)

ND

Masada et al. (2009)

ND

Ko et al. (2008)

51

Continued

2. Enzymatic synthesis of glycosylated flavonoids

4.1

Table 2.1 Summary of the transglycosylation results in flavonoids prepared using enzymatic methods.dcont’d

52

References

2.5% (w/v) naringin, 2.5% (w/v) maltotriose, 10,000 Cu enzyme in 50 mM sodium citrate buffer (pH 6.0) at 55 C for 20 h

ND

Lee et al. (1999)

Leuconostoc mesenteroides B1299CB

70 mM ampelopsin, 150 mM sucrose, 1 U/mL enzyme in 20 mM sodium acetate buffer (pH 5.2) at 28 C for 1 h

ND

Woo et al. (2012)

Deinococcus geothermalis

20 mM baicalein, 40 mM sucrose, 1 mg/mL enzyme in 50 mM Tris-HCl buffer (pH 8) at 30 C for 12 h 2 mg/mL flavone compound, 100 mM sucrose, 1.6 U/mL enzyme in 50 mM TrisHCl (pH 8.0) at 37 C for 24 h

59.1

Kim et al. (2014)

19.6 1.8 86.0 57.0 56.0

Jang et al. (2018)

44

Bertrand et al. (2006)

Enzyme

Enzyme source

Reaction conditions

Naringin

Maltogenic amylases

Bacillus stearothermophilus

Glucansucrase

Amylosucrase

Flavanonol Ampelopsin

Flavone Baicalein

Apigenin Isorhoifolin Luteolin Homoorientin 6,7Dihydroxyflavone Luteolin

Dextransucrase Alternansucrase

L. mesenteroides B512F L. mesenteroides B23192

9 mM luteolin, 120 mM sucrose, 3 U/mL enzyme in 20 mM sodium acetate buffer [pH 5.2, (70%)] and bis(2-methoxyethyl)ether [MEE (30%)] at 30 C for 24 h

Bacillus stearothermophilus

1% (w/v) puerarin, 5.0% (w/v) soluble starch/ maltotriose, 50 U/mL enzyme at 55 C for 45 min.

8

Isoflavone Puerarin

Maltogenic amylase

70

Choi et al. (2010), Li et al. (2004)

Chapter 2 Enzymatic synthesis of flavonoid glucosides

Transfer ratio (%)

Acceptor

2. Enzymatic synthesis of glycosylated flavonoids

53

FIGURE 2.1 Chemical structures of quercetin, astragalin and their glucosides.

astragalin G1 (kaempferol-3-O-b-D-glucopyranosyl-(1 / 6)-O-a-D-glucopyranoside or kaempferol3-O-b-D-isomaltoside) was 21.8% and 2.7%, respectively (Kim et al., 2012) (Fig. 2.1). The lower transfer ratio (7.7%) of astragalin glucoside (kaempferol-3-O-b-D-glucopyranosyl-(1 / 4)-O-a-Dglucopyranoside) was observed when cyclodextrin glucanotransferase from Bacillus licheniformis was used (Choung et al., 2017) (Fig. 2.1). Han et al. (2017) synthesized b-D-galactosyl-(1-6) astragalin (11.6%) and b-D-galactosyl-(1-4)-b-D-galactosyl-(1-6) astragalin by using b-galactosidase from Bacillus circulans with 11.6% and 6.7% transfer ratio, respectively (Fig. 2.1). Recently, Rha et al. (2019) synthesized enzymatically modified isoquercitrin (EMIQ) that contained one to five attacked glucosyl unit to quercetin 3-O-D-glucoside (isoquercitrin) from quercetin 3-O-glucoside using different cyclodextrin glucanotransferase, a-amylase, and amylosucrase. The transfer ratio of quercetin 3-Oglucoside was 72.35%, 74.66%, 76.77%, and 97.64% for cyclodextrin glucanotransferase “Amano,” cyclodextrin glucanotransferase “Toruzyme 3.0,” a-amylase G “Amano L,” and amylosurase, respectively (Table 2.1) (Fig. 2.1).

2.2 Flavanones Flavanones are a group of flavonoids comprising of 2,3-dihydro-2-phenylchromen-4-one backbone. Natural flavanones consist of naringenin, hesperetin, eriodictyol, narirutin (naringenin-7-O-rutinoside), hesperidin (hesperetin-7-O-rutinoside), eriocitrin (eriodictyol-7-O-rutinoside), and naringin (naringenin 7-O-neohesperidose) (Klimczak et al., 2007). Cyclodextrin glucanotransferase from Bacillus sp. was employed to produce the glucosylneohesperidin and oligomaltosylneohesperidin using

54

Chapter 2 Enzymatic synthesis of flavonoid glucosides

FIGURE 2.2 Chemical structures of prunin, naringin, neohesperidin and their glucosides.

neohesperidin as an acceptor and soluble starch as substrate (Kometani et al., 1996). The chemical structure of neohesperidin glucoside was determined as 3G-a-D-glucopyranosyl neohesperidin by NMR (Kometani et al., 1996) (Fig. 2.2). Lee et al. (2017) used naringinase from Aspergillus sojae for the hydrolysis of a-L-rhamnosyl-(1e6)-glucose connected to the 7-OH group of naringin and hesperidin to prunin and hesperidin 7-O-glucoside. Subsequently, prunin and hesperidin-7-O-glucoside were transglucosylated to oligoglucosyl prunin (prunin-a-Gn (0  n  4) (Fig. 2.2) and prunin-a-Gn (n  5)) and oligoglucosyl hesperidin 7-O-glucoside (hesperidin-7-O-a-Gn (0  n  4) and hesperidin-7-O-a-Gn (n  5)) where glucose, maltose, maltotriose, and oligoglucosyl molecules were connected to prunin and hesperidin-7-O-glucoside using cyclodextrin glucanotransferase in the presence of 1% a-cyclodextrin at 55 C for 0.5e1 h (Lee et al., 2017). The transfer ratio of oligoglucosyl prunin and oligoglucosyl hesperidin-7-O-glucoside was 46% and 44%, respectively. Gonzelez-Alfonso et al. (2018) used the same enzyme from different sources for the synthesis of hesperetin-7-O-a-D-glucopyranoside. The optimum transfer ratio (4.1%) was obtained in the presence of 15 mg/mL hesperetin, 180 mg/mL soluble starch, 10% (v/v) CGTase, 30% (v/v) bis(2-methoxyethyl) ether, and 60% (v/v) 10 mM sodium citrate buffer (pH 5.0) at 60 C, 1000 rpm (Gonzalez-Alfonso et al., 2018). Kim et al. (2006) reported that flavonoid 7-O-glycosyltransferase from Arabidopsis thaliana can be converted into eriodictyol and naringenin of flavanone subclass to their glucosides with a transfer ratio of 100% and 71.6%, respectively. In another report, the recombinant flavonoid 3-O-glucosyltransferase UGT706D1 from the rice was reported to transfer glucosyl group to the 7-hydroxyl group of naringenin (Ko et al., 2008). Masada et al. studied the tranglucosylation behavior of liquiritin using

2. Enzymatic synthesis of glycosylated flavonoids

55

liquiritin as acceptor, glucosyltransferase from Catharanthus roseus in the presence of 500 mM acceptor, 2 mM UDP-glucose, and 1e5 mg enzyme in 50 mM Tris-HCl pH 5.2 at 30 C for 4 h (Masada et al., 2009). Naringin is a bitter compound existing in citrus fruits, especially in grapefruit. The mono-, di-, and triglycosylnaringins were synthesized through transglycosylation using maltogenic amylase from Bacillus stearothermophilus employing maltotriose as a substrate (Table 2.1 and Fig. 2.2). The chemical structure of major glycosylnaringin was identified as 6G-a-maltosylnaringin by NMR (Lee et al., 1999) (Fig. 2.2). Kometani et al. (1996) reported that cyclodextrin glucanotransferase from Bacillus sp. can synthesize 3G-a-D-glucopyranosyl naringin using starch as a substrate at pH 10.0 (Table 2.1) (Fig. 2.2).

2.3 Flavanonol Woo et al. (2012) synthesized five different ampelopsin-glucosides using L. mesenteroides B-1299CB4 dextransucrase employing 70 mM ampelopsin, 150 mM sucrose, and 1 U/mL enzyme in the presence of 20 mM sodium acetate buffer (pH 5.2) at 28 C for 1 h. Among the synthesized glucoside products, 55% of total ampelopsin glucosides was ampelopsin-40 -O-a-D-glucopyranoside (ampelopsin-G1) (Woo et al., 2012) (Table 2.1 and Fig. 2.3).

2.4 Flavone The chemical structure of flavone subclass comprises of 2-phenylchromen-4-one as a backbone. Apigenin, baicalein, eupatilin, luteolin, tangeretin, and wogonin are the major flavones found in vegetables, fruits, and herbs. Kim et al. (2014) studied transglucosylation of baicalein in the presence of recombinant amylosucrase from Deinococcus geothermalis with sucrose as a substrate. The reactions yielded 59.1% using 40 mM sucrose, 1 mg/mL amylosucrase, 20 mM baicalein in 50 mM Tris-HCl buffer (pH 8.0) at 30 C for 24 h. The transglucosylation compound of baicalein was determined as baicalein 6-O-a-D-glucopyranoside by NMR (Kim et al., 2014) (Fig. 2.4). Jang et al. (2018) used the same enzyme for transglucosylation of different flavone compounds including apigenin, isorhoifolin, luteolin, homoorientin, orientin, luteolin-30 -7-diglucoside, 7-hydroxylflavone,

FIGURE 2.3 Chemical structures of ampelopsin and its glucoside.

56

Chapter 2 Enzymatic synthesis of flavonoid glucosides

FIGURE 2.4 Chemical structures of baicalein, luteolin and their glucosides.

chrysin, and 6,7-dihydroxyflavone (Jang et al., 2018). Among them, the transglucosylation products with different transfer ratio were found when apigenin (19.6%), isorhoifolin (1.8%), luteolin (86.0%), homoorientin (57.0%), and 6,7-dihydroxyflavone (56.0%) were used as acceptors in the existence of 1.6 U/mL amylosucrase, and 100 mM sucrose in 50 mM Tris-HCl (pH 8.0) at 37 C for 24 h (Table 2.1) (Jang et al., 2018). The highest transfer ratio was 86% when luteolin was used as an acceptor. The chemical structure of luteolin transglucosylation products with amylosucrase was identified as luteolin-40 -O-a-D-glucopyranoside by NMR (Jang et al., 2018) (Fig. 2.4). Luteolin (9 mM) was also used as an acceptor of L. mesenteroides B-512F dextransucrase and L. mesenteroides B-23192 alternansucrase at 3 U/mL with 120 mM sucrose in a mixture of 20 mM acetate buffer pH 5.2 (70%)/ bis(2-methoxyethyl) ether (30%) (Bertrand et al., 2006). The yield of luteolin transglucosylation was 44% and 8% for dextransucrase and alternansucrase, respectively (Table 2.1). Two glucosyl luteolin compounds were purified and determined as luteolin-40 -O-a-D-glucopyranoside and luteolin-30 -Oa-D-glucopyranoside by NMR (Bertrand et al., 2006) (Fig. 2.4).

2.5 Isoflavones Isoflavones are phytoestrogens present in soy and its products, kudzu, and red clover. The chemical structure of the backbone of isoflavone is 3-phenylchromen-4-one (Tapas et al., 2008). Daidzein, genistein, glycitein, puerarin, and biochanin A are major isoflavone compounds (Murphy et al., 1999). Li et al. (2004) screened four different enzymes: maltogenic amylase from B. stearothermophilus, maltosyl-transferase from Thermotoga maritima, 4-a-glucanotransferase from Thermus scotoductus, and Bacillus sp. I-5 CGTase for their glucosyl transfer’s ability with puerarin (daidzein 8-C-glucoside) as acceptor. Among these enzymes, maltogenic amylase was identified to transfer glucosyl residue to puerarin with a yield of 40.9% with 0.5% (w/v) soluble starch, 66.6% using 5% (w/v) maltotriose, and 70% using 5% (w/v) soluble starch in the presence of 1% (w/v) puerarin at 55 C for 45 min (Li et al., 2004) (Fig. 2.5).

2. Enzymatic synthesis of glycosylated flavonoids

57

FIGURE 2.5 Chemical structures of puerarin and its glucosides.

2.6 Physical and biological characterization of glycosylated flavonoids 2.6.1 Solubility Although flavonoids have been informed to have various biological functions, they are limited to be used as active ingredients of foods, cosmetics, and pharmaceutical products due to their low solubility in water, the slow absorption rate in the small intestine, and fast oxidative degradation (Kim et al., 2014). Transglycosylation using enzymes is a common method used for improving the water solubility of flavonoids. In flavonol subclass, quercetin and rutin (quercetin-3-O-rutinoside) exhibit poor solubility in water (Table 2.2). The solubility of flavonol glucosides in water improved from 9.7-fold (rutin) to an impressive 81,489.4-fold (enzymatically modified isoquercetin) compared to quercetin (Choung et al., 2017; Han et al., 2017; Lee et al., 2017; Moon et al., 2007). The solubility in water of flavonol and their glucosides was in the following order: EMIQ (isoquercetin-a-Glcn) > kaempferol 3-O-b-D-glucopyranosyl (1 / 4)-O-a-D-glucopyranoside > quercetin 40 -O-a-D-glucopyranoside > kaempferol-3-O-b-D-glucopyranosyl-(1 / 6)-b-D-galactopyranosyl-(1 / 4)-b-D-galactopyranoside > kaempferol-3-O-b-D-glucopyranosyl-(1 / 6)-b-D-galactopyranoside > quercetin 3-O-glucoside > astragalin > rutin > quercetin. The flavonoids with a-glucosyl linkages showed higher improvement in water solubility (130.39 mg/mL, 21.44 mg/mL, and 5.67 mg/mL) (Table 2.2) compared to flavonoids with b-glucosyl linkages. In flavanone subclass, Lee et al. (2017) reported that oligoglucosyl prunin (prunin-a-Gn (0  n  4) and prunin-a-Gn (n  5)) and oligoglucosyl hesperidin 7-O-glucoside (hesperidin-7-Oa-Gn (0  n  4) and hesperidin-7-O-a-Gn (n  5)) exhibited increase in water solubility from 2.45 mg/mL for prunin and 0.027 mg/mL for hesperetin to 934.22 mg/mL of prunin-a-Gn and 1047.21 mg/mL of hesperidin-7-O-a-Gn, respectively (Table 2.2). Oligoglucosyl prunin and oligoglucosyl hesperetin exhibited improvement in water solubility by 346,007-fold and 387,855-fold compared to hesperetin, respectively. It is apparent that the increase in water solubility is related to the number of glucosyl residues connected to flavonones. The solubility of flavone glucosides in water increased from 5.2-fold to an impressive 387,855-fold compared to hesperetin (Lee et al., 2017) in the following order: hesperetin-7-O-Glc-a-Glcn (n  5) > hesperetin-7-OGlc-a-Glc4 > prunin-a-Glcn (n  5) > prunin-a-Glc4 > hesperetin-7-O-Glc-a-Glc3 > hesperetin-

58

Chapter 2 Enzymatic synthesis of flavonoid glucosides

Table 2.2 Water solubility of glycosylated flavonoids. Subclass Flavonol

Compound a

Flavanoneb

Quercetin Rutin Quercetin 3-Oglucoside Enzymatically modified isoquercetin Quercetin 40 -O-a-Dglucopyranoside Astragalin Kaempferol 3-Ob-D-glucopyranosyl (1-4)-O-a-Dglucopyranoside Kaempferol-3-Ob-D-glucopyranosyl(1 / 6)-b-Dgalactopyranoside Kaempferol-3-Ob-D-glucopyranosyl(1 / 6)-b-Dgalactopyranosyl(1 / 4)-b-Dgalactopyranoside Naringenin Naringin Prunin Prunin-a-Glc1 Prunin-a-Glc2 Prunin-a-Glc3 Prunin-a-Glc4 Prunin-a-Glcn (n  5) Hesperetin Hesperidin Hesperetin-7-O-Glc Hesperetin-7-O-Glca-Glc1 Hesperetin-7-O-Glca-Glc2 Hesperetin-7-O-Glca-Glc3

Water solubility (mg/mL)

Relative water solubility

0.0016 0.0155 0.81

1 9.7 504.4

130.39

81,489.4

5.67

3543.8

Moon et al. (2007)

0.027 21.44

16.88 13,400

Choung et al. (2017)

3.83

2393.8

Han et al. (2017)

3.88

2425.0

0.014 0.37 2.45 40.36 264.49 468.13 815.92 934.22

5.2 137.0 907.4 14,948.2 97,959.3 173,381.5 302,192.6 346,007.4

0.0027 0.0117 0.32 259.19

1 4.3 118.5 95,996.3

594.23

220,085.2

813.04

301,125.9

Reference Lee et al. (2017)

Lee et al. (2017)

2. Enzymatic synthesis of glycosylated flavonoids

59

Table 2.2 Water solubility of glycosylated flavonoids.dcont’d Subclass

Flavanonolc

Flavoned

Isoflavonee

Compound Hesperetin-7-O-Glca-Glc4 Hesperetin-7-O-Glca-Glcn (n  5) Ampelopsin Ampelopsin-G1 Ampelopsin-G2 Baicalein Baicalin Baicalein-6-O-a-Dglucopyranoside Luteolin Luteolin-40 -O-a-Dglucopyranoside Luteolin-30 -O-a-Dglucopyranoside Puerarin a-D-glucosyl-(1-6)puerarin a-D-maltosyl-(1-6)puerarin

Water solubility (mg/mL)

Relative water solubility

945.91

350,337.0

1047.21

387,855.6

0.215 30.15 327.04 0.0054 0.05 0.1419

1.0 140 1521.1 3.2 29.4 83.5

Woo et al. (2012)

0.0017

1.0

Bertrand et al. (2006)

0.0136

8.0

0.0136

8.0

5.1 96.62

1.0 19.0

1552.4

304.4

Reference

Kim et al. (2014)

Li et al. (2004)

a

The relative water solubility was compared with quercetin. The relative water solubility was compared with hesperetin. c The relative water solubility was compared with ampelopsin. d The relative water solubility was compared with luteolin. e The relative water solubility was compared with puerarin. b

7-O-Glc-a-Glc2 > prunin-a-Glc3 > prunin-a-Glc2 > hesperetin-7-O-Glc-a-Glc1 > prunin-a-Glc1 > prunin > naringin > hesperetin-7-O-Glc > naringenin > hesperidin > hesperetin (Table 2.2). With regard to flavanonol subclass, Woo et al. (2012) reported that the water solubility of ampelopsin with two glucosyls and one glucosyl attached to ampelopsin by a-linkages enhanced by 1521.1fold and 140-fold compared to ampelopsin. The solubility of baicalein, lutein, and their glucosides in water is shown in Table 2.2. By attaching glucoside at 6-hydroxyl group of A-ring baicalein, the solubility of baicalein-6-O-a-D-glucopyranoside in water increased by 26.3-fold compared to baicalein and by 83.5-fold compared to luteolin (Kim et al., 2014). Bertrand et al. (2006) reported that the solubility in water of luteolin-40 -O-a-D-glucopyranoside and luteolin-40 -O-a-D-glucopyranoside was increased by eightfold compared to luteolin and the connection of glucosyl residue to B-ring did not affect the glucosylated luteolin solubility in water.

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Chapter 2 Enzymatic synthesis of flavonoid glucosides

The solubility of puerarin of isoflavone in water was 5.1 mg/mL. However, the water solubility of a-D-glucosyl-(1-6)-puerarin and a-D-maltosyl-(1-6)-puerarin which were synthesized using maltogenic amylase and soluble starch or maltotriose as a substrate in the presence of puerarin increased by 19.0-fold and 304.4-fold compared to puerarin (Li et al., 2004).

2.6.2 Biological characterization Flavonoids are well known for their enzyme inhibition (Hua et al., 2018; Walker et al., 2000), antioxidant (Gomes et al., 2016), antiinflammation (Aziz et al., 2018; Le et al., 2018; Middleton, 1998; Yang et al., 2012), antiaging (Lin et al., 2015), neuroprotective (Prakash and Sudhandiran, 2015), antimicrobial (Nguyen et al., 2017), and antitumor activities (Kanadaswami et al., 2005). Most of the research activities have motivated on the enzymatic synthesis of novel flavonoids and their water solubility; there exists not much information about the biological activities of synthesized glycosylated flavonoid compounds. Hence, in the present work, we have reviewed the biological activities of a few enzymatically synthesized glycosylated flavonoids. Antioxidant activity: There are several reports on the antioxidant activity of a-glucosyl flavonoids (Choung et al., 2017; Kim et al., 2012; Moon et al., 2007; Woo et al., 2012). The antioxidant activity of quercetin-a-D-glucopyranoside and kaempferol-3-O-b-D-glucopyranosyl-(1-4)-O-a-D-glucopyranoside was observed to be lower than that of quercetin and kaempferol-3-O-glucoside (Choung et al., 2017; Moon et al., 2007) while an increase was noted from 10.9% for ampelopsin (10 mM) to 23.6% for ampelopsin-40 -O-a-D-glucopyranoside (10 mM) (Woo et al., 2012). The reactive oxygen species (ROS) scavenging activities of astragalin (kaempferol-3-O-glucoside), kaempferol-3-O-b-D-glucopyranosyl(1 / 6)-O-a-D-glucopyranoside, kaempferol-3-O-b-D-isomaltooligosaccharide, and isomaltooligosyl astragalin at 10 mg/mL were 9.2%, 18.6%, 18.5%, and 21.2%, respectively (Kim et al., 2012). Antiinflammation activity: Inflammation is a biological protection mechanism against the attack by pathogens and risk factors involving inflammation of cells, like macrophages. The lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage cells are usually selected for studying the inflammatory activity of natural products and herbal medicine due to their sensitivity to LPS stimulation and macrophagemediated release of various inflammatory mediators, for example, NO, interleukin, and tumor necrosis factor-alpha (Kim et al., 2018). The O-glycosylation of flavonoids decreased NO production, iNOS expression, and NF-k-B activation in LPS-induced RAW264.7 (Bai et al., 2011; Kim et al., 2013). However, two studies reported a-glucosylated flavonoid-mediated improvement in antiinflammatory activity (Choung et al., 2017; Kim et al., 2014). Baicalein-6-a-glucoside led to a reduction in NO making in LPS-treated RAW264.7 cells (Kim et al., 2014). Baicalein-6-a-glucoside activated Nrf2 more efficiently than baicalein, deprived of the facilitation of ROS. Therefore, baicalein-6-a-glucoside was more operative in overwhelming ROS making in LPS-treated RAW264.7 cells. Choung et al. (2017) studied the antiinflammatory activity of kaempferol, kaempferol-3-O-glucoside, and kaempferol-3-O-b-D-glucopyranosyl-(1-4)-O-a-D-glucopyranoside on NO production by LPS-induced inflammation in RAW264.7 cells. These compounds showed inhibition of NO synthesis in LPS-treated RAW264.7 cells depending on a concentration with IC50 values of 15.0, 13.2, and 8.4 mM, respectively. Fructose transporter (GLUT5): Several flavonoids were tested for their inhibitory effect on GLUT5 such as astragalin, rutin, naringin, naringenin, dihydromyricetin, ampelopsin, quercetin, chrysin, astragalin 1-glucoside, astragalin 2-glucoside, and astragalin 6-glucoside. Among them, astragalin with six glucosides connected via a(1-6) bond inhibited GLUT5 with IC50 of 6.8 mM while astragalin, kaempferol-3-O-b-D-glucopyranosyl-(1 / 6)-O-a-D-glucopyranoside (astragalin 1-

2. Enzymatic synthesis of glycosylated flavonoids

61

glucoside), kaempferol-3-O-b-D-isomaltooligosaccharide (astragalin 2-glucoside), and other flavonoids did not exhibit any inhibitory effect (George Thompson et al., 2015). Matrix metalloproteinase (MMP-1) inhibition: The inhibition of astragalin and its glucosides on MMP-1 in human dermal fibroblasts (HDFs) and fibroblast collagenase was studied by Kim et al. (2012). The inhibitory effect against MMP-1 production of kaempferol-3-O-b-D-glucopyranosyl(1 / 6)-O-a-D-glucopyranoside, kaempferol-3-O-b-D-isomaltooligosaccharide, and isomaltooligosyl astragalin was 27.0%, 56.4%, and 61.7% while kaempferol-3-O-glucoside did not exhibit any inhibitory effect (Kim et al., 2012). The researchers concluded that the inhibition against MMP-1 making in HDFs was increased with an increase in the degree of glucosyl linkage to kaempferol-3O-glucoside. Inhibition activity in melanogenesis: The inhibition of melanin formation on melan-A cells by kaempferol-3-O-b-D-glucopyranosyl-(1 / 6)-O-a-D-glucopyranoside, kaempferol-3-O-b-Disomaltooligosaccharide, and isomaltooligosyl astragalin was identified as 3.7%, 8.5%, and 19.7%, respectively, while kaempferol-3-O-glucoside did not exhibit any inhibitory effect (Kim et al., 2012). In another report, Woo et al. (2012) reported that ampelopsin and ampelopsin-40 -O-a-Dglucopyranoside inhibited mushroom tyrosinase in a competitive inhibition pattern with a Kiof 62.6 and 40.2 mM, respectively. Bioavailability: Flavonoid metabolite products in blood frequently exist as glucuronide, sulfate, and methylate conjugates (Matsumoto et al., 2004; Yamada et al., 2006). Yamada et al. (2006) studied that rats administered orally with gastric intubation using 1 mmol/kg of glucosyl hesperidin demonstrated early detection of hesperetin-glucuronide after 15 min and maximum (6.3 mM) at 6 h in serum than rats administered with 1 mmol/kg of hesperidin (after 6 h and minimum 1.3 mM at 9e12 h). The extent under the concentration-time curve (AUC) for hesperetin-glucuronide in the glucosyl hesperidin rat sera was 3.7-fold higher than that of hesperetin rat sera (Yamada et al., 2006). The similar results were obtained when a-oligoglucosyl isoquercetin was orally administered to rats by gavage (Makino et al., 2009). The plasma concentration of quercetin and its methylated metabolite, tamarixetin enlarged quickly and reached to a maximum level within 15 min and 15e30 min, respectively, after oral administration of a-oligoglucosyl isoquercetin; whereas the plasma concentration of isorhamnetin and methylated metabolites increased and extended to a peak after 6 h for quercetin. However, the amount of quercetin and its methylated metabolite in plasma after oral taking of a-monoglucosyl rutin and a-oligoglucosyl rutin was slightly increased and exhibited improvement compared to rutin (Makino et al., 2009). The bioavailability of a-oligoglucosyl isoquercetin in a human was reported by Murota et al. (2010). Compared to quercetin, quercetin glycosides (isoquercetin and rutin) and a-oligoglucosyl isoquercetin demonstrated efficient absorption. The number of quercetin metabolites in plasma was enhanced with a maximum level at 1.5 h after intake (Murota et al., 2010).

2.6.3 Sensory of a-glucosylated flavonoids Glycosylation has been extensively studied to improve the physical characteristics of food materials (Devlamynck et al., 2019). It has been reported that 6-a-maltosyl-naringin and 3-a-D-glucopyranosyl neohesperidin were 10 times less bitter than naringin and neohesperidin, which are the major bitter flavonoid compounds in citrus juices (Kometani et al., 1996; Lee et al., 1999; Shaw et al., 1991).

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Chapter 2 Enzymatic synthesis of flavonoid glucosides

3. Conclusion Enzymatic synthesis of a-glucosyl flavonoids using different enzymes showed increased water solubility, biological activity, bioavailability, as well as sensory improvement. Apparently, they are proposed to have the potential to be used as active components in food, pharmaceutical, and cosmetic applications.

Acknowledgment This work was partially supported by the OTTOGI Corporation through the Research and Publication Project. The present study has been also conducted under the framework of International Cooperation Program (2016K1A3A1A19945059) and by the research grants (2018R1D1A1B07049569, T.T.H. Nguyen, 2018R1D1A1A09083366, D. Kim) of NRF, Republic of Korea, and by the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) through the Agriculture, Food and Rural Affairs Research Center Support Program, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (D. Kim, 710012-03-1-HD220), Republic of Korea.

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CHAPTER

Fish gelatin: molecular interactions and applications

3 Donghwa Chung

Food Technology Major, Graduate School of International Agricultural Technology, Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang, Gangwon, Republic of Korea

1. Introduction Gelatin is a collagen-derived protein that has considerable applications in food, cosmetic, pharmaceutical, and medical industries due to its unique viscoelastic, gelling, melting, interfacial, and biological properties. In food and cosmetic industry, gelatin is often used as a texturizer, moisture absorbent, emulsifier, foaming agent, and emulsion/foam stabilizer (Karim and Bhat, 2009). In pharmaceutical and medical industries, gelatin is used to form a matrix of implants, drug delivery devices, ointments, hard and soft capsules, tablet coating, plasma expanders, and emulsions (Karim and Bhat, 2009; Lin et al., 2017). Gelatin is mostly derived from pigskin, and also from bovine hide, pig/bovine bones, and fish (Lin et al., 2017). However, the use of gelatin from pigs and bovines has been of concern to consumers due to the health- and socioculture-related issues, such as bovine spongiform encephalopathy, vegetarianism, and religious sentiments (Karim and Bhat, 2009; Razzak et al., 2017). The gelatin derived from fish skins, scales, and bones is regarded as a promising alternative to animal-derived gelatin, because it has no consumer concerns mentioned earlier, and can be economically produced from fish processing byproducts (Lin et al., 2017; Razzak et al., 2017). However, the direct applications of fish gelatin require careful considerations because the properties of fish gelatin are strongly influenced by the fish species and manufacturing procedure (Karim and Bhat, 2009). For example, the gelatin derived from cold-water fish species forms a weak gel, which has lower gelling and melting temperatures than that formed from animal-derived gelatins, due to its low proline and hydroxyproline levels (about 30% and 17% for mammalian and cold-water fish gelatin, respectively) (Go´mez-Guille´n et al., 2009; Karim and Bhat, 2009; Yang et al., 2012). An approach to overcome the poor thermal and rheological properties of fish gelatin is to use it in blend with polysaccharides, such as alginate, gum arabic, k-carrageenan, hydroxypropylmethylcellulose, pectin, and chitosan (Chen et al., 2009; Haug et al., 2004; Kołodziejska et al., 2006; Liu et al., 2007; Razzak et al., 2016; Yang et al., 2012). Proteins like fish gelatin interact attractively or repulsively with polysaccharides in aqueous environments, depending on molecular properties (molar mass, charge density, reactive groups, conformation, flexibility, etc.), solvent properties (pH, ionic strength, polarity, etc.), and mixing conditions (mixing ratio, total biopolymer Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00003-5 Copyright © 2020 Elsevier Inc. All rights reserved.

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Chapter 3 Fish gelatin: molecular interactions

concentration, time, temperature, pressure, shear, cross-linkers, irradiation, etc.) (McClements, 2006; Razzak et al., 2016; Schmitt et al., 2009; Turgeon and Laneuville, 2009). The proteinepolysaccharide interactions have gained growing attention in food, pharmaceutical, and cosmetic industries, because they play an important role in developing gels, emulsions, foams, encapsulation devices, edible films, coatings, fat replacers, meat analogues, textures, and protein recovery (Phawaphutanon et al., 2019; Turgeon et al., 2007; Weinbreck et al., 2003; Yang et al., 2012). In this chapter, current knowledge on the molecular interactions of fish gelatin with polysaccharides and their applications is discussed.

2. Gelatin Gelatin molecules exist in a soluble random coil state in warm water and form a viscous solution. The molecules undergo a structural transition from coil to triple helix during cooling to form junction zones, followed by forming a fibril structure by further helical transition and packing, which is collagen-like but not well organized as native collagen (Djabourov, 1988; Karayannakidis and Zotos, 2016) (Fig. 3.1). Finally, the system is transformed to a thermos-reversible, physical gel, that is, a network of gelatin molecules, consisting of physical interactions or bonds, such as van der Waals interactions and hydrogen bonds, with a relatively low bonding energy (Karayannakidis and Zotos, 2016; Karim and Bhat, 2009). The gel strength of gelatin is often expressed as Bloom value. According to the standard of Gelatin Manufacturers Institute of America, the Bloom value is defined as the force in grams (weight) required for a cylindrical plunger (12.7 mm diameter) to deflect the surface of gel by 4 mm without breakage, where the gel was prepared at a gelatin concentration of 6.67% (w/w) and conditioned at 10
C for 16e18 h (Karayannakidis and Zotos, 2016; Lin et al., 2017). Most commercial gelatins have the Bloom values between 80 and 320 g, which are often categorized as follows: low Bloom (220 g) (Karayannakidis and Zotos, 2016). In general, the Bloom value of 250e260 g is most desirable for commercial gelatins (Karayannakidis and Zotos, 2016; Lin et al., 2017). The flow behavior of gelatin solution depends on many factors, including the concentration, temperature, pH, ionic strength, and molecular weight. For example, gelatin solutions show a

FIGURE 3.1 Reversible coil-to-helix transition of gelatin molecules by cooling or heating.

3. Fish gelatin

69

Newtonian behavior with low viscosity under the conditions of low gelatin concentration and high temperature, but a non-Newtonian behavior with high viscosity at high gelatin concentration and low temperature (Karayannakidis and Zotos, 2016). In general, gelatin solutions of low viscosity produce brittle gels, whereas those of high viscosity give relatively strong and elastic gels (Wainewright, 1977). Gelatin forms thermally reversible gels such as casein, agarose, pectin, and carrageenan (Karim and Bhat, 2009). The gelling and melting temperatures of porcine and bovine gelatin gels are typically in the range of 20e25
C and of 28e31
C, respectively (Karim and Bhat, 2009). The melting temperature is a little below body temperature, and the difference between gelling and melting temperatures is relatively small compared to other biopolymer gels. These unique properties enable gelatin gels to have a melt-in-mouth characteristic, which is one of the main advantages of gelatin gels over other biopolymer gels made of alginate, pectin, or carrageenan (Karayannakidis and Zotos, 2016; Karim and Bhat, 2009; Lin et al., 2017). Gelatin is known to have surface-active properties due to the presence of hydrophobic peptide region on the molecules (Cole, 2000). Therefore, gelatin has been widely employed as an emulsifying, foaming, and wetting agents in food, cosmetic, pharmaceutical, and medical industries (Karim and Bhat, 2009). The surface activity of gelatin is in general lower than that of other surface-active biopolymers, such as globular proteins and gum arabic; therefore, gelatin is often used with other surface-active materials or after chemical modifications when used for stabilizing interfaces (Karim and Bhat, 2009).

3. Fish gelatin Gelatin can be economically obtained from the byproducts of fish processing, which account for about 3/4 of the total weight of fish catch (Rustad et al., 2011). They are often dumped in landfills or ocean and cause serious environmental problems (Karayannakidis and Zotos, 2016; Rustad et al., 2011). The reuse of these byproducts is not active but remains in the production of low commercial value products, such as silage, fertilizer, and fish meal (Karayannakidis and Zotos, 2016; Rustad et al., 2011). Therefore, the fish processing byproducts are good sources of gelatin. Fish gelatin has no consumer concerns related to culture, religion, and health, as mentioned earlier. Furthermore, fish gelatin hydrolysates are known to possess various biological functionalities, such as antioxidant, antihypertensive, cryoprotective, anticancer, and antidiabetic activities (Karayannakidis and Zotos, 2016). Despite the merits of fish gelatin, careful considerations must be taken when replacing mammalian gelatin with fish gelatin. This is because fish gelatin yields quite different types of gel depending on fish species and manufacturing procedure, as mentioned earlier. The gelatin from cold-water fish species possesses a much weaker gelling ability than porcine or bovine gelatin; no gel network is formed by some cold-water fish gelatins at room temperature or even at 10
C at which the Bloom value is measured (Karayannakidis and Zotos, 2016). The gelatin from cod, Alaska Pollock, hake, and salmon forms a weak gel having a typical Bloom value of 70e110 g or less (Karayannakidis and Zotos, 2016; Karim and Bhat, 2009). The gelling and melting temperatures of cold-water fish gelatin are typically 5e7
C and 12e14
C, respectively (Chiou et al., 2006), which are much lower than those of mammalian gelatin. This is attributed to the lower fraction of imino acids (proline and hydroxyproline) in cold-water fish gelatin, which are known to play key roles in the formation and stabilization of collagen-like triple helix junction zones (Go´mez-Guille´n et al., 2009; Yang et al., 2012). The gelatin from warm-water fish species, such as tilapia, grass carp, and yellowfin tuna, has higher Bloom values

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Chapter 3 Fish gelatin: molecular interactions

and gelling/melting temperatures than the cold-water fish gelatin. Some warm-water fish gelatins form gels that are stronger or gels as strong as mammalian gelatin (Cho et al., 2005; Gudmundsson and Hafsteinsson, 1997). Unfortunately, the byproducts from warm-water fish processing are less abundant than those from cold-water fish processing. To explore and expand the applications of cold-water fish gelatin, its inferior thermal and rheological properties have been modified by treating with several chemicals: cross-linking chemicals (e.g., formaldehyde, glutaraldehyde), cross-linking enzymes (transglutaminase), plant phenolic compounds (e.g., tannic acid), protease inhibitors, salts, sugars/polyols, and biopolymers (proteins and polysaccharides) (Karayannakidis and Zotos, 2016; Lin et al., 2017). Some physical methods, such as high pressure and UV irradiation, have been also studied for modifying gelatin properties (Lin et al., 2017).

4. Proteinepolysaccharide interactions Proteins are often used as an emulsifying, foaming, gelling, drying, or structuring agent in the development of liquid or solid matrices of foods, delivery devices, films, coatings, and capsules in food, pharmaceutical, and cosmetic industries. Polysaccharides are also extensively used in many industries as a thickening, water holding, gelling, drying, or structuring agent. Proteins interact with polysaccharides either attractively or repulsively in aqueous environments (Fig. 3.2), depending on many intrinsic and extrinsic factors, such as pH, ionic strength, mixing ratio, and total concentration (Razzak et al., 2016). Therefore, controlling proteinepolysaccharide interactions can be a promising

FIGURE 3.2 Interactions between protein and polysaccharide molecules in aqueous environments: IS ¼ ionic strength; C ¼ biopolymer concentration.

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strategy to overcome the disadvantages of using each biopolymer alone and to further enhance their thermal, rheological, and interfacial properties. Attractive proteinepolysaccharide interactions occur primarily via the electrostatic attractions of positively charged region of proteins with negatively charged groups of polysaccharides (McClements, 2006; Turgeon and Laneuville, 2009; Yang et al., 2012). These attractive interactions form soluble or insoluble proteinepolysaccharide complexes; the insoluble complexes are phase separated, forming a two-phase system composed of complex-rich and solvent-rich phases (Fig. 3.2). The transformation of proteinepolysaccharide solution into the two-phase system by the formation and separation of the insoluble complexes is known as “associative separation”; this phase-separation phenomenon is also called “complex coacervation” when the separated insoluble complexes are in liquid state, or “precipitation” when the phase-separated complexes are in solid state. In general, flexible, weakly charged anionic polysaccharides, such as gum arabic, hyaluronic acid, dextran sulfate, and some pectins, are known to have tendency to form liquid-state complex coacervates by relatively mild electrostatic attractions with net-positively charged proteins (Razzak et al., 2017; Turgeon and Laneuville, 2009). On the other hand, rigid, strongly charged anionic polysaccharides, such as gellan gum, alginate, and k-carrageenan, are prone to produce solid-state precipitates by relatively stronger attractions with the proteins; the strong attractions induce the desolvation of proteinepolysaccharide complexes by strong counterion expulsion, leading to precipitation rather than complex coacervation (Razzak et al., 2016; Turgeon and Laneuville, 2009). Repulsive proteinepolysaccharide interactions are attributed to the steric exclusion-associated thermodynamic incompatibility occurring between similarly charged or noncharged biopolymers (Harnsilawat et al., 2006; Phawaphuthanon et al., 2019). These repulsive interactions form a single phase, where the two biopolymers are codissolved, at dilute biopolymer concentrations, but a twophase system, where the two biopolymers are dissolved in different phases, at high biopolymer concentrations (Phawaphuthanon et al., 2019) (Fig. 3.2). Many intrinsic and extrinsic factors influence the molecular interactions between proteins and polysaccharides (McClements, 2006; Razzak et al., 2016; Schmitt et al., 2009; Turgeon and Laneuville, 2009): (1) molecular characteristics of proteins and polysaccharides, such as molar mass, conformation, flexibility, reactive groups, and charge density, (2) solvent properties, such as pH, ionic strength, and polarity, (3) mixing conditions, such as temperature, time, pressure/shear, mixing ratio, total biopolymer concentration, cross-linkers, and irradiation.

5. Molecular interactions of fish gelatin with polysaccharides Fish gelatin is usually obtained from cold-water fish species rather than from hot-water fish species. Unfortunately, as mentioned earlier, cold-water fish gelatin has inferior thermal and rheological properties compared to warm-water fish gelatin and mammalian gelatin. Therefore, the applications of fish gelatin have been explored often in blend with polysaccharides, such as pectin (Liu et al., 2007), kcarrageenan (Haug et al., 2004), chitosan (Kołodziejska et al., 2006), and hydroxypropylmethylcellulose (Chen et al., 2009). So far, only a few studies have investigated the mechanism of molecular interactions between fish gelatin and polysaccharides. These studies showed that fish gelatin actively interacted with anionic polysaccharides, such as sodium alginate, hyaluronic acid, and gum arabic in aqueous solutions, depending mainly on pH, mixing ratio, total biopolymer concentration, and ionic strength, using several

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contemporary techniques, such as turbidimetry, automatic acid titration, spectrophotometric method based on methylene blueepolysaccharide interactions, confocal microscopy, zeta potentiometry, protein assay, dynamic light scattering, and state diagram analysis (Razzak et al., 2016; Razzak et al., 2017; Yang et al., 2012).

5.1 Formation of insoluble complexes The formation of light-scattering insoluble complexes between proteins and polysaccharides can be noted by the appearance of turbidity at around 600 nm (Yang et al., 2012). As illustrated by the schematic turbidity-pH profiles in Fig. 3.3, fish gelatin is known to form insoluble complexes with gum arabic, sodium alginate, and hyaluronic acid in aqueous environments (total biopolymer concentration ¼ 0.05%, 25e40
C); the formation of insoluble fish gelatinepolysaccharide complexes strongly depended on the pH and biopolymer mixing ratio (Razzak et al., 2016; Razzak et al., 2017; Yang et al., 2012). Three types of boundary pH values can be determined from the turbidity profiles: pH41 ¼ the boundary pH below which the turbidity appears, pH42 ¼ the boundary pH below which the turbidity disappears, and pHp ¼ the boundary pH below which the biopolymer aggregates instantly precipitate. The fish gelatinegum arabic system showed peak-like turbidity profiles between two boundary pH values, pH41 and pH42, as illustrated in Fig. 3.3A (Yang et al., 2012). The insoluble complexes formed between the two boundary pHs have a liquid-state complex coacervate structure, in whose structure many water vacuoles exist (Yang et al., 2012). Similar turbidity-pH profiles have been also reported for other proteinepolysaccharide systems, such as whey proteinegum arabic and whey proteine carrageenan systems, where complex coacervation occurred between protein and polysaccharide molecules depending on pH and biopolymer mixing ratio (Weinbreck et al., 2003, 2004). On the other hand, the fish gelatinealginate and fish gelatinehyaluronic acid systems showed incomplete peak-like turbidity profiles between pH41 and pHp (Fig. 3.3B), and no pH42 values were reported (Razzak et al., 2016, 2017). The insoluble complexes formed below pH41 become larger with decreasing the pH due to further aggregation, and visible large aggregates begin to immediately precipitate when the pH reaches pHp, below which the turbidity is no longer measurable. For the fish gelatinealginate system, the insoluble complexes formed below pH41 are precipitates (solid state), where no water vacuoles are contained within their microstructure, rather than complex coacervates (liquid state) (Razzak et al., 2016). The precipitation of proteinepolysaccharide insoluble complexes below pH41 has been also reported for other several proteinepolysaccharide systems; for examples, a system of canola protein isolate and alginate or carrageenan, a system of lentil protein isolate and k-carrageenan or gellan gum, and a system of napin protein isolate and carrageenan (Aryee and Nickerson, 2014; Klassen et al., 2011; Stone et al., 2013, 2014). The fish gelatinealginate precipitates were phase separated to form a gel structure when stored statically for 24 h (Razzak et al., 2016). This electrostatic gel is classified as a coupled network system, in which fish gelatin and alginate molecules are associated together to form a gel network via junction zones, rather than an interpenetration system or a phase-separated network system (Le and Turgeon, 2013; Razzak et al., 2016). For the fish gelatinehyaluronic acid system, the insoluble complexes formed below pH41 are liquid-state complex coacervates, as reported for the fish gelatinegum arabic system (Yang et al., 2012), having a multivesicular microstructure and a dense flowing behavior under gravity (Razzak et al., 2017). These fish gelatinehyaluronic acid complex coacervates, not like those of fish gelatin and gum arabic, were found to be further rearranged to more compact structures, such as lumped

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FIGURE 3.3 Schematic turbidity changes with respect to pH and fish gelatin-to-polysaccharide ratio in (A) fish gelatin-gum arabic system at 40
C and (B) fish gelatin-alginate or fish gelatin-hyaluronic acid system at 25
C, where the turbidity is not measurable below pHp due to the instant precipitation of large biopolymer aggregates. The total biopolymer concentration is 0.05% (w/v). The schematics are drawn based on the results reported by Razzak, M.A., Kim, M., Chung, D., 2016. Elucidation of aqueous interactions between fish gelatin and sodium alginate. Carbohydrate Polymers 148, 181-188; Razzak, M.A., Kim, M., Kim, H.-J., Park, Y.-C., Chung, D., 2017. Deciphering the interactions of fish gelatin and hyaluronic acid in aqueous solutions. International Journal of Biological Macromolecules 102, 885e892; Yang, Y., Anvari, M., Pan, C.-H., Chung, D., 2012. Characterisation of interactions between fish gelatin and gum arabic in aqueous solutions. Food Chemistry 135, 555e561.

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aggregates or precipitates having no water vacuoles, after 24-h static storage (Razzak et al., 2017). However, this structural rearrangement does not lead to gel formation, as reported for the fish gelatinealginate system. The structural rearrangements of the fish gelatinehyaluronic acid complex coacervates, as well as of the fish gelatinealginate precipitates, observed during the static storage, may be attributed to the tendency of the insoluble complexes to achieve better electroneutrality, which could induce further aggregation or coalescence of the insoluble complexes (Razzak et al., 2017).

5.2 Formation of soluble complexes The formation of soluble proteinepolysaccharide complexes can be examined using a spectrophotometric method based on the interactions of polysaccharides with methylene blue (Benichou et al., 2007; Koupantsis and Kiosseoglou, 2009; Yang et al., 2012). Methylene blue, a dye with positive charge, binds to negatively charged polysaccharide to form water-soluble metachromatic complexes, resulting in decrease in the ratio of absorbance at 664 to 615 nm (A664/A615) (Fig. 3.4). The addition of protein can increase the absorbance ratio if the added protein molecules replace the methylene blue molecules, electrostatically bound to the polysaccharide molecules, and cause the release of methylene blue (Fig. 3.4). The methylene blue spectroscopic analysis showed that fish gelatin formed soluble complexes with gum arabic, sodium alginate, and hyaluronic acid even above the isoelectric point (IEP) of fish gelatin (4.8e5.4 depending on temperature), where both fish gelatin and polysaccharide molecules have net negative charges, and no insoluble complexes are formed (Razzak et al., 2016; Razzak et al., 2017;

FIGURE 3.4 Methylene blue spectroscopy for evaluating the formation of soluble protein-polysaccharide complexes.

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Yang et al., 2012). The soluble fish gelatinepolysaccharide complexes formed above the IEP may be resulted from the attractive interactions between the positive charge patches localized on the net negative fish gelatin molecules and the polysaccharide molecules with negative charge. The existence of soluble complexes can be also examined by measuring particle size using dynamic light scattering. Yang et al. (2012) examined the changes in the volume weighted mean diameter (d4,3) of aqueous fish gelatinegum arabic system, prepared with different biopolymer mixing ratios and a total biopolymer concentration of 0.05% (w/v), as a function of pH at 40
C. The d4,3 values of fish gelatinegum arabic system were larger than those of pure fish gelatin and gum arabic solutions (d4,3 ¼ 1.59e1.84 and 7.20e9.01 nm, respectively) in the entire pH range examined (2.5e8.0). Between the pH41 and pH42, the larger size of the mixed biopolymer system is due to the formation of insoluble complexes, whereas above the pH41 or the IEP of fish gelatin, the larger size indicates the formation of soluble biopolymer complexes in the mixed system via electrostatic attractions between the positive charge patches of fish gelatin and the gum arabic with negative charge, as described earlier. The larger size of the mixed biopolymer system below the pH42, where almost no turbidity appears, indicates that the soluble complexes of fish gelatin and gum arabic are also formed in this pH region. In this low pH region, gum arabic molecules are weakly negatively charged (pKa z 2.2; Weinbreck et al., 2003) and may undergo weak attractive interactions with net-positively charged fish gelatin molecules, resulting in the formation of soluble complexes rather than insoluble complexes. Dynamic light scattering also showed that soluble biopolymer complexes are formed above the IEP of fish gelatin in the systems of fish gelatinealginate and fish gelatinehyaluronic acid (Razzak et al., 2016; Razzak et al., 2017).

5.3 State diagram 5.3.1 Effects of pH and mixing ratio The effects of pH and biopolymer mixing ratio on the state of fish gelatinepolysaccharide systems can be seen more clearly in state diagram. Fig. 3.5A shows the state diagram of aqueous fish gelatinegum arabic system, prepared at 0.05% (w/v) total biopolymer concentration and 40
C, where the two boundary pH values, pH41 and pH42, are plotted as a function of the fraction of fish gelatin in total biopolymer content (i.e., biopolymer mixing ratio) (Yang et al., 2012). Between the lines of pH 8.0 and pH41, where fish gelatin is net-negatively charged, the fish gelatinegum arabic system formed a transparent one-phase solution containing individual biopolymer molecules and their soluble complexes formed by weak electrostatic attractions between the positive charge patches of fish gelatin molecules and the negative charge groups of gum arabic molecules. Between the lines of pH41 and pH42, where fish gelatin is net-positively charged, more intense electrostatic attractions occurred between fish gelatin and gum arabic and formed their insoluble complexes (liquid-state complex coacervates); the mixed biopolymer system was phase separated into complex-rich and solvent-rich phases to form a two-phase system. The pH of maximum turbidity (pHmax), at which the formation of insoluble complexes is most favored due to the strongest electrostatic attractions, almost coincided with the IEP of the system, which was measured by zeta potentiometry. This indicates that the most intense attractions between fish gelatin and gum arabic occur under the conditions that the system is electrostatically neutralized (Burgess and Carless, 1984; Ducel et al., 2004; Yang et al., 2012). Between the lines of pH42 and pH 2.5, the system also formed a one-phase transparent solution, in which individual biopolymer molecules and their soluble complexes are contained. This is because, in the

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Chapter 3 Fish gelatin: molecular interactions

FIGURE 3.5 Schematic state diagrams for (A) fish gelatin-gum arabic system at 40
C and (B) fish gelatin-alginate or fish gelatin-hyaluronic acid system at 25
C. The total biopolymer concentration is 0.05% (w/v). The schematics are drawn based on the results reported by Razzak, M.A., Kim, M., Chung, D., 2016. Elucidation of aqueous interactions between fish gelatin and sodium alginate. Carbohydrate Polymers 148, 181e188; Razzak, M.A., Kim, M., Kim, H.-J., Park, Y.-C., Chung, D., 2017. Deciphering the interactions of fish gelatin and hyaluronic acid in aqueous solutions. International Journal of Biological Macromolecules 102, 885e892; Yang, Y., Anvari, M., Pan, C.-H., Chung, D., 2012. Characterisation of interactions between fish gelatin and gum arabic in aqueous solutions. Food Chemistry 135, 555e561.

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region of low pH and high fish gelatin fraction, gum arabic molecules are highly protonated and weakly negatively charged, and thus weakly attracted to the net-positively charged fish gelatin molecules, leading to the formation of soluble complexes. The pH41, pH42, and pHmax show an increasing trend with increasing the fraction of fish gelatin from 0.1 to 0.9 in the state diagram (Fig. 3.5A). This is because more fish gelatin molecules are available per gum arabic molecules when the fraction of fish gelatin is high, and thus, the fish gelatin molecules with lower positive charge at a higher pH are preferred for the formation of complex coacervates with net neutral charge (Weinbreck et al., 2004; Yang et al., 2012). The state diagram in Fig. 3.5B illustrates the effects of pH and biopolymer mixing ratio on the state of aqueous fish gelatinealginate and fish gelatinehyaluronic acid systems, prepared at 0.05% (w/v) total biopolymer concentration of and 25
C (Razzak et al., 2016, 2017). In this state diagram, the values of pH41 and pHp are plotted with respect to the fraction of fish gelatin in total biopolymer content. The increase in pH41 and pHp with the increase of fish gelatin fraction from 0.1 to 0.9 is due to the same reason discussed earlier for the fish gelatinegum arabic system. Between the lines of pH 8.0 and pH41, the two fish gelatinepolysaccharide systems yielded a one-phase transparent solution, in which not only the individual negatively charged biopolymer molecules but also the soluble complexes formed by weak fish gelatinepolysaccharide electrostatic attractions are dissolved, as discussed earlier for the fish gelatinegum arabic system. Between the lines of pH41 and pHp, the fish gelatinealginate system generated a two-phase suspension, in which solid-state precipitates were formed as insoluble complexes due to strong electrostatic attractions. Interestingly, the precipitates were transformed to a gel network when stored undisturbed for 24 h. On the other hand, the fish gelatinehyaluronic acid system formed a two-phase system containing liquid-state complex coacervates as insoluble complexes in this pH region between pH41 and pHp. These complex coacervates were further rearranged to more compact structures (lumped aggregates) after static storage for 24 h, but not to a gel structure. Below the pHp line, instant phase separation of large aggregates (precipitates or complex coacervates) occurred in both the two fish gelatinepolysaccharide systems. The charge of the systems was not necessary to be completely neutralized for the instant phase-separation. For the fish gelatinealginate system, the precipitates instantly phase separated below the pHp were not converted to a gel structure after the 24-h static storage, different from those formed between pH41 and pHp. However, when the total biopolymer concentration increased to 0.2% (w/v) or more, the precipitates formed below the pHp were also transformed to a gel network during the static storage (Razzak et al., 2016). For the fish gelatinehyaluronic acid system, the complex coacervates instantly phase separated below the pHp underwent structural rearrangement during the 24-h static storage to form solid-state precipitates having no water vacuoles within their structure, but no gel structure was formed (Razzak et al., 2017).

5.3.2 Effects of NaCl Fig. 3.6 shows the effects of ionic strength (as NaCl concentration) on the state of fish gelatinealginate system (total biopolymer ¼ 1.0% (w/v), fish gelatin fraction ¼ 80%) and fish gelatinehyaluronic acid system (total biopolymer ¼ 0.05% (w/v), fish gelatin fraction ¼ 70%) at 25
C (Razzak et al., 2016, 2017). In the presence of a low NaCl concentration (20 mM), however, the two boundary pH values decreased with the increase of NaCl concentration, indicating that the addition of NaCl gave an adverse effect on the formation of the insoluble complexes and their aggregates. The addition of a high level of NaCl may further decrease the Debye length, which could screen the short-range attractive interactions of fish gelatin with the polysaccharides (Li et al., 2012; Razzak et al., 2016, 2017).

6. Applications The proteinepolysaccharide interactions could find many valuable food, pharmaceutical, and cosmetic applications, especially for the development of biopolymeric encapsulation or chemical delivery devices, gels, edible films, coatings, emulsions, foams, fat replacers, meat substitutes, and textures (Phawaphutanon et al., 2019; Turgeon et al., 2007; Weinbreck et al., 2003; Yang et al., 2012). The fish gelatinepolysaccharide interactions have been also studied to explore some interesting applications. (1) Gelation The complex coacervate phase separated from a cold-water fish gelatinegum arabic system (total biopolymer concentration ¼ 2% (w/v), pH 3.5, interaction temperature ¼ 40
C, phase-separation

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temperature ¼ 10
C) forms a thermos-reversible gel structure by cooling (Anvari et al., 2015). This coacervate phase has a slightly higher gelling point (3.9
C) but a much stronger gel strength (complex modulus ¼ 85.7 Pa at 3
C) than the alkaline mixture of fish gelatin and gum arabic (gelling point ¼ 3.7
C, complex modulus ¼ 36.7 Pa at 3
C), prepared under the conditions of pH 8.0 and the biopolymer compositions same as those in the coacervate phase (fish gelatin ¼ 7.7%, gum arabic ¼ 7.4%) (Anvari et al., 2015). At pH 3.5, fish gelatin molecules have net positive charges and thus attractively interact with the gum arabic molecules with negative charges to form complex coacervates; whereas, at pH 8.0, both biopolymers are net-negatively charged and not strongly attracted to each other to form complex coacervates. In addition, fish gelatin has an ability to form a cold-set gel, but gum arabic is a nongelling polysaccharide. Therefore, the earlier results implies that the gum arabic molecules may be involved in the formation of fish gelatin gel network via their electrostatic attractions with fish gelatin molecules, resulting in the formation of a more compact gel structure. The cold-set gel of fish gelatinegum arabic complex coacervates becomes more liquid-like as the angular frequency increases during frequency sweep test because of the breakage of gel network (Anvari et al., 2015). Therefore, the complex coacervate gel may be considered as a weak physical gel, which forms its network structure via noncovalent interactions, such as electrostatic interactions, hydrogen bonding, and hydrophobic interactions (Tunick, 2011). The phaseseparation temperature also influenced the gelling behavior of the complex coacervate gel composed of fish gelatin and gum arabic; the complex coacervate phase showed a better gelling tendency and formed a firmer gel when obtained at a lower temperature (Anvari et al., 2015). This may be because the complex coacervate phase prepared at a lower temperature has a lower mobility of molecules and a stronger hydrogen bonding interactions. Furthermore, fish gelatin is known to have a helical molecular structure below a specific temperature (around 15
C, Haug and Draget, 2009), and the molecules of helical structure could develop junction zones more easily than those of random coil structure, leading to the faster and stronger gelation (Anvari et al., 2015). (2) Microencapsulation The fish gelatinegum arabic interactions have been studied for developing coreeshell type microspheres encapsulating fucoxanthin, which is a xanthophyll carotenoid abundant in marine macro- and microalgae (Quan et al., 2013). The schematic of the microspheres developed is shown in Fig. 3.7. This coreeshell-type microspheres are composed of a solid core formed with the lipid mixture of cetyl palmitate and canola oil, in which fucoxanthin is dissolved, and a solid shell formed with fish gelatinegum arabic complex coacervates hardened with tannic acid, in which the polysorbate 80 used as an emulsifier is also contained. Cetyl palmitate, a wax ester of cetyl alcohol and palmitic acid, has been frequently employed to prepare nanostructured lipid carriers or solid-lipid nanoparticles in the development of drug delivery devices (Quan et al., 2013; Sarmento et al., 2007). Cetyl palmitate is a solid lipid having a melting point of about 49
C and a solidification point of about 45
C (Quan et al., 2013), and thus mixed with canola oil to form a sold lipid core to secure the integrity of microspheres, provide better stability to fucoxanthin, and achieve better sustained release performance, via the reduction in molecular mobility. Tannic acid, a hydrolysable tannin, is used as a safe gelatin cross-linking agent, instead of commonly used toxic aldehyde-type cross-linking agents, to transform the shell formed by fish gelatinegum arabic complex coacervation from liquid to gel state (Zhang et al., 2011).

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Chapter 3 Fish gelatin: molecular interactions

FIGURE 3.7 Fucoxanthin-encapsulated microspheres composed of the cetyl palmitate-based solid-lipid core and the fish gelatin-gum arabic complex coacervate shell cross-linked by tannic acid.

The freezeedried microspheres have a find powder form with a diameter of about 87e89 mm and an encapsulation efficiency of about 75%e79% depending on the fraction of cetyl palmitate in the core (30 or 50%) (Quan et al., 2013). The release of fucoxanthin from the microspheres into the simulated gastrointestinal fluids can be slowed down by increasing the cetyl palmitate fraction in the core up to 50% (Fig. 3.8A). The stability of fucoxanthin encapsulated is significantly improved by increasing the cetyl palmitate fraction in the core as shown in Fig. 3.8B, where the fucoxanthin encapsulated within the microspheres of 50% cetyl palmitate in the core retains 80% of its initial loading after 100 day-storage in dark at 0% relative humidity and 40
C (Quan et al., 2013). As the core structure becomes more solidified or crystalline with increasing cetyl palmitate ratio, the mobility of molecules in the core can be more hindered, causing retardation in the release and degradation of fucoxanthin molecules, as well as the microspheres may be more protected against their disintegration in simulated gastrointestinal fluids. (3) Foam formation and stabilization The fish gelatinealginate interactions have been studied for their effects on the formation and stabilization of foams at 25
C by correlating the interface, bulk, and foaming characteristics of the aqueous fish gelatinealginate system prepared at different values of pH and mixing ratio with 0.5% (w/v) total biopolymer concentration (Phawaphuthanon et al., 2019). At pH 7.0, where fish gelatin has net negative charge and weakly attracts to the negatively charged alginate to form soluble complexes, the addition of alginate (increasing the alginate fraction) reduces the surface activity of the fish gelatinealginate system at aireliquid interface (increase in surface tension),

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FIGURE 3.8 Schematic profiles for (A) the release of fucoxanthin from the dried microspheres consisting of solid-lipid core and fish gelatin-gum arabic coacervate shell, exposed to simulated gastrointestinal fluids at 37
C and (B) the stability of fucoxanthin encapsulated in the dried microspheres exposed to 0%e51% relative humidity at 40
C in the dark. The schematics are drawn based on the results reported by Quan, J., Kim, S.-M., Pan, C.-H., Chung, D., 2013. Characterization of fucoxanthin-loaded microspheres composed of cetyl palmitate-based solid lipid core and fish gelatin-gum arabic coacervates shell. Food Research International 50, 31-37.

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Chapter 3 Fish gelatin: molecular interactions

but increases the bulk viscosity and particle size of the system. At pH 3.5, where fish gelatin is netpositively charged and undergoes strong electrostatic attractions with anionic alginate to form more charge-neutralized insoluble complexes, the addition of alginate (increasing the alginate fraction) causes significant increase in the aireliquid surface activity (decrease in surface tension) and particle size, but suppression in bulk viscosity. Compared to the solution of fish gelatin or whey protein concentrate only, the fish gelatinealginate system shows a weaker forming ability, but much greater foam stability during the storage at 25
C (Phawaphuthanon et al., 2019). Alginate is highly viscous, negatively charged, and poorly surface active at aireliquid interface. Therefore, the addition of alginate (increasing the alginate fraction) reduces the aireliquid surface activity (increase in surface tension) and increases the bulk viscosity; this reduces the incorporation of air during foam formation, resulting in the suppression in foaming ability (Makri and Doxastakis, 2007; Phawaphuthanon et al., 2019). The foams prepared at pH 3.5 show weaker suppression in foaming ability upon the addition of alginate than those prepared at pH 7.0. This may be because the strong fish gelatinealginate electrostatic attractions at pH 3.5 (compared to at pH 7.0) and the resulting charge neutralization promote the surface adsorption of the biopolymers (increase in surface activity) and the formation of biopolymer network, leading to the incorporation of a larger amount of air into the system; this could minimize the suppression of foaming ability by the addition of alginate (Phawaphuthanon et al., 2019). In addition, alginate is hygroscopic and has high molar mass. Therefore, the addition of alginate (increasing the alginate fraction) increases water holding ability, forming stiffer and heavier foams, and develops a network structure with fish gelatin in the lamella and plateau border regions of foams; this reduces the coalescence of bubbles and the drainage of liquid, leading to the enhancement of foam stability (Miquelim et al., 2010; Phawaphuthanon et al., 2019; _ Zmudzi nski et al., 2014). The foams produced at pH 3.5 shows higher enhancement in foam stability upon the addition of alginate than those prepared at pH 7.0. This may be because the strong fish gelatinealginate electrostatic attractions at pH 3.5 (compared to at pH 7.0) and the resulting charge neutralization enhance the surface activity and the formation of biopolymer network in the lamellar and plateau border regions of foams, resulting in the inhibition of bubble coalescence and liquid drainage; this could maximize the enhancement of foam stability by the addition of alginate (Phawaphuthanon et al., 2019).

7. Conclusions The molecular interactions between proteins and polysaccharides in aqueous environments could find many interesting food, pharmaceutical, and cosmetic applications: (1) developing novel encapsulation devices, coatings, and edible films, (2) stabilizing foams and emulsions, and (3) engineering smart structures and textures for high quality foods (better appearance, sensory, and stability), including fat replacers, meat substitutes, and various types of colloidal suspensions, gels, and particles. Studies showed that fish gelatin interacts with anionic polysaccharides, such as gum arabic, alginate, and hyaluronic acid, in aqueous environments to form soluble or insoluble biopolymer complexes in the form of complex coacervates (liquid state) or precipitates (solid state) primarily via electrostatic attractions, depending on mixing conditions, such as pH, temperature, total concentration, and ionic

References

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strength. The molecular interactions of fish gelatin with polysaccharides are considered as an effective approach to improve the inferior thermal and rheological properties of fish gelatin, especially derived from cold-water fish species. Studies showed that such interactions are indeed useful for improving the gelling tendency and rigidity of fish gelatin gels, developing the outer shell structure of coreeshelltype microcapsules, and enhancing the ability of foam formation and the stability of foams. Further studies will provide novel fish gelatin-based biopolymeric materials, which are safe and have no religious, cultural consumer concerns, for various purposes in the industries of foods, cosmetics, pharmaceutics, and biomedical devices.

References Anvari, M., Pan, C.-H., Yoon, Y.-B., Chung, D., 2015. Characterization of fish gelatin-gum arabic complex coacervates as influenced by phase separation temperature. International Journal of Biological Macromolecules 79, 894e902. Aryee, F.N.A., Nickerson, M.T., 2014. Effect of pH, biopolymer mixing ratio and salts on the formation and stability of electrostatic complexes formed within mixtures of lentil protein isolate and anionic polysaccharides (k-carrageenan and gellan gum). International Journal of Food Science and Technology 49, 65e71. Benichou, A., Aserin, A., Lutz, R., Garti, N., 2007. Formation and characterization of amphiphilic conjugates of whey protein isolate (WPI)/xanthan to improve surface activity. Food Hydrocolloids 21, 379e391. Burgess, D.J., Carless, J.E., 1984. Microelectrophoretic studies of gelatin and acacia for the prediction of complex coacervation. Journal of Colloid and Interface Science 98, 1e8. Chen, H.-H., Lin, C.-H., Kang, H.-Y., 2009. Maturation effects in fish gelatin and HPMC composite gels. Food Hydrocolloids 23, 1756e1761. Chiou, B.-S., Avena-Bustillos, R.J., Shey, J., Yee, E., Bechtel, P.J., Imam, S.H., Glenn, G.M., Orts, W.J., 2006. Rheological and mechanical properties of cross-linked fish gelatins. Polymer 47, 6379e6386. Cho, S.M., Gu, Y.S., Kim, S.B., 2005. Extraction optimization and physical properties of yellowfin tuna (Thunnus albacares) skin gelatin compared to mammalian gelatins. Food Hydrocolloids 19, 221e229. Cole, C.G.B., 2000. Gelatin. In: Francis, F.J. (Ed.), Encyclopedia of Food Science and Technology, second ed. John Wiley & Sons, New York, NY, pp. 1183e1188. Djabourov, M., 1988. Architecture of gelatin gels. Contemporary Physics 29, 273e297. Ducel, V., Richard, J., Saulnier, P., Popineau, Y., Boury, F., 2004. Evidence and characterization of complex coacervates containing plant proteins: application to the microencapsulation of oil droplets. Colloids and Surfaces A: Physicochemical and Engineering Aspects 232, 239e247. Quan, J., Kim, S.-M., Pan, C.-H., Chung, D., 2013. Characterization of fucoxanthin-loaded microspheres composed of cetyl palmitate-based solid lipid core and fish gelatin-gum arabic coacervates shell. Food Research International 50, 31e37. Go´mez-Guille´n, M.C., Pe´rez-Mateos, M., Go´mez-Estaca, J., Lo´pez-Caballero, E., Gime´nez, B., Montero, P., 2009. Fish gelatin: a renewable material for developing active biodegradable films. Trends in Food Science and Technology 20, 3e16. Gudmundsson, M., Hafsteinsson, H., 1997. Gelatin from cod skins as affected by chemical treatments. Journal of Food Science 62, 37e47. Harnsilawat, T., Pongsawatmanit, R., McClements, D.J., 2006. Characterization of b-lactoglobulin-sodium alginate interactions in aqueous solutions: a calorimetry, light scattering, electrophoretic mobility and solubility study. Food Hydrocolloids 20, 577e585.

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Haug, I.J., Draget, K.I., 2009. Gelatin. In: Phillips, G.O., Williams, P.A. (Eds.), Handbook of Hydrocolloids, second ed. Woodhead Publishing Limited, Cambridge, pp. 142e163. Haug, I.J., Draget, K.I., Smidsrød, O., 2004. Physical behavior of fish gelatin-k-carrageenan mixtures. Carbohydrate Polymers 56, 11e19. Karayannakidis, P.D., Zotos, A., 2016. Fish processing by-products as a potential source of gelatin: a review. Journal of Aquatic Food Product Technology 25, 65e92. Karim, A.A., Bhat, R., 2009. Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocolloids 23, 563e576. Klassen, D.R., Elmer, C.M., Nickerson, M.T., 2011. Associative phase separation involving canola protein isolate with both sulphated and carboxylated polysaccharides. Food Chemistry 126, 1094e1101. Kołodziejska, I., Piotrowska, B., Bulge, M., Tylingo, R., 2006. Effect of transglutaminase and 1-ethyl-3-(dimethylaminopropyl) carbodiimide on the solubility of fish gelatin-chitosan films. Carbohydrate Polymers 65, 404e409. Koupantsis, T., Kiosseoglou, V., 2009. Whey protein-carboxymethylcellulose interaction in solution and in oil-inwater emulsion systems. Effect on emulsion stability. Food Hydrocolloids 23, 1156e1163. Le, X.T., Turgeon, S.L., 2013. Rheological and structural study of electrostatic cross-linked xanthan gum hydrogels induced by b-lactoglobulin. Soft Matter 9, 3063e3073. Li, X., Fang, Y., Al-Assaf, S., Phillops, G.O., Yao, X., Zhang, Y., Zhao, M., Zhang, K., Jiang, F., 2012. Complexation of bovine serum albumin and sugar beet pectin: structural transitions and phase diagram. Langmuir 28, 10164e10176. Lin, L., Regenstein, J.M., Lv, S., Lu, J., Jiang, S., 2017. An overview of gelatin derived from aquatic animals: properties and modification. Trends in Food Science and Technology 68, 102e112. Liu, L., Liu, C.-K., Fishman, M.L., Hicks, K.B., 2007. Composite films from pectin and fish skin gelatin or soybean flour protein. Journal of Agricultural and Food Chemistry 55, 2349e2355. Makri, E., Doxastakis, G.I., 2007. Surface tension of Phaseolus vulgaris and coccineus proteins and effect of polysaccharide complexes at their interfaces. Food Hydrocolloids 24, 398e405. McClements, D.J., 2006. Non-covalent interactions between proteins and polysaccharides. Biotechnology Advances 24, 621e625. Miquelim, J.N., Lannes, S.C.S., Mezzenga, R., 2010. pH Influence on the stability of foams with proteinpolysaccharide complexes at their interfaces. Food Hydrocolloids 24, 398e405. Phawaphuthanon, N., Yu, D., Ngamnikom, P., Shin, I.-S., Chung, D., 2019. Effect of fish gelatin-sodium alginate interactions on foam formation and stability. Food Hydrocolloids 88, 119e126. Razzak, M.A., Kim, M., Chung, D., 2016. Elucidation of aqueous interactions between fish gelatin and sodium alginate. Carbohydrate Polymers 148, 181e188. Razzak, M.A., Kim, M., Kim, H.-J., Park, Y.-C., Chung, D., 2017. Deciphering the interactions of fish gelatin and hyaluronic acid in aqueous solutions. International Journal of Biological Macromolecules 102, 885e892. Rustad, T., Storrø, I., Slizyte, R., 2011. Possiblitities for the utilization of marine byproducts. International Journal of Food Science and Technology 46, 2001e2014. Sarmento, B., Martins, S., Ferreira, D., Souto, E.B., 2007. Oral insulin delivery by means of solid lipid nanoparticles. International Journal of Nanomedicine 2, 743e749. Schmitt, C., Aberkane, L., Sanchez, C., 2009. Protein-polysaccharide complexes and coacervates. In: Phillips, G.O., Williams, P.A. (Eds.), Handbook of Hydrocolloids, second ed. Woodhead Publishing Limited, Cambridge, pp. 420e476. Stone, A.K., Cheung, L., Chang, C., Nickerson, M.T., 2013. Formation and functionality of soluble and insoluble electrostatic complexes within mixtures of canola protein isolate and (k-, i- and l-type) carrageenan. Food Research International 54, 195e202.

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Stone, A.K., Teymurova, A., Dang, Q., Abeysekara, S., Karalash, A., Nickerson, M.T., 2014. Formation and functional attributes of electrostatic complexes involving napin protein isolate and anionic polysaccharides. European Food Research and Technology 238, 773e780. Tunick, M.H., 2011. Small-strain dynamic rheology of food protein networks. Journal of Agricultural and Food Chemistry 59, 1481e1486. Turgeon, S.L., Laneuville, S.I., 2009. Proteinþpolysaccharide coacervates and complexes: from scientific background to their application as functional ingredients in food products. In: Kasapis, S., Norton, I.T., Ubbink, J.B. (Eds.), Modern Biopolymer Science. Academic Press, London, pp. 327e357. Turgeon, S.L., Schmitt, C., Sanchez, C., 2007. Protein-polysaccharide complexes and coacervates. Current Opinion in Colloid and Interface Science 12, 166e178. Wainewright, F.W., 1977. Physical test for gelatin and gelatin products. In: Ward, A.G., Courts, A. (Eds.), The Science and Technology of Gelatin. Academic Press, London, pp. 507e534. Weinbreck, F., de Vries, R., Schrooyen, P., de Kruif, C.G., 2003. Complex coacervation of whey proteins and gum Arabic. Biomacromolecules 4, 293e303. Weinbreck, F., Nieuwenhuijse, H., Robijn, G.W., de Kruif, C.G., 2004. Complexation of whey proteins with carrageenan. Journal of Agricultural and Food Chemistry 52, 3550e3555. Yang, Y., Anvari, M., Pan, C.-H., Chung, D., 2012. Characterisation of interactions between fish gelatin and gum Arabic in aqueous solutions. Food Chemistry 135, 555e561. Zhang, Z.-Q., Pan, C.-T., Chung, D., 2011. Tannic acid cross-linked gelatin-gum Arabic coacervate microspheres for sustained releases of allyl isothiocyanate: characterization and in vitro release study. Food Research International 44, 1000e1007. _ Zmudzi nski, D., Ptaszek, P., Kruk, J., Kaczmarczyk, K., Ro_znowski, W., Berski, et al., 2014. The role of hydrocolloids in mechanical properties of fresh foams based on egg white proteins. Journal of Food Engineering 121, 128e134.

CHAPTER

Peptides as biopolymersdpast, present, and future

4

Advaita Ganguly1, 2, Kumakshi Sharma3, Kaustav Majumder4 1

Comprehensive Tissue Centre, UAH Transplant Services, Alberta Health Services, Edmonton, AB, Canada; 2Health Sciences Education and Research Commons, University of Alberta, Edmonton, AB, Canada; 3Health, Safety and Environment Branch, National Research Council Canada, Edmonton, AB, Canada; 4Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, United States

1. Introduction Peptides isolated or identified from various sources have been long recognized as biomaterials and well known for its biological applications specifically in biomedical and food industries. About 60 FDA-licensed peptide-based drugs in the United States are fully available for clinical applications, and more entities are in the process of entering different phases of clinical trials and commercialization (Fosgerau and Hoffmann, 2015). Reports suggest that close to 150 peptide molecules are in various stages of investigation and regulatory approval around the world (Fosgerau and Hoffmann, 2015; Kaspar and Reichert, 2013). The peptide-based therapeutic and biomedical applications are considered more robust than the applications involving antibodies, but extensive investigations and validation studies are needed to develop more potent peptideebased products and making them relevant in biomedical applications. Multiple peptideebased therapeutics is in clinical phases wherein peptide research is reliant on novel targets and combination of underlying mechanisms. Peptideedrug conjugates and peptides with adaptive attributes studies are currently prevalent (Kaspar and Reichert, 2013; Engel et al., 2012; Kaspar and Reichert, 2013). The developments of peptides with more effective chemical functions are being studied with broader pathogenic target domains. According to research by Reichert and colleagues, the development of novel peptideedrug conjugates can be supplemented by the knowledge gained in the studies of antibody-based conjugates (Reichert, 2011). Future peptideebased therapeutic studies would be more focused on peptideedrug conjugates (Kaspar and Reichert, 2013). A substantial amount of research exhibited that peptide conjugates can target various cellular processes (Sawyer, 2009). Further validations of these results are needed and investigations to better understand the action of the peptide-based conjugates on the in vitro and in vivo systems. There is a robust prevailing propensity to improve the understanding of fundamental principles of active and passive drug diffusion to design useful bioactive peptideedrug cohorts with enhanced functionalities (Kaspar and Reichert, 2013). The peptide therapeutic market has been witnessing a lot of support and investments from major pharmaceutical and drug manufacturers in the recent years (Fosgerau and Hoffmann, 2015; Kaspar and Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00004-7 Copyright © 2020 Elsevier Inc. All rights reserved.

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Reichert, 2013; Reichert, 2011; Sheridan, 2012). Major thrusts and focus to expedite growth in developing peptideedrug conjugates stem from the goal to create bioactive peptideebased therapeutics targeting debilitating and potentially fatal diseases (Okarvi, 2008). The peptide-based therapeutics has been found to have relatively high safety standards as well as efficacious in their modes of action (Fosgerau and Hoffmann, 2015). The future emphasis would involve precise engineering and modifications of peptide features and focusing on improving the limitations and effectively generate peptide conjugates with an enhanced field of action (Fosgerau and Hoffmann, 2015). Nevertheless, it is essential to note the effects of host immune implications against the bioactive peptide conjugates and their role in the effectiveness of the peptide therapeutics (Zhou and Bohn, 2014). Research should also target novel applications beyond the traditional peptide targets and peptide conjugates capable of cell penetration which would help to add to the existing know-how of peptides, antibodies, and other molecule passage through cellular membranes (Fosgerau and Hoffmann, 2015). Along with the development and commercialization of peptideedrug conjugates and other bioactive peptideebased therapeutics, the delivery mechanisms involved in transferring the peptide entities to the site of action should also be comprehensively studied. The biological activity or properties of peptides are also explored in various food applications. Most of the primary research has now identified various peptide sequences through different in vitro studies. However, the commercial application of these food-derived bioactive peptides with health beneficial properties have been delayed and the three primary reasons for these delays are (1) lack of understanding about the molecular mechanisms of action of these peptides, (2) lack of well-conducted clinical trials, and (3) absence of scale-up technologies. This chapter briefly highlights the codevelopment and future potentials of peptides and biopolymers and their increasing importance in the biomedical and food-processing industries. Currently, the use of natural polymers, peptides, and their conjugates are being considered owing to their functional and safety features. Peptide features and polymer developments are also discussed along with biopolymer modification mechanisms to suit applications in food packaging as well as in clinically therapeutic roles. Block copolymerization, blending, peptide engineering, and conjugations are also discussed.

2. Peptides with biomedical applications In the recent past, peptideedrug conjugates have reached the clinical trial stages and extensive studies are undertaken to generate more potent peptides with varying residues and elucidating their action mechanisms. Enhancing peptide functionality and developing them against diverse biomarkers are absolutely crucial in the development of clinically efficacious therapeutic conjugates. Antibodies as targeting vehicles have been successfully validated as in the case of certain cytotoxins (Reichert, 2011). Scientists have successfully validated the effectiveness of antibodyedrug conjugates like gemtuzumabeozogamicin, brentuximabevedotin, and trastuzumabeemtansine. These antibody-based conjugates obtained approval by FDA, for clinical use based on key parameters including the targeting moiety, the corresponding linker, and the specific drug in the conjugate. The gemtuzumabeozogamicin combination was, however, subsequently deregulated. The research knowhow from developing antibodyedrug combinations can definitely be transferred in designing effective peptide conjugates, thereby expediting the process. Some of the peptideedrug entities in different

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stages of clinical trials are NGR-hTNF (Phase III), AEZS108 (Phase II), and EP100 (Phase II). These bioactive peptideebased moieties comprise of a protein (tumor necrosis factor), doxorubicin, and a peptide as a part of the conjugate. More studies would help develop more varied combinations of peptide-based conjugates and fast-tracking clinical trial and ultimately clinical use. Dublin-based Shire (currently Takeda Pharmaceuticals) and Arrowhead Pharmaceuticals had earlier collaborated to design peptideedrug conjugates merging their respective peptide and therapeutic cohort platforms against a vast array of therapeutic targets. Designing peptides with active conformations can lead to candidates with high-affinity and demonstrating structural stability and resistant to enzymatic degradation, significantly enhancing their functional attributes. A precise structural design would help develop peptides with more effective cellular membrane passage functions, thereby increasing potency and furthering the ability to target more therapeutically relevant cellular pathways. A number of therapeutic and pharmaceutical companies have come up with platforms able to design and develop constrained peptides as therapeutic applications. Constrained peptides have for long been of considerable interest to researchers. These peptides can combine antibody and small molecule functions in a single entity. Bicycle Therapeutics Inc. has developed cysteine-containing peptides with two macrocycles from phage display libraries (Rentero and Heinis, 2013). Clones with specific binding ability are isolated and sequenced to obtain the desired peptide. A urokinase-type plasminogen activator antagonist peptide showed stability in vivo studies, and potentially could be combined with an albumin-binding peptide (Angelini et al., 2012) or an immunoglobulin (Angelini et al., 2012) to generate protease-resistant molecules with higher shelf life. Another commercial enterprise used hydrocarbon staples to insert alpha helices in the peptides (Henchey et al., 2008; Guo et al., 2010; Sawyer, 2009). This technology enabled considerable enhancement of the pharmacokinetic features of an HIV fusion antagonist (Bird et al., 2010). Such peptides have had success in clinical trials as well. Similarly designed peptides are also been comprehensively studied as anticancer therapeutic choices. Chang and coworkers demonstrated that ATSP-7041 a peptide developed using hydrocarbon staples was able to inhibit both MDM2 and MDMX, in a xenograft oncologic animal model (Chang, 2012). ATSP-7041 preserved the active a-helical conformation and also exhibited druglike characteristics, like cell penetration, high-affinity binding to both target proteins, and enhanced in vivo stability. The development of constrained peptides specifically targeting cellular processes is being investigated for a while but due to limited knowledge and understanding of cellular entry mechanisms have restricted progress in this domain (Sawyer, 2009). Bioactive peptides derived from food enrich nutrition retain biological characteristics and also possess therapeutic potential in some chronic health conditions. Food-derived bioactive peptides and their diverse health benefits have led to a deluge in commercial research and applications globally. From peptide incorporation in functional foods, as dietary add-ons, as well as pharmaceuticals to ameliorate of chronic disease conditions, applications involving bioactive peptides have been on a growing rapidly. In a recent report by Chatterjee and group (Chatterjee et al., 2018), the potential of soybean-derived peptides in facilitating chronic disease management has been thoroughly reviewed. More comprehensive knowledge about transfer mechanisms of drugs and druglike entities across cells would benefit the development of more effective peptide candidates.

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2.1 Therapeutic peptides and biopolymers Several peptides based therapeutic candidates demonstrated in vitro potential but considerably lower functionality in vivo experiments. This may be due to limitations in movement across cellular membranes. Reduced in vivo efficacy of therapeutic drug candidates also owes to suboptimal pharmacokinetics or systemic toxicity. Further research is needed to significantly ameliorate the pharmacokinetic profiles of drug cohorts. Peptide-based biomaterials show considerable potential for efficient in vivo delivery of pharmaceuticals (Hart and Gehrke, 2007). Polymers as macromolecular transporters have the ability to circumvent transfer barriers that generally limit drug delivery to, especially tumors (Langer, 1998; Putnam and Kopecek, 1995; Duncan, 1992; Jain, 2001). Polymer constructs commonly used for drug delivery are large hydrophilic molecules conjugated to a therapeutic moiety. These conjugates target tumors passively through the EPR effect as well as actively employing a stimulus or affinity toward the therapeutic site (Duncan, 1992; Ringsdorf, 1975; Tomlinson, 1985). These large macromolecular carriers have a higher half-life, lower systemic toxicity, and retain functional characteristics against diverse drug-resistant cell lines, and also improve the solubility of drugs. These features have resulted in increased anticancer therapeutic efficiency for passively targeted polymer conjugates (Duncan, 1992; Ghandehari and Cappello, 1998; Kopeceket al., 2000; 2001; Lukyanov and Torchilin, 2004; Torchilin et al., 2003; Nan et al., 2005; Weissig et al., 1998; Kabanov et al., 2005). Progress in peptide-based polymer developments resulted in polypeptide delivery vehicles targeting anticancer biomarkers. There are considerable advantages of using polypeptides; the foremost being the composition of natural amino acid residues upholds biocompatibility and stability during degradation forming metabolites cleared through normal metabolic routes (Cappello, 1990). Genetically encoded peptides have uniform molecular weight and sequences, which are important attributes influencing pharmacokinetic control, transport, biodistribution, and degradation (Ghandehari and Cappello, 1998; Nagarsekar and Ghandehari, 1999). Short peptide sequences as targeting candidates can be inserted at the gene level at relevant and functional locations. Amphiphilic polypeptides are also capable in encapsulating drugs in self-assembling structures like micelles and vesicles, thereby mimicking synthetic polymers. Peptide-based biomaterials also have a range of applications in the domain of regenerative medicine. Tissue engineering applications of elastin-based polypeptides, extracellular matrixederived collagens, fibrins, spider silk proteins, etc. (Langer and Tirrell, 2004; Lutolf and Hubbell, 2005; Cappello and Ghandehari, 2002; Foo and Kaplan, 2002; van Hest and Tirrell, 2001) have been studied extensively. Their utility stems from their stable chemical compatibility in aqueous solutions, efficient in vivo biocompatibility, and controlled degradation rate in vivo. These moieties can readily break down into natural amino acids, which are metabolized by the body. They also exhibit considerably low cellular toxicity, immune response, and inflammation (Karle and Urry, 2005; Urry, 1999; Urry et al., 1998; Nicol et al., 1992, 1993; Altman et al., 2003; Horan et al., 2005). Biopolymers can also readily be functionalized to improve their cellular interactions providing optimal cellular activities and tissue functions. Peptide-based biopolymers for tissue engineering are commonly used as injectable scaffolds forming gels. These biomaterials essentially provide a minimally invasive mode to deliver nanoscale tissue scaffolds. The liquidlike precursors can be modified and then injected at specific sites having tissue imperfections. The polymers form a hydrogel after injection that provides structural support to the embedded cells as well as tissue repairing functions for tissue repair, helps in tissue reconstitution,

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and controls drug release. Different gelation mechanisms include self-assembly by environmental factors such as temperature, pH, or ionic strength, by chemical cross-linking using enzymes, radiation, radical polymerization, or photopolymerization, as well as physical cross-linking methods (Chow et al., 2008).

2.2 Biopolymers and peptides targeting the cardiovascular system Hypertension is considered a major risk for developing myocardial infarction, congestive heart failure, and arteriosclerosis. The enzymes, angiotensin-converting enzyme I (ACE I) and renin, help in the regulation of blood pressure and salt water balance within the renin angiotensin aldosterone system. The ACE I is one of the primary targets in blood pressure management and drugs such as captopril is used in the treatment. These pharmaceutical drugs though exhibit complex side effects like sleep disorders and angioedema. To overcome such manifestations, food-derived bioactive peptides are being regularly used as antihypertensive agents (Table 4.1) (Hayes and Tiwari, 2015). In another approach, naturally derived polymer-based biomaterials have had applications as cardiac patches to assist in cardiac regeneration. Naturally derived biopolymers consisting of polysaccharide and protein are able to interact with the extracellular matrix. This helps in cell adhesion and proliferation and allays any immunogenic reaction when compared to synthetic polymers. The natural biopolymers also have a very high safety profile and get readily absorbed by the body ensuring no toxicity as well as enhancing heart (Coutu et al., 2009). Delivery of therapeutics targeted at the cardiovascular system widely uses polymer systems to enhance pharmacokinetic properties and reduce toxicity (Geldenhuys et al., 2017). Table 4.2 highlights some cardiovascular delivery of therapeutic agents using different polymer systems.

3. Food industry applications Developments in nanotechnology have considerably progressed to ameliorate encapsulation and delivery issues encountered in the food industry, particularly about functional value addition in the food products (Aditya et al. 2015; Aditya et al., 2014; Patel and Velikov, 2011). Polymers, polymer Table 4.1 Food-derived antihypertensive peptides. Peptide

Bioactivity/Source

Reference

LKPNM

Antihypertensive (peptACE)/Bonito Antihypertensive (Valtyron)/Sardine Antihypertensive (Evolus)/Milk Stress relief (Lactium)/ Milk Blood pressure management/Whey proteins

Fujita and Yoshikawa (1999) Kawasaki et al. (2000)

VY IPP and VPP YLGYLEQLLR Whey peptides

Siltari et al. (2012) Nagai et al. (2006) Arihara (2006)

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Table 4.2 Cardiovascular therapeutics using polymer-based delivery. Polymer system Liposome PLGA PEG Exosomes

Therapeutic agent/ Disease siRNA/Atherosclerosis Pitavastatin/ Atherosclerosis Peptides/ Atherosclerosis siRNA/Inflammation

Reference Guo and Huang (2014) Kim and Bynoe (2015) Harder et al. (1995) Shtam et al. (2013)

conjugates, proteins, carbohydrates, and also fats have been studied and applied as materials to functionally enhance products (Patel and Velikov, 2011; Yao et al., 2015). But these materials are more adept in delivering bioactive entities characteristically hydrophobic in nature (McClements et al., 2016; McClements and Xiao, 2014). In the pharmaceutical industry liposomes, solid lipid nanoparticles, and polymeric nanoparticles are used for hydrophobic pharmaceuticals with significantly less bioavailability. Hydrophilic pharmaceutical agents pose considerably lower problems as they can be altered with different formulations. This kind of modifications cannot be implemented in the food industry as standards related to appearance and sensory factors are negatively impacted. A specific way to safeguard functional bioactive agents, e.g., peptides, and prevent their breakdown during different stages of development and simultaneously preserving the sensory and other parameters of the functionally enhanced food is very important (Fabra et al., 2014).

3.1 Biopolymer nanoparticles as delivery systems Functional groups present on proteins get charged due to factors such as pH changes, temperature, etc. Supramolecular structures generated by charged macromolecular interactions can enable the encapsulation of bioactive agents and delivery. These supramolecular entities are also deemed safe and appropriate to be used in the food industry. The intermacromolecular interactions are affected by physicochemical factors, e.g., molecular weight, charge density, biopolymer concentration, ionic strength, pH deviations, pressure, temperature fluctuations, and shearing (Schmitt and Turgeon, 2011). The process of electrostatic complex formation includes dissolution of biopolymers in the solvent followed by polymer mixing and ultimately acidification (Aditya et al., 2017). Acidification affects the size of the biopolymer complex. Postblending acidification is considered more favorable and results in particles relatively smaller in size (Be´die´ et al., 2008). Enforcing enhanced shear can also generate small-sized particles thereby preventing certain preblending limitations. Particle size reduction was also achieved in the case of sodium caseinateechitosan complexes by means of preblending acidification and sonication (Kurukji et al., 2016). Biopolymer-based nanoparticles demonstrate inherent characteristics to both safely encapsulate as well as delivering bioactive agents such as peptides, proteins, pharmaceuticals, and also dietary components. Chapeau and coworkers demonstrated encapsulation of hydrophilic vitamin B9 coacervates of oppositely charged milk proteins b-lactoglobulin (blg) and lactoferrin (Chapeau et al., 2015). The particles can have

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applications as texturizing agents in different formulations. Catechinechitosan complex colloidal particles considerably lowered the degradation of catechin in the intestine bypassing glucuronidation (Zhang and Kosaraju, 2007). Particle complexes developed as a result of electrostatic interactions are more susceptible to degradation with changes in factors like pH, etc. Controlled and targeted release of bioactive agents can be carried out as a result of such degradations in the gastrointestinal tract. These complexes are usually formed in acidic pH and, therefore, can afford safe passage of bioactive agents under acidic conditions of the stomach and degrade in the intestine under alkaline conditions. This phenomenon helps in targeted release and increased absorption (Jones et al., 2010). When a targeted approach is not essential biopolymer-based particles can be generated by heat-based dissociation of proteins and complexes are developed with polysaccharides by electrostatic interactions. The nanoparticles, as a result, become more immune to environmental changes and have considerable applicable benefits in the food industry (Jones et al., 2010; Aditya et al., 2017).

3.2 Peptides as value enhancers There have been extensive studies about value-adding agents in functional foods to further enhance nutritional and health benefits. Bioactive peptides from various sources with antioxidant activity, antimicrobial activity, anticancer, and cardiovascular disease prevention (Lopez-Fandino et al., 2006) have been validated. But adding peptides to food products can have a bitter flavor (Cho et al., 2004), which can be avoided by proper encapsulation methods. Encapsulation and controlled release mechanisms would also be beneficial for omega-3 fatty acids, which are increasingly consumed and have proven dietary advantages. Omega-3 fatty acids as functional food enhancers can have a few limitations and can be prone to oxidative degradation (Frankel, 2005). Phytosterols are plant-based bioactive compounds, which are also incorporated in functional foods due to their ability to reduce the levels of total cholesterol concentration in humans by hindering the absorption of dietary cholesterol (Ostlund, 2004). Similar to omega-3 fatty acids, proper encapsulation of phytosterols could effectively enhance their oxidative stability and robustness.

3.3 Biopolymers in food processing and packaging Different kinds of meat and fish have been the primary dietary components of animal protein globally. Higher bioavailability and comprising of essential amino acids enhance its nutritional advantage over other forms of dietary protein (Sanchez-Ortega et al., 2014). The quality of meat, poultry, and fish severely changes during storage as the inherent proteins and lipids undergo oxidation, which also affects flavor. The magnitudes of these alterations are influenced by storage conditions like temperature, duration of storage, exposure to oxygen, etc. Oxidation induces alterations in amino acids  resulting in variations in water retaining ability and nutritional content (Popova et al., 2009; Sojic et al., 2014). Contamination with microbes can also be a crucial factor and leads to faster degradation of meat by bringing about changes in pH value, altered flavor, altered appearance, structural break down, as well as slime build up (Dave and Ghaly, 2011). Routine and usual methods adopted to enhance shelf life and reduce degradation are salting, drying, smoking, fermentation, and even canning. Novel implementable procedures to maintain quality and nutritive content of animal proteins have been studied (Dave and Ghaly, 2011). Biopolymer-based packaging has been adopted to maintain the highest food safety and standards.

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These new packaging procedures follow aseptic conditions and controlled environments. Biopolymers are also being used to packaging meat and other food products prior to storage. For meat packaging purpose, composite biopolymer films are generated from two biopolymers (chitosanestarch, gelatinealginate, fish protein isolate or fish skin gelatin), hydrocolloids and lipids (sunflower oil), or biopolymers and synthetic polymers like chitosaneLDPE, chitosanePVA, CMCePVA, starchePVA, etc. (Ali Arfat et al., 2015; Chatli et al., 2014; Kazemi and Rezaei, 2015; Lu et al., 2013; Muppalla et al., 2014; Reesha et al., 2015; Vargas et al., 2011; Wu et al., 2010). The composite biopolymer films afford enhanced protection and sustain the quality of food products. There are also other procedures to develop biopolymer-based films for packaging animal proteins and other food products such as extrusion blowing, heat pressing, adding nanoparticles in the polymer matrix, electrospinning, etc. (Dehnad et al., 2014; Gudjonsdottir et al., 2015; Park et al., 2010; Reesha et al., 2015). Research with incorporating bioactive components showed a considerable and significant lowering of microbial growth and degradation of food products especially animal proteins. Among several procedures available to incorporate bioactive compounds, adding bioactive components in filmogenic solutions is routinely used and has proven advantages in muscle protein products. Among other methods, product addition in bioactive compounds in liquid phase followed by biopolymer solutions (Bazargani-Gilani et al., 2015), mixing of natural bioactive ingredients in liquids, powder, or encapsulated into meats and other food products, and ultimate incorporation in filmogenic solutions (Kenawi et al., 2011) and also essential oil vapors are used (de Oliveira et al., 2013). Mixing different active compounds with treatment for enhancing film-hindering features can afford greater protection and shelf life to animal proteinebased products while in storage (Ali Arfat et al., 2015; Guo et al., 2014; Nagarajan et al., 2015; Rodriguez-Turienzo et al., 2011, 2012; Vimaladevi et al., 2015; Weerasinghe et al., 2013). Regulatory requirements concerning biopolymers and other polymeric components are quite robust. Some threshold features include ease of processing, more robust and effective barrier characteristics especially to gases and moisture, a strong barrier to smaller organic molecules, enhanced chemical, preferential permeation. The polymeric materials need to be highly tolerant to humid conditions and recycling and also possess robust biodegradability properties. Robust barrier polymers generated comprise aliphatic polyketones copolymers (Bonner and Powell, 1997; Lagaron and Powell, 2000). These semicrystalline polymers have greater mechanical, thermal, and barrier properties, unlike some ethylene vinyl alcohol-based copolymers. The polymers also demonstrate increased chemical resistance and greater humidity tolerance barrier features. These strong characteristics enable a wide range of applications in packaging. Novel biopolymers for food-packaging applications are also developed from biomass (Fig. 4.1 displays the major resources to obtain biopolymers). Biomass-derived agents are environment-friendly and can be recycled and biodegraded quickly (Petersen et al., 2001; Weber et al., 2002). These biopolymers also possess enhanced barrier parameters to gases. Barrier effectiveness though can significantly reduce in humid conditions like in chitosan-based particles. Biodegradable polyhydroxyalkanoates possess high water barrier features and therefore multilayer systems in combination with chitosan particles can be generated. Certain biopolymers, such as polylactide and starch, exhibit greater preferential permeability to carbon dioxide than oxygen compared to other mineralbased polymers. This feature can be very beneficial in diverse food-packaging applications where oxygen exposure needs to be strictly hindered. Carbon dioxide escape provisions should also be adhered to prevent package deformation. More studies and research with biopolymer are still required

4. Conclusion

Biomass From Agricultural Sources (Eg: Polysaccharides, Proteins and Lipids)

95

Microbial Production (Eg: Poly Hydroxyalkanoates)

Biopolymers

Chemically Synthesized From Agricultural Sources (Eg: Poly lactic Acid)

Chemically Synthesized from Fossil Sources (Eg: Aromatic and Aliphatic copolyesters)

FIGURE 4.1 Four major resources to obtain biopolymers. Adapted from Valde´s, A, Mellinas, A.C., Ramos, M., Garrigo´s, M. C., Jime´nez, A., 2014. Natural additives and agricultural wastes in biopolymer formulations for food packaging. Frontiers in Chemistry 2 (6).

especially in lowering relatively higher costs in comparison to traditional food-packaging materials. There has been considerable progress in biopolymer developments. There are multilayer systems generated by combining different biopolymers using lamination and coinjection methods. Aluminummetalized polymeric films obtained by vacuum deposition and aluminum or silicon oxides coated with polymeric films are widely used in the food-packaging industry. Traditional plastics coated with vacuum-deposited aluminum are also used and help enhance barrier features to gases, humidity, and organic vapors. Further, vacuum-deposited aluminum technology is more environmental-friendly than the application of aluminum foil, which is difficult to recycle. There are certain drawbacks associated with coating technology. Additional metal coating tends to relatively lower flexibility and thermoformability in comparison to solo application of biopolymer films. Metal coatings can lead to nonmicrowaveable food package and also making them nontransparent. To mitigate these problems, oxide-coated polymer films are developed which act as strong barriers and negate package opacity. On the contrary oxide coating can induce microcracking during different stages of production. Mixing polymers and other nanocomposites can also generate robust barrier systems for use in the foodpackaging industry. Fig. 4.2 summarizes the advantages of using biopolymers as a packaging material. However, further research is required to evaluate the efficacy and efficiency of different bio-based polymers as packaging material.

4. Conclusion Novel biopolymers, peptides and their combinations, new molecular targets along with the use of efficient encapsulation and targeting have shown promising results in both clinical and industrial applications. For example, antihypertensive peptides have been effective against molecular targets and further research are being carried out to have better results with peptide therapy regimens aimed at cardiovascular disease management. Encapsulating peptides with polymer-based nanoparticles can

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Edible, Biodegradable and Inexpensive

Improved nutrition and organoleptic features

Improved Shelf-life, Microencapsulation and Controlled release

Multilayer packaging and facilitate additives (antioxidants, antimicrobials, etc)

Controlled passage of gases and moisture within package components

FIGURE 4.2 Advantages of biopolymer-based packaging in food industry. Adapted from Rhim, J.W., Ng, P.K.W., 2007. Natural biopolymer-based nanocomposite films for packaging applications. Critical Reviews in Food Science and Nutrition 47 (4), 411e433.

significantly enhance targeting them to action sites along with the controlled release. Protein molecules just like cellulose can be used to derive biopolymers possessing characteristics such as toughness and shear strength. Soy protein, keratin, etc., have characteristic elastic features comparable to natural polymers. Protein-derived biopolymers are modified and engineered to alter their mechanical features

Table 4.3 Polymers having the food industry and biomedical use. Polymer

Application

Reference

g-Polyglutamic acid

Vaccine delivery, drug delivery, biosensors, diagnostics

Gellan gum

Drug delivery, food, or pharmaceuticals

Dextran

Vascular applications, drug delivery, tissue engineering, dental uses Drug delivery, stabilizer in food industry

Candela and Fouet. (2006), Candela et al. (2009), Buescher and Margaritis (2007) Hamcerencu et al. (2008), Rajinikanth and Mishra (2009) Naessens et al. (2005), Brondsted et al. (1998), Robyt et al. (2008) Becker et al. (1998), Becker et al. (2009)

Xanthan

4. Conclusion

97

Table 4.4 Plant-based biomaterial in the food industry. Type

Application

Reference

Chia and whey protein

Structuring component increases tensile strength Film generation, antioxidant properties Film development and cell viability Encapsulation and structuring Ice cream stabilizer

Munoz et al. (2012), Soukolis et al. (2018)

Quince seed Flax seed Chia seed and flax seed Basil seed gum Yellow mustard and flax seed

Glycemic regulator

Jouki et al. (2014) Bustamante et al. (2015) Bustamante et al. (2017) Bahramparvar and Goff (2013) Kay et al. (2017)

Table 4.5 Applications of mixed-polymers and copolymers. Type

Function

References

Carboxymethylcellulose/soy protein Keratin and chitosan

Increased tensile strength Increases strength and flexible film formation Water-insoluble characteristics Enhanced tensile strength Greater tensile strength

Su et al. (2010)

Whey protein isolate with cellulose Soy and agar Keratin and poly(ethylene oxide) Polyisoprene-bpoly(epsilonbenzyloxycarbonyl-Llysine) PI-b-PZLys and polyisoprene-b-poly(Llysine) PI-b-PLys block copolymers Polyethylene glycol and amino acid block polymers Poly(ethylene oxide)e peptides copolymers

Tanabe et al. (2002) Aluigi et al. (2008) Tien et al. (2000) Tian et al. (2011)

Copolymer nanostructures

Babin et al. (2005), Gupta and Nayak. (2015)

Anticancer drug encapsulation

Bae and kataoka (2009)

Targeted and controlled release of drugs

Van Domeselaar et al. (2003)

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to enhance their suitability. These alterations contribute to their applications in drug delivery, tissue engineering, and as packaging biomaterials of food products. New combinations and blends involving polymers and peptides are constantly being studied and newer applications are being pursued. We have highlighted the applications of some peptides and polymers and their combinations in the biomedical and food industry (Tables 4.3e4.5). The food industry has comprehensive applications of peptides and polymers to enhance sustainable production. There are a few impediments to greater use of these biomaterials such as cost of processing biomaterials and peptide isolates, regulatory safety assessments, etc. The prevailing and upcoming set of bioactive peptides and polymer-based biomaterials must be thoroughly assessed employing various assays and analytical methods to better elucidate parameters related to their functional diversity. Toxicity studies along with mechanisms of absorption and metabolism should also be carefully studied while adopting these smart biomaterials in diverse applications.

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CHAPTER

Microbial production of biopolymers with potential biotechnological applications

5

Madan L. Verma1, Sanjeev Kumar2, John Jeslin3, Navneet Kumar Dubey4 1

Centre for Chemistry and Biotechnology, Deakin University, Geelong, VIC, Australia; 2Department of Biotechnology, Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India; 3Department of Biotechnology, St. Joseph’s College of Engineering, Chennai, Tamil Nadu, India; 4School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan

1. Introduction Biopolymers have occupied a major position in the field of biotechnology (Ventorino et al., 2017; Santiago et al., 2018). They are biocompatible and eco-friendly in nature. Biopolymers are a heterogeneous, amenable group of materials synthesized from biological origin or from any basic building units such as amin‘o acids, lipids, and sugars (Tang et al., 2012). Microbial biopolymers such as polyesters, polyamides, and polysaccharides are successfully produced from pure cultures, mutants selected from the laboratory, or genetically modified organisms. The biopolymers range from viscous fluids to bioplastics. The molecular mass and composition of the biopolymer decide its physical properties (Plank, 2004). Biopolymer from bacteria has created unprecedented recognition in a wide range of biomedical and other industries. It has been found to possess varying biological functions with distinct properties. The fundamental understanding of its biopolymer synthesis and its regulations has encouraged protein and metabolic engineering to develop tailored biopolymers with modified properties to serve as a renewable source (Rehm, 2010). The genetic modification of biopolymer-producing microorganisms facilitate the production of economically valuable tailored biopolymers that act as high-value products for tissue engineering and drug delivery applications (Mezule et al., 2015; Liguori et al., 2016). Biopolymers are obtained from a broad array of microorganisms (Fig. 5.1). Microbial polymers are produced by fermentation or by the chemical polymerization of monomers. Synthetic, nonbiodegradable plastic materials can be substituted by an eco-friendly, biodegradable, renewable source that can be transformed into dissimilar commodities such as biochemicals obtained by sugar fermentation (Mezule et al., 2015; Liguori et al., 2016). Precursor molecules of biopolymers such as succinic acid (Ventorino et al., 2016, 2017) and 2,3-butanediol (Saratale et al., 2016) have been explored technologically. Microorganisms produce biopolymers by amassing extracellular compounds such as exopolysaccharides (EPSs) and are used in the food, chemicals, cosmetics, and wrapping industries as adhesives, absorbents, and lubricants (Pepe et al., 2013). Pasteur identified dextran-based biopolymer Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00005-9 Copyright © 2020 Elsevier Inc. All rights reserved.

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FIGURE 5.1 Benefits of microbes for biopolymer production.

in 1861, and Van Tieghem identified an efficient dextran-producing bacteria, Leuconostoc mesenteriodes, in 1878 to produce a microbial biopolymer. Soluble or insoluble exopolysaccharides are secreted into media by a variety of microorganisms with varying physiochemical properties with varying compositions and functions. The exopolysaccharides are made of sugars as well as nonsugar moieties such as pyruvate, acetate, succinate, and phosphate (Llamas et al., 2012). Biopolymers have an extensive range of applications and can potentially substitute man-made materials that can be synthesized from a vast renewable source under variable conditions. This reduces costs related to their biosynthesis (Huang et al., 2018; Raza et al., 2018) and can solve the difficulty of waste management after their production. It is important to select a suitable microorganism that does not endanger human health. This issue is at the heart of the global anxiety concerning expansion of the development of biopolymers as an alternative source of synthetic polymers in the environment. The increasing realization of the disadvantages of synthetic polymers in the environment along with emerging legislation prohibit their applicability in consumer products (Lee, 1996). Biopolymer replace petrochemical plastics; they are usually biodegradable, nonpolluting, ecofriendly, and economically valuable polymers (Rawte and Mavinkurve, 2001). The biopolymer becomes entirely split into various molecules such as carbon dioxide, methane, water, and biomass. This bioplastic serves as a possible extreme source rather than synthetic plastics; it would reduce environmental hazards and high energy use. In the case of bacterial biopolymers, it acts as a reserve material against external stress conditions. Based on biochemical reactions, microbial biopolymers are divided into capsular, storage, and extracellular polysaccharides that are essential for their pathogenicity and biofilm development (Schmid and Sieber, 2015). Different biopolymers can be synthesized from any natural or genetically engineered microorganisms such as polylactic acid (PLA), polyhydroxyalkanoate (PHA), polysaccharide, carboxylic acid, and butanediol. The efficacy of

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biopolymers and their precursor molecules are enhanced by systems biology and metabolic engineering approaches (Xu et al., 2013). This chapter discusses a plethora of microbial biopolymers. The production strategy for a microbial polyhydroxyalkanoate biopolymer is discussed in detail.

2. Microbial biopolymer production The plethora of biopolymer applications leads to the production of polymers with modified properties from a diverse range of microorganisms such as xanthan, pullulan, cellulose, glucan, and polyhydroxyalkanoate.

2.1 Xanthan The first commercial polysaccharide successfully produced from bacteria was xanthan gum (Kumar et al., 2018a,b). This copolymer complex is produced through fermentation. Five distinct sugar groups form the repeating units or building blocks of this biopolymer. Xanthomonas campestris is a bacterial species capable of producing xanthan gum; it is preferred for genetic modifications. Genetic modification of this species leads to about a 50% increase in xanthan gum production through recombinant DNA technology. It can also lead to a modified biosynthetic pathway in the host system (Dai et al., 2017). Corn syrup and molasses are widely used feedstocks for the large-scale production of xanthan from X. campestris through fermentation technique (Schmid et al., 2015). Upon polymerization, the xanthan gum is exuded from the bacteria and precipitated out by alcohol precipitation with subsequent removal of the bacterial biomass. The major hindrance in xanthan production is the higher energy requirement for proper mixing to compensate for the oxygen required for maximum microbial growth as it becomes difficult owing to the increase in the viscosity of the culture broth by polymer production (Zhang et al., 2016). However, the unique mechanical and physical properties of this biopolymer find applications in different industries such as food (60%) as well as nonfood (40%). Some examples of its industrial applications include growth stimulators in the agricultural field, controlling the viscosity of drilling mud fluid during the recovery of oil, acting as a biocide in processing mineral ore, acting as a modifier in the paper industry, for controlled and sustained drug release in the pharmaceutical industry, and for controlled dust release in the cosmetic industry. In the food industry, it acts as a gelling agent for making puddings, ice creams, and so on. It is also used to formulate clear gel toothpaste. With regard to the production volume, bacterial xanthan occupies a major place in polysaccharide or biopolymer production (Becker and Vorholter, 2009; Garcia-Ochoa et al., 2000).

2.2 Dextran The dextran is the generic name given to the diverse family of microbial-derived polysaccharides (Xu et al., 2017). It is produced by a polymerization reaction in the outer cell by the enzyme dextran sucrase. It is the fuel source of bacteria and yeast, which is made up of the monomers of simple sugar. Dextran is commercially produced from Leuconostoc mesenteroides and sucrose is used as the major feedstock for its enzymatic conversion or for the fermentation process. It can be produced mainly through enzymatic filtration or in industrial large-scale fermenter systems. Enzymatic filtration is

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highly preferred over the other method because of its uniformity in the product’s quality and owing to increased product yield. Dextran produced through this method can easily be purified, whereas production conditions can be controlled in both methods to yield products with varying molecular weights (Ye et al., 2019a,b). Dextran has a wide application in the medical field, especially in wound dressing, blood volume expanders, medical sutures, anemia treatment and vascular occlusion treatment (Plank, 2004). The oxygen export capacity of the dextranehemoglobin complex enables it to be used for blood substitutes. It is also employed as a plasma expander. The chemical modification of dextran leads to the formation of modified dextran-like dextran sulfate, which is used as an anticoagulant and antiulcer agent (Yang et al., 2019).

2.3 Pullulan Aureobasidium pullulans and different species of yeast are capable of producing water-soluble polysaccharides in the outer cell (Hilares et al., 2019). The linear polymer of pullulan consists of monomers of three glucose molecules. Chemical modifications of pullulan reduce its solubility, making it partially or fully soluble. It can also modify its electrical and thermal properties. This biopolymer is readily biodegradable and is highly resistant to heat. The versatile nature of pullulan makes it useful for various applications. In industry, pullulan is selectively preferred as a plastic material that mimics the properties of polyvinyl chloride and polystyrene in terms of its transparency, hardness, strength, luster, and toughness (Xue et al., 2019). However, it is not elastic. The decomposition of pullulan occurs above 200
C without the ejection of toxic gases. Its odorless, tasteless, and nontoxic nature makes it useful in the food industry as an additives to increase texture and bulk quantity. It has no caloric value because it is not degraded by digestive enzymes. Therefore, it is used in low-calorie food materials and drinks, replacing starch and other filler molecules. It can also be used as a preservative, inhibiting moisture retention and the growth of fungi in food materials stored long-term. Its transparency and impermeability to oil, grease, water, and oxygen molecules also enable pullulan to be used in food packaging applications. Etherified or esterified pullulan is employed to coat food and drug molecules to prevent the solubility and permeability of oxygen and water molecules by forming an airtight membrane (Hezarkhani and Yilmaz, 2019). Derived pullulans possess qualities similar to gum arabic, such as an adhesive nature. The process of polymerization decides its adhesive nature and viscosity. Highly viscous pullulan is used to extract the fibers from it, which has applications in various fields. It is also used as a stationary paste, as binders, and in nonwoven fabric production. In the medical field, it is used as adhesives, drug carrier molecules, and a plasma extender, and is devoid of any offsite reactions (Prasongsuk et al., 2018).

2.4 Glucans ¨ zcan and O ¨ ner, 2018). Glucans are the polymers made up of the monomers of glucose molecules (O The diverse glucan family includes pullulan, cellulose, and yeast glucan. Saccharomyces cerevisiae is the major source of glucan molecules because it is the main cell wall component of the yeast (12%e14% of its cell dry weight). It is also found in wide varieties of microorganisms such as lichen, fungi, and bacteria, as well as in higher plants (Barcelos et al., 2019). The insoluble glucan is extracted from the yeast by hot alkali treatment and is easily purified to produce glucan of varying molecular weights from higher to lower. Glucan has wide applications in the medical and food industries. In the

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medical industry, it is used as an antiinfectious, antitumor, and radioprotective agent (Plank, 2004). It is also employed for the controlled and sustained release of drugs. In the food industry, it is exploited as a thickener in noncaloric food. Apart from this, it is used as a solid support for chromatographic technique (Xia et al., 2018).

2.5 Gellan Four molecules of sugar monomeric repeats (glucoseeglucuronic acid glucoseerhamnose) make up the gellan polymer (Barcelos et al., 2019). The plant-derived bacterium Pseudomonas elodea produces this gellan polymer. The production of gellan is similar to the fermentation process of xanthan polymers. The chemical modification of gellan can induce changes in properties, such as reduced viscosity in the increased temperature by a hot caustic method. A strong gel of polymer is produced by cooling it in the presence of cations (Rehm, 2010). It also occupies a major position in the food industry, such as in icings, frostings, jellies, and jams (Anderson et al., 2018).

2.6 Alginate The polymer alginate is devoid of branching patters or monomeric repeats like any other polymeric compounds (Anderson et al., 2018). It is made of two unique elements: as a-L-guluronic acid (C5epimer) and b-D-mannuronic acid. It is generally produced from brown algae. Azotobacter and Pseudomonas also possess the biosynthetic pathway of producing alginate. The biosynthetic pathway initiates with guanosine diphosphate-mannuronic acid, a cytosolic precursor that upon polymerization gives poly-mannuronic acid, which travels through the cytoplasmic membrane. The bacterial strategy of producing alginate undergoes epimerization or acetylation to modify it enzymatically before transporting from the inner to the outer environment. The tailored alginate biopolymer engrosses wide industries with its distinct properties. Alginates are negatively charged biopolymers that contribute to material characteristics such as the viscosity of the solution to the gel structure of particles in the presence of cations. In biotechnology, alginates are highly preferred for the encapsulation process by means of self-assembly. The ability to produce micro- or nanosized particles attracts the biomedical field for drug delivery applications (Barcelos et al., 2019).

2.7 Cyanophycin The polyamide molecule cyanophycin consists of a backbone made of poly(aspartic acid) and the amine group of arginine residues associated with the eCOOH group of aspartic acid (Altun et al., 2018). In the host organism, cyanophycin acts as the storage molecule that provides an energy, carbon, and nitrogen source (Diniz et al., 2006). Cyanophycin synthetase is the key enzyme accountable for the formation of cyanophycin polymer. The gene encoding this enzyme can be successfully incorporated into the prokaryotic organisms to modify the organism genetically for the enhanced production of the biopolymer (Lippi et al., 2018). Apart from the bacterial source, yeast and higher plants also produce this biopolymer in sufficient quantity. Intracellular and extracellular degradation of this biopolymer are characterized by bacterial cyanophycinases (CGPase), intracellular cyanophycinases (CphB), or the extracellular cyanophycinase (CphE). The product of biodegradation is dipeptides, which upon further intracellular degradation by dipeptidase yield amino acids. The cyanophycin occupies a major position in different chemical, biomedical, and other global industries (Sallam et al., 2009).

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2.8 Poly-g-glutamic acid Poly-g-glutamic acid is peculiar anionic polypeptide molecules composed of L- and/or D-glutamic acid that is polymerized by means of g-amide linkage (Cao et al., 2018). This polymer is optically active in nature, in which all of glutamate units possess a chiral center. D-Glutamate homopolymer and Lglutamate homopolymer are copolymerized to form random units of D- and L-glutamate. This biopolymer attracts many biomaterial industries that would serve a healthy life (Inbaraj et al., 2006). Certain properties of this biopolymer, including biodegradability, multifunctionality, no-toxicity, biocompatibility, and its edible nature, occupy a major position in the food and medical industries. Some applications of this biopolymer include as a chelating agent in wastewater treatment, a moisturizer in the cosmetic industry, a thickener in the food industry, a hydrogel formation in a carrier molecule for drug and gene delivery, in liquid crystal displays, and in conductive materials (Xu et al., 2005). Kumar et al. (2018a,b) studied promising poly(g-)glutamic acid (g-PGA) from microorganisms, which acts as a defense mechanism against stress conditions. g-PGA is a biodegradable poly-amino acid that is soluble in water. It is primarily revealed as a Bacillus anthracis capsule; it was discovered by Ivonovics and Bruckner (Shih and Van, 2001) and later was found mostly in natto. g-PGA of higher molecular weight restricts built-up applications owing to high viscosity, uncontrollable rheology, and hard modification (Shih and Wu, 2009). The production of g-PGA by a microbial source has gained consideration because of its low production cost and diverse relevance with high compatibility and high biodegradability. Optimization of the growth medium for the production of gPGA from microbes has been concerned with the conditions for their high production yield. The medium producing g-PGA by dissimilar bacteria is vital in deciding the g-PGA yield, because it directly influences the characteristics of g-PGA. The concentration of sodium chloride in the medium affects the yield of g-PGA and secretes g-PGA of different molecular masses. To analyze the consequence of ionic strength for producing g-PGA by Bacillus licheniformis ATCC 9945a, sodium chloride in the medium was dispersed at 0%e4%. Results showed that increasing the concentration of sodium chloride increased the molecular mass of the g-PGA formed, which enhanced its production, properties, and applications by a factor of 1.8 (from 1.26,106 to 2.26,106 kDa). g-PGA of higher molecular mass is the most prominent source for various industrial applications. Alsaheb et al. (2016) studied polyglutamic acid (PGA), a biologically degradable polymer produced by microbial fermentation. PGA was identified by Bruckner (1937) when a capsule of B. anthracis was unconfined in the culture medium upon sterilization (Hezayen et al. 2001) and was also produced by Bacillus subtilis sawamura (Singer et al., 2000). PGA is primarily produced by grampositive bacteria, including the genus Bacillus. Moreover, some gram-negative bacteria (Fusobacterium nucleatum) are efficient in producing g-PGA (Candela and Fouet, 2006). The biosynthetic pathway of glutamic acid leads to the formulation of PGA by endogenous or exogenous production. During the endogenous mode of L-glutamic acid synthesis, a carbon source becomes converted through the tricarboxylic cycle through acetyl-CoA (Rehm, 2009), whereas the exogeneous mode of synthesis undergoes the conversion of L-glutamic acid to L-glutamine in the presence of an enzyme called glutamine synthase. The processes of PGA consist of four divergent stages: racemization, polymerization, regulation, and degradation (Bajaj and Singhal, 2011). The common genus used to generate PGA is Bacillus and various strains such as B. licheniformis and B. subtilis by microbial fermentation. The PGA is structurally diverse during its formation along with its coproducts, biopolymers. B. subtilis growth in a higher ammonium sulfate content resulted in

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the formation of super highemolecular weight PGA of 26 kDa, which is abundant in L-glutamate. The bacterium meant for this PGA production is distinguished into two classes based on the nutrient constraints for growth, such as L-glutamic acidedependent and independent bacteria. The first category includes bacterial species such as B. subtilis, B. licheniformis 9945. B. subtilis CGMCC 0833 (Potter et al., 2010), B. subtilis ATCC 15,245 (Bhat et al., 2013), B. subtilis C10 (Yao et al. 2012), and Bacillus amyloliquefaciens LL3 (Cao et al., 2011). In this mode of PGA synthesis, the greater the Lglutamic acid absorption, the greater will be the PGA yield.

2.9 Levan The homopolysaccharide levan is made up of residues of D-fructofuranose molecules linked by 2,1 linkage of two to six diverse branches (Erkorkmaz et al., 2018). This biopolymer is highly employed in different industries, such as in the pharmaceutical, cosmetic, food, and feed fields (Poli et al., 2009). In the food and feed industry, it acts as additives serving as a hypocholesterolemic and prebiotic agent. In the cosmetic industry, it acts as a moisturizer and skin alleviating and blending agent and also supports cell proliferation (Dom_zał-Kedzia et al., 2019). Derivatives of this polymer act as an anti-AIDS agent, such as acetylated, sulfated, and phosphate levan. In protein engineering, it is used in a two-phase partition for molecular separation and purification and as a carrier molecule for drug delivery. The simplicity of the purification process and its unaltered chemical nature in the solution make it as a vital source in different industries (Mantovan et al., 2018).

2.10 Hyaluronic acid Hyaluronic acid (HA) is a biopolymer produced from animal tissue, such as through bacterial fermentation or from rooster comb (Westbrook et al., 2018). Because of the presence of infectious agents and the susceptibility to contamination in the animal source, there are drawbacks to the production of HA from an animal source (Liu et al., 2011). Therefore, fermentation is the widely chosen process for producing this biopolymer. Streptococcus Group C in batch fermentation produces a viscous titer value of 5e10 g/L. This high-value biopolymer is commercially used in various cosmetic and pharmaceutical industries (Chong et al., 2005). The cost of the initial substrate is the factor that drives the value of this biopolymer. The process and the strain development are essential for the enhanced production of the biopolymer. HA synthase is the key enzyme responsible for the complete polymerization and translocation of HA (Chahuki et al., 2019). Increased resources resulted in highemolecular weight biopolymer production. This highemolecular weight biopolymer is also produced in heterologous host systems such as Escherichia coli, B. subtilis, and Lactobacillus lactis by genetic engineering techniques. Various omics technologies and metabolic engineering pave way for the enhanced production of this biopolymer (Kogan et al., 2007).

2.11 Bacterial cellulose The cellulose biopolymer is made up of linear homopolymers of glucose units (Basu et al., 2019). The ribbon-shaped bacterial cellulose is extruded from the cell surface of bacteria such as Acetobacter, with the formation of an interconnected fiber network (Keshk et al., 2006). Initially, this cellulose was extracted from a plant source that widely used in the paper and pulp industries (Ye et al., 2019a,b).

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The cellulose is produced through agitated fermentation in the presence of a rich growth medium supplemented with corn steep liquor, glucose, chelators, growth enhancers, and salts. Commercial fermenters (50,000 gallons) produce more than 0.2 g of cellulose at a high rate (Do´rame-Miranda et al., 2019). The bacterial cells are disrupted by hot caustic treatment to release the inner waterinsoluble fibers of cellulose. The surface area of the cellulose from bacteria is 200-fold higher than that of cellulose obtained from wood pulp. The mechanical properties of cellulose derived from bacteria have enhanced features compared with that of wood pulp cellulose. It is used as a pseudoplastic thickener because it can absorb water sixfold higher than traditional cellulose. Even at a low cellulose concentration, higher water absorption can be accomplished by its larger surface area. Coating paper with bacteria-derived cellulose can increase the smooth surface of the paper, reducing its moisture absorption capacity (Barcelos et al., 2019).

2.12 Organic acid fermentation for polymer synthesis Until now, petrochemical polymers have had a major position in global industries. However, the production of polymers from renewable sources is essential to prevent the exploitation of nonrenewable sources because of environmental hazards and increased oil demands. The fermentation of organic acids of 3e4 carbon molecules such as fumaric acid, lactic acid, acrylic acid, aspartic acid, and succinic acid is widely employed in biopolymer production on a large scale (Barcelos et al., 2019). Optimized production is also investigated by metabolic engineering techniques for increased biopolymer production (Ullrich, 2009).

2.13 Microbial exopolysaccharides Exopolysaccharides or extracellular polysaccharides are economically valuable polysaccharides produced from microorganisms along with intracellular and structural polysaccharides (Barcelos et al., 2019). Extracellular polysaccharides are composed of monosaccharides along with a few noncarbohydrate moieties. The non-carbohydrate moieties include succinate, acetate, phosphate, and ¨ zcan and O ¨ ner, 2018). Exopolysaccharides are used commercially for diverse applications pyruvate (O in different pharmaceutical and food industries (Ullrich, 2009). The novel importance of exopolysaccharides in these industries is in the production of advanced microbial exopolysaccharides from nature (Xia et al., 2018). Research activities had mainly focused on extremophilic bacteria for the production of biopolymers, because they could withstand extremely stressful conditions through certain modified metabolic pathways. Caruso et al. (2018) focused on synthesizing biopolymers from spongeassociated Antarctic bacteria. Bacteria that grow under extremely cold conditions produce new biomolecules with novel functionality (Lo Giudice and Fani, 2015). Highly efficient polymeric extracellular molecules replace chemical polymers owing to the advantages of nontoxicity and biodegradability and do not cause environmental pollutants (More et al., 2014). Different bacterial strains such as Winogradskyella sp. strains CAL384 and CAL396, Colwellia sp. strain GW185, and Shewanella sp. strain CAL606) are cultured on agar plates for mucoid signs, resulting in the formation of EPS with 15%e28% carbohydrate, 3%e24% protein, and 3.2%e11.9% uronic acid content. The carbohydrate possesses glucose, galactose, galactosamine, and mannose units when subjected to chemical hydrolysis. The impending biological appliance in the biotechnology field subjects EPS from

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Winogradskyella sp. CAL384 as an efficient emulsifying agent on hydrocarbon. When allowed to grow in a culture medium supplemented with carbohydrate, the EPS-producing bacterium also stimulates its growth in the medium containing a high cadmium and mercury content. Another bacterial strain, Halomonas almeriensis M8T, halophilic bacteria, produce exopolysaccharide with extensive biotechnological applications (Llamas et al., 2012). The exopolysaccharides are a carbohydrate-rich polymeric molecule excreted by certain bacteria or fungi into the extracellular environment. This molecule acts as an adhesin component for cellular association and interaction within the microorganism to support gene and metabolite transfer. It also acts as a protective agent against certain viral and protist attacks. In addition, it acts as a survival component that supports growth during nutrient stress conditions (Wolfaardt et al., 1999; Sutherland, 2001). It has had an impact on the field of biotechnology in the pharmaceutical, food, and cosmetics, and petroleum industries (Freitas et al., 2011). A substantial number of EPS have been synthesized by various bacterial species excluding xanthan from X. and gellan from Sphingomonas paucimobilis (Sutherland, 2002). The bacterial strain M8T secreted 1.7 g/L of EPS under optimal nutrient and growth conditions. The EPS molecule is composed of two highe and lowemolecular weight carbohydrate fractions (6.3  106 and 1.5  104 Da, respectively), as represented by anionic exchange and size exclusion chromatography. Among the two fractions, the higheremolecular weight fraction consists of carbohydrate constituents of mannose (72% w/w), glucose (27.5% w/w), and rhamnose (0.5% w/w). The latter is composed of mannose (70% w/w) and glucose (30% w/w). The emulsifying capacity of EPS molecules depends on the protein content and M8T strain resulting in the protein content of 1.1% w/w able to emulsify hydrophobic substrates. The exopolysaccharides produced by Halomonas almeriensis M8T cultured in an optimal intensification environment directs its use as an emulsifier, detoxifier, and other biologically valuable molecules.

2.14 Polyhydroxyalkanoates Polyhydroxyalkanoates are polyesters produced from bacteria during nutrient stress conditions. They are made of (R)-3-hydroxy fatty acid molecules. This type of biopolymer acts as stored energy and a carbon source for bacteria during the stress condition. It is stored in an insoluble globular inclusion known as PHA granules. The granules consist of a phospholipid-protein layer with a polyester core. PHA synthase mediates the formation of PHA from the precursor molecules (Lu¨tke-Eversloh et al., 2002). The granuledependent proteins help in the depolymerization of the PHA synthaseepolyester complex, which also regulates stabilization of the structure. PHA is widely used as micro- or nanobeads; in other fields, it is valuable for its biodegradable and biocompatible nature (Ballistreri et al., 2001).

3. Biosynthesis of microbial polyhydroxyalkanoates Biodegradable PHAs polymers are mainly produced naturally by microorganisms in the shape of insertion bodies; they act as storage materials within asexual cells (Marang et al., 2018; Santiago et al., 2018). Among biopolymers, PHA is preferred by industries because of its eco-friendly nature, biocompatibility, renewable precursor molecule, and biochemical diversity (Shah et al., 2008). Generally, PHA consists of monomeric units of 600e35,000 [R]-hydroxy fatty acid (Fig. 5.2) (Khanna and Srivastava, 2005). Almost 150 different PHA molecules have been identified to date; the number is

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FIGURE 5.2 Chemical structure of polyhydroxyalkanoate (PHA). The nomenclature and carbon number for PHA compounds are determined by the functional alkyl R group. Asterisk denotes the chiral center of the PHA building block. Adapted from Tan, G.Y.A., Chen, C.L., Li, L., Ge, L., Wang, L., Razaad, I.M.N., Li, Y., Zhao, L., Mo, Y., Wang, J.Y., (2014). Start a research on biopolymer polyhydroxyalkanoate (PHA): a review. Polymers 6, 706e754.

increasing with ongoing research into modification of existing PHA molecules, enhancing the physiochemical properties of the biopolymer (Zinn and Hany, 2005), and using genetically engineered organisms to create a PHA biopolymer with modified functional groups (Escapa et al., 2011). The modified properties of PHA enable them to be used in various applications ranging from biodegradable

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packaging materials to medical products. PHA molecules are also employed as an active pharmaceutical component to treat deadly diseases such as AIDS and cancer (De Roo et al., 2002; Rai et al., 2011). The identification of varying PHA materials, along with their enhanced properties and downstream applications, was reported by Chen (2010), Olivera et al. (2010) and Rai et al. (2011). Polyhydroxyalkanoates replace existing petroleum synthetic polymers because they are biodegradable and because associated molecules such as thermoplastics and elastomers have an important position in environmental applications. The toxicity of polymeric compounds can be reduced by using agroindustrial debris, and the application of PHA in the medical field should be highly pure enough to meet the requirements (Koller, 2018; Peptu and Kowalczuk, 2018). Fig. 5.3 represents the production of intracellular PHA by bacteria. PHA granules as a carbon or energy component are deposited in the cytosol of the organism during conditions of nutrient disparity. This deposition occurs when an essential nutrient component in the growth media, such as carbon, is in excess compared with another. The synthesized polyester covers vast applications in industries because PHA is a thermoplastic polyester that efficiently substitutes for synthetic petroleum plastics. A wide variety of bacteria including gram-positive and gram-negative, aerobic, anaerobic, photosynthetic bacteria, as well as lithotrophs and organotrophs, which are capable of accumulating intracellular PHA for their carbon and energy source, are listed in Table 5.1 (Sudesh et al., 2000). This

FIGURE 5.3 Microbes produce polyhydroxyalkanoate (PHA) using waste materials. Adapted from Nielsen, C., Rahman, A., Rehman, A.U., Marie, Walsh, K., Miller, C.D., (2017). Food waste conversion to microbial Polyhydroxyalkanoates. Microbial Biotechnology. 10(6), 1338e1352.

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Table 5.1 List of PHA-producing bacteria (Tan et al., 2014, Mohapatra et al., 2017). Microorganism

PHA monomer

PHA content (% CDM)

Reference

Malt waste

P3HB

70.1

Yu et al. (1998)

Sucrose

P3HB

50.0e88.0

Fructose, glucose

P3HB

76.5e79.4

Glucose

P3HB

24.8

Xylose Glycerol Fructose, glucose, sucrose Lauric acid, myristic acid, oleic acid, palmitic acid, stearic acid Glucose

P3HB P3HB P3HB

58.4 31.3 50.4e59.0

P3HB

1.0e69.0

Yamane et al. (1996a,b), Wang and lee (1997), Grothe et al. (1999) Gomez et al. (1996) Lasemi et al. (2012) Pan et al. (2012) Zhu et al. (2010) Gomez et al. (1996) Chee et al. (2010)

P3HB

18.3

Fructose, glucose

P3HB

67.0e70.5

4Hydroxyhexanoic acid Corn oil, oleic acid, olive oil, palm oil Acetate, butyrate, lactic acid, propionic acid CO2

P3HB

76.3e78.5

P3HB

79.0e82.0

Fukui and Doi (1998)

3HB, 3HV

3.9e40.7

Chakraborty et al. (2009)

P3HB

88.9

Hydrolyzed starch

P3HB

56.0

Lactose, sucrose

P3HB3HV

20.2e62.5

Hydrolyzed whey and valerate

P3HB3HV

40.0

Sonnleitner et al. (1979) Quillaguama´n et al. (2005) Povolo et al. (2013) Koller et al. (2007)

Substrate

Gram-negative bacteria Azohydromonas australica Azohydromonas lata

Azotobacter beijerinckii Burkholderia cepacia

Burkholderia sp. USM

Caulobacter vibrioides Cupriavidus necator H16

Halomonas boliviensis LC1 Hydrogenophaga pseudoflava

Qi and Rehm (2001) Gomez et al. (1996) Valentin et al. (1994)

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Table 5.1 List of PHA-producing bacteria (Tan et al., 2014, Mohapatra et al., 2017).dcont’d Microorganism

Substrate

PHA monomer

PHA content (% CDM)

Methylobacterium extorquens M. extorquens

Methanol

P3HB

40.0e46.0

Methanol

P3HB

35.0e62.3

Methylocystis sp. GB25 a Novosphingobium nitrogenifigens Y88 Paracoccus denitrificans Pseudomonas aeruginosa

Methane

P3HB

51.0

Glucose

P3HB

81.0

n-Pentanol

P3HV

22.0e24.0

Cane molasses, fructose, glucose, glycerol, sucrose Oil and wax products from polyethylene (PE) pyrolysis Terephthalic acid from polyethylene terephthalate (PET) pyrolysis 1,3-Butanediol, octanoate 1,3-Butanediol, octanoate 4Hydroxyhexanoic acid Styrene

P3HB

12.4e62.0

mcl-PHA

25.0

Guzik et al. (2014)

mcl-PHA

24.0

Kenny et al. (2008)

scl-mcl-PHA, mcl-PHA scl-mcl-PHA

11.9e31.4

Lee et al. (1995)

13.5e19.3

Lee et al. (1995)

scl-mcl-PHA

18.6

Valentin et al. (1994)

mcl-PHA

31.8

mcl-PHA

36.4

NikodinovicRunic et al. (2011) Ward et al. (2006)

mcl-PHA

27.0e0.005,

Kenny et al. (2008)

mcl-PHA

23.0

Kenny et al. (2008)

mcl-PHA

2.0e28.0

Lageveen et al. (1988)

P. aeruginosa PAO1

Pseudomonas frederiksbergensis GO23 a Pseudomonas marginalis Pseudomonas mendocina Pseudomonas oleovorans Pseudomonas putida

P. putida GO16a

P. putida GO19a

P. putida GPo1

Styrene from polystyrene pyrolysis Terephthalic acid from PET pyrolysis Terephthalic acid from PET pyrolysis Alkenes, n-alkanes

Reference Bourque et al. (1995) MokhtariHosseini et al. (2009) Wendlandt et al. (1998) Smit et al. (2012) Yamane et al. (1996a,b) Tripathi et al. (2012)

Continued

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Table 5.1 List of PHA-producing bacteria (Tan et al., 2014, Mohapatra et al., 2017).dcont’d Microorganism

P. putida KT2440

P. putida F1

P. putida mt-2

Pseudomonas sp. strain-P(16). Thermus thermophilus HB8

PHA content (% CDM)

Substrate

PHA monomer

n-Alkenoates scl-mcl-PHA, mclPHA 5.0e60.0 NG Nonanoic acid 4Hydroxyhexanoic acid Glucose Benzene, ethylbenzene, toluene Toluene, p-xylene

scl-mcl-PHA, mcl-PHA

5.0e60.0

mcl-PHA mcl-PHA

26.8e75.4 25.3e29.8

mcl-PHA mcl-PHA

32.1 1.0e22.0

Davis et al. (2013) Nikodinovic et al. (2008)

mcl-PHA

22.0e26.0

Nikodinovic et al. (2008) Shahid et al. (2013)

Acetic acid, citric acid, glucose, glycerol, octanoic acid, pentanoic acid, succinic acid Rice bran

mcl-PHA

4.0e77.0

PHA

90.9

Whey

scl-mcl- PHA

35.6

Reference Gross et al. (1989), Elbahloul and Steinbuchel (2009) Sun et al. (2007) Valentin et al. (1994)

Aljuraifani et al. (2018) Pantazaki et al. (2009)

Gram-positive bacteria Bacillus aryabhattai Bacillus cereus SPV B. cereus

Sucrose, glucose, andfructose Glucose

PHA

57.62

3HB and 3HV

38.00

Glucose

PHB-3HHX

13.77

Glucose

PHB

53.01

Glucose

PHB

70.00

Contreras et al. (2013)

Glucose

PHB

57.20

Glucose

PHB

76.32

Bacillus sp.

Glucose

PHB

68.85

Bacillus sp.

Raffinose

P(3HB)

60.57

Aarthi and Ramana (2011) Ramana and Narayanan (2012) Joshi and Jaysawal (2010) Singh et al. (2011)

Bacillus licheniformis Bacillus megaterium uyuni S29 Bacillus mycoides DFC1 B. mycoides DFC1

Tanamool et al. (2013) Valappil et al. (2007) Abinaya et al. (2012) Dash et al. (2014)

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Table 5.1 List of PHA-producing bacteria (Tan et al., 2014, Mohapatra et al., 2017).dcont’d Microorganism

Substrate

PHA monomer

PHA content (% CDM)

Bacillus sp.

Glucose

PHA

80

Bacillus sp. Bacillus sp. SW1-2

Sucrose Glucose

PHAs PHB

51.49 36.00

Bacillus sp. Ti3

Starch

PHB

58.73

Bacillus subtilis

Glucose

PHB

69.01

B. subtilis (KP172548) Bacillus thuringiensis B. thuringiensis IAM12077

Fish solid waste

PHB

1.620

Glucose

PHB

11.30

Glucose

PHB

64.16

Lysinibacillus sp. 3HHX Paenibacillusdurus BV-1 Bacillus megaterium

Glucose

PHB

80.94

Fructose

PHB

0.93 g/L

Citric acid, glucose, glycerol, succinic acid Acetate, nalkanoate, 3hydroxybutyrate, propionate, sucrose, valerate Acetic acid, citric acid, glucose, glycerol, succinic acid Acetate, glucose

P3HB

9.0e50.0

3HB, 3HV, 3HHx

2.2e47.6

Valappil et al. (2007)

P3HB, mcl-PHA

4.0e32.0

Shahid et al. (2013)

3HB, 3HV

8.0e21.0

Glucose

3HB, 3HV

20.0e30.0

Haywood et al. (1991) Akar et al. (2006)

Acetate, succinate

3HB, 3HV

7.0e20.0

Various Bacillus spp. type strains

Corynebacterium glutamicum

Corynebacterium hydrocarboxydans Microlunatus phosphovorus Nocardia lucida

Reference Mohapatra et al. (2014) Raj et al. (2014) Berekaa and Thawadi (2012) Israni and Shivakumar (2013) Mohapatra et al. (2015) Mohapatra et al. (2017) Patel et al. (2011) Gowda and Shivakumar (2013) Mohapatra et al. (2016) Hungund et al. (2013) Shahid et al. (2013)

Haywood et al. (1991)

3HB, poly (3-hydroxybutyrate); 3HV, poly(3-hydroxyvalerate); CDMs, cell dry mass; mcl-PHA, medium chainelength polyhydroxyalkanoates; PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate; scl-mcl-PHA, small chainelength to medium chainelength polyhydroxyalkanoates. Adapted from Tan, G.Y.A., Chen, C.L., Li, L., Ge, L., Wang, L., Razaad, I.M.N., Li, Y., Zhao, L., Mo, Y., Wang, J.Y., (2014). Start a research on biopolymer polyhydroxyalkanoate (PHA): a review. Polymers 6, 706e754.

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(A)

(B)

FIGURE 5.4 Fluorescence microscopic view exhibiting bright yellowish-orange color (A) and (B). Electron microscopic view showing polyhydroxyalkanoate granules inside cells of Pseudomonas sp. strain-P (16). Adapted from Aljuraifani, A.A., M.M. Berekaa, Ghazwani, A.A.,. (2018). Bacterial biopolymer polyhydroxyalkanoate production from low-cost sustainable sources. Microbiology Open . 2018, e755.

PHA molecule is generally produced by the bacterial fermentation of lipid or carbohydrate as a linear polyester molecule. The polyester consists of three repetitious units of hydroxyalkanoic acid with a chain length of varying carbon groups (Wang and lee, 1997). Apart from the ability to act as the carbon and energy source, the PHA molecule helps in the sporulation of microbial species (Rawte and Mavinkurve, 2001). The synthesis of PHA molecules under nutrient-deficient conditions of limited nitrogen, phosphorous, oxygen, sulfur, and magnesium elements is shown in Fig. 5.4. PHA molecules exist in bacterial species such as E. coli, Azotobacter vinelandii, and B. subtilis and also exist in the microsomes and mitochondria of eukaryotes in the form of molecules containing calcium ions and polyphosphates (2:1:2) (Reusch, 1992). Aljuraifani et al. (2018) assayed bacterial biopolymer polyhydroxyalkanoate generation from an inexpensive origin such as Pseudomonas sp. The generation of PHA by Pseudomonas sp. was studied in the occurrence of dissimilar economical carbon sources such as rice bran, date, and soy molasses. The maximum PHA generation (i.e., 90.9%) occurred when the medium was supplemented with a carbon source of rice bran at 15 g/L (w/v) concentration with molasses; the greatest PHA production of 82.6% was obtained at 20 g/L concentration; for soy molasses, the maximum PHA production was 91.6% at 20 g/L concentration. It can be inferred from these three carbon sources that the greater PHA yield of 15e20 g/L can be obtained. Huang et al. (2006) obtained an ultimate PHA of 55.6% from Haloferax mediterranei with rice bran as the carbon source. Solaiman et al. (2006) generated PHA at 5%e17% by means of 2%e5% (w/v) soy molasses as the carbon precursor molecule. Meanwhile, Full et al. (2006) formed 25.4% PHA using 2% soy molasses as the initial carbon source. Another approach to producing bioplastic was studied by Ali et al. (2017) from a bacterial strain isolated from locally available soil and organic debris. The physiochemical properties of polyhydroxybutyrate (PHB), such as its mechanical properties, biodegradability, and eco-friendly nature,

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enable it to substitute for synthetic petroleum plastics. An applicable method known as sodium hypochlorite chloroform extraction induces PHB separation and purification with a maximum yield of 74 mg/L from a microbial biomass (Jacquel et al., 2008; Kunasundari and Sudesh, 2011). Modifications of the plasma membrane permeability are the two main steps to the release and solubilization of polyhydroxyalkanoates, and then precipitation of PHA (Jacquel et al., 2008). Different solvents such as acetone, methanol, and ethanol are successfully employed to extract and precipitate PHB from the cellular biomass (Jiang et al., 2006; Ramsay et al., 1994). The PHA or PHB molecules are generally produced as an intracellular inclusion that can be extensively produced from the cellular dry biomass of about 90% in terms of its growth against adverse environmental conditions (Verlinden et al., 2007). Some of the microbial origin for 50%e80% PHA production includes Ralstonia eutropha, Alcaligenes latus, and Pseudomonas oleovorans effectively extracted from their cellular biomass (Pei et al., 2011). The ability of Bacillus sp. to be efficiently produced in growth-mediated and nonegrowth mediated PHA components is a step toward the significant development of polymeric compounds in industries (Mohapatra et al., 2017). The capability of Bacillus sp. to grow under intense environmental conditions with the involvement of cheaper carbon substrates and an easy purification strategy makes it a suitable organism for higher PHA yields in optimal conditions (Lee et al., 1999; Borah et al., 2002). The enzyme responsible for hydrolysis that is found in Bacillus sp. suppresses the production of the PHA molecule. Bacillus megaterium was actively used to secrete the PHA molecule into cytosol (Lemoigne, 1926). About 90% of PHA molecules are successfully produced by various Bacillus sp. in a nutrient disparity environment (Madison and Huisman, 1999). Most bacterial species have the capacity to synthesize PHA during the log phase and stationary phase (Lee, 1996; Borah et al., 2002). The microbial biosynthetic pathway of PHA production varies according to the species involved in the process of accumulation of polymers in themselves. There about eight biosynthetic pathways for PHA biosynthesis (Chen, 2010). Among them, three are well-studied for efficient PHA monomer production. One mechanism involves the conversion of carbohydrates such as glucose and fructose to form homopolymers of PHA molecules. Current studies of PHA formation involve the model described by Griebel et al. (1968). This study involves two thiol groups of the enzyme called PHA synthase, in which one thiol group bond is on the loading site and other is on the elongation site. In terms of enhancing PHA synthesis by genetically modifying wild strains, E. coli is generally preferred as the standard organism (Nielsen et al., 2017). Certain strains of E. coli use lactose as the primary precursor carbon source for PHA production. The economically valuable whey protein is essentially used as a lactose source for the carbon utilization by modulating PHA-producing genes called pha operon. Certain strains such as XL1-Blue, JM, or DH5a are not able to employ lactose as the carbon source. Therefore, genetically engineering the wild-type strains, E. coli GCSC6576 (pSYL107) would preferentially use the lactose for the large-scale production of PHA molecules in the fed batch system, resulting in the production of 79% PHA molecules with a higher dry cell biomass weight (87 g/L). Modulating the time period of PHA synthesis by a genetically modified E. coli strain GCSC6576 (pSYL107) enhanced the PHA formation with an 80% dry cell mass weight. The pha operon are engineered from A. latus and Cupriavidus necator species. The studies revealed that the strain CGSC4401 is an efficient organism for PHB production with a dry cell mass weight of 119.5 g/ L; a PHB yield of 80.5% was effectively obtained (Ahn et al., 2000). Squillaci et al. (2017) studied the pigment-producing and PHA capacity of halophilic archaeon Haloterrigena turkmenica when it was grown under particular situations. The growth medium was

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supplemented with 1% (w/v) glucose for high mobility group box 1 protein growth. Growth was found to be associated with external stress conditions (Dawes, 1984) and the maximum biopolymer yield was found with nitrogen starvation, which also altered the stationary phase during growth. PHA production can also be confirmed by means of Nile Blue A staining. The PHA molecule produced by halophilic bacteria is polyhydroxybutyrate (PHB). The Nile Blue A staining technique can determine the specificity of the produced PHB molecule (Ostle and Holt, 1982; Tsuge, 2002). Osman et al. (2016) studied biopolymer synthesis by Microbacterium sp. WA81 in batch fermentation. Assimilation of biodegradable polymers in the plastic industry has become a necessity owing to the negative environmental impacts of plastics. Microbial polymer was confirmed to be the most latent polymer for use in the polymeric industry because it surpasses nondegradable synthetic polymers such as thermoplastics (Madison and Huisman, 1999). When supplementing with this cheap and efficient carbon and nitrogen source, the bacteria can effectively convert it into water vapor and carbon dioxide through an enzymatic reaction (Farrin, 2005; Vroman and Tighzert, 2009). A bacterial strain, Microbacterium sp. WA81, was recognized to be able to produce about 18 mg/L of the polymer. The formed polymer was extracted, purified, and characterized; it had a low molecular weight, which is a valuable property. To lessen the cost of manufacture, a number of economical materials such as molasses, whey, and ground sesame were used as potential media components to maximize the output by the bacterial isolate. A consequent response surface methodology algorithm was useful to find out the exact optimum combination to make the most of the polymer productivity. The finest combination was determined to be 9.21, 8.49, and 8.12 g/L for molasses, whey, and ground sesame, respectively, leading to a significant boost in polymer productivity (up to 660 mg/L). When this procedure was taken to the bioreactor cultivation level, the quantity of polymer formed rose to 2.5 g/L, a rise of more than 250%. Kundu et al. (2015) analyzed the synthesis of PHAs by various microbes and provided a detailed outlook into the act of different nonfastidious, inexpensive, naturally available microorganisms over low-cost, carbon-rich substrates for the biosynthesis of polyhydroxyalkanoates. The biosynthesis of PHA is carried out by microorganisms grown in variable resources such as glucose, starch, and sucrose. The particularity of PHA syntheses determines the monomers included in the polymers (Chen and Wu, 2005). To exploit the full latency of microbial systems for PHA production, it is important to alter biochemical pathways in particular microorganisms to ensure the maximal acquiescence of PHAs. The carbon source, PHA synthase, and the metabolic pathways involved have a vital role in shaping the type of PHA that can be produced by a picky microorganism (Chen, 2010; Po¨tter and Steinbu¨chel, 2006). PHA production and deprivation are controlled by a related pathway established in strains of Aeromonas hydrophila, Pseudomonas stutzeri, R. eutropha, and P. oleovorans (Sudesh et al., 2000). PHA deprivation is catalyzed by PHA depolymerase, dimer hydrolase, 3-hydroxybutyrate dehydrogenase, and acetoacetyl-CoA synthase. Solid, nonmetallic macromolecular biopolymers consist of repetitive units of monomeric components (Priyadharshini et al. 2015; Kumar et al., 2011). The monomeric units decide the physiochemical properties of the resultant polymeric component, such as its viscous nature, hardness, modulus elasticity, and semicrystalline nature. The major advantage of polymeric components is their biodegradable nature, which surpasses synthetic polymers, which need about 40e90 days for complete degradation. Thus, polymeric components prevent the emission of carbon dioxide and other toxic gases into the environment and are sustainable in nature (Kumar et al., 2011). The microorganisms

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123

R. eutropha, B. megaterium, Pseudomonas sp., recombinant E. coli, and Enterobacter aerogenes have been used to manage biopolymers. The enzyme catalyzed reaction of converting a carbon and nitrogen source into CO2 and steam leads to effective biopolymer accumulation (Lu et al., 2009a,b; Kumar et al., 2011). PHAs are separated into two groups based on the degree of carbon atoms linked in the polymeric chain: i.e., short-chain length (SCL), three to five C atoms; and medium-chain length (MCL), six to 14 C atoms. Variability in the carbon chain is due to the action of the key enzyme PHA synthase that acts on the 3hydroxyalkanoic acid (Bonartsev et al., 2007). The crystallinity index of the produced biopolymer also varies. For example, the SCL-PHA molecule is high crystalline whereas the MCL-PHA is low crystalline in nature (Raza et al., 2014). For instance, PHB is an attractive polyester containing less than 50% of crystallinity index with a melting temperature of 180
C (Bhuwal et al., 2013). PHB production involves three basic enzymatic reactions in the chemolithoautotrophic bacteria R. eutropha. The reaction starts with the substrate acetyl coenzyme A, in which the two molecules are converted to acetoacetyl-CoA molecules through the condensation of b ketothiolase (PhbA) enzyme. Furthermore, the resultant molecule undergoes a reduction reaction by stereospecific acetoacetyl-CoA reductase (PhbB) to R-()-3-hydroxybutyryl-CoA. Finally, the reduced component is polymerized to form PHB molecules with the help of the enzyme PHB synthase (PhbC) by the subsequent release of CoA molecules (Santhanam and Sasidharan, 2010). The formation of PHB can be modulated by the nutrient starvation of one mechanism with an excess carbon source and another limiting nutrient contents such as P, N2, Mg, or S, and another mechanism in bacterial species such as Alcaligenes eutrophus, Protomonas extorquens, and Protomonas oleovorans. Another mechanism includes production without considering nutrient starvation in bacterial species such as A. latus, A. vinelandii, and genetically modified E. coli (Bonartsev et al., 2007). Biopolymer formation from the bacterium Pseudomonas fluorescens was successfully studied by using Fourier transform infrared (FTIR) and differential scanning calorimetric (DSC) analysis (Priyadharshini et al., 2015). The study was carried out by microbial fermentation using Sudan Black B staining and the biopolymer produced was successfully extracted by means of sodium hypochlorite extraction, yielding 55% of biopolymer in a 72-h production process (Shamala et al., 2012). The Sudan Black B staining technique determines the capability of P. fluorescens (pf01) to induce the production inner biopolymer granules. The formation of black colonies confirms the presence of biopolymer granules in the inner cells by staining (Bhuwal et al., 2013). Thus, 55% of biopolymer chitosan was successfully extracted from the bacteria; the maximum growth of the organism was measured using a UV-Vis spectrophotometer. The obtained biopolymer was a white crystalline structure analyzed using characterization techniques (FTIR and DSC) (Shamala et al., 2012). A similar technique was used by Sathianachiyar and Devaraj (2013) to extract a biopolymer, PHA, from Bacillus and Pseudomonas bacteria isolated from the rhizosphere soil of Jatropha curcas. A similar staining method, Sudan Black B, was employed to detect the formation of PHA from the bacterial species. The former bacterial species produced a biopolymer of about 13.3 g/100 mL, whereas Pseudomonas sp. yielded 11.2 g/100 mL at pH 7. The production process involved the use of 2% (v/v) crude glycerol plus methanol as the feedstock. A similar feedstock with varying concentrations gave a considerable yield of grams of PHB per grams of methanol þglycerol (Dobroth et al., 2011; Braunegg et al., 1999; Ibrahim and Steinbuchel, 2010; Cavalheiro et al., 2009). In certain studies, crude glycerol and methanol are used for efficient biopolymer production in which microbes prefer methanol initially followed by glycerol at a higher oxidation state. Dobroth et al. (2011) stated

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Chapter 5 Microbial production of biopolymers

that a greater hydraulic residence time helps in constructive escalating microbial expansion for PHB manufacture under nutrient stress conditions. Different bacterial strains such as Enterobacter sp., Pantoea agglomerans, Pseudomonas sp., and B. subtilis were isolated from Brassica plants (cauliflower, cabbage, and lettuce) (Sivakumara et al. 2013). Mucoid colonies from the isolated strains were chosen for the advanced studies. Biopolymers such as EPS (cellulose, alginates, dextran, etc.), and intracellular and capsular polysaccharides were finely extracted from these bacterial strains. This microbe-derived EPS components were employed as a stabilizer, flocculant, emulsifier, and biosorbing medium for heavy metal absorption (Noghabi et al., 2007), according to their physiochemical properties. A large number of microbial biopolymers are identified, but only a limited biopolymers are potentially employed in the industries (Sutherland, 2001). In this study, glucose is primarily used as the carbon source for efficient biopolymer synthesis by isolated bacterial strains. Among the isolated strains, Pseudomonas sp. exhibited a maximum biomass yield based on the dry cell weight (DCW) after fermentation. The more viscous the medium is, the more the biopolymer yield will be. This was found to be greater at an optimal condition of 2% (w/v) glucose, 0.4 M sodium chloride, and 35
C incubation (Allison and Goldsbrough, 1994). Biopolymer accumulation was 2.2, 2.17 and 2.05 mPa from P. agglomerans, B. subtilis, and Enterobacter sp., respectively. The accumulated biopolymer was then dried to give a maximum yield of 3.17 g/L by Pseudomonas sp. Tanamool et al. (2011) studied the biosynthesis of polyhydroxyalkanoate by Hydrogenophaga sp. during batch fermentation. They screened sucrose using microbes from soil to assess the capability for the buildup of biopolymer of polyhydroxyalkanoate. Polyhydroxyalkanoate, a major kind of biodegradable plastic classified as polyester, was the first biomaterial discovered in B. megaterium and was characterized in 1925 by a French microbiologist (Lemoigne, 1926). The polyester may contain proportions of 3-hydroxyacids (Braunegg et al., 1998). The bacteria can be produced by PHAs from a number of substrates and assembled in their cells as a carbon source and energy reserve (Anderson and Dawes, 1990). To consider PHA production, the separate was full-grown in the medium containing sucrose as an exclusive carbon under controlled conditions of 35
C and pH 7. The maximum DCW and PHA production were obtained at 3.61 and 2.41 g/L after 36 and 42 h batch fermentation, respectively. The most PHA was 68.15% (gPHA/gDCW), giving a maximum PHA yield (YP/S) of 0.17 (gPHA/gSucrose) and an efficiency of 0.057 gPHA/L$h. This highlights the prospective of microbial assets in a soil environment and may be a usable application for the industrial production of PHA. Hydrogenophaga sp. was cultivated in a sucrose medium and its growth was monitored. PHA was produced and accumulated in its granules after 30 h and the maximum PHA assembly was obtained at 2.41 g/L within 42 h of batch fermentation. The results indicated that PHA accumulated during the mid-log phase (30 h) to the stationary phase (48 h) due to carbon and nitrogen appeared to discrepancy condition in fermentation media. In the case of sucrose uptake in the culture medium, it was at 14.73 g within 48 h. The maximum PHA inclusion in microbial cell was 68.15% (gPHA/ gDCW), giving a maximal PHA yield (YP/S) of 0.17 (gPHA/gSucrose); the volumetric efficiency for PHA amounted to 0.057 gPHA/L$h. The growth conditions of biopolymer-producing microbes were optimized to strengthen the enhanced production of biopolymers by Basnett and Roy (2010) in which the polyhydroxyalkanoates as reserve molecules during stress conditions were established by the microorganism during adverse conditions (limiting N2 in an excess carbon environment) (Madison and Huisman, 1999). Among different precursors used for PHA production, lactic acid was identified as the effective precursor for

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125

biopolymer formation. This lactic acid was induced by the fermentation of any hexose molecule to lactic acid by lactic acideproducing microbes. The economically valuable carbon source promotes the production of PLA from the purified lactic acid (Sodergard and Stolt, 2002). The biosynthetic production of polyhydroxyalkanoates occurs by three major pathways. The first pathway is described in C. necator, in which 3HB monomeric molecules are initially produced through acetyl-CoA condensation to give acetoacetyl-CoA by means of the enzyme PhbA. Furthermore, 3hydroxybutyryl-CoA is produced from acetoacetyl-CoA catalyzed by the enzyme acetoacetyl-CoA reductase. The 3-hydroxybutyryl-CoA is further polymerized via esterification by means of the enzyme called PHA synthase to form poly(3-hydroxybutyrate) (P[3HB]) (Sudesh et al., 2000). The second pathway of PHA synthesis involves fatty acid oxidation generally explained by Pseudomonas aeruginosa. This synthetic pathway includes hydroxyacyl substrate formation from fatty acid. The formed substrate is then subjected to a polymerization reaction to produce polyhydroxyalkanoates with the presence of an enzyme called PHA synthase enzyme (Philip et al., 2006). The third synthetic pathway undergoes de novo synthesis, which involves the conversion of sugar molecules into subsequent bioproducts. In this pathway, the intermediate, (R)-3-hydroxyacyl, is transformed into CoA by the enzyme, acyl-ACP-CoA transacylase (encoded by phaG) from the acyl carrier protein substrate. The enzyme responsible for this enzymatic conversion acts as the key component between PHA and fatty acid biosynthesis (Philip et al., 2006). P(3HB) yield can be enhanced by controlled environmental conditions such as in surplus carbon and restricted nitrogen concentration by fermentation. Various carbon sources are actively used for production. Among them, molasses (sugarcane), whey protein, and rapeseed cake are widely employed carbon sources in the form of glucose and sucrose. A technically variable fermentation technique was investigated by EleSayed et al. (2009) involving a two-stage, fed-batch, and batch fermentation process in C. necator. This mode of PHA synthesis forms the initial multigene biosynthetic pathway for PHA synthesis (Pouton and Akhtar, 1996). Intracellular P(3HB) molecules were also successfully produced from gram-negative bacteria (e.g., A. latus) and from a genetically engineered E. coli strain (Lee et al., 1999). P(3HB) molecule was successfully extracted by means of chloroform and by sodium hydroxide digestion. The endotoxin levels varied by adding chloroform and by the further addition of sodium hydroxide from more than 10 to 104 endotoxin units (EU)/g of P(3HB). However, the endotoxin was drastically decreased by the extended time of digestion by sodium hydroxide (i.e., to 1 EU/g) (Pouton and Akhtar, 1996). P(3HB) was successfully used as thin sheets, films, and fibers. X-ray diffraction studies revealed a crystallinity index of about 50%e80% (Khanna and Srivastava, 2005). Thus, it can be inferred from these studies that optimization of the fermentation processes enhances microbial biopolymer production.

4. Conclusion The exploration of efficient bacteria for large-scale PHA production is limited, although numerous organisms have been reported for it. The key concern is in effectively improving strains to serve the purpose of improving the quantity and quality of the biopolymer produced. Therefore, an effort has to be made to explore more bacterial species to enhance PHA production.

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Biopolymers have diverse applications such as in wastewater treatment, the medical and food industries, cosmetics, fuel cells, hydrogels, and drug delivery system owing to their unique properties. They are also used in the tissue engineering field. Therefore, they have an impact on the identification of novel methods to increase polymer quality and quantity.

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Further reading Faraco, V., Ambrosanio, A., Viscardi, S., Pepe, O., 2017. Bio-Based succinate production from Arundo donax hydrolysate with the new natural succinic acid-producing strain Basfia succiniciproducens BPP7. Bioenergy Research 10 (2), 488e498. Rekha, M.R., Sharma, C.P., 2007. Pullulan as a promising biomaterial for biomedical applications: a perspective. Trends in Biomaterials and Artificial Organs 20 (2), 116e122. Remminghorst, Rehm, 2009. Microbial production of alginate: biosynthesis and applications. In: Microbial Production of Biopolymers and Polymer Precursors. Caister Academic Press, ISBN 978-1-904455-36-3. Sutherland, I.W., 2011. Microbial polysaccharides from gram-negative bacteria. International Dairy Journal 11, 663e674.

CHAPTER

Animal-derived biopolymers in food and biomedical technology

6 Varsha Wankhade

Department of Zoology, Savitribai Phule Pune University, Pune, Maharashtra, India

1. Introduction Biopolymers are the polymers produced by living organisms. They are polymeric biomolecules. They are biodegradable chemical compounds and the most organic compound in the ecosphere. Biopolymers are made up of monomer units; these units form larger structures through covalent bondings. Biopolymers are also called as natural polymers as they are synthesized during the growth of living organism (Vijayendra and Shamala, 2013). Some examples of biopolymers are proteins, carbohydrates, DNA, RNA, lipids, nucleic acids, peptides, and polysaccharides (such as glycogen, starch, and cellulose). Biopolymers can be broadly classified as natural and synthetic biopolymers. Synthetic biopolymers are of two types: nondegradable and degradable (Table 6.1). Biopolymers on the basis of monomers used and the structure of the biopolymer formed are classified into three main classes, viz. polynucleotides, polypeptides, and polysaccharides. Polynucleotides (RNA and DNA) are made up of nucleotide monomers. Polypeptides are made up of

Table 6.1 Broad classification of biopolymers with examples. Natural

Proteins

Biopolymers

Polysaccharides

Synthetic

Degradable Nondegradable

Collagen Fibrinogen Soy protein Silk Cellulose Chitin Hyaluronic acid Starch Hemicellulose Pectin PGA, PLA, PDS, PCL, PHB, PPF PE, PP, PC, PVC, PMMA, PTFE

PC, Polycarbonate; PCL, Polycaprolactone; PDS, Polydioxanone; PE, Polyethyleneglycol; PGA, Polyglycolic; PHB, Polyhydroxybutyrate; PLA, Polylactic acid; PMMA, Polymethylmethacrylate; PP, Polypropylene; PPF, Poly(propylene fumarate); PTFF, Polytetrafluoroethylene; PVC, Polyvinyl chloride. Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00006-0 Copyright © 2020 Elsevier Inc. All rights reserved.

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amino acids and are short polymers. Polymers of polysaccharides are carbohydrate structures and regularly bonded linear (Chandra and Rustgi, 1998; Kumar et al., 2007; Meyers et al., 2008; Mohanty et al., 2005). Rubber, suberin, melanin, and lignin are the few other examples of biopolymers. The biomaterials that are made from proteins, polysaccharides, and synthetic biopolymers do not have mechanical properties and stability in aqueous environments. These properties are required for applications in medical field. The industrial applications of polymers depend broadly on the plants or marine algae-based materials. Natural polymers possess a large variety of applications in food and pharmaceutical industries and are also being used for several industrial functions such as petroleum well drilling, explosives, photography, making shampoos, in paper industry, fire fighting, textiles, etc. Similarly, they also play an important role as protective agents in the set up of symbiosis, energy reserve materials, osmotic adaptation, aid in cell functioning, and to support the microbial genera to adapt, function, multiply, and survive in the varying conditions of the environment. Approximately, every year 140 million tons of synthetic polymers are synthesized globally. The synthetic polymers like waste plastics and water soluble synthetic polymers in sewage cause environmental pollution. Degradation cycles of polymers in the biosphere are unlimited as they are enormously stable (Gopalrao et al., 2014). Plastics and polymers have become an inseparable part of our daily life. However, due to its stability and resistance to degradation, plastics and polymers build up in the surroundings at the rate of about 8% by weight and 20% by volume of the landfills. Day by day the biopolymers are demanded as pioneer industrial material because of the flexibility in the usage. Therefore production of new biodegradable polymers is need of the time. During growth cycles of all organisms, huge numbers of biodegradable polymers are synthesized. The main benefits of biopolymers are that they can be used in such conditions where they raise the functionality and deliver additional profits. These materials could be used in living systems for decreasing the proliferation of chronic inflammation or immunological reactions and toxicity that develops usually after the implantation of synthetic polymer (Yates and Barlow, 2013). Natural polymers could be modified for specific requirements. Biopolymers might be the materials occurring naturally synthesized from animals, plants, bacteria, and fungi or could be the polymers which are produced chemically by using biological materials like sugars, amino acids, oils, or natural fats (Augustine et al., 2013). Biopolymers such as glycolic acid, starch, lactic acid, cellulose, etc., could be used in the synthesis of biodegradable products. These biopolymers have the potential to be used in medical appliances, water treatment, packaging, food additives, cosmetics, clothing fabrics, absorbents, industrial plastics, biosensors, and even data storage elements. Biopolymers, because of its considerable biocompatibility and biodegradability, have an important role in biomedical applications, especially in the area of drug delivery systems, tissue engineering, wound treatment, dialysis membrane, and biosensors that have a prospective role in human health improvement (Augustine et al., 2013). The smart biopolymers have plenty of biomedical applications like drug delivery, tissue engineering, and gene delivery. The usefulness and nonexploitability of smart polymeric materials make biopolymers a predominant interface of chemistry and biology. In recent times, polymers and hydrogels have become more attractive in biotechnology and medicine due to their stimulusresponsive behavior in aqueous solution. Considering the versatile applications of biopolymers, there is a need to spread awareness and invent new methodologies with biomedical and agricultural

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applications. Biopolymers, with edible and biodegradable properties, are one of the practical substitutes for food packaging and for various applications in biomedical science. Very limited work has been reported on animal biopolymers and their application in food and biomedical sciences. In this context, the present chapter is focused on the animal biopolymers and their applications in food and biomedical engineering.

2. General classifications of biopolymers Biopolymers are classified as polyesters (polyhydroxyalkanoates and polylactic acid), proteins (silk-alginate, collagen/gelatine, elastin, polyamino acids, resilin, serum albumin), polysaccharides of animal origin (hyaluronic acid, chitin/chitosan), lipids/surfactants (surfactants, acetoglycerides, emulsan, waxes), polyphenols (humic acid, tannin, lignin), specialty polymers (synthetic polymers, poly-gamma-glutamic acid, from natural fats), and others (Onar, 2014) (Fig. 6.1).

FIGURE 6.1 General classifications of biopolymers.

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3. What are animal and natural biopolymers? Natural polymers, also known as biopolymers, involve four groups, such as lipids, polysaccharides, polynucleotides, and polypeptides. Among them polysaccharides are produced in nature by plants, wood, fungi, bacteria, and algae. Whereas polysaccharides synthesized from animals are chitin, chitosan, glycosaminoglycans (GAGs), and hyaluronic acid (HA).

4. Types of animal biopolymers Biopolymers are the animal proteinebased biopolymers like gelatine, silk, wool, polysaccharides, and collagen. Chitosan is a polysaccharide synthesized by the deacetylation of chitin. It has antimicrobial activity which is well studied. It is widely used in different fields inter alia agriculture, waste management, food, and medicine. It has biological properties like anticholesterolemic, biodegradability, biocompatibility, antimicrobial, and mucoadhesive and has permeation enhancement effects. It has several applications in therapeutic delivery. Chitosan is useful in specific functions like formation of antibacterial/anti-biofouling coatings, hydrogels; in nanofiltration; in controlled release coatings and microcapsules, tissue engineering scaffolds; and in drug delivery (Nigmatullin et al., 2009). It is used in coating form in targeting drug delivery vehicles to specific tissue and also in providing a stimuluscontrolled release response. Chitosan is a multipurpose molecule and possess potential applications in many fields. It is able to form polymeric complexes by electrostatic interactions of chitosan NHþ 3 groups with COO or SO-3 groups in polyanions or proteins (e.g., pectin and alginate). Chitosane protein or chitosanepolyanioneprotein complexes may be recovered as the suspended proteins in waste effluents, fermentation media, and other industrial streams (Torres et al., 1999). Glycosaminoglycans (GAGs) or mucopolysaccharides are made up of repeating disaccharide unit. These are unbranched and long polysaccharides. It is made up of the repeating units of amino sugar (Nacetylglucosamine or N-acetylgalactosamine) with a uronic sugar (glucuronic acid or iduronic acid) or galactose. Generally, it is present in all connective tissues, extracellular matrix, and also on the surfaces of many cell types. Hyaluronic acid (HA; conjugate base hyaluronate), also known as hyaluronan, an anionic, nonsulfated glycosaminoglycan disseminated extensively throughout connective, epithelial, and neural tissues. It is a nonsulfated form with very huge, molecular weight and present in the plasma membrane except in the membrane of Golgi apparatus (Fraser et al., 1997). HA is one of the important components of the extracellular matrix and contributes notably to proliferation and migration of cell. It might be involved in the development of few types of malignant tumors. The biopolymers are classified as “1st (First) class bio-based polymers” and “2nd (Second) class bio-based polymers.” First class bio-based polymers are naturally derived biomass polymers while 2nd class bio-based polymers are bioengineered. Artificially synthesized biodegradable polymers are not included in these classes (Nakajima et al., 2017). Biodegradable polymers include naturally as well as artificially synthesized polymers. Sometimes it is also called as bio-compostable polymers, especially in agricultural, fishery, waste, and construction industries. It also consists of naturally derived building blocks called as bioabsorbable polymers because they are used for pharmaceutical, medical, and bioengineering applications.

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5. Silk Silkworm produces the raw silk thread. Its core is made up of fibroid and is covered with sericin. Fibroin is water insoluble. It has a triangular cross-section. The thread of silk shows a characteristic shine, pleasant handling, and an elegant drape. Core silk fibroin fibers exhibit comparable biocompatibility. Sericin has wide applications (Fig. 6.2). It is used as an anti-frosting agent to coat a film on the surface of refrigerators, deep freezers, and refrigerated trucks and ships. Environment-friendly biodegradable polymers can be produced by blending sericin with other resins. Sericin coatings enhance the functionality of materials. It is used in skin cosmetics as a moisturizing agent and in nail cosmetics and is a valuable natural ingredient in food industry due to its antioxidant properties. It is also a good moisture absorbent and used in manufacture of a blended hydrogel. Sericin is also used in medical textiles in medical materials and wound dressing (Fig. 6.2).

6. General applications of biopolymer The biopolymers are tremendously used in many applications such as energy storage, protection, and cellular construction and transfer of genetic information. • • •

Polymers based on sugar: These are synthesized in human body. There are no side effects of it which are harmful. Hence it is used especially in surgical implants. Biopolymers made from starch can form conventional plastics. Biopolymers-based synthesized polymers are used to create substrate mats.

FIGURE 6.2 Uses of sericin.

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Cellophane and other biopolymers made from cellulose are used as packaging materials. Many chemical compounds are used to make thin wrapping films, food trays, and pellets for sending fragile goods by shipping.

7. Formulation of different biopolymers Microorganisms are used to obtain a large amount of biopolymers. Possible applications of biopolymers have been increased quickly which include applications in food additives and biomedical field, biodegradable plastics from renewable resources (Kreyenschulte et al., 2012). Beside these, the biopolymers have huge applications. But the introduction of biopolymers into market in large scale is many times restricted due to nonefficient and complex downstream processing and its cost of productions. Generally, the manufacture of by-products is undesirable as it can influence the downstream processing and reduce the overall yield of product because of inefficient conversion of nutrients. The mixed biopolymer hydrogels synthesized have a bilayered structure. Both the layers have their specific biological activity, porosity, and chemical composition. The two layers can cross-link together chemically and can interpenetrate mechanically with each other (Spiro and Liu, 2004). The biopolymers or mixed polymers prepared by a ring-opening oxidization reaction result into HA with active aldehyde groups which are linked to the sugar chains (Rehakova et al., 1996). Cross-linking is a regular way to subdue the limitations of biomaterials. The functions of the crosslinker are to interconnect the molecules, increase molecular weight, provide higher mechanical properties and improve stability. However, cross-linking also helps to minimize the degradability, reduce the availability of functional groups in the cross-linked polymer, and alter the rheology of the polymers, which lead to succeeding processing problems and probable augmentation in cytotoxicity (Reddy et al., 2015). Martinez et al. (2014) stated that depending upon the kind of biopolymer to be cross-linked for that purpose many cross-linkers and cross-linking techniques are available. Plentiful chemical crosslinkers are used, among them glutaraldehyde is the predominant due to its reactivity with the functional groups in both carbohydrates and proteins. It also offers materials with considerable improvement in tensile properties. Glutaraldehyde improves mechanical properties but glutaraldehyde-cross-linked materials may show cytotoxicity (Reddy et al., 2015). Gelatine water-soluble proteinaceous substance was synthesized by destroying the tertiary, secondary, and to some amount the primary structure of the native collagens. For this the incomplete hydrolysis of collagen derived from the white connective tissue, bones, and skin of animals was carried out (Morrison et al., 1999). Other hydrocolloids differ from it, as most of them are polysaccharides. Gelatine contains all the essential amino acids except tryptophan and is a digestible protein. Gelatine could be synthesized from the collagen derived from fish, pig skin, cattle bones, etc. (Lobo, 2002; Mariod and Adam, 2013). It can be used as a wetting agent in food, foaming, pharmaceutical, emulsifying agent, and in medical and technical applications (Lobo, 2002). Payen discovered collagen in 1838. Collagen is a primary structural material of vertebrates. It is the most plentiful protein of mammal, which is about 20%e30% of total body proteins (Harkness, 1961; Lee et al., 2001). It is produced by fibroblasts, which generally originate from pluripotential adventitial ˚ long and about 15 A ˚ wide molecule. The cells or reticulum cells. Collagen is a rod-shaped, 3000 A molecular weight of collagen is around 300 KDa. It can be absorbed easily in the body, and it presents

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less antigenicity. Similarly, it does not show toxicity and is biocompatible and biodegradable. Tensile strength of collagen is high and shows high affinity for water.

8. Applications of biopolymers in food industries Due to the loss of toughness and quality of food it is necessary to use biodegradable polymers in order to increase life of food. Edible and biodegradable natural polymeric materials can be used in food custom. These are synthesized from polysaccharides, lipids, and proteins. These biopolymers reduce the cost that would otherwise need to be spent on protection of environment (Grujic et al., 2017). Biodegradable natural polymeric materials are also used as packaging materials, edible films and coatings, carriers of antimicrobial and antioxidant materials, etc. It can be used as tanks for the controlled release of drugs or fungicides. These natural polymers are used in many food industries. Intensive research is being carried out on the polysaccharides to understand its applicability in food packaging (Kristo and Biliaderis, 2006; Grujic et al., 2017). When the pectin in the form of film (biopolymers) is used in combination with different food grade proteins like soybean proteins and gelatine, pectin forms edible hydrogels that can be used for wrapping and foods packaging. However, bacteriocidins, like nisin, or other antibacterial substances like allyl isothiocyanate could also be preincorporated into the gels. The biopolymer mixtures can protect overall food quality ( Farris et al., 2009: Liu et al., 2012). Fennema et al. (1993) studied the control of moisture migration between regions with different water activities developed by methyl and hydroxypropyl methyl cellulose films. Films having paraffin, beeswax, stearic acid, or hydrogenated palm oil showed less water permeability than low-density polyethylene. Films and coatings can be made from proteins as a structure making constituent with added lipids and plasticizers to enhance their efficiency (Torres et al.,1999). Gelatine and collagen are the materials that have been used for packaging sausages and other products made from meat (Wang and Padua, 2005). Plausible applications of certain other proteins such as soy protein, wheat gluten, whey protein, corn zein, and casein have been studied. Proteins and polysaccharides can be used to prepare edible coatings. It may help to maintain high preservative concentrations on food surfaces. This will restrict the microbial growth that lowers the shelf life of many products (Torres et al., 1999). Raw materials which are renewable such as polysaccharides, proteins, and lipids are used to synthesize edible wrappers. These biopolymers might be used separately or in combination (Jensen et al., 2015). The selection of materials for packaging will depend on the needed applications of the product. Polysaccharides are the natural polymers and nontoxic and quite broadly accessible ( Grujic et al., 2017). Several types of polysaccharides and their derivatives such as konjac, alginate, starch, carrageenan, chitosan, pectin, and cellulose have been screened to develop edible/biodegradable films. Polysaccharide-based films are useful in preventing the leakage of gas. Polysaccharide-based films can keep out aromatic materials and lipids. However, they cannot form a fine obstacle for water vapors. These water vapors can pass through them easily. Chitosans exhibit antimicrobial properties and thus may restrain the development of microorganisms on the surface of the product. Chitosans are used to manufacture paper, carton, and cellophane wrapping (Kjellgren et al., 2006; Bordenave et al., 2007). Packing of eggs, fruits and vegetables, meat, and other kinds of food can be done with the help of this protein. Films of collagen and gelatine are

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lucid; however, they are permeable to moisture. The possible ways to produce biodegradable films by using vegetable proteins and milk casein have been profoundly studied. The usage of renewable sources in food wrapping is being increased due to high demand of natural sources (Gabor and Tita, 2012). Natural compounds are used in large amount to increase the duration and quality of all kinds of foods with enhanced preservation and security from oxidation and microbial spoilage. The application of synthetic films may create great ecological issues as these materials are nonbiodegradable and may cause several problems in environment (Sabiha-Hanim and SitiNorsafurah, 2012). The natural biopolymers can be formed from the replenishable resources and are biocompatible and biodegradable. Such natural biopolymers can be used in food packaging (Gabor and Tita, 2012). Animal connective tissues contain collagen as a primary protein element. It is developed from different polypeptides, which contain mostly glycine, proline, hydroxyproline, and lysine. There might be difference in the amino acid sequence of collagen derived from diverse sources. These collagens have been used for the purpose of food packaging to increase their shelf life. In addition, bones and skin collagen is hydrolyzed to form a protein, gelatine (Go´mez-Guille´n et al., 2009). Mammalian (porcine and bovine) gelatine is of great demand. Chitosan has different usage in food and nutrition because of its great nutritional quality (Talens et al., 2012). Further, it also shows strong antimicrobial properties against fungi, bacteria, and viruses. Its biocide properties are also well known.

9. Biopolymers and its application in biomedical sciences Chitosan and chitin are well-recognized biodegradable natural polysaccharide polymers which are recovered from different animals and plants. Chitin is found extensively in cell walls of yeasts, fungi, and molds and in the cuticle and exoskeletons of invertebrates. Chitin and its most important derivative, chitosan, possess several functional chemical and physical properties such as nontoxicity, biodegradability, and high strength. The chitin family of polymers is being generally used in agriculture, waste treatment, and medicine (Onar, 2014). Chitosan is a useful agent to speed up the process of blood clotting. Chitin compounds are biodegradable and thus are mainly appropriate in drug delivery systems. It is also used as slow carriers that can lead to release drugs slowly into the body. This kind of potential is tremendously important in cancer chemotherapy as the agents are often highly toxic and need more time for administration. The chitosan compounds are also studied for their potential as an inhibitor of the AIDS virus. From medical perspective, chitosan is used in bandages and treatment for wound healing, as it forms hard, oxygen permeable, water-absorbent, and biocompatible film. Stable HA hydrogels are used in the transplants in tissue engineering technology, like would healing, artificial skin, cartilage repair, soft tissue augmentation, and facial intradermal implants (Elia et al., 2010; Burdick & Prestwich, 2011; Liu et al., 2012). This becomes possible by covalently cross-linking the polysaccharide with various multifunctional reagents, like a group of epoxides, divinylsulfone, tresyl chloride, or hydrazide derivatives, or to synthesize hybrids with other biopolymers, like collagen, and mineralized collagen. The combinations showed potential candidate having functional properties of the extracellular matrix of human tissues (Dickerson et al., 1997; Liu et al., 2002).

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HA could be used as a disease indicator. It can be used in identification of arthritis, presence of liver cirrhosis, tumors, and scleroderma. It is used in the ear surgery as a scaffold material, in healing of tympanic membrane perforations. It is also used in eye surgery. It protects tissues of cornea and is used in reattachment of retina. It stimulates tissue repair and thus helps in wound healing. It is used in tendon surgery. It helps in repair of flexor tendon lacerations. It is used in degenerative joint disease in animals. It is also used as an antiadhesive and in scar controls in general surgery (Onar, 2014). The biological materials, such as leather, wool, cellulose, and silk, are conventional commodity polymers (Onar, 2014). Natural polymers with specific properties can be synthesized by using recent advanced biotechnological techniques such as recombinant, advancing genetic engineering, bacterial fermentation, and nanotechnology and thus exceptional biopolymers can be developed. Biopolymers play important roles in our body such as holding cells together to form tissues. Further, it gives chemical signals to the cells and thus regulates their behavior. It also plays role in maintaining skin’s hydration and elasticity and acts as a lubricant in joints. Biopolymers can assemble into the mucus gel which covers eyes and respiratory tract and protects these organs from pathogens (Pattanashetti et al., 2017). These polymers also could have role in gene delivery which is helpful in the treatment of several types of genetic diseases. Tissue engineering now has become a promising area for repair of organs and tissues, which are damaged by injury or disease. Its main aim is to redevelop or restore biologically damaged or diseased tissue or generate replacement of organs for a broad range of medical conditions like diabetes, heart diseases, osteoarthritis, cirrhosis, disfiguration, and spinal cord injury. In the field of tissue engineering, biopolymers are widely used for various applications (Ward and Georgiou, 2011). The biopolymers are the most important agent, which can be used in medical applications (Yadav et al., 2015). Biopolymers are used in wound closure, surgical implant devices, drug delivery systems, bioresorbable scaffolds, and in healing products for tissue engineering. Chitosan can modulate peroxide production and show in vivo stimulatory effect on nitric oxide production. In the patients undergoing hemodialysis, chitosan is found to inhibit activation of neutrophil and also oxidation of serum albumin, thus chitosan can have role in lowering of uremia-associated oxidative stress (Ueno et al., 2001). In addition, DNA can be protected by chitosan and can lengthen the gene’s expression period. Collagen may be used in tissue-based devices such as prosthetic heart valves and vascular prosthesis which reduces the risk of infection and lengthen the cardiac patient’s life (Lee et al., 2001). However, it could be used in collagen-based drug delivery systems as collagen film or sheet or for the treatment of tissue infections like infection of corneal tissue or in cancer of liver. Whereas, for the treatment of infection in corneal tissue along with antibiotic agents, like gentamycin and tetracycline, the soluble ophthalmic combined as film or wafer and used as drug delivery system. Collagen sponges have a potential in the treatment of severe burns and as a dressing for severe types of wounds (Lee et al., 2001). Collagen dressings are developed in many forms such as membrane sheets, sponges, and powder which have necessary biological properties essential for such application. In solid tablets, cellulose ether can be used in swelling-driven release of the drug after contact of the tablet with physiological fluids (Stabenfeldt et al., 2006). Comparable biocompatibility is shown by the core silk fibroin fibers with other frequently used biomaterials like collagen and polylactic acid. Altman et al. (2003) studied the synthesis of a wire rope matrix for formation of autologous tissue-engineered anterior cruciate ligaments (ACL) by using

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FIGURE 6.3 General medical applications of polymers.

patient’s own adult stem cells with silk. The materials modified with sericin and sericin composites can be used as degradable biomaterials, fibers, functional membranes, biomedical materials, and fabrics (Zhang, 2002). Alginates are mainly used as absorbing wound dressings. Alginates easily develop hydrogels by forming complex with calcium ions, so they are appropriate for delivery of drugs and for cell entrapping. In animal body dextranases slowly degrade dextrans (Vert, 1987). After considerable loss of blood due to injury they are used as isotonic plasma substitutes to regenerate the volume of body fluids. HA is rapidly biodegraded in human body where it is normally regenerated. Biopolymers have varied applications such as in skin tissues repair, formation of antimicrobial membrane, in biosensors and biodiagnostics, in vascular grafts, in medical implants, in drug delivery, in making bioscaffolds, etc. (Fig. 6.3). Biopolymers such as poly(1,4-dioxan-2-one), poly(a-hydroxy acids), polyglyconate, collagen, and poly(a-cyanoacrylate) are used as binding materials. Biopolymers such as poly-L-lactide (PLLA), polyglactin, and hydroxyapatite are used as bone-setting materials. Biopolymers such as gelatine are used as antiadhesive and matrix for tissue culture materials. Biopolymers such as PLLA and polyglactin are used as fibers in artificial tendon and ligament and porous material in artificial blood vessel. Biopolymers such as collagen, chitin, polyglactin, and poly-L-leucine are used as wound cover attributes. Biopolymers such as collagen are used to make synthetic skin. All degradable polymers are also used in drug delivery system in the form of microcapsule, microsphere, needle, hollow fiber, etc. (Onar, 2014) (Fig. 6.4).

11. Conclusion

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FIGURE 6.4 Biomedical applications of biopolymers.

10. Some environmental benefits of use of biopolymer Biopolymers can always be renewed. They are composed of living materials so they are sustainable. Biopolymers can decrease the level of carbon dioxide in the atmosphere and reduce carbon emissions. Biodegradation of these chemical compounds release carbon dioxide which can be reabsorbed by crops grown as a substitute in their place. Biopolymers are compostable and cause less environmental pollution. Use of biopolymers decreases the reliance on fossil fuels which are nonrenewable. These are easily biodegradable and thus can help in decreasing air pollution. Use of biopolymers would reduce the use of plastics thus greatly reducing the harmful effect of plastic use on the environment (Fig. 6.5).

11. Conclusion The biopolymers play versatile role in food industry and biomedical science. Biopolymers are diverse group of polymers, and their usage in food packaging, wrapping, and biomedical science is diverse and multiple. Biopolymers, especially the chitosan and chitin from animal origin, could be promising agents for biomedical application. Due to its biodegradability, stability, low toxicity, and renewable nature the biopolymers play an important role as catalyst. Animal biopolymers are used in food industry and in biomedical science. Biopolymers can be used in implants, tissue engineering, drug delivery systems, wound covers, and also to increase food life. Although there are a number of applications of biopolymers as biosensors in electronic and optical fields, the present chapter is focused on applications of biopolymers in food and biomedical science.

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FIGURE 6.5 Environmental benefits of biopolymers.

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Pattanashetti, N.A., Heggannavar, G.B., Kariduraganavar, M.Y., 2017. Smart biopolymers and their biomedical applications. Procedia Manufacturing 12, 263e279. Reddy, N., Reddy, R., Jiang, Q., 2015. Crosslinking biopolymers for biomedical applications. Trends in Biotechnology. https://doi.org/10.1016/j.tibtech.2015.03.008. Rehakova, M., Bakos, D., Vizarova, K., Soldan, M., Jurickova, M., 1996. Properties of collagen and Hyaluronic acid composite materials and their modification by chemical cross-linking. Journal of Biomedical Materials Research 30, 369e372. Sabiha-Hanim, S., Siti-Norsafurah, A.M., 2012. Physical properties of hemicellulose films from sugarcane bagasse. Procedia Engineering 42, 1390e1395. Spiro, R.C., Liu, L.S., 2004. Collagen/Polysaccharide Bilayer Matrix. United State Patent 6,773,723, August 10 ,. Stabenfeldt, S.E., Garcia, A.J., LaPlaca, M.C., 2006. Thermoreversible laminin-functionalized hydrogel for neural tissue engineering. Journal of Biomedical Materials Research Part A 77 (4), 718e725. Talens, P., Pe´rez-Ması´a, R., Fabra, M.J., Vargas, M., Chiralt, A., 2012. Application of edible coatings to partially dehydrated pineapple for use in fruitecereal products. Journal of Food Engineering 112 (1e2), 86e93. Torres, J.A., Dewitt-Mireles, C., Savan, V., 1999. Two food applications of biopolymers: edible coatings controlling microbial surface Spoilage and chitosan Use to recover proteins from aqueous processing wastes. ACS Symposium Series. American Chemical Society, Washington, DC. https://doi.org/10.1021/bk-19990723.ch017. Ueno, H., Mori, T., Fujinaga, T., 2001. Topical formulations and wound healing applications of chitosan. Advanced Drug Delivery Reviews 52, 105e115. Vert, M., 1987. Design and synthesis of bioresorbable polymers for controlled release of drugs. In: Davis, S.S., ILLUM, L. (Eds.), Controlled Release of Drugs from Polymeric Particles and Macromolecules. Wright lOPPub1.Ltd., Bristol, pp. 117e125. Vijayendra, S.V.N., Shamala, T.R., 2013. Film forming microbial biopolymers for commercial applications-a review. Critical Reviews in Biotechnology. https://doi.org/10.3109/07388551.2013.798254. Wang, Q., Padua, G.W., 2005. Properties of zein films coated with drying oils. Journal of Agricultural and Food Chemistry 53, 3444e3448. Ward, M.A., Georgiou, T.K., 2011. Thermoresponsive polymers for biomedical applications. Polymers 3, 1215e1242. Yadav, P., Yadav, H., Shah, V.G., Shah, G., Dhaka, G., 2015. Biomedical biopolymers, their origin and evolution in biomedical sciences: a systematic review. Journal of Clinical and Diagnostic Research 9 (9), 21e25. Yates, M.R., Barlow, C.Y., 2013. Life cycle assessments of biodegradable, commercial biopolymers. A critical review, Resources, Conservation and Recycling 78, 54e66. Zhang, Y.-Q., 2002. Applications of natural silk protein sericin in biomaterials. Biotechnology Advances 20 (2), 91e100.

CHAPTER

Application of CRISPR technology to the high production of biopolymers

7

Hyo Jin Kim1, 2, Timothy Lee Turner3 1

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea; 2Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea; 3Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

1. Introduction CRISPR/Cas9 technology has had a great impact on biology, medicine, microbiology, and food microbiology. In particular, the highly sophisticated base pairelevel gene editing technique enabled more precise manipulation of genes to allow for the insertion or removal of several genes quicker and easier than conventional DNA manipulation techniques. Additionally, the ease of deletion of gene targets in polyploidy organisms made a major improvement on research in the life sciences across a variety of eukaryotic systems. Ultimately, CRISPR/Cas9 will promote the development of the biological sciences for a variety of organisms and will facilitate the production of numerous industrially relevant substances, particularly biopolymers. This chapter covers microbial metabolic engineering via the CRISPR/Cas9 technology and focuses on the application of the CRIPSR/Cas9 technology to create various types of biopolymers, including exopolysaccharides (EPSs), produced from food microorganisms.

2. Application of CRISPR/Cas9 in various research The purpose of the CRISPR (clustered regularly interspaced short palindromic repeats) sequence, found in the genomes of bacteria and archaea, remained unknown when it was first discovered by Yoshizumi Ishino at Osaka University, but the function was uncovered years later by scientists at Danisco Company (Barrangou et al., 2007; Ishino et al., 1987). Numerous domesticated bacteria widely used in fermentation and biotechnology processes are often susceptible to phage attack. Barrangou et al. (2007) found that CRISPR together with associated cas genes were involved in resistance against phages, and they showed that the phage resistance indeed was conferred on the host by the CRISPR sequences (Barrangou et al., 2007). In 2012, Emmanuelle Charpentier at Umea˚ University, who is now affiliated with the Max Planck Institute for Infection Biology in Berlin, in collaboration with Jennifer Doudna at the University of California (UC) in Berkeley, has shown that Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00007-2 Copyright © 2020 Elsevier Inc. All rights reserved.

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(A)

Double-strand breakage

Gene disruption

(B)

Double-strand breakage

Gene addition

FIGURE 7.1 (A) Programming Cas9 with crRNA (CRISPR RNA):tracrRNA (transactivating crRNA). (B) Programming Cas9 with single-guide RNA.

the action of dual-RNA (crRNAs and transactivating crRNAs, Fig. 7.1A) and endonuclease Cas9 can generate a double-stranded breakage in target DNA (Jinek et al., 2012). They emphasized the fact that this tool could edit genomes precisely with great ease, and this proved to be true later in other studies (Ceasar et al., 2016). This programmable, dual-RNA-guided DNA endonuclease was found to be much more convenient and widely applicable than conventional restriction enzyme systems, TALEN (transcription activatorelike effector nuclease) nuclease, or zinc-finger nuclease. In addition, the George Church group at Harvard University and his former postdoc Feng Zhang’s group of the Broad Institute succeeded in simultaneously applying the CRISPR/Cas9 system to human cells (Cong et al., 2013; Mali et al., 2013). In particular, the Church group generated the fusion of dual-RNA to enable a more convenient system (Fig. 7.1B) (Mali et al., 2013). The success of this mammalian system facilitated manipulation of eukaryotic genetics, which was previously difficult and labor-intensive, and allowed subsequent CRISPR studies of fungi, fishes, insects, plants, and animals in various fields (Gantz and Akbari, 2018; DiCarlo et al., 2013; Vyas et al., 2015; Hwang et al., 2013; Jiang et al., 2013; Guo and Li, 2015). More importantly, the application of the CRISPR/Cas9 technique for gene therapy has had tremendous medical implications for treating patients with genetic disorders. People suffering from genetic disorders due to mutations of a critical gene for normal cellular functions could potentially be rescued by use of the CRISPR/Cas9 technique by rendering a correction of the mutated

2. Application of CRISPR/Cas9 in various research

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gene sequence. A similar approach has recently shown the possibility of AIDS treatment by introducing a mutation in a coreceptor for HIV entry to confer resistance against HIV infection (Schumann et al., 2015). This is an important improvement in AIDS research, as this outcome would be truly difficult without the precision afforded by the CRISPR/Cas9 technology. However, further development of CRISPR/Cas9 technology will be needed before it is ready for widespread use in human disease treatment. Using the CRISPR/Cas9 technique enables knock-in and knock-out of target genes. The target gene can be disrupted, or a heterologous gene (or an endogenous overexpression target) can be introduced through the CRISPR/Cas9 technique. The knock-out of the target gene by CRISPR/Cas9 is driven by a molecular mechanism called nonhomologous end joining (NHEJ, Fig. 7.2A). On the other hand, the gene knock-in of the CRISPR/Cas9 technique introduces the target gene at the position where double-strand breakage occurs by the homology-directed repair (HDR, Fig. 7.2B) mechanism utilizing repair DNA as a template. In general, it is known that gene knock-in by the CRISPR/Cas9 technique that is required

(A)

Cas9

sgRNA

(B)

dCas

sgRNA

RNAP

Gene disruption

RNAP block RNAP

FIGURE 7.2 (A) Nonhomologous end joining (NHEJ), including proteins such as Ku repair double-strand breaks without a repair template. In the CRISPR/Cas9 technology, NHEJ results in variable length of indels. (B) Homologydirected repair (HDR) double-strand breaks by means of a repair template with homology arms. The knockout and knock-in can be performed precisely in the organisms whose HDR pathway is activated.

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for precise gene editing occurs at a low frequency, although the CRISPR/Cas9 technique allows gene knock-out at a high frequency (Chu et al., 2015). The knock-in and knock-out efficiency of the target gene of CRISPR/Cas9 by NHEJ and HDR can be influenced by the activity of the enzymes involved in the NHEJ and HDR machineries of the corresponding organisms. In the organisms where genes in the HDR pathway are more enhanced than NHEJ, the gene knock-in efficiency can be increased as compared to that of the organisms where the genes are suppressed. In addition to the gene knock-in problem, the off-target effect is repeatedly raised in studies using the CRISPR/Cas9 system. The offtarget effect is a problem in that guide RNA not only recognizes a 20-nucleotide sequence in the target gene but also nucleotide sequences in other positions in the genome. The incorrect recognition in the other sites of the genome can lead to unwanted or “off-target” breakage. This is a phenomenon that can occur more easily when the genome size is large or if a target sequence contains similar sequences in the genome. There is concern to overestimate an off-target effect of the CRISPR/Cas9 technique. However, it is necessary to estimate whether the mutations are caused by the CRISPR/ Cas9 technique through appropriate experimental control and statistical evaluation (Akcakaya et al., 2018; Willi et al., 2018). To overcome the limitation of the CRISPR/Cas9 technique, scientists have searched for better DNA nucleases possessing similar functions as Cas9. The Feng Zhang group discovered Cpf1 nuclease likely to be more precise and convenient than Cas9 nuclease (Zetsche et al., 2015). Later, Jin Su Kim’s group proved that the Cpf1 detected and cut the target sequence more precisely than Cas9, implementing the digenome-seq technique (Kim et al., 2016). In spite of the study claiming the superiority of the Cpf1 nuclease over Cas9, the CRISPR/Cas9 system has been preferentially used. While the application of the CRISPR/Cas9 technology into the metabolic engineering of the microbial cell factory has grown rapidly, the approaches to food microbes using the CRISPR/Cas9 technique are also noteworthy. Our group utilized CRISPR/Cas9 technology to decrease the production of carcinogenic chemical ethyl carbamate (EC) from the yeast strain Saccharomyces cerevisiae (Chin et al., 2016). EC is a carcinogen that potentially attacks human cells by mutating the Kras oncogene (Cazorla et al., 1998). The fermented foods, in particular alcoholic beverages, are prone to forming EC by reaction of ethanol and urea from yeast or other food microbes (Chin et al., 2016). In the study of our group, the key enzyme in the urea synthesis pathway was inactivated via the CRISPR/ Cas9 technology. The complete deletion of the target gene or inactivation induced by the nonsense mutation in the upstream of the target gene was executed by the CRISPR/Cas9 technique. The highly accurate and redundant alterations of the sequences on the target gene were conducted in the yeast system by the CRISPR/Cas9 technique, leading to the dramatic decrease of the precursor urea and EC (Chin et al., 2016). The Jay Keasling group also successfully employed the CRISPR/Cas9 technology to improve the fermentation process of alcoholic beverages (Denby et al., 2018). They introduced genes relevant to the “hoppy” flavor into a beer yeast strain. The introduction of the hoppy flavor appeared to simplify the beer-making process by eliminating the additional step of the addition of hops. As described before, the CRISPR/Cas9 technology has been utilized for and an influencer of various fields such as genetics, cell biology, gene therapy, food technology, etc. The CRISPR/Cas9 system on metabolic engineering, however, is one of the most important applications because the production of biopolymers through microbial cell factories appears primed to rely on the CRISPR/ Cas9 technique in the future.

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3. CRISPR/Cas9-based metabolic engineering Accurate genome editing is one of the main advantages of the CRISPR/Cas9 technique for metabolic engineering. The introduction of the nonsense mutation by the CRISPR technique induces the inactivation of the target gene to control and rewire metabolic circuits. Although the simple deletions of the genes involved in the competing pathways are the primary selection to decrease unwanted flow, the delicate control by modulating the expression levels could show an optimized result. This delicate control can be executed by modulating the levels of the gene employing the promoter containing the appropriate synthetic sequence. In addition, the desired phenotype obtained by global transcription machinery engineering (gTME) can be reintroduced into another host strain by altering the sequence of its homologous transcription factor to the desired sequence by the CRISPR/Cas9 technique. Although the delicate sequence change into the desired form has been accomplished by site-directed mutagenesis so far, the CRISPR/Cas9 technique is more convenient and amenable for implementation when high accuracy is needed. For example, some proteins enhance the fermentation performance of the host cell when the proteins are truncated (Kim et al., 2013). The precise truncation can be executed by the alteration of the target sequence on site of the genome through utilization of the CRISPR/Cas9 technique. The CRISPR/Cas9 technique can be used to induce multiple mutations on different genes, which usually require multiple markers and is inconvenient when conducted by conventional DNA manipulation. Since the coordination of multiple genes is important for the pathway optimization to acquire the desired phenotype of the host cells, the CRISPR/Cas system appears to broaden coverage in metabolic engineering. The usefulness of the coordination of the genes involved in various metabolic pathways by the CRISPR/Cas9 technique has been well documented in yeast systems. For example, the mevalonate pathway in budding yeast plays a critical role for production of the many important natural products such as isoprenoids (Asadollahi et al., 2010; Kirby et al., 2008; Verwaal et al., 2007; Engels et al., 2008). Multiple knock-in and knock-out of the genes involved in the mevalonate pathway is required for optimizing the productivity and yield for the desired products. Since limited selection markers can be used in yeast gene manipulation, recycling selection markers has been used for multiple gene coordination (Jakociunas et al., 2015). However, the recycling selection markers can cause unwanted chromosomal rearrangements by internal recombination between flanking homologous repeats (Solis-Escalante et al., 2014; Jakociunas et al., 2015). One of the important advantages of CRISPR/Cas9 is the ability to avoid unwanted chromosomal rearrangements. Corresponding to this, the Jay Keasling group successfully implemented CRISPR/Cas9 technology to modulate multiple genes involved in the mevalonate pathway in yeast to enhance the production of mevalonate, a key intermediate for the industrially important isoprenoid biosynthesis pathway (Jakociunas et al., 2015). They employed CRISPR/Cas9 technology for multiplex genome engineering of the knock-out of four target genes (BTS1, ROX1, YPL062W, and YJL064W) and the knock-down of one target gene (ERG9) to obtain a strain capable of highly producing mevalonate. In this study, when strain development was performed through all 31 possible target gene combinations, a strain with a 41-fold increase of mevalonate production, compared to the wild-type strain, was developed (Jakociunas et al., 2015). Similarly, Mans and his colleagues constructed the guide RNA vector for multiplex genome engineering using Gibson assembly for the CRISPR/Cas9-based approach (Mans et al., 2015). In addition to the multiplex genome engineering by gene knock-in and knock-out (Fig. 7.3A), CRISPR interference (CRISPRi, Fig. 7.3B) utilizing the nuclease-deactivated Cas9 (dCas9) is useful for metabolic engineering (Qi et al., 2013). CRISPRi by the dCas system can regulate genes involved in

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(A)

(B) Cas9

Cas9

crRNA

Single-guide RNA

tracrRNA FIGURE 7.3 (A) Normal CRISPR/Cas9 system. (B) CRISPR interference by dCas. RNA polymerase (RNAP) is blocked by the dCasesgRNA duplex, leading to the inhibition of the transcription.

the metabolic pathway transiently or constitutively to improve fermentation performance (Cho et al., 2018). The dCas9 is especially useful for the bacterial system because the normal Cas9 nuclease can confer programmed cell death to the bacterial strain by the breakage of the chromosomal DNA. The Cas9 DNA nuclease appears to be lethal to most bacterial species. The bacteria without appropriate DNA repair machinery, such as Ku and LigD, are unable to repair their chromosome from the doublestrand breakage of the chromosome (MacNeill, 2005). Therefore, the deactivated dCas9 interferes with the target genes by attaching to target sequences with guide RNA and can be efficiently exploited for engineering bacterial systems, such as E. coli, lacking adequate repair machinery. Another important advantage of the CRISPR/Cas9 system is the efficient and convenient gene knock-out in the diploid cells to generate a homozygous genotype. This function is critical when the gene is dominant and the loss of one allele of the gene has a negligible effect. In particular, it is difficult to generate a homozygous genotype in polyploidy cells possessing more than two complete sets of chromosomes, such as fungus and plant cells. The Yong-Su Jin group at the University of Illinois at Urbana-Champaign successfully implemented CRISPR/Cas9 technology to construct a quadruple auxotrophic mutant of an industrial polyploid S. cerevisiae strain (Zhang et al., 2014). They subsequently applied the CRISPR/Cas9 technique to the probiotic Saccharomyces boulardii that appeared to have a diploid nucleus (Liu et al., 2016; Hudson et al., 2014). They successfully developed a quadruple auxotrophic mutant of S. boulardii for further metabolic engineering. In the study of filamentous fungi such as Aspergillus sp., Christina Nødvig and her colleagues utilized the CRISPR/Cas9 system to mutagenize six species, of which one had not been genetically engineered before (Nodvig et al., 2015). Due to the difficulties of genetics in polyploid organisms, advances in research requiring genetic manipulation, such as metabolic engineering in eukaryotes, have been hampered. Moreover, if the breeding to generate the homozygous cells is challenging, the organism would be avoided as a host cell to produce the target molecule. Therefore, the advantage of the CRISPR/Cas9 technique in the genetic study of polyploidy cells can diversify options for selection of the host organism to generate desired products.

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4. CRISPR/Cas9-based genome editing in biopolymer production from prokaryotes Numerous bacteria have been harnessed for the production of biopolymers such as polylactic acid (PLA), polyhydroxyalkanoate (PHA), EPS, etc. The studies for PHA biosynthesis have been extensively studied so far to replace the need for the petroleum-based plastic. Many research groups have attempted to increase the metabolic flux toward PHA synthesis. The Guo-Qiang Chen group at Tsinghua University has successfully exploited the CRISPR/Cas9 system for PHA production from E. coli and the extremophile Halomonas spp. (Table 7.1) (Chen and Jiang, 2018). They regulated multiple essential genes in a PHA biosynthesis pathway using the CRISPRi system, allowing them to avoid the need for laborious and time-consuming multiple step gene manipulation (Lv et al., 2015). Repression of the genes in competing pathways by CRISPRi efficiently directed flux to the target monomer 4-hydroxybutyrate. They also engineered morphology of E. coli using CRISPRi to enhance PHA accumulation intracellularly. CRISPRi allowed the down-regulation of genes involved in the cell wall synthesis allowing the E. coli cell to become more elastic, creating more space for PHA accumulation (Zhang et al., 2018). In addition to engineering E. coli for enhanced production of PHA, the extremophile Halomonas spp., an industrially promising bacterial chassis, was harnessed to produce poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV), another common PHA (Tan et al., 2014; Chen et al., 2017). They repressed morphology-related gene ftsZ and PHA synthesis-related genes (gltA and prpC) in Halomonas spp. by CRISPRi to enhance the production of PHBV (Tao et al., 2017). In this study, the efficient repression by CRISPRi directed the flux to the competing pathways and balanced appropriate production of PHBV monomers. To obtain an optimized result, guide RNAs binding the different positions of the target genes were constructed and estimated the effect on the levels of the transcription of the target genes (Tao et al., 2017). They also were able to use the CRISPR/Cas9 system for the metabolic engineering of Halomonas sp. in further studies (Qin et al., 2018; Ling et al., 2018). As compared to the CRISPRi system, the CRISPR/Cas9 system can

Table 7.1 Biopolymers created by engineered microbes using CRISPR technology. Strains

Target products

CRISPR systems

References

E. coli

Poly(3-hydroxybutyrateco-3-hydroxyvalerate) Poly-3-hydroxybutyrate Poly(3-hydroxybutyrateco-3-hydroxyvalerate) Poly(3-hydroxybutyrateco-4-hydroxybutyrate) and poly(3hydroxybutyrate-co-3hydroxyvalerate) Glycan Hyaluronan Hyaluronan

CRISPRi

Lv et al. (2015)

CRISPRi CRISPRi

Zhang et al. (2018) Tao et al. (2017)

CRISPR/Cas9

Ling et al. (2018)

CRISPR/Cas9 CRISPRi CRISPRi

Ru¨tering et al. (2017) Westbrook et al. (2018a) Westbrook et al. (2018b)

E. coli Halomonas bluephagenesis H. bluephagenesis

P. polymyxa B. subtilis B. subtilis

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delete the desired genes permanently. While the constitutive deletion by the CRISPR/Cas9 system has various advantages in metabolic engineering, suitable expression level of a target enzyme by the CRISPRi system can be beneficial depending on the host cells. In addition to the intensive implementation of the CRISPR/Cas9 system in PHA production, CRISPR-Cas9-mediated genome engineering was conducted to diversify the EPS variants from Paenibacillus polymyxa (Ru¨tering et al., 2017). The CRISPR/Cas9-mediated gene deletion led to the alteration of monomer compositions and rheological features of EPSs from the mutants. EPSs are high-molecular-weight carbohydrate biopolymers possessing potential for application in medicine and the food industry, and other industrial purposes (Moscovici, 2015). Various bacteria, including lactic acid bacteria, are amenable for producing EPSs in suitable conditions (Zannini et al., 2016). In the food industry, EPSs from food microbes can determine rheological properties of foods (Zeidan et al., 2017). In addition, some EPSs appear to harbor biological functionality beneficial in biomedical, pharmaceutical, and cosmetic applications (Caggianiello et al., 2016). The Perry Chou group at the University of Waterloo implemented the CRISPRi system in Bacillus subtilis to enhance the production of the high-value biopolymer hyaluronic acid (HA) (Westbrook et al., 2018a). They controlled the membrane cardiolipin distribution by repressing the ftsZ gene, encoding a cell division initiator protein, via CRISPRi in order to improve the HA titer (Westbrook et al., 2018a). They also partially diverted the carbon flux from central metabolism into HA synthesis by reducing the expression of pfkA and zwf genes in the glycolytic and pentose phosphate pathways via CRISPRi, leading to a substantial improvement of the HA titer (Westbrook et al., 2018b). Although EPSs are valuable and promising, there is a lack of metabolic engineering studies for the production of EPSs from microbes. One of the possible reasons could be the involvement of numerous genes in EPS biosynthesis. Furthermore, it is a major hurdle to sequence the genome and to develop gene manipulation techniques individually for an EPS-producing microbe. Combined with recently evolved and increasingly cost-effective nextgeneration sequencing (NGS) techniques, the CRISPR/Cas9 system could be an excellent option for developing an engineered system to improve the EPS-producing microbes toward desired phenotypes (Fig. 7.4).

FIGURE 7.4 Metabolic engineering of the EPS-producing microbes using the CRISPR/Cas9 technology.

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Public concerns about genetically modified organism (GMO) safety impede the research of CRISPR/Cas9 techniques in food microbes. As the study of the CRISPR/Cas9 systems started originally with lactic acid bacteria, however, metabolic engineering of EPS-producing bacteria using endogenous CRISPR/Cas9 systems may be an attractive option. It is highly plausible, as many genomics studies focusing on EPS-producing bacteria have shown the presence of an endogenous CRISPR system in bacterial genomes (Wallace et al., 2014; Wu et al., 2014; Marcotte et al., 2017). Otherwise, it would be also possible to produce EPSs for food or medical purposes using a heterologous CRISPR/Cas9 system, followed by approval from regulatory bodies. This approach, however, can be time-consuming and complicated depending on government policy and consumer sentiment.

5. CRISPR/Cas9-based genome editing in biopolymer production from eukaryotes The metabolic engineering for lactic acid production in yeast can be applied to the usage as a monomer in PLA production (Ozaki et al., 2017). Mans and colleagues (Mans et al., 2017) attempted to identify transporters to export lactate in S. cerevisiae. This investigative study provided useful information for future metabolic engineering for lactate production through the deletion of putative lactate transporters utilizing the CRISPR/Cas9 technique (Mans et al., 2017). The Akihiko Kondo group at Kobe University successfully performed metabolic engineering of fission yeast via the CRISPR/Cas9 system to produce lactic acid from a glucose and cellobiose mixture (Ozaki et al., 2017). Using the CRISPR/ Cas9 system, they disrupted genes encoding two pyruvate decarboxylases: an L-lactate dehydrogenase, and a minor alcohol dehydrogenase to attenuate ethanol production. They also overexpressed acetylating acetaldehyde dehydrogenase and D-lactate dehydrogenase to increase the cellular supply of acetyl-CoA and D-lactic acid production (Ozaki et al., 2017). The engineered fission yeast efficiently produced D-lactic acid from both glucose culture and mixed culture with glucose and cellobiose. This study will provide useful information for the advancement of the PLA industry. There have been several efforts to produce PHA from S. cerevisiae (de Las Heras et al., 2016; Portugal-Nunes et al., 2017; Sandstrom et al., 2015). The budding yeast could be an alternative host to E. coli because of its own advantages as a workhorse over other microbial hosts: easy genetic manipulation, the simplicity of culture, food-grade status, posttranslational modification mechanisms, the presence of cellular compartmentation, etc. Introduction of the heterologous polyhydroxyalkanoate synthase gene allowed the production of PHA from S. cerevisiae (Portugal-Nunes et al., 2017). Presently, few studies, if any, employed the CRISPR/Cas9 system to engineer S. cerevisiae for PHA production, although the CRISPR/Cas9 system could be relatively easily implemented in S. cerevisiae to produce PHA. The utilization of the CRISPR/Cas9 system in S. cerevisiae is well established and DNA transformation is relatively easy as compared to other model organisms. The knock-out of the target genes can be efficiently performed via the CRISPR/Cas9 system. In the budding yeast system, however, efficiency of the knock-in of the target gene is also very high due to the HDR mechanism. Accurate genome editing is also very successful in S. cerevisiae. Therefore, the multiplex genome engineering for biopolymer production in budding yeast is possible through the CRISPR/Cas9 technique. In addition, the versatility of the CRISPR/Cas9 system can be applied to diverse fungal organisms as a biopolymer producer (Mahapatra and Banerjee, 2013). The endogenous synthesis pathways of

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biopolymers in numerous fungi can be employed. However, differences in the implementation of the CRISPR/Cas9 system are expected. Since the NHEJ pathway is enhanced in filamentous fungi, the gene knock-out via CRISPR/Cas9 appears to be operational. The precise genome editing in the filamentous fungi, however, may not as efficient as with budding yeast. By paying attention to several features of the fungal CRISPR/Cas9 system, a major impact on the production of biopolymers from fungi may be achieved.

6. Conclusions and perspectives Recent renovation in the scientific approach to genetic manipulation in microbes has been led by genome editing systems utilizing DNA nucleases such as zinc-finger nuclease, TALEN nuclease, and CRISPR/Cas. Among them, the CRISPR/Cas9 system is relatively convenient and widely applicable. The CRISPR/Cas9 technique is precisely editable and is capable of coordinating multiple genes. In addition, it is advantageous to manipulate the genome of polyploidy organisms, proving the CRISPR/Cas9 system can be an excellent genetic tool for eukaryotic genome manipulation. While numerous applications via the CRISPR/Cas9 system have been performed in all fields of life sciences, the CRISPR/Cas9 techniques have been heavily, and successfully, applied to microbial metabolic engineering. The multiplex genome editing via the CRISPR/Cas9 technique is suitable for the control of multiple genes involved in metabolic circuits. Not only knock-in and knock-out of target genes, but also repression of genes using CRISPR interference have had a profound impact in the metabolic engineering field. The CRISPRi systems are especially effective in organisms omitting genes necessary for DNA repair pathways. PHA and EPS production from prokaryotes has been facilitated by the CRISPR/Cas9 system, in particular CRISPRi. The CRISPR/Cas9 system has prosperously established itself not only in the conventional model organisms such as E. coli and B. subtilis but also in undomesticated organisms such as Halomonas sp. In eukaryotic microbes, the CRISPR/Cas9 technique has also been thoroughly implemented. Although few studies have illustrated the usage of the CRISPR/ Cas9 system for engineering eukaryotic microbes for the production of biopolymers, it appears to have great potential. The CRISPR/Cas9 system in budding yeast can be conveniently and efficiently executed to knock-in and knock-out target genes. Moreover, accurate genome editing is easily available in budding yeast via the CRISPR/Cas9 system. These benefits may eventually be utilized for the production of biopolymers in budding yeast. In addition to the benefits in the yeast CRISPR/ Cas9 system, the CRISPR/Cas9 technique may facilitate regulating genes in numerous pathways for biopolymer production in filamentous fungi. Collectively, the CRISPR/Cas9 system has already made a significant mark on the study and production of microbe-based biopolymers, and this impact is likely to rise in the coming years.

Acknowledgments This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07051143) and by the Ministry of Science and ICT (NRF-2018M3C1B5052439).

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CHAPTER

Biomedical and food applications of biopolymer-based liposome

8

Sayantani Dutta, Jeyan A. Moses, C. Anandharamakrishnan Computational Modeling and Nano Scale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, India

1. Introduction Polymers have been an integral part of our lives, particular post industrial revolution. Polymers, unlike metals, are light in weight and offer modalities of processing and application that are otherwise difficult. Biopolymers are natural polymers that are either naturally available or synthesized from natural sources by chemical methods. Besides being natural and thus greatly biocompatible, biopolymers also offer plethora of reasons to be a favorite for food and biomedical applications. The advantages offered by this class of molecular blocks are broadly nontoxicity, biodegradability, and easy availability, in addition, economic processing and procurement costs (Jacob et al., 2018). The commonest biopolymers perhaps would be cellulose, chitosan, starch, and gums that are present all around our ecosystem and have been integral part of our lives since long. Specifically, their attributes such as high biocompatibility, durable mechanical properties, low immunogenicity, nonmutagenicity, and homocompatibility, adsorbing ability, besides certain biopolymers being edible and their nonirritant nature ascribe to their preference. In fact, in terms of compatibility to human body, they exhibit “biomimetic” properties. These attributes are quite rare and resemble native extracellular matrix which avert graft rejections in biomedical transplantations (Kumar et al., 2018; Velema and Kaplan, 2006). Liposomes are well-known bilayered vesicles produced by dispersion of polar liquids in aqueous solvents, forming a coat around the core, i.e., encapsulated compound and polar phase being surrounded by nonpolar phase, thus an encapsulate. Liposomes are an integral part of delivery vehicles today and also help to protect sensitive bioactives from environmental stresses and storage losses. Liposomes also help in targeted release with considerable sample loading (Dutta and Bhattacharjee, 2017). They are an effective mode of delivery of biological biopharmaceuticals and nutraceuticals and have been effectively employed in various domains of food and biomedical sciences. They have a unique ability to allow release of core compound at specific site that allows tailored release phenomenon, for both water and lipid soluble moieties (Benech et al., 2002; Were et al., 2003). Accordingly, liposomes have been very useful and popular for delivery of miniature molecules (microparticles, nanoparticles), antioxidants, antimicrobials, flavors, and bioactives. Liposomes being small sized, they (50 nme50 mm) enjoy greater bioaccessibility into blood stream and body fluids. Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00008-4 Copyright © 2020 Elsevier Inc. All rights reserved.

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Liposomes also are viable (to an extent) when the ionic conditions of the surrounding environment and themselves are different (Gomez-Hens and Fernandez-Romero, 2006; Taylor et al., 2005; Xu et al., 2007). Biopolymer-based liposomes are an interesting combination of the two very useful delivery modalities. The need to include biopolymers is owing to inherent limitations of liposomes that include curtailment of entrapment in vesicles owing to coalescence due to change in surface charge of liposomes, leakage of core material with prolonged storage, limited mechanical tensility with respect to physical shear and stress, inability to produce neutral or positive surface charge liposomes owing to common deployment of phosphatidylcholine instead of phosphatidylethanolamine (since the latter is expensive). These factors also reduce the durability of loaded liposomes and raise concern regarding their versatility (Laye et al., 2008). Besides, liposomes are desired to be responsive to changes in external stimuli, controlling their mobility and action. This phenomenon is essential to govern controlled release of encapsulated molecules in liposomes; however, with liposomes alone, it is challenging (Rao et al., 2011; Wang et al., 2015). Addition of a new subunit to a liposome was first reported with amphiphilic polyelectrolytes (Seki and Tirrell, 1984). These authors reported that at high acidity, the liposomes destabilize due to polymer collapse and hydrophobic bonding between alkyl groups of polymer and lipid. Thus, engineering to produce durable and, if required, designing tailor made liposomes with suitable alternate additives is the prerequisite today. This chapter garners the key findings in the domain of biopolymers in liposomal delivery of bioactives in biomedical and food sectors. Most of these biopolymers are either from food sources or natural reserves and are used in biomedical and food engineering applications. The various biopolymers and their applications have been summarized in Table 8.1 and described in subsequent sections. The exposition would categorically describe the frontier areas of focus, and the scientific achievements that have been made, along with their impact on current understanding.

2. Starch Starch is one of the commonest biopolymers that we know and forms a regular part of our daily diet. In a research work, starch-coated magnetic nanoliposomes have been employed for delivery of antipulmonary arterial hypertension drug, fasudil in nasal passage for pulmonary therapy (Nahar et al., 2014). The miniaturized particles resemble body cells in size and could be a useful candidate for delivery of the drug to lungs for pulmonary disorders. The magneto-nanoparticles are iron oxide and are biocompatible, biodegradable, and have paramagnetic properties. They are often conjugated with starch and other biopolymers. Such particles when encapsulated in liposomes load appreciable quantity of hydrophobic or hydrophilic drug molecules, in the core or at the lipid bilayers (Laouini et al., 2012). In fact, coating with iron oxide with surfactants reportedly also averts coalescence followed by gravitational sedimentation phenomenon that is often observed in liposomes with progressive storage (Shubayev et al., 2009). Nahar et al. further reported appreciable drug release, improved cellular uptake of drug, antiproliferative effect, and prolonged sustenance of drug’s effect (by drug half life analysis). As elaborated by the authors, the beauty of this intervention was that the magnetic particles could be kept at the specific field by external magnetic field force with sustained release of the drug.

Table 8.1 Applications of liposomes coated with biopolymers (other than chitosan). Encapsulated compound

Wall material(s)

Coating material(s)

Egg or dipalmitoylphosphatidylcholine

Human fibronectin

Insulin and growth hormone

Dipalmitoylphosphatidylcholine

Carboxymethyl chitin

Acetylsalicylic acid

Dipalmitoylphosphatidylcholine

Acaciaegelatin

Acetylsalicylic acid

Phosphatidylcholine and cholesterol

Poly(ethylene glycol)

e

Dipalmitoylphosphatidylcholine Hydrogenated phospholipids and cholesterol

e

Sodium alginate and cytochrome-c Carboxyfluorescein

Collagen/hexyl derivative/lauryl derivative of collagen Alginate

5(6)carboxyfluorescein

Egg phosphatidylcholine

Nisopropylacrylamide

Pyranine

Reference

Sustained release of drug; collagen base can provide cell attachment and proliferation for treatment of wounds or burns Stability and sustained release of drug was higher when chitin was added during preparation of liposome compared to post preparation coating Improved stability of liposome; sustained prolonged release of drug Coating hinders protein interaction with the surface of the liposome; improved stability of liposome Sustained release of alginate and cytochrome c Increased stability of liposome after coating with improved blood elimination and liver uptake Egg phosphatidylcholine and cholesterol liposome was found to be more resistant toward the leakage, and it improved the drug retention within the gel Increasing copolymer can significantly improve in vitro release; leakage can be decreased when copolymer is attached by incubation with liposome

Weiner et al. (1985)

Dong and Rogers (1991)

Cunji Dong and Rogers (1993) Torchilin et al. (1994)

Monshipouri and Rudolph (1995) Jose´Fonseca et al. (1996)

Takagi et al. (1996)

Zignani et al. (2000)

2. Starch

Egg phosphatidylcholine/ egg phosphatidylcholine and cholesterol

Impact of the process

169

Continued

170

Table 8.1 Applications of liposomes coated with biopolymers (other than chitosan).dcont’d Coating material(s)

Phosphatidylcholine, cholesterol

Stearylamine, phosphatidic acid, phosphatidylserine, and/or polyethylene glycol

111-InCl3

Phosphatidylcholine

Alginate

Bee venom peptide

Egg phosphatidylcholine, cholesterol, 1,2-distearoylsn-glycero-3phosphatidylethanolamineN-[poly(ethylene glycol)2000] (PEG) Soy phosphatidylcholine

Alkylated copolymer of Nisopropylacrylamide, methacrylic acid, and N-vinyl-2-pyrrolidone

e

g-Zein

e

Egg phosphatidylcholine

Alginate and crosslinking cation such as Ca2þ, Ba2þ, and Al3þ

Bovine serum albumin (BSA)

Phosphatidylcholine and cholesterol

Poly (methacrylic acid-con-alkyl methacrylate)

e

Impact of the process

Reference

PEGylated liposome showed increased circulation time and decreased liver accumulation, whereas incorporation of charged compounds increased clearance and liver accumulation; PEG was able to change the effects of charge on liposome Specific delivery of drug to colon The liposomeepolymer complex was stable at neutral pH but released the contents at acidic condition; improved circulation time was observed for PEGylated complex Increased stability and permeability of liposome membrane; suggests a mechanism of g-zein deposition inside maize protein Encapsulation rate for Ba2þ was highest among all formulations, and it showed improved stability of BSA Concentration, molecular weight, alkyl group content, and alkyl chain length of the polymer control the size and structure of the bilayer of the vesicles; stability of liposome improved with the polymer

Levchenko et al. (2002)

Xing et al. (2003) Roux et al. (2003)

Kogan et al. (2004)

Dai et al. (2005)

Cho et al. (2007)

Chapter 8 Biomedical and food applications of biopolymer-based liposome

Encapsulated compound

Wall material(s)

Polydiacetylenedoped calcium alginate fibers

e

Phosphatidylcholine and cholesterol

Chitosan/gelatin/ chitosanegelatin

Camptothecin-11-HCl

Phosphatidylcholine and cholesterol

Poly (methacrylic acid-co-stearyl methacrylate)

Calcein/ a-ketoglutaric acid

Octadecyl-quaternized lysinemodified chitosan and cholesterol

Folate-PEG

Calcein

Dipalmitoylphosphatidylcholine Phosphatidylcholine and cholesterol

Alginate

Alkaline phosphatase

Tremella coating followed by acidinduced alginate

Inactivated H5N3 vaccine

1,2-Dioleoyl-sn-glycero-3phospho-(10 -rac-glycerol) sodium salt, 1,2-distearoyl-snglycero-3-phosphocholine, cholesterol Dipalmitoylphosphatidylcholine

1,2-Distearoyl-snglycero-3phosphoethanolamineNmPEG-2000

Polyethylenimine along with rhodamine

Methoxypoly(ethylene glycol)b-poly(N-2hydroxypropylmethacrylamide-cohistidine)-cholesterol using biotin2-PEG as cross-linker

Doxorubicinhydrochloride

“Raman response” has been developed to measure PDA response against external stimuli such as heat, solvent, and chemical Improved stability of liposome; liposome coated with chitosan alginate showed highest entrapment efficiency and controlled release Improved drug delivery to human keratinocytes; improved production of procollagen at human dermal fibroblasts Enhanced encapsulation efficiency and sustained release of drug; improved uptake by MCF-7 cells Prolonged and sustained activity of the enzyme Improved resistance to acidic pH; improved mucosal antiviral secretary immunoglobulin A response Study on MCF-7 cells revealed low toxicity, improved cellular uptake, enhanced control over biological interaction kinetics Improved reduction of adsorption of proteins such as HSA, IgG, and fibrinogen; increased stability of drug; improved uptake and accumulation of liposome in HCT116 colon cancer cells

Kauffman et al. (2009)

Shende and Gaud (2009)

Cho et al. (2009)

Wang et al. (2010)

Smith et al. (2010) Cheng et al. (2011)

Sunoqrot et al. (2011)

Chiang et al. (2013)

2. Starch

10,12Pentacosadiynoicdiacetylenes

171

Continued

172

Table 8.1 Applications of liposomes coated with biopolymers (other than chitosan).dcont’d Coating material(s)

Encapsulated compound

Impact of the process

Reference

Egg yolk phosphatidylcholine, cholesterol, 1,2-distearoylsn-glycero-3phosphoethanolamine-N[methoxy(polyethyleneglycol)2000], 1,2-distearoylsnglycero-3phosphoethanolamine-N[amino(polyethyleneglycol)2000] 1,2-Dipalmitoyl-sn-glycero-3phosphocholine, cholesterol and 1,2 disteroyl-sn-glycero3-phosphoethanolamine-N[methoxy (polyethylene glycol)-2000] Egg yolk phosphatidylcholine

Cisplatin-sodium alginate conjugate

Epidermal growth factor

Targeted delivery of drug; sustained release of drug; improved antitumor efficacy; reduced nephrotoxicity and body weight loss in mice

Wang et al. (2014)

Anionic and cationic iron oxide and starch

Fasudil

Nahar et al. (2014)

3-Methylglutarylated poly (glycidol)

Ovalbumin, IFN-gencoding plasmid DNA

Phosphatidylcholine

Gelatin

Usnic acid

Phosphatidylethanolamine

Aldehyde-modified xanthan gum

e

Threefold greater uptake of liposome at pulmonary arterial smooth muscle cells; sustained drug release; 40% reduction in cell proliferation; improved stability of liposome Improved delivery of drug to murine dendritic cell line; liposomeeDNA complex promoted delivery of drug to tumors Controlled release of drug to burn wounds in male pigs; scar repair potency comparable or better than conventional dressing or ointment Ease in biodegradation of liposome by papain; enhanced viability of encapsulated cells; can be used as smart cell carrier or cell culturing scaffold

Yuba et al. (2015)

Nunes et al. (2016)

Ma et al. (2016)

Chapter 8 Biomedical and food applications of biopolymer-based liposome

Wall material(s)

5. Collagen

173

3. Zein Zein, the maize protein, has been utilized in designing biopolymer-based nanoliposomes, post hydrolysis with enzymes such as papain and alcalase (Kong and Xiong, 2006). The zein hydrolysate was the core in liposomes composed of soybean phosphatidylcholine. Lipid peroxidation was induced in liposomal system by iron redox cycling, as mentioned by authors. When examined, it was revealed that alcalase-hydrolyzed zein administered liposome resisted oxidation significantly more than papainhydrolyzed zein-administered liposome. This alcalase-hydrolyzed zein-administered liposome further exhibited significant radical scavenging activity (antioxidant potency). The said liposome assembly also had markedly improved performance with respect to synthetic antioxidants such as BHA, and natural antioxidants such as a-tocopherol and ascorbate solution. In addition to oxidative stability rendered to liposome, zein is also a source of amino acids that include alanine, leucine, and proline, which is a bonus. This liposome could be used as a natural and stable preservative in food systems replacing currently used preservatives.

4. Xanthan gum Xanthan gum is biocompatible, biodegradable, and safe and offers flexibility in developing different biomedical aids. In an indagation, the naturally occurring xanthan gum has been employed for developing biomedical aids, with liposome-based delivery (Ma et al., 2016). Herein, xanthan was subjected to aldehyde modification before injecting into the phosphatidylethanolamine liposome. The combined biopolymereliposome unit was a xanthan gumebased liposome hydrogel, wherein, modified xanthan gum acted as a matrix material and liposome as a cross-linker. The researchers’ envisaged embedding liposome in modified xanthan via Schiff’s base linkage, thereby making it possible to have controlled released of drugs. Notably, the liposomal assembly was syringe injectable and therefore could be engaged in administration of injectable drugs, with a good integrity of the hydrogel microstructure. The liposome also showed self-healing property and a 3D cell culturing ability. These capabilities showcase ability of the liposome to become an in vivo cell carrier of bioactives and biomaterials and a biological scaffold, with multiresponsive character. Particularly, heat, chemical, or mechanical stimuli could be used to trigger decomposition of the hydrogel.

5. Collagen Collagen, the most abundant protein in the human body, is also a biopolymer and has been employed in many studies. Researchers have explored opportunity of coating unilamellar liposome with collagen for enhanced entrapment and site-specific time-delayed release of carboxyfluorescein (biomarker) in liver (Jose´Fonseca et al., 1996). The optimized half life at 7 h was obtained for liposomes prepared with saturated phospholipids at a dose of 2 mmol. The drug uptake in this liposomal assembly was 1.5e2.0 folds higher than in conventional noncoated liposomes, examined in vitro. The formulated liposome was also found to facilitate molecule uptake in vivo after 16 h in rats, when the biomarker was found in Kupffer cells of their liver. Such liposomes have been opined to be of significant importance in therapy involving macrophages, invoking drug-mediated activation in them for antitumor, antimicrobial, and antiviral activities.

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Another liposomeecollagen gel matrix for effective drug delivery has been worked out by a separate group of investigators. The scientists engaged delivery of insulin and growth hormone in such liposomal assembly through injection route of administration (intramuscular or subcutaneous). The liposomeecollagen hydrogel matrix was prepared by critical point drying technique (Weiner et al., 1985). The authors propose that slow release mechanism could facilitate higher batch loading eliminating repeated dosages of drugs; as well as cells exposed to the liposomal therapy could be attached to the hydrogel, while being exposed to an external stimuli that would often regulate release behavior.

6. Chitin Chitin is a very ubiquitous biopolymer mostly recovered from marine arthropods’ exoskeleton. It has been used in multiple forms in various application domains of interest. In practice, for liposomal delivery, often subcutaneous, intravenous, and intraperitoneal routes are employed. However, the viability of drugs when liposomes are administered orally becomes a challenge. The biopolymer chitin has been successful in such entrapments, by not yielding to the acidic environment of the gastric tract. In a study, scholars have prepared liposomes entrapping acetylsalicylic acid, in sodium cholate and coated liposome with carboxymethyl chitin, for oral delivery. The transmission electron microscope (TEM) image has been shared by said researchers that delineates the effect of chitosan coating on liposome (Fig. 8.1) (Mady et al., 2009). The liposomes prepared were of dipalmitoylphosphatidylcholine (DPPC). The authors describe that addition of chitosan enhanced the overall size of liposome. Also the zeta potential of the liposome was found to increase post coating, owing to more cationic particles at liposomal surface, indicating a better stability. This observation was made up to 0.3% chitosan concentration. The coated liposome showed pseudoplastic flow behavior with an

FIGURE 8.1 TEM image of (A) noncoated and (B) chitosan-coated liposome (Mady et al., 2009).

7. Gelatin

175

enhanced yield stress (the minimum stress needed to cause a Bingham plastic to flow) compared with noncoated liposome. Biomedical application has been reported by few authors as well for the said liposome and coat (Dong and Rogers, 1991). Sodium cholate is present endogenously in gastric tract; hence, the authors had included the same in the assembly to counter the gastric environment. The release of the drug was also sustained in comparison with noncoated liposome. While noncoated liposome released 85% of drug in 20 min, the polymer-coated liposome released 70% drug in 20 min. For the best outcomes, authors recommend adding chitin at the liposome formation stage itself, so that the polymer associates with each bilayer of liposome well.

7. Gelatin Gelatin is yet another biopolymer that is widely used in biomedical and food industries. Acacia gelatinemicroencapsulated liposomes have been developed by few authors who intended to entrap the drug, acetylsalicylic acid, in DPPC in acacia gelatin, using coacervation technique (Cunji Dong and Rogers, 1993). On an average, the coat thickness was 0.1e0.4 mm, at 1.5%e5% coat concentration of biopolymer, while the final particle size was between 1 and 7 mm. Remarkably, there was no effect on encapsulation efficiency of the drug, and the liposome was also stable in sodium cholate at pH 5.6. The optimized concentration of polymer was 3% acacia gelatin. The drug release was 25% in 6 h at 23 C and 75% release in 30 min at 37 C, much slower than in control samples. Gelatin coat has been employed on liposome with usnic acid as drug and has been found to be quite effective in controlling dermal burn injury (first degree) in porcine (pig) animal model creating a lesion (Nunes et al., 2016). The authors prepared the liposome by casting method and tested its performance against two commercially available medications. In the liposomal system, the drug release increased initially at 38.7% occurring in first 4 h, and thereafter release occurred in a controlled manner until 24 h, releasing 98% of the entrapped drug. Of course, gelatin incorporation was a welcome maneuver because gelatin has been attributed with ability to facilitate adherence and migration of fibroblasts throughout the film matrix and is highly biocompatible and biodegradable, besides being hygroscopic (Wang et al., 2012; Yeh et al., 2011). It has been pointed out that a moist environment around the wound improves healing though migration of epithelial cells, which prevent infection from external environment (Neel et al., 2013); hence, hygroscopicity of gelatin is a boon. The formulated liposome of aforesaid researchers (Nunes et al., 2016) also exhibited antiinflammatory action on the wound that was absent when using commercial interventions. The liposome also facilitated wound shrinkage, scar repair, and increased collagen deposition, indicative of healing, with time. Combining biopolymers for versatile and flexible usage has always intrigued researchers. In one of the endeavors, scientists have coated liposomes with biopolymers developed from single and combined biopolymer matrices (Shende and Gaud, 2009). They designed the liposomes with phosphatidylcholine and cholesterol and capped the liposome with chitin or chitosan or gelatin or combination of them. The entrapped molecule was an anticancer drug, camptothecin (CPT)-11eHCl (irinotecan HCl). The authors report a hierarchy in the size of liposomes (capped) as noncoated liposome < chitosan-coated liposome < gelatin-coated liposome < combination of chitosan and gelatin coated liposome. Their zeta potentials (all positive), encapsulation efficiency, and stability were in reverse order. The freeze fracture micrograph shows their topographical differences (Fig. 8.2). A minimum of 0.1% polymer concentration has been reported to prevent flocculation that destabilizes

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Chapter 8 Biomedical and food applications of biopolymer-based liposome

FIGURE 8.2 Freeze fracture micrographs of (A) noncoated, (B) chitosan-coated, (C) gelatin-coated, and (D) blend of chitosan- and gelatin-coated liposomes (Shende and Gaud, 2009).

the liposome. Best encapsulation efficiency was 91.65% in the mixed biopolymer. Furthermore, the release study of the drug delineated that noncoated liposome released almost the entire drug in 5 h. However, coated liposomes showed controlled release and restrained the flow of drug by allowing only 73%e91% of drug outflow in 40 h. These coated liposomes however did release 20% of the drug in first 3 h. The authors have explained that such a remarkable achievement in drug entrapment and controlled release was made possible owing to the inherent surface chemistry of liposomes and coats. Liposome surface was negatively charged while coats were positive, yielding a neutral surface charge. Increased size of liposome and its viscosity and preceding factors facilitated a controlled release behavior. The in vitro and in vivo release behaviors followed the hierarchy as that of encapsulation efficiency. Maximum restraint in release (by half live of the drug) was achieved in combinational coat (gelatin and chitosan) at 56.8 h, while minimum was observed for noncoated liposome at 0.27 h.

8. Methacrylate In another attempt to work out alternative biopolymers for biological activities and response sensitive liposomes, scientists have focused on methacrylate as coat. This biopolymer was used in the form of poly (methacrylic acid-co-(n)-alkyl methacrylate) by a group of researchers when they examined the effect of its changes in concentration and that of lipid components, while designing assembly of liposomes entrapped in biopolymer unit (Cho et al., 2007). They designed liposome with phosphatidylcholine/cholesterol and engaged the polymer by high pressure homogenization. The variations in ingredients were studied for obtaining a stable liposome. Beyond a threshold of polymer

9. Calcium alginate

177

concentration, the liposomal system became homogenous with uniform bilayers. Concomitantly, vesicle size increased and so did the fluidity. The authors emphasized that polymer coatings improve stability of liposomes in buffered or emulsified environments. Such an observation plays critical role in appreciating the role of polymers since sustenance of liposome in target environment is the first prerequisite for object-oriented time desired delivery, especially for a sustained release. Another important observation suggested that polymer molecular weight is the key criterion in deciding the vesicle size in the liposomeepolymer complex, and not the structure of the polymer. In an equivalent study on polymereliposome complex, methacrylate was employed as coat on phosphatidylcholine/cholesterol liposome. The group working on the subject attempted in vitro delivery of calcein or a-ketoglutaric acid, using the aforesaid delivery complex. The examination revealed an enhanced delivery of the biomolecule into keratinocytes, than uncapped liposomes, as well as higher production of procollagen compared with uncapped counterpart (Cho et al., 2009). These authors have recommended using the liposome complex for cytoplasmic delivery of bioactives. It has also been stated that biopolymers are amphiphilic and their hydrophobic ends help them stick to liposome surface. In turn, these polymers which cover the outer surface of the vesicle respond to external stimuli. Because many biopolymers are resistant to a broad spectrum of pH, they facilitate release of active compound even when the environment is acidic (as an endosome), which otherwise would not have been possible.

9. Calcium alginate Calcium alginate, or commonly alginate, is yet another biopolymer that has been extensively used for encasing liposomes. In a study, alginateesucrose and alginateechitosan microbeads have been engaged for delivery of resveratrol, a nutraceutical (Balanc et al., 2016). The authors report increase in size of liposome from 412 to 471 nm, with addition of sucrose as cryoprotectant. Having a polymer coat allowed controlled release from liposome and resulted in increase in mass transfer resistance by one order of magnitude higher (106 s/m) than in noncoated liposomes. The particle size were alginateechitosan > alginate > alginate sucrose, diffusivity from three sample sets were alginatee sucrose > alginate > alginate liposome; mass transfer resistance was alginate > alginatee chitosan > alginateesucrose. The stress endurance capacity of the beads of alginate and alginatee sucrose were almost similar, while alginateechitosan endured more. However, the hardness values followed the pattern, alginate-chitosan > alginate-sucrose > alginate in formulated beads. Same was the observation for elastic modulus of beads. The size was least for chitosan beads because shorter alginate chains diffused into longer polymer chains, thus making a compact dense microstructure with smaller size. However, the same compactness of chitosan beads has been attributed by the authors for reduced efficiency (in fact, the least among all sample sets) owing to lower sample loading capacity. In addition, this reason could have caused leakage, because the diffusion path of the resveratrol is shorter in chitosan bead due to its compact structure. In another study, scholars capped liposome carrying inactivate H5N3 vaccine first with tremella (a nontoxic bioadhesive) and then by acid-induced alginate, i.e., triple encapsulated, and finally administered it orally to mice (Cheng et al., 2011). Transepithelial electrical resistance analysis revealed that capped liposomes were able to deliver the antigen (attenuated H5N3). The mode was

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Chapter 8 Biomedical and food applications of biopolymer-based liposome

particularly useful because most of proteins in oral vaccines cannot survive the acidic environment of the gastric tract. As a result, there was enhanced mucosal secretary Ig-A response in the body of mice. However, owing to multiple coatings, the encapsulation efficiency was low at 35%. As expected, the alginates showed more stability in gastric fluids compared with intestinal fluids and allowed a slow release. The encapsulation yielded an improved immune response compared with noncoated vaccine, both administered orally. Alginate-based encasement of bovine serum albumin (BSA) in liposomal assembly has also produced noteworthy results. In an endeavor, the in vitro release study showed that in the first 5 days about 75% of BSA was released from noncapped liposome. However, in capped liposome in the same duration, the release was only 25%e30%, especially when Ba2þ was incorporated in the liposomal complex. The release was also without a burst phenomenon commonly observed in liposomes at the outset (Dai et al., 2005). The researchers further stated that in the coated liposome, 75% of protein was reportedly released only after 18 days, whereas in 30 days 85% of protein was liberated. Modifying the technique of encapsulation, these authors subsequently employed multivesicular liposomes prepared by double emulsification technique (Dai et al., 2006). The core and coat were the same as before. The process was successful in bringing out better results than before with entrapment efficiency up to 95% with Ba2þ, with spherical and small particles of the encapsulate (mean diameter 10 mm). In addition, maximum in vitro drug release in controlled manner was observed in over 2 weeks, without an initial burst. In another attempt, layer by layer assembly of alginate by forming its nanoparticles has been worked out, using BSA as encapsulated biomolecule. The scientists initiated adsorption of several layers of biopolymers to provide stability to the complex, using anionic alginate and cationic chitosan on cationic phospholipid vesicle (Haidar et al., 2008). The resulting particles were 345 nm in size, with a positive zeta potential. Coated liposomes (3 or 5 polymer-layered coating) could load about more than double than that of the noncoated liposomes. In fact, a 10-layered coating could load 80% of the protein, much higher than noncoated liposome. Examining the release behavior revealed that noncoated liposomes released 60% of protein in only 3 days, while coated liposomes displayed controlled, linear, and sustained release, releasing 88% of protein in over a month’s time. Lipoinsulin-encapsulated alginateechitosan capsule has been explored for antidiabetic effect through intestinal delivery in rats (Ramadas et al., 2000). The formulation complex was able to deliver insulin at intestine by passing though the acidic gastric tract. The liposomal assembly was 800e1000 mm, spherical in shape, with sample loading up to 21.5%. The formulation did not release insulin in simulated gastric fluid (SGF), while being exposed for 3 h; however, it did release insulin in a controlled manner in simulated intestinal fluid (SIF). The half life of insulin was about 3.5 h. This resulted in reduction in blood glucose level in rats from 3 h post administration, and the levels returned to normal after 6 h from dosage. Thereafter, the relapse of glucose in blood to diabetic level occurred in noncoated liposomes within 10 h. But in coated liposomes, the increase was observed much slower, indicating presence of insulin in blood owing to controlled release phenomenon. In another work, cisplatinealginate conjugate liposomes have been developed for targeted delivery of epidermal growth factor (EGFR) to ovarian cancer cells, using cisplatin as a drug (Wang et al., 2014). Herein EGF liposomes were modified to especially target EGFR expressing tumors. The liposomal conjugate did target EGFR-positive SKOV3 cells and penetrated tumor cells. The said liposome also arrested the growth of tumor cells for longer (compared with uncapped liposomes), besides inhibiting proliferation and migration of tumor. The authors delineate further the cause of

10. Chitosan

179

enhanced antitumor activity. According to them, there was a significant decrease in Ki-67-positive cells, in tumors of mice, along with heightened apoptosis in tumor cells. The capped liposome also showed reduced nephrotoxicity of the drug than is otherwise witnessed as a side effect. Using bee venom peptide as a model drug, researchers in another find have explained the feasibility of developing a colon-specific drug delivery system, in vitro. The objective was to improve bioavailability of proteins and peptides administered orally (Xing et al., 2003). In this work, the model drug in a beaded form (owing to coating of liposomal complex) survived the environment of stomach and small intestinal, and only released the drug in colon, in a controlled manner. The optimized ingredients for the liposomal complex were sodium alginate 4% that could delay the release of bee venom by 50%. The scanning electron microscope image revealed beads to be of size 0.95e1.10 mm, with a dense cross-linking of gel structure post drying (possibly delaying release). Increase in alginate concentration also meant increase in encapsulation efficiency, which was achieved by maintaining a ratio of liposome:sodium alginate at 1:2. The release from the complex was scanty with only 16% being released in 4 h. In aggregate, about 90% of the bee venom was released in 8 h in vitro. For in vivo assessment, the authors used g-scintigraphy with a tracer molecule. The assay revealed a consensus between the beads’ colon arrival time and bee venom release time, establishing targeted delivery of the drug. This phenomenon was further in compliance with in vitro data. Working on protection of alkaline phosphates activity, a model bioactive protein, in gastric environment, scientists have devised a liposomal complex capped with alginate for sustenance in acidic environment and targeted delivery (Smith et al., 2010). The size of the coated vesicle was twice that of noncoated counterpart. The alginate-coated liposomes also exhibited a higher zeta potential than noncoated liposomes, conforming presence of alginate at the surface of the microstructure. Evaluating release behavior in SGF (pH 2.0), capped liposome released only 13% of the enzyme, while noncapped vesicle lost 20% of the enzyme in 2 h at 37 C. In the same conditions, in the next 48 h, noncapped liposome released about 70% of enzyme, while capped liposome released a meager 30% of the enzyme. In addition, regarding the activity of the released enzyme, the study reported retention of 80% of original activity in enzyme recovered from capped liposome and 55% in that recovered from noncapped liposome, at the end of 2 h, in the gastric condition defined before. This was reasoned to acid insoluble nature of alginate which protected the enzyme activity more in capped liposome.

10. Chitosan Chitosan is produced by deacetylation of chitin. It has been of great importance to multitude of domains in food and biomedicine, and a conspectus of the same has been provided in Table 8.2. Chitosan has widely been used to protect liposomal vesicles from environmental stresses, as well as targeted and controlled release, as would be described in this section. In a study, Coenzyme Q10 has been encapsulated in liposomal vesicles and further coated with trimethyl chitosan (Zhang and Wang, 2009). The objective was to treat selenite-induced cataract in suckling rats. The coated liposome showed significant ocular tolerance. Owing to chitosan coat, the enzyme showed about 4.8 times increased residence time in precorneal zone. Because of addition of chitosan, the encapsulation efficiency was, however, not changed and was at 98%. Curcumin has been subjected to the combined encasement of liposome and biopolymer. Past researches have shown potential use of curcumin for many ailments, which renders it the importance of a

180

Table 8.2 Applications of liposomes coated with chitosan and its derivatives. Coating material(s)

Encapsulated compound

Impact of the process

Reference

Egg L-a-phosphatidylcholine alone or with L-aphosphatidyl-DL-glycerol Dipalmitoylphosphatidylcholine and dicetyl phosphate Egg yolk phosphatidylcholine and cholesterol

Chitosan

e

Improved stability

Henriksen et al. (1994)

Chitosan

Insulin

Takeuchi et al. (1996)

Chitosan and alginate

Insulin

Lecithin

Chitosan

FITC-dextran

Egg phosphatidylcholine

Chitosanb-glycerophosphate (CGP)

Carboxy-fluorescein

Soy lecithin, stearylamine, phosphatidylglycerol, and cholesterol Lecithin and cholesterol

Chitosan

Superoxide dismutase (SOD)

Chitosan

Leuprolide

Chitosan/carbopol

Calcitonin

Chitosan

Calcitonin

Significant decrease in the blood glucose level of rats during 12 h The system could deliver insulin to intestine with increased absorption and bioavailability Increased stability of liposome in simulated gastric fluid Controlled release over at least 2 weeks; liposome-C-GP rapidly gelled at body temperature Liposome targeting mucosal tissues was prepared Increment in particle size and positive zeta potential Chitosan-coated liposome showed 2.8 times and carbopolcoated liposome showed 2.4 times higher plasma calcium concentration in rats Prolonged retention in the intestinal mucosa

Dipalmitoylphosphatidylcholine and stearylamine

Ramadas et al. (2000)

Filipovic-Grcic et al. (2001) Ruel-Gariepy et al. (2002)

Rengel et al. (2002)

Guo et al. (2003)

Takeuchi et al. (2003)

Takeuchi et al. (2005)

Chapter 8 Biomedical and food applications of biopolymer-based liposome

Wall material(s)

Distearoylphosphatidylcholine, dicetyl phosphate, and cholesterol Chitosan

improving the enteral absorption of drug Fluorescein sodiumeloaded chitosan nanoparticle

Distearoylphosphatidylcholine, dicetyl phosphate, and cholesterol

Low-molecular weight and high-molecular weight chitosan

Elcatonin

Cholesterol and one of the below-mentioned combination: dipalmitoylphosphatidylcholine/ distearoylphosphatidylcholine/ distearoylphosphatidylcholine and dipalmitoylphosphatidylserine Soy lecithin

Chitosan nanoparticles

e

Chitosan

e

Octadecyl-quaternized carboxymethyl chitosan and cholesterol

e

Quantum dots, superparamagnetic nanoparticles, Vincristine

1,2-Dipalmitoyl-sn-glycero-3phosphocholine and cholesterol

Chitosan and alginate

Bovine serum albumin

Improved stability for 45 days Increased encapsulation efficiency, improved physical and thermal stability, prolonged release of drug over 2 weeks Sustained linear release of the drug; extended shelf life and decreased loss of drug

Huang et al. (2005)

Thongborisute et al. (2006)

Diebold et al. (2007)

Laye et al. (2008) Liang et al. (2008)

Haidar et al. (2008)

Li et al. (2009) Continued

181

Diclofenac sodium

Decreased release, prolonged circulation, and higher bioavailability of drug Low-molecular weight chitosan showed significantly prolonged effectiveness to reduce blood calcium concentration and effective protection to the drug The nanosystem retained in the mucus layer of eye before entering conjunctival cells

10. Chitosan

Soy phospholipids, cholesterol, and chitosan

182

Table 8.2 Applications of liposomes coated with chitosan and its derivatives.dcont’d Coating material(s)

Soy phosphatidylcholine, cholesterol, phosphatidylserine

Low-molecular weight chitosan

Phosphatidylcholine and cholesterol

Chitosan

Vitamin E

Dipalmitoylphosphatidylcholine

Chitosan

e

Soy phosphatidylcholine, cholesterol

Trimethyl chitosan

Coenzyme Q10

Dilauroylphosphatidic acid and dimyristoyl phosphatidylcholine

Chitosan, dextran sulfate, deoxyribonucleic acid (DNA)

1-hydroxy pyrene-3,6,8trisulfonic acid, alendronate, and glucose

Distearoylphosphatidylcholine, dicetyl phosphate, and cholesterol

Chitosaneaprotinin conjugate

Calcitonin

Egg phosphatidylcholine and cholesterol/egg phosphatidylcholine, phosphatidylglycerol and cholesterol/distearoyl glycero-

Chitosan

Rifampicin

Impact of the process Prolonged precorneal retention of liposome in rabbit; enhanced penetration for trans corneal delivery Improved loading efficiency and increased shelf-life Improved resistance to detergent; change in natural membrane permeation 4.8 times increase in precorneal resistance time of liposome; anticataract effect evaluated by morphological examination Suppressed release of compounds; temperature-dependent release was observed using DNA as coating material Increased efficacy of drug; capable of inhibiting trypsin in vitro Negatively charged liposome provides increased vesicle stability during nebulization; showed

Reference

Liu and Park (2009)

Mady et al. (2009)

Zhang and Wang (2009)

Fukui and Fujimoto (2009)

Werle and Takeuchi (2009)

Zaru et al. (2009)

Chapter 8 Biomedical and food applications of biopolymer-based liposome

Encapsulated compound

Wall material(s)

phosphatidyl-choline and cholesterol/distearoyl glycerophosphatidyl-choline, phosphatidylglycerol, and cholesterol Dipalmitoylphosphatidylcholine

highly improved muco adhesive property

L-a-phosphatidylcholine, cholesterol, stearylamine, and dicetyl phosphate

Chitosan

Ciprofloxacin hydrochloride

Soy phosphatidylcholine, cholesterol, and Da-tocopheryl polyethylene glycol 1000 succinate

N-trimethyl chitosan

Curcumin

1,2-Dipalmitoyl-sn-glycero-3phosphocholine, 1,2dipalmitoylsn-glycero-3phosphoethanolamine-N-[4-(pmaleimidomethyl) cyclohexane-carboxamide] L-a-phosphatidylcholine

Chitosanethioglycolic acid or S-protected chitosanethioglycolic acid

Salmon calcitonin

Chitosan derivatives: using quaternary ammonium groups/Ndodecyl groups/both

Curcumin

Chitosan/gelatin hydrogels cross-linked

Calcein

Phosphatidylcholine

Increased positive zeta potential resulting in more stable liposome; prolonged and controlled drug release Longer drug residence in the eye of Albino rabbits and prolonged drug release The coated liposomes displayed different pharmacokinetic parameters and enhanced bioavailability of curcumin Increased oral bioavailability of calcitonin by Sprotected thiomers

Liposome containing both quaternary ammonium and Ndodecyl groups showed better penetration into cell membrane and controlled release of curcumin; this was toxic for murine melanoma (B16F10) cell line Improved release behavior of calcein and

Mady and Darwish (2010)

Abdelbary (2011)

Chen et al. (2012)

Gradauer et al. (2013)

Karewicz et al. (2013)

Ciobanu et al. (2014) Continued

183

Doxorubicin

10. Chitosan

Chitosan

184

Table 8.2 Applications of liposomes coated with chitosan and its derivatives.dcont’d Coating material(s)

Phosphatidylcholine

by glutaraldehyde and sodium sulfate/sodium tripolyphosphate Chitosan

Clotrimazole

Dipalmitoylphosphatidylcholine and cholesterol

Chitosan nanofiber

Gentamicin

Phosphatidylcholine and cholesterol

Chitosan

Curcumin

Soy phosphatidylcholine, cholesterol, and vitamin E

Chitosan/sodium alginate and chitosan

Vitamin C

Egg yolk phospholipid

Chitosan

Lycopene, b-carotene, lutein, and canthaxanthin

Phospholipon 90NG

Sodiumealginate microbeads or beads coated with sucrose/ chitosan

Resveratrol

Impact of the process

Reference

improved stability of liposomes Increased clotrimazole tissue retention at the vaginal site of pregnant sheep Sustained release of drug during 16 h with antimicrobial activity Decreased release rate of curcumin at higher temperature Release of vitamin C by pancreatic enzyme digestion was prevented Improved protection against heat, gastrointestinal stress, and centrifugal sedimentation; additional layer of chitosan displayed higher protection to b-carotene and lutein compared to lycopene and canthaxanthin Chitosan coating decreased the encapsulation efficiency of resveratrol; all coatings provided increased barrier to resveratrol diffusion

Jøraholmen et al. (2014)

Monteiro et al. (2015)

Liu et al. (2015)

Liu et al. (2016)

Tan et al. (2016)

Balanc et al. (2016)

Chapter 8 Biomedical and food applications of biopolymer-based liposome

Encapsulated compound

Wall material(s)

10. Chitosan

185

food-based drug. Encapsulated curcumin in liposome has been further encased in n-trimethyl chitosan chloride for oral delivery (Chen et al., 2012). The authors were interested to understand the in vitro and in vivo release and pharmacokinetic behavior of such a complex in male Wistar rats. The liposomes considered were composed of phosphatidylcholine, cholesterol, and D-a-tocopheryl polyethylene glycol 1000 succinate. The aggregate displayed maximum drug loading capacity of 2.33%, encapsulation efficiency of 86.67%, with a particle size of 657.7 nm for the biopolymer-coated liposome and a positive zeta potential (a negative zeta was reported for noncoated liposome). Although the in vitro release profiles from coated and noncoated liposomes were similar, there was a stark difference in the in vivo pharmacokinetic parameters. The maximum available curcumin at target site of intestine was significantly higher for coated liposome, and the coated liposome had comparatively higher half life, over noncoated liposome. This suggested much greater bioavailability of curcumin for a longer period of time. The authors opine that better results in coated liposome were due to adherence of chitosan to intestinal mucosal surface, protection from acidic gastric juices. In another study, investigators contemplated preparing chitosan derivatives with modifications in chitosan’s native form, for improved stability and delivery of biomolecules. In the said work, cationic biopolymer of chitosan (by adding quaternary ammonium groups), hydrophobic chitosan biopolymer (by addition of N-dodecyl groups), and cationic hydrophobic chitosan biopolymer containing both forms of modifications described were designed (Karewicz et al., 2013). Liposomes were efficiently coated and protected in all the three forms. However, the biopolymer with combined modification, the third type, was found to be most promising. The attributes for the said preference have been described as ability to penetrate cell membrane and second exhibition of controlled delivery of curcumin at target (release being studied in oleic acid as a model mimicking cell membrane environment). The release had no burst effect, indicating controlled release phenomenon with curcumin diffusing into the oil linearly up to 90 min. The plateau phase of release was only achieved after 10 h. This liposomal complex also had highest zeta potential and, remarkably, the minimum particle size among all those tested. Besides, the particles of the said third type modification were nonaggregating, indicating stability. The complex has further been shown to be nontoxic on murine fibroblast NIH3T3 cell line (normal cells), but toxic on murine melanoma B16F10 cell line (cancerous cells). The find clearly indicates possible usage of the biopolymer-coated liposomal assembly in anticancer therapy. The final optimized liposomal complex was cationic, anchor decorated, and modified with both functionalities. Vitamin E is a well-known essential amine required by the body, and lack of it causes nutritional deficiency disorders (neuronal abnormality and muscular dysfunction), and its extreme deficiency can cause hemolytic anemia. Hence, the availability of the said vitamin in the blood stream is important. Considering the requirement, Liu and Park carried out entrapment of the same in liposomal complex, encased in a biopolymer, chitosan (Liu and Park, 2009). Different ratios of phosphatidylcholine and cholesterol were worked out for maximum loading of vitamin E, and best combination was found with ratio of 40:60 of phosphatidylcholine:cholesterol. The chitosan nanocoated phosphatidylcholinee cholesterol nanoliposome had a size of 144 nm, noncoated liposome being of 133 nm size. The carrying capacity (payload) of coated liposome was 27%, loading efficiency was 99%, and its zeta potential was positive. The said capped liposomes also exhibited positive surface charge that would facilitate adherence to intestinal epithelium for delivery of bioactives/drugs. Thus, it could also be employed in antitumor therapy, if need be. A 3-month storage study revealed that in the coated complex, vitamin E retained 97% of original concentration, while in noncoated liposome the same was 60%, both samples being stored at 4 C.

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Considering the importance of temperature on structure, stability, and release of active constituent from liposomes, Liu et al. worked out a methodology to understand the said effect (Liu et al., 2015). The liposomes were created using phosphatidylcholine:cholesterol ¼ 5:1, while in liposome phosphatidylcholine:curcumin was 10:1 (w/w), and chitosan concentration was 0.5% (w/w). The mean particle diameters were 93.2 and 332.7 nm for noncoated and chitosan-coated liposome, respectively. Whereas the zeta potential of curcumin liposome was negative, and that of chitosan-coated liposome was positive, indicating better stability owing to high electrostatic repulsion among particles. Chitosan-coated liposome also had higher encapsulation efficiency at 52.8% compared with 41.42% for noncoated liposome. The comparatively low efficiency in either sets was reasoned on the fact that curcumin could only be loaded at the hydrophobic bilayers which is rather thin, while the core of the liposome is hydrophobic. While curcumin noncoated liposome released 61% curcumin in 360 min at 23 C, chitosan-coated liposome released only 41% in the same temperature and duration. At an enhanced temperature of 60 C, the release from noncoated liposome was 67%, while coated liposome released only 43%. Temperature wise, the coat did protect the liposome with higher viability in temperature between 60 and 90 C, due to effect of biopolymer resisting liposome disruption. These data were also supported by differential scanning calorimetric results, as claimed. The release mechanism of curcumin was also noted by authors, which was found to follow Higuchi model at 23 C. At 60 C, the observation was different, and the liposomes followed RetgerePeppas model. In all experiments on release, non-Fickian (anomalous) transport was reported, depicting release by both diffusion and dissolution phenomena. The authors observed that the liposomal structure collapsed at 50 C by X-ray diffraction. The impact of operational environment on sustenance of liposome is also important. Researchers are interested to know what the consequences of increasing coats on a liposome are, is one coat enough or do we need to increase the same. To expound such theory, scientists have experimented with liposomes (encapsulating Vit. C), which have been encased in chitosan, alginate, and mixture of both in layer-by-layer assembly, vis-a`-vis noncoated liposomes, with varying conditions. The variables considered included ionic strength of solution, pH, and storage duration (Liu et al., 2016). The data were rather interesting and showed an obvious increase in particle size with coat(s); however, the surface charge was negative (in combinational complex). This was due to the sequence of layering, where chitosan was positive and alginate was negatively charged. The combined coat liposome was more stable under low pH conditions (pH 1.5), with least change in appearance, size, and shape with time of exposure. The authors argue that at such conditions, alginates dissociates from the complex, making the outermost layer of chitosan alone and thus provide positive zeta potential, and higher endurance to acidic environment. Surprisingly, with fall in pH to 5.5e7.4, reverse surface charge was observed. The effect of ionic strength using NaCl as model ion provider resulted in an observation that the particles shrunk in size with increasing ionic strength, indicating leakage of water from the core of liposome. The liposomes released the active consequent more in SIF than in SGF indicating protection of liposomes from acidic gastric juices. For delivery of carotenoids to specific targets orally, Tan et al. have probed developing a method employing liposomes and biopolymers for potential use in nutraceuticals’ delivery and in developing efficient functional foods (Tan et al., 2016). The core materials encapsulated in liposomes were four types of carotenoid namely lycopene, b-carotene, lutein, and canthaxanthin. The shell of encapsulate consisted of phospholipidechitosan, chitosan at 0.50 mg/mL. The liposomal complex has been reported to be highly stable owing to rigidity offered by interaction of chitosan and liposomal membrane.

10. Chitosan

187

TEM images of the particles illustrate well-formed spherical particles in the size range of 70e100 nm, and the figure by DLS technique (inserted in the main figure) showcases similarity to some extent with microscopic image (Fig. 8.3). Notably, the figure reveals that chitosan loading b-carotene and lutein display only one sharp peak meaning a homogenous distribution. However, two peaks have been observed in lycopene and canthaxanthin. The particle sizes of coated liposome followed the order canthaxanthin > lutein > b-carotene > lycopene. In terms of carotenoid loading capacity and encapsulation efficiency, the order was lutein > b-carotene > lycopene > canthaxanthin. In addition, authors mentioned about electrostatic and hydrophobic interactions occurred within the liposomal complexes that render compactness to the liposomal complex. Experiments validated that the coat complex was better able to protect b-carotene, lutein than lycopene, and canthaxanthin. Besides

FIGURE 8.3 TEM photographs of chitosan-coated liposomes with (A) lycopene, (B) b-carotene, (C) lutein, and (D) canthaxanthin (Tan et al., 2016).

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mechanical and chemical tensility, chitosan also provided thermal endurance to the liposomal assembly. The in vitro release profile depicted a pattern in SGF wherein the release rates occurred in the order canthaxanthin > lycopene > b-carotene > lutein for over 4 h. In SIF, the release order was canthaxanthin (80%) > lycopene (68%) > b-carotene (57%) > lutein (49%), after 10 h of incubation, indicating a controlled release and better protection to encapsulates to acid extremities of gastric tract. In terms of instability index, authors concluded that encapsulates of b-carotene and lutein fared better in comparison with their counterparts.

11. Future prospects Although many biopolymers have been worked on, few challenges still remain. Work still remains in furtherance of encapsulation efficiency, greater drug loading, and better control over controlled release. Biopolymers could also be chemically or mechanically modified to better adsorb onto liposomes. Liposomes could in turn be made more flexible and durable so that addition of biopolymers could further sustain unit operations, if any. This could be useful when these polymer-coated liposomes would be an ingredient in food and therapeutic supplements. More research could be warranted for encapsulating higher molecular weight nutraceuticals, molecules, flavors, peptides, and antibodies. Owing to amphipathic nature of liposomes, many areas of application open up for such initiatives those are different from the existing products. Nutraceutical-coated liposomes as a food supplement (directly) could also be pondered on, and designing newer liquid-based and drug formulatedebased interventions and food supplements could be worked out.

12. Conclusion Liposomes though are very useful candidates for delivery of sensitive nutraceuticals and pharmaceuticals, they are fragile when considering their targeted action at the site desired considering physiological limitations. In addition, their release needs to be controlled which cannot be accomplished unless regulated. Biopolymers those have been discussed have shown considerable promise in mitigating both these broad concerns. They have also been attributed with rendering positive charge to the surface of liposomal complex, indicating stability. Research shows suitability of even incorporating multiple biopolymers for enhanced protection and delayed release. These attributes greatly circumvent the existing concerns about drug/nutraceutical delivery. The treatise presented the findings on various biopolymers, and the modes and domains of their applications. Encapsulation of various drugs, bioactives, and proteins has been presented that showcases the versatile realms of usage of biopolymers.

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CHAPTER

Nanosized magnetic particles for cancer theranostics

9

Sadaf Hameed, Pravin Bhattarai Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, Haidian, China

1. Introduction The materials having at least one of its dimensions within 1e100 nm are usually termed as nanosized materials (Anselmo and Mitragotri, 2015). Such nano-moieties have gained prominence due to their exceptionally high surface-area-to-volume ratio and fascinating physiochemical properties (Huang and Lovell, 2017). These unique properties exert a considerable impact in tuning the mechanical, optical, and magnetic functioning of nanomaterials for the diagnosis and detection of ailment (Almeida et al., 2011). Particularly, magnetic nanoparticles (MNPs) are the major class of nanosized materials having wide range of applications in the biomedical field, such as drug delivery, magnetic resonance imaging (MRI), and hyperthermia-induced cancer therapy (Revia and Zhang, 2016). A large interest on the MNPs can be simply linked to their high magnetic susceptibility, stability, biocompatibility, and relatively easier synthesis methods (Angelakeris, 2017). Moreover, MNPs are easily manipulated by an alternating magnetic field, which provides an opportunity to deliver the therapeutic drugs at a specific site and at a specific rate, thereby overcoming the problems of traditional diagnostic and therapeutic procedures (Gobbo et al., 2015). MNPs are usually classified into pure metals and metal oxides. The most popular MNPs include but not limited to Fe, Co, Ti, Ni, and iron oxides (Kumar and Mohammad, 2011). Among them, iron oxides (Fe2O3 or Fe3O4) are preferred due to their minimal toxicity (Estelrich and Busquets, 2018). The successful application of MNPs for cancer diagnosis and treatment is related specifically with their distinct magnetic behavior (Martinkova et al., 2018). Such magnetic properties are usually governed by the type of MNP; the synthesis protocol; the size, shape, and surface charge of NPs; the interaction between the NPs; and the biodistribution (Nam et al., 2013). In this context, a suitable synthesis route must be adopted to achieve a precise and specific performance (Zhu et al., 2017). In regard to previous findings, MNPs which are more susceptible to the quickest change in their magnetic state with respect to an external magnetic field are mostly preferred. Therefore, MNPs must associate high magnetic susceptibility and lack of magnetization with the retraction of an external magnetic field (Singamaneni et al., 2011). This unique ability of MNPs to be guided by an external magnetic field has been extensively utilized for various biological applications. Moreover, further “functionalization” of these MNPs with bioactive agents, such as peptides and nucleic acids, forms distinct therapeutic and diagnostic modalities that can Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00009-6 Copyright © 2020 Elsevier Inc. All rights reserved.

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specifically penetrate the cells and tissue barriers with enhanced efficacy, while simultaneously reducing the side effects. Likewise, the functionalization of MNPs with appropriate biocompatible polymer is also ruled not only by their inherent magnetic properties but also by their stability, nontoxicity, and biocompatibility (Durymanov et al., 2015). Several polymers, such as poly(ethylene glycol) (PEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(caprolactone) (PCL), chitosan, dextran, and so on, have already been approved by FDA (Assa et al., 2017). This biofunctionalization of MNPs decreases the risk of blood capillary obstruction and prevents self-aggregation. Among leading classes of nanomaterials in disease management, MNP largely contributes as an excellent MRI contrast agent for in vivo biological and medical imaging (Xie et al., 2011). MRI, owing to three-dimensional resolution, high penetration depth, and excellent soft tissue contrast, has emerged as one of the most extensively used noninvasive clinical diagnosis tool (Wu et al., 2018). A considerable amount of research has focused on the design and synthesis of MNPs as MRI contrast agents that provide better delineation between the diseased and healthy tissues (Wu et al., 2018; Yu and Zheng, 2015). MNPs are being developed for a therapeutic purpose such as drug vehicle and hyperthermia (McCarthy and Weissleder, 2008). MNPs-based drug delivery system can be precisely controlled by an external magnetic field, which is crucial for targeted drug and gene delivery (Mody et al., 2014). An excellent example of such an interesting capacity of MNPs was reported by Chiang et al. (Chiang et al., 2016). The authors have synthesized the Trastuzumab-functionalized doxorubicin (DOX)-encapsulated MNPs through a simple double emulsion method that portrayed a key paradigm shift in cancer nanovehicle design and cancer treatment in preclinical investigations. Furthermore, MNPs are also being investigated rigorously for heat-activated drug delivery applications and hyperthermia-induced cancer therapy. Thanks to the excellent heat generating ability of MNPs in the presence of a high-frequency magnetic field (Lahiri et al., 2017). This approach represents lower side effects as compared to traditional chemotherapy and radiotherapy (Tay et al., 2018). In short, outstanding magnetic properties and appropriate safety profile make MNPs an excellent candidate for tumor imaging, targeted drug and gene delivery, and image-guided therapy that hold strong potential for future clinical applications.

2. Synthesis techniques of magnetic nanoparticles The synthesis of MNPs ranges from nanoscale biomedical applications, e.g., diagnosis and therapy to macroscale industrial applications such as storage media, magnetic ink for jet printing, and so on. The variations in the MNPs applications are strongly related to the adopted synthesis procedure because of the dramatic affinity of magnetic properties with the morphology and structure of MNPs. However, from a fundamental scientific and technical point of view, the synthesis of MNPs with controllable size, shape, and crystal defects is important for biomedical applications and is generally challenging. Over the years, several synthesis methods have been proposed to obtain MNPs with uniform size, shape, magnetic characteristics, surface chemistry, and reproducibility. These methods are generally classified into physical and chemical methods: (1) physical methods, such as electron beam lithography and gas-phase deposition; and (2) chemical methods, such as oxidation, chemical coprecipitation method, solgel synthesis, hydrothermal, thermal decomposition, flow injection synthesis, aerosol/vapor-phase method, and electrochemical methods. Among the two synthesis approaches, chemical methods are more effective and preferred to obtain monodispersed MNPs with controllable

3. Physiochemical properties of MNPs

195

Table 9.1 Summary of various synthesis methods of magnetic nanoparticles (MNPs).

Physical methods

Chemical methods

Methods

Advantages

Disadvantages

Reference

Electron beam lithography method Gas-phase deposition method Oxidation method

Controllable interparticle spacing

Expensive and highly complex

Hicks et al. (2005)

Easy preparation method Narrow size distribution High yield and high magnetization

Difficult to control the morphology of NPs Small-sized ferrite colloids Broad size distribution and irregular morphologies Products contain solgel matrix at the surface

Binns et al. (2005) Woo et al. (2004) Laurent et al. (2008)

Coprecipitation method Solgel synthesis

Hydrothermal method

MNPs of desired shape and morphology, particularly useful for the synthesis of magnetic nanohybrids Controllable particle size and shape, narrow size distribution

Thermal decomposition

Monodispersed MNPs with a high level of size and shape control

Microemulsion

Shape and sizecontrolled MNPs Good size control

Electrochemical methods

Medium reaction yield, require high reaction conditions, such as high pressure and temperature Complicated procedure, require inert atmosphere and high temperature Low yield Low reproducibility

Lu et al. (2002)

Lu et al. (2007)

Hao et al. (2010)

Tartaj et al. (2003) Cabrera et al. (2008)

magnetic properties. A brief overview of the most commonly used method is listed in Table 9.1 (Binns et al., 2005; Cabrera et al., 2008; Hao et al., 2010; Hicks et al., 2005; Laurent et al., 2008; Lu et al., 2007; Lu et al., 2002; Tartaj et al., 2003; Woo et al., 2004).

3. Physiochemical properties of MNPs The well-standardized synthesis route not only yields highly functional MNPs but also governs the physiochemical triads: size, shape, and surface properties of MNPs (Nandwana et al., 2015). For instance, these physiochemical triads are largely responsible for identifying if these MNPs are ideally suitable for diagnostic and therapeutic purposes in vivo and are often rate-limiting factors in the clinical translation of MNPs. Likewise, the biological behavior of MNPs, such as biodistribution and clearance, has been thought to originate from the size and shape of MNPs as well as their

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polydispersity, surface charge, and nature of the surface coating (Issa et al., 2013). Therefore, these key parameters have to be taken into consideration for developing MNPs for biomedical application.

3.1 Size of MNPs The size of MNPs determines their behavior with the biological system, such as interaction with blood proteins, mode of endocytosis, and cellular uptake, which consequently affects the biodistribution of MNPs (B. H. Kim et al., 2011; Nemati et al., 2018). Apparently, decreasing the size of MNPs leads to an exponential increase in the surface-area-to-volume ratio, thus making the surface of MNPs more reactive on its own (aggregation) and also the biological counterparts (Gal et al., 2017). Therefore, a slight change in the size of MNPs leads to an obvious alteration in cellular responses and receptor cross-linking (Tong et al., 2017). It is generally agreed by the investigators that MNPs with a diameter ranging from 10 to 100 nm are considered optimal for cancer therapy with acceptable pharmacokinetics, whereas MNPs smaller than 5 nm and larger than 200 nm are either removed by the renal clearance or sequestered by the spleen. Ultrasmall and ultrafine MNPs ( 20 nm) that are more suitable for the T2
contrast enhancement (Park et al., 2017).

3.2 Shape of MNPs The next important “passive” parameter of MNPs that influence their tumor extravasation and accumulation is the shape of NPs (Duan and Li, 2013). In general, the shape of NPs usually confers spherical shapes but may vary to wormlike, star-shaped, rod-shaped, cylindrical, cubes, and some specialized flexible shape-changing nanomaterials (Wei et al., 2018). The shape of NPs dictates their biodistribution mainly due to the differential uptake of NPs via mononuclear phagocytic system (MPS) in the organs such as spleen and liver, accumulation in the targeted regions of the tumor, in vitro cellular uptake, and

3. Physiochemical properties of MNPs

197

renal clearance in vivo (Veiseh et al., 2010). It has been observed that the elongated MNPs with high aspect ratios have fewer chances to be internalized by the phagocytic system than spherical particles (Park et al., 2008). In addition to extended blood circulation time, the large surface area and high aspect ratio of elongated MNPs bestow them with superior tumor accumulation (Duan and Li, 2013). In spite of the fact that the use of elongated MNPs could improve their attachment with the tumor endothelium and tumor accumulation, it seems quite complicated to identify the appropriate geometry of MNPs for their effective delivery to tumor cells (Veiseh et al., 2010).

3.3 Surface properties and biopolymer-functionalized MNPs Surface charge and hydrophobicity of MNPs can limit or enhance their interaction with plasma proteins, adoptive immune system, nontargeted cells, and extracellular matrix. First, the hydrophobic MNPs are often found to have short blood circulation life due to instantaneous opsonization followed by the RES recognition and clearance (Chen et al., 2016; Davis, 2002). Moreover, MNPs are also susceptible to agglomeration upon injection, leading to the faster clearance from blood particularly due to the RES (Chouly et al., 1996). Second, the surface charge of MNPs has subtle differences during the process of cellular interaction and internalization. For instance, MNPs decorated with positively charged surfaces have a higher affinity to negatively charged cells, resulting in the nonspecific internalization of cationic MNPs via adsorptive endocytosis (Kievit and Zhang, 2011). However, it is quite essential to minimize the nonspecific interactions of MNPs. For this, efforts have been directed toward reengineering the surfaces of MNPs by deploying biofunctionalization strategies (Kang et al., 2017). Surface modification of MNPs with hydrophilic polymers (i.e., PEG) increases their stability, reduces opsonization, and improves the blood circulation life (Harris and Chess, 2003). Moreover, surface functionalization can also protect the targeting and therapeutic agents during circulation inside the biological system, avoid their undesired accumulation in vital organs, and improve their delivery efficiency. The most investigated surface modifier includes polymers or copolymers of PEG (Kievit et al., 2009), dextran and its derivatives (Kernstine et al., 1999), antifouling polymers (Lee et al., 2006), and proteins (Xie et al., 2010). The use of biopolymers not only renders iron oxides as a biocompatible agent but also allows additional roles in drug delivery and related applications (Davodi et al., 2019). Recently, chitosan from a class of natural biopolymers has been widely used in the design and synthesis of MNPs because it offers advantages such as cross-linkage to entrap drug molecules, stabilize, and control the diameter of NPs within a range that minimizes uptake by the RES and increases the half-life in blood (Assa et al., 2017). For example, Li et al. (2015) prepared carboxymethyl chitosanecoated Fe3O4 NPs for loading anticancer drug, rapamycin, by solvent evaporation technique. The biopolymer-functionalized MNPs facilitated optimal loading, controlled tumor-specific delivery of rapamycin and good magnetic responsivity in vitro. However, the premature burst release of rapamycin which could be either due to improper surface coating or faster degradation of chitosan at particular pH could be deleterious for effective therapy. Huang et al. present more robust layer-by-layer (LBL) assembly of milk protein casein on the surface of iron oxide NPs further loaded with two different anticancer agents: DOX and ICG inside the polymeric layer (Huang et al., 2015a) (Fig. 9.1A and B). The assembly depicted excellent stability in the acidic gastric environment followed by the slow and controlled release of the drugs after decomposition of casein mainly due to intestinal proteases (Fig. 9.1C). Therefore, such MNPs are quite promising as orally administered clinical MRI agents that can possibly resist the degradation in the

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FIGURE 9.1 (A) Schematic illustration of layer-by-layer assembled casein-coated MNPs loaded with drug (DOX/ICG). (B) Representative TEM images of CN-DOX-IO NPs. (C) Comparison of release profiles of DOX at different pH (2.0 and 7.0). (D) Representative T2-weighted MR images of mice before and 3 h after oral administration of CNICG-IO NPs (Huang et al., 2015b).

stomach but show enhanced bioactivity in the small intestine (Fig. 9.1D). Most recently, Carvalho et al. reported the one-pot green synthesis of ultrasmall MNPs modified by a naturally biocompatible carboxymethylcellulose (CMC) (Carvalho et al., 2019). The presence of higher hydroxyls and carboxylate functional groups in the polymeric chain provided an additional advantage to link DOX covalently (Fig. 9.2A). Hence, these ultrasmall coreeshell hybrid MNPs could be used effectively to treat glioblastomas via combined magnetic hyperthermia (MHT) and chemotherapy (Fig. 9.2B). Several other intriguing applications encompassing the use of natural biodegradable polymers such as gemcitabine-loaded MNPs for the treatment of nonsmall lung cancer, combination of dextran-coated MNPs with hyaluronic acid and cisplatin for the treatment of prostate cancer, amylose-modified MNPs for in vivo MRI tracking of stem cells in acute ischemic stroke are being explored further (Table 9.2) (Hamarat Sanlier et al., 2016; Lin et al., 2017; Unterweger et al., 2014).

3.4 Biocompatibility and safety The potential toxicity of NPs is the most critical concern for its limited use in biomedical applications. In contrast to organic counterparts, inorganic NPs are often challenged by the issues of toxicity, especially MNPs (Ehlerding et al., 2016). Few intracellular and in vivo investigations highlighted that the MNPs can generate reactive oxygen species (ROS) after their internalization at high dose, resulting in the toxicity against various cell lines mainly via alterations of cell proliferative genes level expression (Mahmoudi et al., 2011b). Notably, it was further confirmed that the surface coating of MNPs could overcome such unwanted toxicity issues and contributed largely to establishing the safe

3. Physiochemical properties of MNPs

199

FIGURE 9.2 (A) DLS and ZP (x) results obtained from MION@CMCeDOX. Expanded views: details of the interactions of CMCeDOX_MION. (B) MTT assays in glioma (U87) cells incubated with MIONeCMC nanocolloids comparing the effect of DOX and the magnetic hyperthermia treatment in cell viability response (Carvalho et al., 2019).

yield of MNPs for clinical applications. Subsequent studies on mice have clearly shown that the biocompatible coating reduces the long-term toxicity of MNPs in various organs to some extents (Mahmoudi et al., 2011a). For instance, while nontargeted MNPs (i.e., iron oxide NPs) mostly accumulated in spleen (6.2%) and liver (86%), within 1 h after intravenous injection (Weissleder et al., 1989), urokinase plasminogen activator receptor (uPAR)-targeted MNPs showed negligible systemic toxicity during the acute phase (3 days) and no apparent toxicity was observed even after long-term intravenous administration of NPs (up to 3 months) (Chen et al., 2015). These studies greatly support the safety, biodistribution, and pharmacokinetics of MNPs and stimulate a number of clinical applications in human. For instance, the FDA-approved ferumoxytol (Feraheme) MNPs have been successfully used as an MRI contrast agent in the detection of lymph node metastases and also in the

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Table 9.2 Summary of Biopolymer-Coated MNPs used in cancer treatment. Biopolymers

Synthesis method

Size

Applications

References

Starch

Chemical coprecipitation

6 nm

Kim et al. (2003)

Dextran

Van der Waals force/ hydrogen bond/ electrostatic interactions Coprecipitation and esterification Metaleligand complex coordination Desolvation and chemical coprecipitation In situ coprecipitation method

13 nm

In vivo magnetic resonance imaging (MRI) of the rat brain. The MRI of the rabbit liver, marrow, and lymph.

Dextran and hyaluronic acid Fucoidan

Albumin

Chitosan

77 nm 365 nm

56 nm

103 nm

Magnetic drug targeting for cancer therapy. pHestimuli-responsive drug release and efficient magnetic hyperthermia. Curcumin-loaded magnetic nanoparticle for drug delivery. pH-responsive targeted delivery of DOX in tumor.

Hong et al. (2008)

Unterweger et al. (2014) Santha Moorthy et al. (2017) Nosrati et al. (2018)

Unsoy et al. (2014)

treatment of anemic patients (Hetzel et al., 2014; Schwenk, 2010). Results from phase II/III clinical trials revealed that it is safe to administrate 2.9 mg/kg MNPs (30 nm size) in human patients (Harisinghani et al., 2007). Therefore, MNPs-based drug carriers will likely manifest minimal safety concerns compared with other nanosized metallic materials for the development and translation of novel cancer theranostics agents (Zhu et al., 2017).

4. Magnetic nanoparticles for cancer imaging Noninvasive tumor imaging is widely preferred to accurately access the intertumoral efficacy of NPsmediated drug delivery in patients since the human tumor is highly heterogeneous and NPs face obstruction from biological barriers. Such noninvasive imaging approaches allow timely diagnosis and assessment of treatment strategies for cancer patients. Many imaging approaches have already been employed for the cancer diagnosis, including ultrasound, X-ray, MRI, positron-emission tomography (PET), and computed tomography (CT) (Kernstine et al., 1999). Among these, researchers and clinicians are mostly benefitted from the MRI-based imaging approaches because of its ability to acquire the functional and anatomical information noninvasively and also with relatively high spatial and temporal resolution in three dimensions (Weissleder et al., 2014). Taking the advantage of exclusive physicochemical properties of MNPs, extensive research efforts have been made to develop and explore new theranostics MNPs, as MRI contrast agent, in preclinical settings (Wei et al., 2017). Among many kinds of MNPs, superparamagnetic iron oxide NPs (SPIONs) have been extensively investigated as MRI contrast agent due to their strong effect on T2 relaxation (Liu et al., 2017). These NPs can differentiate the cancerous cells from the surrounding healthy cells through either passive

4. Magnetic nanoparticles for cancer imaging

201

targeting or active targeting mechanism. Passive targeting mechanism relies on the physicochemical and anatomical differences between the healthy and tumor tissues and generates enough contrast between them. On the other hand, active targeting depends on the covalent or noncovalent conjugation of targeting ligands on the surface of SPIONs. These ligands specifically bind to the overexpressed surface biomarkers on the cancer cells. For example, different proteins and antibodies that can bind with higher specificity to its corresponding receptors on the cancer cells, such as EGFR, uPAR, avb3 integrin, MUC-1, and prostate-specific membrane antigens, are attached to the surface of SPIONs for active targeting, as a consequence SPIONs preferentially accumulate and retain at tumor site and generate a strong contrast (Huang et al., 2017). Besides, unlike other conventional nonmagnetic NPs, such MNPs further allow the use of an external magnetic field to accumulate at the tumor site (Shanavas et al., 2017). Upon accumulation in the tumor through any of the above-stated mechanism, SPIONs offer contrast enhancement by shortening the transverse relaxation time (T2 and T2
) and longitudinal relaxation time (T1) of surrounding protons. However, the shorting process of T1 is influenced by the close interaction between T1 agents and protons that can be impeded by the thickness of MNP coating. The shortening of relaxation time by SPIONs is associated with high susceptibility difference between the NPs and the surrounding environment resulting in magnetic field gradient. The SPIONs are primarily used to offer negative (hypointense) contrast enhancement by using T2-weighted images (Lee et al., 2013). Recently, Yan et al. reported the potential of protoporphyrin IX (PpIX)coated SPIONs as a contrast agent in preclinical MRI (Yan et al., 2018). The SPIONs were intravenously injected at a dose of 5 mg Fe/kg of body weight and T2-weighted MR images were acquired after 24 h. The T2-weighted MR images revealed a gradual loss of signal at the tumor site, thus indicating the accumulation of SPIONs in the tumor (Yan et al., 2018). In another study, Chee et al. reported the fabrication of peptide-coated ultrasmall SPIONs (USPIONs) as a robust MRI contrast agent that can possibly outweigh the limitations of other commercially available probes. The relatively higher relaxivity ratios (>90) make these SPIONs competent for T2 contrast MRI (Fig. 9.3) (Chee et al., 2018). Until now, a vast majority of MNPs-based contrast agents are in preclinical investigations and five formulations have already been approved for clinical use, namely ferumoxytol (Feraheme; with particle diameter about 30 nm), ferucarbotran (Resovist; with particle diameter about 60 nm), ferumoxides (Feridex; with particle diameter about 120e180 nm), ferumoxsil (Iumiren; with particle diameter about 300 nm), and ferristene (Abdoscan; with particle diameter about 300 nm) (Finn et al., 2017; Wang, 2011; Wang et al., 2001). Another growing trend in MNPs-mediated cancer diagnosis is the development of synergistic integration of multimodal contrast agents. The MNPs can also facilitate the addition of fluorescent molecules and radioisotopes for multimodal imaging of tumor cells. Up to now, several multimodal MNPs have been developed and investigated for MRIePET/SPECT, and MRI-optical dual-modality (Hu et al., 2018; H.-Y. Lee et al., 2008) or PET/MRI/NIRF triple-modality imaging (Xie et al., 2010) to provide more accurate and detailed imaging information in vivo. Very recently, Jung et al. prepared 64 Cu-deposited Fe3O4@Au coreeshell NPs to provide triple-mode MRI/PET/PA contrast agents. The individual components, Fe3O4 offered MRI contrast, 64Cu used as PET isotope, whereas Au facilitated PA (photoacoustic) imaging of natural-killer cells in the tumor (Jung and Chen, 2018). Similarly, polyaspartic acid (PASP)-coated iron oxide NPs have been reported for PET/MRI dual-modal imaging of avb3 integrin expression. For integrin targeting, cyclic arginineeglycineeaspartic (RGD) peptide was conjugated with PASP-coated iron oxide NPs. Moreover, 64Cu was used to label iron oxide NPs

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FIGURE 9.3 In vivo MRI with the 2 PG-S
VVVT-PEG4-ol-coated USPION contrast agent. (A) In vivo MR images in coronal (top) and the transverse (bottom) plane of NCr nude mice at 0, 0.5, and 1 h after intravenous injection of 2PGS
VVVT-PEG4-ol-coated USPIONs. (B) Quantification of liver contrast collected at 0, 0.5, and 1 h after accumulation of peptide-coated USPIONs in NCr nude mice at a dose of 1.0 mg Fe/kg. (C) In vivo MR images in coronal (top) and the transverse (bottom) plane of orthotopic xenograft liver tumor model at 0, 0.5, and 1 h after intravenous injection of 2PG-S
VVVT-PEG4-ol-coated USPIONs. Dashed circle indicates the tumor location. (D) Quantification of CNR of tumor-to-liver contrast at 0, 0.5, and 1 h after accumulation of 2PGS
VVVT-PEG4-ol-coated USPIONs at a dose of 2.0 mg Fe/kg. (E, F) Histopathological analysis of liver of mice after 1 h post-intravenous injection of 2PG-S
VVVT-PEG4-ol-coated USPIONs at a dose of 2.0 mg Fe/kg. Scale bar is 100 mm (Chee et al., 2018).

for PET. PET/MRI dual-modal imaging showed integrin-specific uptake of NPs by the tumor (Lee and Chen, 2009). The success of these multimodal imaging approaches allows the accurate detection of tumor and provides further insight into the molecular and anatomical characterization of cancer cells.

5. Magnetic nanoparticles for cancer therapy 5.1 Chemotherapy Clinically used low-molecular-weight chemotherapeutic drugs can easily penetrate the healthy cells along with the tumor cells and thereby, evenly distribute inside the body, have short circulation

5. Magnetic nanoparticles for cancer therapy

203

half-life, and have relatively high clearance rate. Consequently, the amount of chemotherapeutic drug that reaches the targeted tumor site is significantly lower, resulting in the decreased therapeutic efficacy and increased risk of drug resistance. Therefore, in order to overcome the limitations of conventional chemotherapeutics, MNPs have been extensively studied as a carrier for loading and unloading of chemotherapeutic drugs and genes. Until now, several hydrophilic and hydrophobic drugs, including paclitaxel (PTX), doxorubicin (DOX), small interfering RNA (siRNA), as well as DNA, have been delivered to the tumor site via MNPs. These MNPs-based drug delivery systems are designed either by attaching the therapeutic drugs on the surface of MNPs or encapsulated into the matrix of polymeric MNPs. This approach protects the therapeutic drugs and genes until reaching the desired tumor sites, where drug molecules tend to release and offer therapeutic effects. However, for the development of MNPs-based drug carrier, it is necessary to consider loading and conjugation efficiency as well as the mechanism of drug release. Therefore, MNPs are synthesized with various compositions, morphologies, and structures in an attempt to improve their loading capacity and therapeutic efficacy. For example, mesoporous Fe3O4@SiO2 NPs or hollow Fe3O4 NPs have been designed to improve their loading capacity (Cheng et al., 2009; Zhu et al., 2010). Zhu et al. reported the higher loading capacity of Fe3O4@SiO2 NPs for an anticancer drug, DOX. In addition, the sustained release pattern of DOX from Fe3O4@SiO2 induced fairly higher cytotoxicity against HeLa cells in vitro as compared to its free counterpart (Zhu et al., 2010). The most recent MNPs that are commonly used to deliver various therapeutic drugs are summarized in Table 9.3. Moreover, the magnetic properties of MNPs can also be utilized for magnetic-guided controlled drug release in response to an externally subjected magnetic field. In a recent study, Qiu et al. demonstrated that external magnetic field facilitated the extravasation of drug molecules by temporarily disrupting the endothelial adherent junctions through internalized iron oxide NPs (Fig. 9.4). The technique of magnetic activation is quite inexpensive and reliable to achieve tumor-specific drug delivery (Qiu et al., 2017). However, several key parameters must be considered in magnetic-guided tumor targeting and drug delivery. Notably, magnetization saturation (at least higher than 20.0 emu g1) is one of the fundamental parameters that determines the response rate of MNPs against an applied external magnetic field. The magnetization saturation can be easily tuned by modifying the composition, size, and morphology of NPs (Liu et al., 2011). In addition to magnetization, the strength and gradient of the magnetic field as well as the physiological factors, including tumor volume, the strength of the drugeMNPs bond, blood half-life, and infusion route, are other essential parameters for improved magnetic-guided drug delivery system (Ulbrich et al., 2016). Magnetic-guided drug delivery system not only improves the drug accumulation at the tumor site but also enhances the drug distribution in nearby healthy tissue, increasing the risk of possible side effects on these normal tissues (Bhattacharya et al., 2011). Therefore, the concept of targeted drug delivery is under extensive development and already introduced for clinical applications (Jalalian et al., 2013). The efficiency of drug delivery through MNPs can further be enhanced by targeting ligands which can be specifically bound to the receptors that are overexpressed on the tumor surface. These targeting ligands improve the internalization of MNPs into the tumor cells or facilitate the adhesion of MNPs onto the cell surface. To date, diverse antibodies and small targeting ligands such as folic acid (FA), aptamers, LyP-1 (CGNKRTRGC), and RGD peptide have been reported to maximize the therapeutic efficacy of MNPs (Bhattacharya et al., 2011; Fan et al., 2011; Jalalian et al., 2013). For instance, Yu et al. designed a nanoplatform based on SPIONs and further modified with FA, a highaffinity ligand for folate receptor, to penetrate the blood-brain barrier through receptor-mediated

204

Table 9.3 Magnetic nanoparticles (MNPs) for drug delivery and cancer therapy.

Nanoparticles

Size

Loaded drug

(3-Aminopropyl) triethoxysilane (APTES)functionalized Fe3O4 nanoparticle (MNP-APTES)

7 nm

Telmisartan (TEL)

No

Prostate cancer cells (PC-3)

Magnetic iron oxide nanoparticles (MIONs)

95%, respectively, in encapsulated matrix after 5h of SGI digestion and available for colon delivery was achieved. Similarly, colon-specific delivery of curcumin was achieved by encapsulation in Eudragit S100, a pH-sensitive polymer as microspheres. Plain curcumin administered orally showed 65% in initial concentration in 2 h in stomach and little in intestine and no curcumin found in colon. Only 28.9% of total curcumin administered reached colon after 8h. Whereas in encapsulated form, more than 80% curcumin delivery in colon was achieved (Jithan et al., 2012). This type of colon-specific delivery system can overcome rapid metabolism in upper GI tract and rapid declination in plasma level and thus increase its bioavailability of nutraceutical compounds. Thus, custom designing of encapsulation matrix is proven to improve the bioavailability and health benefits of nutraceutical compounds.

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Chapter 14 Nanoencapsulation of nutraceutical ingredients

7. Conclusion Nutritional compounds derived from, or part of, natural foods which have proven preventive action and therapeutic benefits against chronic diseases are referred as nutraceuticals. Rising number of chronic diseases and aging population demand development of functional foods and dietary supplements. Although food is rich source of nutrition, the oral bioavailability of nutraceutical compounds is limited by various factors including its liberation from food matrices, instability under GIT, and lower epithelial permeability. Additionally, its sensitivity to light, heat, oxygen, and chemical environment hinders its incorporating in functional foods. Encapsulation of nutraceuticals in suitable matrix at tunable size has evolved as the solution to increase bioavailability and stability of the active compounds. Various techniques available for encapsulation of active compounds are discussed. Encapsulation is proven to enhance temperature, pH, and light stability, solubility, bioaccessibility, and permeability of the nutraceuticals compounds. Range of wall material including biopolymers, carbohydrates, and lipids and formulation at different size (micro and nano) and structure (particles and fibers) are proven to influence the bioavailability of nutraceutical compounds. Although there are many claims in effective protection of nutraceutical compounds by micro- and nanoencapsulation, accessing the behavior of encapsulated molecules in real food processing condition needs to be emphasized for functional food development. Evolving technological developments help in realizing the potential of encapsulated nutraceutical compounds as an alternative to drugs/traditional medicine and in preventive care at commercial scale.

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CHAPTER

Nutraceutical encapsulation and delivery system for type 2 diabetes mellitus

15

Navneet Kumar Dubey1, 2, Abhinay Kumar Singh1, 2, Rajni Dubey3, Win-Ping Deng1, 2, 4 1

School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 2Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 3Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan; 4Graduate Institute of Basic Science, Fu Jen Catholic University, New Taipei City, Taiwan

1. Introduction The diabetes mellitus (DM) is a common, noncommunicable, and chronic metabolic disorder characterized by hyperglycemia. It has been approximated to rise from 6.4% in 2010 to 7.7% in 2030, of which more than 90% will suffer from type 2 diabetes mellitus (T2DM) (Yu et al., 2016). The uncontrolled DM has been reported to exhibit diabetic complications such as cardiomyopathy, retinopathy, and neuropathy (Navneet Kumar Dubey, 2018). During the last century, the lifestyles and food habits have changed dramatically. Specifically, the consumption of diets including junk food had made our body susceptible to DM. Therefore, the management of diabetes through supplying nutritional diet in the form of virtuous food and medicinal supplements popularly known as nutraceuticals is an essential step of controlling not only hyperglycemia but the entire metabolism, leading to inhibition of further associated complications (Kumar Chellappan et al., 2012). These nutraceuticals contain appropriate amount of nourishing food components like vitamins, proteins, carbohydrates, minerals, or essential nutrients, depending on their specific needs, in addition to their pharmaceutical effects. Nutraceuticals are also referred to bioactive phytochemicals that have disease preventing, health promoting, and medicinal properties. Guar gum and oat fiber contain certain type of fibers, which act as nutraceuticals and appear to be significant with respect to insulin resistance, while psyllium also produce a similar result. Coffee is another good source of nutraceutical, which comprises of chlorogenic acid that acts as antioxidant. Based on this kind of properties of nutraceutical, the American Diabetes Association stated that nutraceutical possess tremendous capability to control diabetes (Ames et al., 1993).

2. Encapsulation of nutraceuticals and their characterization Encapsulation is a process to entangle one vital agent among the other substances (i.e., wall material). The encapsulated substances, except active agents, are known as the core, fill, vital, or payload phase. Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00015-1 Copyright © 2020 Elsevier Inc. All rights reserved.

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The substances encapsulated are often called matrix, shell, or coating membrane. Encapsulation is a useful technology to deliver nutraceuticals (i.e., bioactive molecules). In most cases, nutraceuticals are encapsulated and therefore protected from physical barrier, and stability of bioactive molecules is preserved. As a consequence, these molecules retain a property to degrade slowly through encapsulation procedure. Thus, the nutraceutical molecule of functional components kept as fully functional. There are a number of encapsulation techniques available for encapsulation of bioactive molecules (Expo´sito-Molina et al., 2011), which have been discussed below.

2.1 Spray drying Spray drying is a method to encapsulate bioactive molecules. Spray drying is a process to produce a particle by converting its aqueous solution into dry form benefiting from hot gaseous medium. The process of turning liquid into solid product in one step has found broad application in the food, pharmaceutical, chemical, and nanotechnology industries. The spray drying process has been considered as a possible method for drying concentrated brines generated in the space life support system for water recovery (Elena Heras-Ramı´rez et al., 2011). The spray drying of bioactive compounds is often obtained by dissolving, dispersing, or emulsifying the active molecule in a fluid carrier material; thereafter, atomization and spraying of mixture is done in a hot chamber (Fig. 15.1) 1. The spray drying process occurs through mode of convection. The basis of working principle of spray drying is based on the removal of moisture content through applying heat to feed product and maintaining the humid nature of drying medium. The unique property of this technique is

FIGURE 15.1 Illustration of spray drying process.

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stimulated removal of moisture by material spraying into a heated atmosphere leading to an enhanced drying rate. The mechanistic insight of spray drying could be well understood, if the process is divided into its component unit operations. When an aqueous feed enters to spray dryer, it undergoes a sequence of transformation prior to its conversion into a powdered phase. Firstly, aqueous feed is pumped to an atomizer, breaking it into a spray of fine droplets. Secondly, the droplet moisture content vaporized, when they enter into a drying gas unit, resulting in the formation of dry particles. Eventually, by employing a suitable device for segregation of dried particles from the drying state, it is gathered in a tank. There are three conjoint stages, namely atomization, droplet to particle, and collection of particle, which when processed impact the yield of not only spray drying but also the final characteristics of particles. The initiation of spray drying process occurs with atomization of aqueous feed in small droplets owing to decline in surface tension. This step is very important for next steps, particularly while exposition of drying chamber. Infeed atomization with a rotatory atomizer or high pressure nozzle and its operation with a particle impart very low overheated concurrent flow of air. This is crucial when the molecules are sensitive toward heat or somewhat volatile such as aromas. The atomizing droplet size depends on the factors which include pressure drop, surface tension, velocity of the spray, and viscosity of the liquid. The atomized droplet size also decides the particle size and drying time (Vehring, 2008). Spray drying mechanism following the atomization advance with particle formation stage, a critical step marked by episodes which are droplet drying and spray-air contact step, leading to evaporation of the droplets’ solvent content and their transformation into dry form of particle. In drying chamber, atomized droplets pass through a hot gas, following rapid moisture evaporation. The temperature of hot gas initiates a heat exchange from itself to droplets, whereas the vapor pressure difference causes movement of moisture in the opposite direction, as a result, dry form of particles is collected. This intimates a segregation procedure, in which dried particles are distinguished from the drying gas. Generally, this segregation occurs in two phases. In the first phase, the densest particles are recovered at the bottom of drying chamber, whereas, in second phase, the smaller particles are moved to external chamber, where particles are segregated from humid air (Nandiyanto & Okuyama, 2011).

2.2 Spray chilling Similar to spray drying, the spray chilling is also a kind of encapsulation technique, in which particles are solidified through cool air instead of hot air to solvent evaporation. This technique is suitable for encapsulation of thermostable materials. Carriers employed in spray chilling are mostly lipids like triacylglycerols, oils, blends, waxes, and fatty acids of these materials. Mostly, the active molecule is diffused through the molten carrier forming suspensions, in which solidification of droplet occurs, when cooling chamber atomizes the mixtures. Lipid is chosen as a carrier material according to the temperature stability of the active molecules. Organic solvents are not essential for spray chilling; therefore, this is a low-cost technique as compared to others and is easy to scale up (Jin Park, 2005). Various nutraceuticals encapsulated in form of microparticles through spray chilling technique. Some functional ingredients include vitamins, prebiotics, minerals, flavors, and probiotics microorganisms (Favaro-Trindade, 2013).

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Phenolic compounds such as curcumin, resveratrol, and rosmarinic acid are mostly encapsulated into lipid nanoparticle and lipid matrices. Many different types of lipid materials have also been employed as carriers to form microparticles through spray chilling (Sillick & Gregson, 2012). Aspirin and a-tocopherol-like active molecules had been successfully encapsulated in a carrier agent of vegetable oil, respectively. Further, gallic acid and its derivatives which act as natural antioxidants have been applied to prevent rancidity induced by lipid peroxidation in food, cosmetics, and pharmaceuticals (Medina-Torres et al., 2013; Whitman, 2001). Spray drying and complex concerted similar techniques are also used for microencapsulating these compounds.

2.3 Fluidized bed Dried nutraceuticals in form are particulate substances such as granule, crystal, and powder. For processing of these substances, fluidization is usually used in upstream and downstream during which air is driven toward upward along a bed of a particulate at a rate that raises and suspends particles. Fluidization is a method where air is driven upwardly through a bed of a particulate at a rate that raises and suspends particles. These particles are free to drive about and the particle bed acts as an aqueous solution. In many fluidizing systems, most of the air is drawn through the bed, which generates a negative pressure compared to the atmosphere and helps in confining matter during process. Fluidization can be associated with spraying to coat or agglomerate particles to synthesize larger granules. These processes are usually referred as fluid bed coating or granulating processes. In these systems, the process air in addition to fluidize particles removes and evaporates the volatile vehicle of coating or granulating system or solidifies the spray in molten state on the surface of particles. The work of Dr. Dale E. Wurster at the University of Wisconsin in 1950s and 1960s evolved fluid bed coating system, which is commonly being used nowadays. His work disclosed a many of ways to cycle particles through spray area of a nozzle. Further, microencapsulation of products with unpleasant taste has been used for decades. A huge number of nutraceutical compounds are reactive at many regions in the chain of production or have a bad taste during consumption. Further, the protection of water-soluble nutraceutical against moisture is very crucial. In this regard, microencapsulation with hot melt has shown good results. A good example is iron, having a metallic taste, and rusty appearance to the finished product (Huang et al., 2010). It leads to bioavailability of iron in the form of ferrous sulfate, a tremendous candidate for taste masking by microencapsulation. Antioxidants such as ascorbic acid will improve the desired effect, following thermal cycle in food process. Effective microencapsulation will preserve the active compound and get released at right time (Huang et al., 2010). The chemical leavening agents, like sodium bicarbonate, when mixed with the acid component, will react quickly to generate carbon dioxide to maintain porous structure of most of baked foods. Microencapsulation in a fluidized bed process with lipids, proteins, or starch might impart ideal baking characteristics (Gosak et al., 1998). Various nutraceutical compounds are quick responsive to acidic condition in the stomach; therefore, the microencapsulation possesses great potential to preserve these compounds at the time of enteric exposure. Most nutraceutical compounds are difficult to pass through intestinal wall in their active form. Generally, crystalline molecules require lot of energy while their dissolution, as they are unable to be absorbed by intestinal epithelial cells. Majority of nutraceuticals released in the small intestine has been a target. The environment for breakdown of the most of lipids in the upper gastrointestinal tract is excellent and completely breaks the wall of triglyceride-based material and

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assists in the production of micelles for better absorption. The enteric coatings derived from nonlipid polymers, like shellac, must be designed for release by the pH over 6, primarily in the ileum (Boonme et al., 2006). They can only be absorbed in the lower part of small intestine and will not be degraded by enzymatic activity.

3. Potential of nutraceuticals and their delivery for treating T2DM Insulin resistance or impaired secretion of insulin leads to a hyperglycemic condition in case of T2DM. Sometimes, controlling this condition appears to be even after administering high doses of oral antidiabetic agents and insulin. Alteration in signal molecules involved in insulin signaling plays a major role in T2DM (Kadowaki, 2000). In recent years, several major insulin-sensitizing agents have been developed, included like metformin, which exert their therapeutic effect via activation of such molecules involved in insulin signaling pathways. The field of nutraceuticals and their biological activities has been rapidly expanding nowadays as a therapeutic agent for T2DM. In recent years, enormous scientific studies have been conducted on the heterogeneous class of molecules known as a phytochemicals or nutraceuticals. These molecules are widely distributed among vegetables, fruits, beverages, and herbal remedies. Several studies have reported that nutraceutical has potential targets for antidiabetic effect (Mbikay, 2012). Some of the nutraceutical compounds like flavonoids, polyphenolics, tannins are present in most of the edible fruits and vegetables constitute a significant portion of diet and have emerged as potential alternative for treating T2DM by suppressing oxidative stress, through multiple pathways.

3.1 Polyphenolic compounds 3.1.1 Quercetin Our muscle cells play vital role in energy balance by mediating insulin-stimulated uptake of glucose by cells and tissues. Approximately 75%e80% glucose uptake occurs by the stimulation of insulin in T2DM (Bailey & Turner, 2004). Quercetin has demonstrated to stimulate AMPK signaling pathway and increase the glucose uptake by enhancing twofold production of AMP to ADP. AMPK pathway is also activated by mechanism independent of changing ratio AMP to ADP (Hedeskov, 1980). Allosteric modification of AMP, phosphorylation of alpha-subunit by upstream kinase like tumor-suppressor kinase, and calcium calmodulinemediated protein kinase (CaMKK) 50e100 fold activates AMPK pathway. CaMKK is an important signaling molecule involved in activation of AMPK. Quercetin upregulates the gene expression of CaMKK and increases in cytosolic calcium level, which suggest that it is involved in AMPK phosphorylation at Thr-172. Quercetin may cause inhibition of respiratory complexes and might be activated AMPK pathway (Hauge-Evans et al., 1999) (Fig. 15.2). Quercetin has also a significant role in upregulation of mRNA expression level of AMPK and downregulation of P38 MAPK. The mechanistic action of quercetin is similar to resveratrol that promotes translocation of GLUT through the activating phosphorylation of both AMPK and AKT pathways, resulting in stimulation of glucose uptake by skeletal muscles cells (Standl et al., 1999). Quercetin also significantly enhances the intracellular calcium ion levels that participate in quercetininduced insulin secretion. ERK1/2 participated in the regulation of insulin secretion through potentiating ERK1/2 phosphorylation. Increased b-cell apoptosis and its deficit are commonly found in

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FIGURE 15.2 Schematic representing action of quercetin. Quercetin enhances the AMP to ATP which can transit change in mitochondrial membrane potential. There is also association with intracellular calcium level, which resulted in the activation of AMPK and downregulation of P38 MAPK. Further, Akt is also upregulated, which infer that quercetin mechanism of action follow AMPK pathway, which overlap with insulin signaling.

T2DM, which could be restored by quercetin-mediated antiapoptotic effect. These therapeutic potentials of quercetin could not only inhibit the activation of caspase-3/9/12 but also increase the ratio of Bcl-2/BAX and reverse impaired mitochondrial membrane potential. Therefore, the quercetin is a beneficial nutraceutical for the treatment of T2DM (Muniyappa et al., 2008). Alpha-glucosidase is a vital enzyme responsible for digestion of starch. This activity is inhibited by quercetin which leads to hypoglycemic condition in T2DM, which indicate that quercetin could be useful to maintain the blood glucose level in T2DM patients. In diabetic condition, the over production of ROS-mediated oxidative stress and reduced synthesis of the antioxidant molecules occurs, which activate proinflammatory nitric oxide synthase and inactivate antiatherogenic enzyme, prostacyclin synthase (Du et al., 2006). However, the quercetin through its antioxidant potential directly scavenges free radicals and ROS and enhances the levels of antioxidant enzymes like SOD, CAT, and GSH-Px (Hanasaki et al., 1994). Thus, quercetin could suppress oxidative stress, thereby effectively reducing the complications of DM.

3.1.2 Silymarin Silymarin is extracted from a seeds of Silybum marianum and has shown a therapeutic effect on T2DM of human and animals by improving glucose level and insulin secretion and preventing weight loss. This hypoglycemic effect exerted by silymarin occurs via restoring the effect of pancreatic cell function and enhances the production of insulin (Lorber, 2014; Polyak et al., 2013).

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In T2DM, silymarin also control the increased level of low-density lipoproteins, cholesterol, triglycerides, and very lowedensity lipoprotein-C. Chronic hyperglycemic condition impairs the mitochondrial respiratory chain and induced the oxidative damages that are involved in progression of T2DM. However, the treatment with silymarin as a nutraceutical reverses the adverse effect of oxidative stress by normalizing the level of malondialdehyde, oxidative stress index, and total oxidative stress and enhances the status of total antioxidant (Hussain, 2007; Ramachandran et al., 2012).

3.1.3 Resveratrol Insulin action proceed through a series of episodes involving receptor autophosphorylation, in which insulin binds with transmembrane receptor, and phosphorylation of intracellular insulin receptors occurs, leading to activation of effector proteins. Resveratrol treatment promotes glucose transport in insulin-resistant condition of T2DM. This occurs by two-way events associated with GLUT4. Firstly, resveratrol increases the translocation of GLUT4 to the plasma membrane of muscle cells. Secondly, it increases the expression of GLUT4 genes and improves mitochondrial b oxidation and promotes mitochondrial biogenesis (Tan et al., 2012). This results in enhanced fatty acid oxidation and reduced content of intramuscular lipid. Resveratrol also exerts beneficially effects in muscle tissues by changing the expression or activity of two intracellular regulators, the silent information regulator 1 (SIRT1) and AMPK. SIRT1 is involved in many processes such as inflammation, stress resistances, glucose imbalance, mitochondrial biogenesis, apoptosis, and intracellular metabolism (Kim et al., 2011). SIRT1 activity or expression is decreased in T2DM patients, and therefore this enzyme is being considered as target for antidiabetic drug combined with nutraceuticals (Fig. 15.3).

FIGURE 15.3 Indirect and direct actions of resveratrol in increasing the secretion of insulin in type 2 diabetes mellitus.

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Resveratrol also reduces pancreatic triglyceride contents in T2DM. Taken together, the resveratrol possesses numerous beneficial effects such as antihyperglycemic actions by protecting pancreatic b cells in type 2 diabetic condition (Um et al., 2010).

3.1.4 Lipoic acid Lipoic acid could efficiently inhibit the glucose-induced AGE formations in T2DM (Muellenbach et al., 2008). The possible mechanism behind antihyperglycemic activity of lipoic acid is inhibition of AGE formation through a several events which include the following: a. b. c. d.

Preventing glycation with free sugar by blocking amino group of protein, Blocked carbonyl group of reducing sugars, Inhibited synthesis of Amadori product through blocking the Schiff’s base, Blocking Amadori and dicarbonyl intermediates which may reduce glycation, and AGE formation, e. Preventing glycoxidation of Amadori product and autooxidation of glucose (Fig. 15.4). The glycation reaction of protein generates numerous AGE-like pentosidine and cross-links, which are implicated in a T2DM complication. Serum AGEs are higher in T2DM and are also associated with collagen, extracellular matrix protein. Therefore, preventing the formation of AGE is an efficient way to suppress the potential consequences of diabetic complications through inhibiting the glycation cascade (Navneet Kumar Dubey, 2018; Sun et al., 2012). Lipoic acid has been reported to significantly inhibit the formation of Amadori product, i.e., fructosamine in a time-dependent fashion either through competing with sugar moiety or rescuing protein amino groups from the nucleophilic addition of carbonyl group of sugar molecules (Chen et al., 2012). The reaction between protein and carbonyl group from freely reversible Schiff’s base generates a stable ketoamine or fructosamine. In such condition, reducing sugar autooxidizes itself in the presence of transition metal and generates various kinds of highly reactive superoxide free radicals and hydroxyl radicals. These deleterious radicals further accelerate the glycation process to form AGE. In addition, fructosamine also generate AGE after reacting with proteins. Thus, decrease in formation of fructosamine would be beneficial for suppression of AGE production, and in therapeutic prevention of T2DM and their complications. FIGURE 15.4 Biological functions of lipoic acid.

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Furthermore, lipoic acid suppressed iron release from heme molecules of myoglobin during the myoglobineglucose glycation. This “mobile reactive iron” could catalyze HabereWeiss reaction in which free radicals such as hydroxyl radicals release and increase cellular oxidative stress (Ziegler & Gries, 1997). Lipoic acid possesses antioxidant properties which are attributed to various factors, including capacity to directly scavenge ROS by reviving endogenous antioxidants, like vitamin E, C, and glutathione. These antioxidant properties of a-lipoic acid have recently been documented in protecting oxidative injuries among various diseases, including neurodegenerative disorders (Evans & Goldfine, 2000). The a-lipoic acid has also exerted beneficial effects in the treatment of diabetes due to its insulinemimetic and antiinflammatory action (Ziegler et al., 2006). Lipoic acid decreases the activity of the protein kinase activated by the AMP (AMPK) and acts as a sensor in the activated cell with reduced energy. The activation of hypothalamic AMPK alters the effects of lipoic acid during intake of food and release of energy. 2-Deoxyglucose-induced hyperphagia is reversed by the inhibition of AMPK. The hypothalamic AMPK plays a key role in the regulating food intake and energy release; in addition, lipoic acid exhibits antiobesity properties through suspension of such activity in the hypothalamic AMPK (Thirunavukkarasu et al., 2004).

4. Conclusion and future prospective Encapsulation of nutraceuticals increases their stability and bioavailability, with help of matrix molecules and various kind of encapsulation technique. This technique preserves the matrix molecules without influencing the molecular structure of bioactive compound and serves as vehicle to target site, where it has to be delivered. In target delivery system, this kind of novel approach is important to employ different enzyme concentration and bioactivity in various part of gut. As compared to pH or time-dependent approach, this technique seems to be more successful. It is highly versatile in nature, as the encapsulating molecule could be applied as dry product or hydrogel. Most of the encapsulation techniques act via on pH or time-dependent release of their content in gastrointestinal tract due to variations in intestinal pH and transit time. This kind of obstacles could be overcome by using matrix molecule that withstand the environment changes in the gastrointestinal tract and reach to their target sites. Overall, the utility of encapsulation of nutraceutical or its analogue seems to be highly useful for prevention of T2DM in humans.

References Gosak, D., Hraste, M., Jalsenjako, I., 1998. Fluid-bed microencapsulation of ascorbic acid. AU - Knezevic, Z Journal of Microencapsulation 15 (2), 237e252. https://doi.org/10.3109/02652049809006853. Ames, B.N., Shigenaga, M.K., Hagen, 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 (17), 7915e7922. Bailey, C.J., Turner, S.L., 2004. Glucosamine-induced insulin resistance in L6 muscle cells. Diabetes, Obesity and Metabolism 6 (4), 293e298. https://doi.org/10.1111/j.1462-8902.2004.00350.x. Boonme, P., Krauel, K., Graf, A., Rades, T., Junyaprasert, V.B., 2006. Characterization of microemulsion structures in the pseudoternary phase diagram of isopropyl palmitate/water/Brij 97:1-butanol. AAPS PharmSciTech 7 (2), E45. https://doi.org/10.1208/pt070245.

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Chen, W.L., Kang, C.H., Wang, S.G., Lee, H.M., 2012. Alpha-Lipoic acid regulates lipid metabolism through induction of sirtuin 1 (SIRT1) and activation of AMP-activated protein kinase. Diabetologia 55 (6), 1824e1835. https://doi.org/10.1007/s00125-012-2530-4. Du, X., Edelstein, D., Obici, S., Higham, N., Zou, M.H., Brownlee, M., 2006. Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation. Journal of Clinical Investigation 116 (4), 1071e1080. https://doi.org/10.1172/JCI23354. Elena Heras-Ramı´rez, M., Quintero-Ramos, A., Camacho, A., Barnard, J., Talama´s-Abbud, R., Vinicio TorresMun˜oz, J., Salas-Mun˜oz, E., 2011. Effect of Blanching and Drying Temperature on Polyphenolic Compound Stability and Antioxidant Capacity of Apple Pomace, vol. 5. Evans, J.L., Goldfine, I.D., 2000. Alpha-lipoic acid: a multifunctional antioxidant that improves insulin sensitivity in patients with type 2 diabetes. Diabetes Technology & Therapeutics 2 (3), 401e413. https://doi.org/10.1089/ 15209150050194279. Expo´sito-Molina, I., Reineccius, G.A., Lo´pez-Herna´ndez, O., Pino, J.A., 2011. Influence of spray-dryer air temperatures on encapsulated Mandarin oil. AU - Bringas-Lantigua, Madai Drying Technology 29 (5), 520e526. https://doi.org/10.1080/07373937.2010.513780. Favaro-Trindade, C., 2013. Technological Challenges for Spray Chilling Encapsulation of Functional Food Ingredients, vol. 51. Hanasaki, Y., Ogawa, S., Fukui, S., 1994. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radical Biology and Medicine 16 (6), 845e850. Hauge-Evans, A.C., Squires, P.E., Persaud, S.J., Jones, P.M., 1999. Pancreatic beta-cell-to-beta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2þ and insulin secretory responses of MIN6 pseudoislets. Diabetes 48 (7), 1402e1408. Hedeskov, C.J., 1980. Mechanism of glucose-induced insulin secretion. Physiological Reviews 60 (2), 442e509. https://doi.org/10.1152/physrev.1980.60.2.442. Huang, Q., Yu, H., Ru, Q., 2010. Bioavailability and delivery of nutraceuticals using nanotechnology. Journal of Food Science 75 (1), R50eR57. https://doi.org/10.1111/j.1750-3841.2009.01457.x. Hussain, S.A., 2007. Silymarin as an adjunct to glibenclamide therapy improves long-term and postprandial glycemic control and body mass index in type 2 diabetes. Journal of Medicinal Food 10 (3), 543e547. https:// doi.org/10.1089/jmf.2006.089. Jin Park, H., 2005. Recent developments in microencapsulation of food ingredients. AU - Desai, Kashappa Goud H Drying Technology 23 (7), 1361e1394. https://doi.org/10.1081/DRT-200063478. Kadowaki, T., 2000. Insights into insulin resistance and type 2 diabetes from knockout mouse models. The Journal of Clinical Investigation 106 (4), 459e465. https://doi.org/10.1172/JCI10830. Kim, S., Jin, Y., Choi, Y., Park, T., 2011. Resveratrol exerts anti-obesity effects via mechanisms involving downregulation of adipogenic and inflammatory processes in mice. Biochemical Pharmacology 81 (11), 1343e1351. https://doi.org/10.1016/j.bcp.2011.03.012. Kumar Chellappan, D., Lakshmana Prabu, S., Timmakondu, S., SureshKumar, S., T, R., 2012. Nutraceuticals: A review 46. Lorber, D., 2014. Importance of cardiovascular disease risk management in patients with type 2 diabetes mellitus. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 7, 169e183. https://doi.org/10.2147/ DMSO.S61438. Mbikay, M., 2012. Therapeutic potential of moringa oleifera leaves in chronic hyperglycemia and dyslipidemia: a review. Frontiers in Pharmacology 3 (24). https://doi.org/10.3389/fphar.2012.00024. Medina-Torres, L., Garcı´a-Cruz, E.E., Calderas, F., Gonza´lez-Laredo, R., Sanchez-Olivares, G., GallegosInfante, J., Ramı´rez, J., 2013. Microencapsulation by Spray Drying of Gallic Acid with Nopal Mucilage (Opuntia ficus Indica), vol. 50.

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Muellenbach, E.A., Diehl, C.J., Teachey, M.K., Lindborg, K.A., Archuleta, T.L., Harrell, N.B., Henriksen, E.J., 2008. Interactions of the advanced glycation end product inhibitor pyridoxamine and the antioxidant alphalipoic acid on insulin resistance in the obese Zucker rat. Metabolism 57 (10), 1465e1472. https://doi.org/ 10.1016/j.metabol.2008.05.018. Muniyappa, R., Lee, S., Chen, H., Quon, M.J., 2008. Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. American Journal of Physiology. Endocrinology and Metabolism 294 (1), E15eE26. https://doi.org/10.1152/ajpendo.00645.2007. Nandiyanto, A., Okuyama, K., 2011. Progress in Developing Spray-Drying Methods for the Production of Controlled Morphology Particles: From the Nanometer to Submicrometer Size Ranges, vol. 22. Navneet Kumar, D., H.-J, W., Sung-Hsun, Y., David F, W., Joseph R, W., Yue-Hua, D., Feng-Chou, T., Peter D, W., Win-Ping, D., 2018. Adipose-derived Stem Cells Attenuates Diabetic Osteoarthritis via Inhibition of Glycation-Mediated Inflammatory Cascade. 0-. https://doi.org/10.14336/ad.2018.0616. Polyak, S.J., Ferenci, P., Pawlotsky, J.M., 2013. Hepatoprotective and antiviral functions of silymarin components in hepatitis C virus infection. Hepatology 57 (3), 1262e1271. https://doi.org/10.1002/hep.26179. Ramachandran, S., Rajasekaran, A., Manisenthilkumar, K.T., 2012. Investigation of hypoglycemic, hypolipidemic and antioxidant activities of aqueous extract of Terminalia paniculata bark in diabetic rats. Asian Pacific Journal of Tropical Biomedicine 2 (4), 262e268. https://doi.org/10.1016/S2221-1691(12)60020-3. Sillick, M., M. Gregson, C., 2012. Spray Chill Encapsulation of Flavors within Anhydrous Erythritol Crystals, vol. 48. Standl, E., Baumgartl, H.J., Fuchtenbusch, M., Stemplinger, J., 1999. Effect of acarbose on additional insulin therapy in type 2 diabetic patients with late failure of sulphonylurea therapy. Diabetes, Obesity and Metabolism 1 (4), 215e220. Sun, L.Q., Chen, Y.Y., Wang, X., Li, X.J., Xue, B., Qu, L., Lu, J.M., 2012. The protective effect of alpha lipoic acid on Schwann cells exposed to constant or intermittent high glucose. Biochemical Pharmacology 84 (7), 961e973. https://doi.org/10.1016/j.bcp.2012.07.005. Tan, Z., Zhou, L.J., Mu, P.W., Liu, S.P., Chen, S.J., Fu, X.D., Wang, T.H., 2012. Caveolin-3 is involved in the protection of resveratrol against high-fat-diet-induced insulin resistance by promoting GLUT4 translocation to the plasma membrane in skeletal muscle of ovariectomized rats. The Journal of Nutritional Biochemistry 23 (12), 1716e1724. https://doi.org/10.1016/j.jnutbio.2011.12.003. Thirunavukkarasu, V., Anitha Nandhini, A.T., Anuradha, C.V., 2004. Effect of alpha-lipoic acid on lipid profile in rats fed a high-fructose diet. Experimental Diabesity Research 5 (3), 195e200. https://doi.org/10.1080/ 15438600490486778. Um, J.H., Park, S.J., Kang, H., Yang, S., Foretz, M., McBurney, M.W., Chung, J.H., 2010. AMP-activated protein kinase-deficient mice are resistant to the metabolic effects of resveratrol. Diabetes 59 (3), 554e563. https:// doi.org/10.2337/db09-0482. Vehring, R., 2008. Pharmaceutical particle engineering via spray drying. Pharmaceutical Research 25 (5), 999e1022. https://doi.org/10.1007/s11095-007-9475-1. Whitman, M., 2001. Understanding the perceived need for complementary and alternative nutraceuticals: lifestyle issues. Clinical Journal of Oncology Nursing 5 (5), 190e194. Yu, S.H., Dubey, N.K., Li, W.S., Liu, M.C., Chiang, H.S., Leu, S.J., Deng, W.P., 2016. Cordyceps militaris treatment preserves renal function in type 2 diabetic nephropathy mice. PLoS One 11 (11), e0166342. https:// doi.org/10.1371/journal.pone.0166342. Ziegler, D., Gries, F.A., 1997. Alpha-lipoic acid in the treatment of diabetic peripheral and cardiac autonomic neuropathy. Diabetes 46 (Suppl 2), S62eS66. Ziegler, D., Ametov, A., Barinov, A., Dyck, P.J., Gurieva, I., Low, P.A., Samigullin, R., 2006. Oral treatment with alpha-lipoic acid improves symptomatic diabetic polyneuropathy: the SYDNEY 2 trial. Diabetes Care 29 (11), 2365e2370. https://doi.org/10.2337/dc06-1216.

CHAPTER

Pickering emulsions stabilized by nanoparticles

16 Soma Mukherjee

Department of Veterinary Medicine School, Mississippi State University, Mississippi State, MS, United States

1. Introduction Pickering emulsions are prepared without surfactants and stabilized with solid particles (Aveyard et al., 2003; Binks, 2003; Binks and Horozov, 2006). This possess the basic characteristics of classical emulsions and any type of emulsions either water-in-oil (w/o), oil-in-water (o/w) or multiple emulsions can be prepared with this formulation. The stabilization with solid particles attributed the most positive benefit of high resistance to coalescence. The surfactant-free characteristics made this type of emulsions attractive for the use of the cosmetic and pharmaceutical industry because these do not cause any adverse effects such as irritancy and hemolytic behavior. The solid stabilizing particles need to be very small particles and smaller than oil/water formed droplet. Smaller insoluble particles of submicron size can stabilize micromillimeter size droplets of emulsions as well as few millimeter droplets to larger droplets of emulsions. Millimeter-sized droplet stabilization is a supplementary benefit compared to the classical emulsion formulations with a surfactant. The higher sized droplet stabilization occurs due to high stability against coalescence. The stabilization of emulsions with surfactants takes place due to absorption of solid particles at the surface or droplets of emulsions. Basic difference with oil and water emulsion and Pickering emulsion is in Pickering emulsion the solid particle need not be amphiphilic. Just the outer surface would show some wet ability causing a stable docking of insoluble particles at the space within oil and water phase.

2. History of pickering emulsion S.U. Pickering first reported this type of emulsion in his paper (Pickering, 1907), and Pickering emulsions are named after his name. Actually, Ramsden (Ramsden and Gotch, 1903) reported the absorption of solid particles (Pickering cited his name in this paper) in the water interface causing foaming, and he recovered insoluble materials forming a solid layer at the exterior part of the liquid. He mentioned in his paper that solid particles focusing an organic particle named as proteins reorganizes after alignment or docking at the two immiscible phases of oil and water. These particles cannot be considered as real solid particles rather it was treated as pliable or delicate insoluble particles. It was claimed that these particles have a potency to be absorbed in the oilewater interface but could not establish his speculative theory with experimental proof. Pickering established this theory by Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00016-3 Copyright © 2020 Elsevier Inc. All rights reserved.

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stabilizing paraffin oil and insoluble materials at the interface. He also explained the physiology of this emulsion prominently with evidence of the absorbed particles at the same location and showed better stability over surfactant-based emulsions. Although Pickering’s paper showed better stability and also he reported that other solid particles can be stabilized using the same technique, it was not been used or reported by other researchers for a long period of time. Till the 18th century, a few isolated papers of academic interest have been published (Newman, 1914; Schlaepfer, 1918; Moore, 1919; Briggs, 1921; Scarlett et al., 1027; Schulman and Leja, 1954). Long-term projects of Pickering emulsions (Levin and Sanford, 1985; Levine et al., 1989; Tambe and Sharma, 1994) were launched by several researchers. In 1983’s edition of Encyclopedia of Emulsion Technology, this technology has been discussed but it could not catch attention to the mass (Lucassen-Reynders and Temple 1963; Rousseau, 2000). At the same time, some researchers discovered that silica particles, when surround water droplets act as solid spherical balls carrying 95% of water (Rousseau, 2000). During 2004, Degussa company commercialized water-in-air emulsion under the trade name “dry water” (Chevalier and Bolzinger, 2013). Apart from this, many emulsion formulations have been discovered, which follows the same theory, but failed or did not acknowledge Pickering emulsion. As for example, in food emulsion formulation with solid fat crystals has been seen to be adsorbed at the surface of emulsion droplet (Rousseau, 2000). Material scientists also reported polymer particles with attached inorganic particles at their surface and similarly did not mention about Pickering emulsion. Pickering emulsion issues mostly have been addressed while crude oil demulsification process was going on. This process was difficult because the oil-in-water emulsion was restored by solid particles, and also several polymeric particles act as surfactants. Two other soluble and insoluble compounds present in crude oil are maltenes and asphaltenes, respectively. In several cases, crude oil emulsion is Pickering emulsion where solid nanosized particles of waxes and asphaltenes are adsorbed at the surface of a water droplet (Schorling et al., 1999; Spiecket et al., 2003). Because of the highly resistant nature of coalescence in Pickering emulsion, the demulsification process showed technical difficulties and for this reason, this field of research caught attention to the emulsion scientists all over the world.

3. Physical chemistry of pickering emulsion The major objectives to prepare Pickering emulsion successfully followed the same rule as another traditional emulsion. The major criteria are to stabilize emulsion against destabilizing events such as coalescence, coagulation, and Ostwald ripening. The formulation process and design are the most important factors that determine the stability of the emulsion system. The physical chemistry of Pickering emulsion includes adsorption of particles, droplet stabilization of adsorbed particles, kinetics of emulsification process, and the rheological properties of the emulsion.

3.1 Globule size and formation criteria In Pickering emulsion, insoluble materials can form a compact single layer, and the interfacial area between two immiscible phases covered by the solids (insoluble material) is determined by the amount of solids. The geometrical parameter of the solids such as droplet diameter and mass ratio establishes the successful formulation of the dispersed phase:

3. Physical chemistry of pickering emulsion

Diam ¼

6 MðoilÞ  roil asolid MðsolidÞ

367

(16.1)

where roil represents density of oil, asoild is the space between two phases occupied by per mass of the insoluble particle. This type of relationships has often been confirmed by the experiments (Arditty et al. 2004, 2005). The mechanism of stabilized emulsions may not be satisfied by Eq. (16.1). Indeed, there are many reports of which describe microscopic pictures depicting incomplete adsorption area of the droplet covered by the solid particles (Binks and Kirkland, 2002). There is also report of the association of the insoluble materials at their globular exterior surface. Another geometric relationship has been given by Frelichowska et al. relating an insoluble particle to the fraction of material associates formed by the solids. The high ration of M (oil)/M(solid) also deviates from Eq. (16.1). In the case of microemulsion, a small amount of solid particles forms very large droplets as compared to nanoemulsion, and limited covered space between two immiscible phases becomes steady and can sustain their integrity. Formation of nanoemulsion usually requires high energy and splits apart large droplet into minute nanosized droplets but once the energy is switched off the droplets start to coalescence. Coalescence process may stop if the interfacial area expands the corresponding area engaged by the particles (Arditty et al., 2003). Partial coalescence occurs even emulsion is steady in the course of maturing. Partial emulsification results unbound oil release and dissociation stops. Very fine emulsions can be prepared with a high amount of solid particles following very high efficient emulsification process. When the emulsification process cannot create sufficient interfacial space that the insoluble particles can occupy, partial adsorption of insoluble particles results and the unabsorbed particles are dispersed as suspended materials in the dissociation phase of the emulsification process (Chevalier and Bolzinger 2013). Pickering emulsions are formed of three distinct phases. In the first phase, the emulsification process basically fails with limited insoluble material. In the second phase, the ratio of Msoild and Moils determines the droplet or globule size according to Eq. (16.1). Finally, in the third phase, the emulsification process reaches with high ratio of Msoild and Moils. Interface and particle adsorption: Adherence of insoluble nanoparticles at the interface needs partial hydrophilic interaction of the insoluble materials either by one of the liquid phase. Three types of interfacial energies are formed: solidewater (gsw ), soildeoil (gs0 ), and oilewater (gow ). When the adhesion energy of water is positive, wetting of the solid by water partially happens. Positive adhesion energy EAdh (w/o) of water results in partial hydration of the delivered insoluble material with water and also requires spreading coefficient, S (w/o) to be negative: EAdh ðW = OÞ ¼ gso þ gow > 0 ¼ gso þ gow > 0 Sðw=oÞ ¼ gso  gow  gsw < 0

(16.2) (16.3)

An equivalent point is established between positive adhesion energy that can result in incomplete hydration of the insoluble material and spreading coefficient S (o/w) shows negative: EAdh ðW = OÞ ¼  SðW = OÞ ¼ gso  gow  gsw < 0   W S ¼  EAdh ðW = OÞ ¼ gso  gsw < 0 O

(16.4) (16.5)

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Chapter 16 Pickering emulsions stabilized by nanoparticles

The extremely hydrophilic exterior face of the insoluble materials is entirely wetted by water; as a result, solid particles cannot adsorb and remain suspended or dispersed in the aqueous phase. Two hydrophobic particles are fully wetted by oil. The contact angle of water (qw Þand oil (qo Þat the partial wetting condition follows Yang’s Law (qo ¼ p  qw Þ: g  gsw Cos ðqw Þ ¼ so gwo (16.6) g  gso Cosðqw Þgow ¼ sw gwo Partial wetting condition facilitates wetting of the solid particles and thus solid particles adsorb strongly. Interfacial tension and volume of insoluble globules are determinants of the free energy of adsorption. The free energy of adsorption of insoluble delivered globule with radius R is as follows: Dads F ¼  pR2 gow ð1  cosðqw Þ for qw < 90



Dads F ¼  pR2 gow ð1 þ cosðqw Þ for qw > 90



(16.7) (16.8)

The contact angle, 90 degrees, shows the maximum adsorption of solid particles and also confers the highest stability of the emulsion in most formulations (Binks and Lumsdon, 2000). Adsorption free energy is high for large particles as contacting area of oil and water is high. Nanoparticles strongly adsorb to the oil and water interface. According to the equation, theoretically no minimum limit for droplet size because free energy formation always exceeds thermal energy, so particle size does not have any impact on this free energy equation. As example, for a spherical solid particle size of 5 nm radius (2  5 diameter), having a 90-degree contact angle, the adsorption free energy is 1.6  1017 J, which is basically much higher than kT (4 1021 J at 293K). Furthermore, the diameter of 1 nm, which is considered as the lower limit of nanoparticle size, gives adsorption energy excessively larger than its thermal energy. The adsorption energy that  is calculated by the interfacial area and covered by the adsorbed particle per unit area is Dads F pR2 , and this is independent of particle size. Additionally, many measurements in experiments showed surface inactivity of the solid particles (Vignati et al., 2003; Dong and Johnson, 2003).

3.2 Stabilizing particles Pickering emulsions have been stabilized by numerous organic and inorganic particles. These particles can be partially wetted by commonly used oils. Most commonly used particles are as follows: calcium carbonate and barium sulfate (Levine et al., 1989), morilonite and laponite (Levine et al., 1989; Ashby and Binks, 2000; Abend and Lagaly, 2001), magnetic particles (Melle et al., 2005), carbon blocks and nanotubes (Wang and Hobby, 2003), latex (Binks and Lumsdon, 2001), and block copolymer like micelles (Laredj-Bourezg et al., 2012). Stabilized Pickering emulsion has been formulated using some less commonly used particles such as cationic nanocrystals (Schelero et al., 2009), bacterial spores, or bacteria (Dorobantu et al., 2004; Wongkongkatep et al., 2012). Adding some supplementary properties such as temperature sensitivity in the emulsion has been successfully introduced by incorporating temperature sensitive polymer (N-isopropyl acrylamide) (Ngai et al., 2005; Brugger and Richtering, 2007; Tsuji et al., 2008; Monteux et al., 2010; Destribats et al., 2012). In the same way, pH sensitive, Pickering emulsions have been prepared using pH sensitive particles (Fujii et al., 2005; Gautier et al.,

3. Physical chemistry of pickering emulsion

369

2007). Finally, Pickering emulsions have been successfully prepared with proteins claiming protein as a solid particle. Protein stabilized emulsions look similar to monolayer-adsorbed proteins, and also no modification of the structure of the protein like cross-linking has been observed in formulating Pickering emulsion of solid protein particles (Fujii et al., 2009). Several of these particles represented submicron-sized (diam ¼ 01e1 mm) particles and successfully stabilizes. The two most two wellknown examples are latex and fumed silica. Sometimes too hydrophilic inorganic particles require incomplete hydrophobic coasting to achieve incomplete hydration by liquid phase. Silica is an ideal example of an inorganic stabilizing agent for formulating Pickering emulsion. The exterior phase of the insoluble materials can be modified to enhance its hydrophobicity. Partial wetting can be reached by surface modification of some particles of interest. Several strategies are involved to modify the surface of solid particles. Two important strategies are chemical grafting of organic molecule or adsorption of large-sized molecules such as carbohydrate, fat, or protein molecule. Chemical grafting is more stable because grafting is stronger and molecules are tightly attached by forming chemical bonds. On the other hand, in adsorption, the adsorbed particles are in equilibrium with loosely attached particles and a large portion of the phase can go through deadsorption under any change of pH, dilution, and the addition of oil or water. However, in chemical grafting, surface modification occurs due to the reaction of the surface group of the solid surface. The aqueous medium is not suitable for molecular grafting. In most cases, these need to be performed in dry power of solid and suspended in a dry organic solvent to avoid this limitation or constraint. The most chemical reaction cannot be performed in an aqueous medium. Chemical bonds need to be stabilized against hydrolysis. Exclusion of residual molecules and unchanged molecules is quite difficult. Adsorption of solid particles can be performed without any constraint before emulsification. Different types of molecules have been incorporated for preparation of Pickering emulsion, which includes surfactants and similar minute molecules, polymers, and ions with more than one valence. Surfactants allow transformation of hydrophilic into the hydrophobic surface, and thus solids exhibit partial wetting condition, which is the required condition of absorbability of solid particles in Pickering emulsion (Gelot et al., 1984; Hassander et al., 1989; Binks and Whitby, 2005; Drelich et al., 2010; Cui et al., 2011). Surfactant (cationic and anionic) allows adsorption of solids by electrostatic binding of oppositely charged sites of the solid materials (Binks et al., 2007; Binks and Rodrigues, 2003). Residual surfactant ensures adsorption equilibrium in the aqueous phase and also takes part in the interfacial adsorption. As a matter of fact, the measurement of the individual contribution of adsorbed insoluble molecule and surfactant in the complete emulsification process in contributing stability is tough. Use of phospholipid like lecithin is a smart choice as these are insoluble in water and oil, and they may only be adsorbed at oilewater interface (Tan et al. 2007; Eskander et al., 2011) (Fig. 16.1). Another alternative approach is the application of surfactant like organic particles (hydrotrophs). They are not surface active at the reaction site and also can turn the surface more hydrophobic (Midmore 1998, 1999). However, researchers are not sure till now about the mechanism of alteration of exterior part of droplets and union of insoluble particles due to polymer cross-linking. After a long effort, Pickering emulsion has been stabilized by multivalent ions of oppositely charged particles with respect to the rigid outer exterior part. Flocculation of the insoluble materials possibly occurred due to induced charge neutralization at the reaction site. Many manufacturers (Woker, Avonik and Cabot) made silica partially hydrophobic that are called fumed silica by grafting with organosilanes. During the grafting process with dichlorodimethysilane and hexamethyldisilazane, hydrophobic dimethylsilyl group is attached to the silica particle.

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Chapter 16 Pickering emulsions stabilized by nanoparticles

FIGURE 16.1 Freeze fracture-scanning electron micrography of paraffin water emulsions without (A) silica nanoparticles in the oil (B) and water (C) phases. Adopted from Ghauchi et al. (2011) with permission. Copy right 2011, Elsevier.

The degree of grafting depends on the surface covered by the left or attached dimethylsilyl group, which imparts hydrophobicity to the solid silica particles and its wet ability in oil or water. These fumed silica particles are a small nanosized particle, which is permanently stuck together by during synthesis. These unbreakable molecules are 100 nm sized and considered as submicron particles rather than nanoparticles. Similarly, titanium dioxide is synthesized by molecular grafting. Silica particles cannot be stabilized in emulsion with commonly used oils, only Pickering emulsions can be stabilized with bare silica in polar oils (Frelichowska et al., 2009a,b). However, these polar oils with limited solubility in water separated two phase coexisting together under operative condition. However, finally, successful Pickering emulsion has been made with bare silica lowering the interfacial tension below 15 m/Nm.

3. Physical chemistry of pickering emulsion

371

3.3 Stable emulsion formation by adsorbed solid particle Phase separation occurs in two consecutive phases: coagulation and coalescence. Modification of the exterior part forming a layer with insoluble molecules can prevent coalescence. A rigid coating formation around the droplet formed of insoluble molecules make a protective barrier for each droplet and also forms a colloidal suspension that actually prevents flocculation. This mechanical barrier is formed due to rearrangement of exterior insoluble materials and thus droplets cannot unite together. Gathering or uniting of the insoluble molecules at the surface of droplet increases the mechanical strength. Insoluble molecules are united in the interface due to strong cooperation creating a force of forming a strong coating. To be very precise, capillary dynamism are distinct from each droplet at the interface. Stability of the Pickering emulsion is influenced by all type of interaction that involves dispersion, electrostatic . and overall summation of all interactive forces. The forces that are not interfacial also can influence by their presence in oil and water interface contributing to emulsion stability. Charged particles contribute electrostatic repulsion and maintain dielectric continuity between oil and water. Behaviors of electrostatic repulsion do not follow the general rule instead show some peculiar behaviors that are not the behavior of dielectric continuity (Leunissen et al., 2007; Wang et al., 2012). Coagulation process is also contributed by solid particles. Electrostatic repulsion forces between droplets contributed by charged particles behave the same as ionic particles do. Particle bridges formed between droplets prevent than to join together and thus stops coagulation. Electrostatic repulsion mechanism and formation of the thick continuous phase of the emulsion mostly contribute to the stability. A group of scientists reported the creation of electrostatic repulsion of the connecting system of united clay molecules. This has been confirmed by X-ray microscopy study (Thieme et al., 1999; Neuhausler et al., 1999).

3.4 Parameters of different types of emulsions Partial hydration of the insoluble adsorbed molecules determines the emulsion type. In the traditional emulsion, hydrophilic emulsifiers determine the type of emulsion. Hydrophilic emulsifiers drive the emulsification process towards o/w emulsion type by orienting the surface interactive forces and hydrophobic emulsifiers do the same to w/o emulsion. In the case of Pickering emulsion, hydrophobic particles produce o/w emulsions. Contact angle q (Fig. 16.2) of emulsion below 90 degrees determines o/w emulsion. Conversely, above qw 90 degrees determines w/o emulsion. It specifically follows the Bancorft rule of emulsifiers. Hydrophilicelipophilic balance (HLB) value scale had been proposed by Kruglyakov on the basis of Bancroft’s rule (Kruglykov, 2000; Kruglykov et al., 2004). HLB value is calculated on the HLB, which is a fractional ratio of the hydrophobic part to the hydrophilic part of surfactant. The maximum durability of the emulsification of the w/o emulsion ranges between 3 and 6 and o/w is between 9 and 15. The value near 8, either any type (w/o or o/w), is stable. The value of contact angle near 90 degrees solid particles can be stabilized in Pickering emulsion regardless of their wetting properties. Relative water and oil content in initial emulsion formulation, as well as wetting behavior, enhance phase inversion of the emulsion. In surfactant-based emulsion, phase inversion is not very common; however, Pickering emulsion shows a strong inclination towards phase inversion (Binks and Rodrigues, 2003).

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Chapter 16 Pickering emulsions stabilized by nanoparticles

FIGURE 16.2 Contact angle, ɵ, in water on a even surface (left: an oil droplet adhered to A silica particle) and the surface of an immiscible particle (right: adsorbed by incomplete hydration at the oilewater junction. Adopted from Frelichowska et al. (2009a,b) with permission. Copy right 2009, Elsevier.

More than one type of emulsions can be created when more than one type of emulsions are mixed together in one droplet. Hydrophobic solid particles play an important role in stabilizing multiple emulsions internally with w/o droplets whereas external surface is stabilized by hydrophilic solid particles. In multiple emulsion formulation, the diffusion of hydrophobic and hydrophilic surfactants disrupts the emulsion triggering the destabilization process in internal and external droplets. Strong adsorbed solid particles at the interface remain stable and facilitate the fabrication of multiple emulsions: the classical example of stabilized primary emulsion with dimethylsilyl group grafted on silica solid particle (covered 49%) and medium chained triglycerides. This primary emulsion is again dispersed in water using hydrophilic silica particles fabricated with dimethylsilyl with coverage of 21% (Barthel et al. 2003).

3.5 Unbound particles and consequence of rheology The concentration of surfactant in emulsion formulation ranges from 0.1% to 1%. An excess amount of surfactant does not take part to cover the entire droplet. As a result, the surplus amount of surfactant remains in the water phase. This residual concentration takes part to reach equilibrium to be taken up to the exterior part of the droplet. In Pickering emulsion, partial wetting condition creates high affinity for the interface stabilizing solid particle adsorbing entirely. No remaining insoluble molecules are left for the second regime (Frelichowska et al., 2010). In the latter regimen, only droplet size is determined by the concentration of the stabilizing particles. Excessive particle imparts thickening of the emulsion in the latter phase of emulsification. Partially wet solid particles with hydrophobic surface undergo aggregation. Thus fumed silica particles that are often used as a stabilizer in formulating Pickering emulsion now commercially sold as a thickener (Barthel, 1995). Thickening of the emulsions is important because it increases the viscosity of the emulsions and in turn reduces the destabilization phenomena. As a result stabilized emulsions can be used for practical utilities. Thickening imparts stability to the emulsion. Moreover, in very highly efficient cases, emulsions are gelled, which confers long-term stability of the emulsion. Two types of solid particles can heterogeneously coagulate. This was explained by Abend and Lagaly as a percoagulate connecting system of united insoluble molecules that were partially absorbed to the oil of the droplet in emulsion (Abend and Lagaly, 2001; Abend et al., 1998; Thieme et al., 1999, Neuhausler et al., 1999). A potential advantage of gelation of the emulsion is the

3. Physical chemistry of pickering emulsion

373

prevention of destabilization events and gelation also holds Pickering emulsion efficiently (Binks and Lumsdon, 2000). The interesting fact of the fate of residual solid particles is that its flocculation is adopted by the physicochemical parameters and it also controls the consistency of the entire system. As for example, residual silica molecules tend to aggregate, but the negative electrical power of the remaining silanol group produces electrostatic repulsion force and stabilizes the emulsion. Addition of electrolytes like sodium chloride reduces electrostatic repulsion and causes aggregation but increases the viscosity. Above the concentration threshold (2 104 mol L1 NaCl) for a particular emulsion noticeable change in flow rate behavior (increasing viscosity, yield stress, and elasticity) have been reported. At above the threshold, the elastic modulus G0 increases and as a result creaming process slows down. In the liquid-based emulsion, below the ionic strength threshold globules with oil can hold insoluble molecules and unbound surplus solids are scattered in the water phase. Dense and bulky emulsion with relatively higher viscosity electively holds all solid particles as all are aggregated together with oil droplet forming microsized droplet with a size range of 2e5 mm. Supplementary stabilization is contributed by thickening of the emulsion and strong thickening and elastic behavior (Fig. 16.3). Flow is noticeable when only more pressure is created to break the particles to exceed yield tension and the entire connecting system is broken due to high pressure. Calculative selections of ionic strength can control yield stress and successfully can maintain the stabilization property as well as practical applicability. As the network of the flocculated solid critically stabilizes the emulsion, disruption of the gel applying shear force may cause coalescence of the emulsion system (Whitby et al., 2011).). However, Lu et al. (2017) demonstrated using zeta potential (z) (Table 16.1), which is electro potential difference in interface and the stable layer of the liquid attached to the immiscible particles (dispersed particles). Value of z from 40 to 60 is considered as stable emulsion (Fig. 16.4).

FIGURE 16.3 Rheological changes in emulsion prepared at 0.0001M NaCL on shearing: (A) Variation in the median drop diameter (d, •) and uniform scattering (U, o) with the shearing duration (t) on shearing at a rate of 10 s1. (B) Variation in the lag time (tlag, •) and time taken to double the droplet size (t, o) with shearing rate. Adopted from Whitby et al. (2011) with permission. Copy right 2011, Elsevier.

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Table 16.1 Zeta potential range and stability performance of the colloidal emulsion. Zeta potential [millivolt (mV)]

Stability performance of the colloid

0 to 5 10 to 30 30 to 40 40 to 60 Above 61

Instant coagulation and flocculation Nascent instability Medium stability Good stability Best stability

Zeta potential (mv)

(A) -0

(B) Emulsified oil dropsets

-30 -60 -90

-120

0

2

4

6 8 pH value

(C)

10

12

14

(D)

(E)

FIGURE 16.4 Zeta potential of emulsion droplets at different pH levels (A), image under microscope of emulsified oil wastewater (B), and microscopic images of the mixture of magnetic nanoparticle sample (S4) and emulsified oil wastewater at various pH levels: (C) 4.0, (D) 7.0, (E) 10.0. Adopted from Lu¨ et al. (2017) with permission. Copy right 2017, Elsevier.

4. Applications

375

4. Applications Applicability of Pickering emulsions is large. Many types of emulsion can be replaced by Pickering emulsion. The distinctive feature of Pickering emulsion without surfactant makes this emulsion different. The use of Pickering emulsion started unintentionally initially. The appropriate example is a reinforced composite material that was later recognized as Pickering emulsion. The polymer emulsion of immiscible polymers where one polymer is dispersed in a continuous phase of other polymer is a type of Pickering emulsion. The same for the inorganic materials can be either partly dispersed or can be adsorbed at the two phases. This type of interfacial adsorption is Pickering-like emulsion (Fenouillot et al., 2009). This adsorption effects either emulsion morphology or/and stability (Elias et al., 2007, 2009). Stimuli responsiveness of Pickering emulsion is the prime research field for the applicability of the Pickering emulsions stabilized by nanoparticles. Two types of applications are booming. The following are two illustrative examples: drug delivery by Pickering emulsion formulation and the manufacture of nanomaterials from Pickering emulsion.

4.1 Potential application to the life science and drug delivery Specific features of Pickering emulsion made this type emulsion popular. The adverse effect of surfactant (irritancy, cytotoxicity, hemolytic behavior)-based emulsion can be avoided in Pickering emulsion. The thick coating of the droplets prevents the diffusion of delivered solid particle, and the system behaves as the encapsulation system (Tan et al., 2009). Surface coating with insoluble materials and then attaching cells can facilitate the targeting drug. Successfully insoluble hydroxyapatite as a coating substance to form various polymers, homopolymers, and gas-filled globules has been used (Fujii et al., 2009; Fujii et al. 2012). Solid inorganic coating of these globules made then resistant to various stresses environment and made then suitable than surfactant-based monolayer. These solidly layered globules can be dried for the preparation of solid forms and showed high potentiality for the biomedical field (Aranberri et al., 2009; Simovic et al., 2009). Drug delivery in dermatological medicine loaded with Pickering emulsion is a very prominent field of application. However, there is a subtle difference between Pickering (coated with silica nanoparticles and emulsifiers) and traditional surfactant-loaded emulsions in skin application. As for example, caffeine has been shown faster adsorption with similar droplet size and viscosity behavior in vivo skin diffusion experiments. However, silica nano-particle diffusion into the skin is disallowed after stratum corneum after emulsions were exposed for 24 h. These effects were not concurrent with other observation in o/w Pickering emulsions filled with medium polar molecules such as fluorescent probe and methyl salicylate (Eskander et al., 2010; Marku et al., 2012). Dermatological application of all-trans retinol compounds has been applied. Retinol compounds are hydrophobic in nature and can accumulate in the oil concentrated area of the stratum corneum. This accumulation occurs more efficiently than traditional surfactant formulations. However, the low transfer rate of this retinol has been confirmed to the epidermis and dermis area. A possible explanation of lesser transfer has been substantiated by previous adhesion of droplets at the exterior region of the skin like w/o-based emulsion. This also signifies the holding capacity of retinol by the Pickering emulsion in the stratum region. Larger accumulation of retinol in outer part also facilitates the silica particles to penetrate and this silica particle presumably plays a part in stabilizing retinol in the interior part of the skin.

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Concurrently, dermatological delivery of retinol with medium chain triglycerides, lecithin, and fumed silica (in nanometer size) showed a similar result (Simovic et al., 2011). Furthermore, retinol showed higher stability under UV radiation when delivered with oil-based emulsion formulation (Eskander et al., 2009). Biodegradability is a most invited and accepted property in all types of emulsion application that includes food, pharmaceutical, and cosmetics. Different environment-friendly perishable organic compounds have been delivered formulating Pickering emulsion. The most common examples of organic particles delivered loaded with Pickering emulsion are carbohydrate, fat crystals, chitin, and block copolymers micelles (Dickinson, 2010).

4.2 Application in material science using polymerization template Current research advancement in nanoemulsion formulation and the development of nanofunctional emulsion or incipient emulsion, and Janus emulsion has freshened up polymerization of emulsion with solid material. Pickering system acquires recognized benefits over conventional emulsion that includes minimum foam production, recycling ability, and cost affectability (Schrade et al., 2013). The novel approach of stimuli responsiveness to these emulsions has demanded definite control of instant stabilization and destabilization of this system of the emulsion as well as the capability of polymerization, simple purification of the products, and emulsifiers using extraneous stimulus. Alkaline lignin has been used to polymerize styrene (Wei et al., 2012). Moreover, the authors demonstrated the recycling procedure successfully. Lignin is a waste product in the paper industry, and it can be reused in the value-added product or in functional ingredients in various industries. Moreover, this technology can be used in producing or fabricating various novel coating nano-materials. As for example, Janus particle has been prepared with Au and Pt nanoparticle by cross-linking acrylic acid ([poly (polyacrylic acid-b-polystyrene)] (Wang et al. 2014). More stable emulsion formation has been observed in the acidic environment in terms of pH-sensitive polymersomes. Interestingly, the addition of metal nanoparticles triggers these particles to move towards the interior side of the water phase of the droplet, and they are restrained in the inward interior position, covering the front side of the polymersomes. Neutralization of the pH condition results in destabilization of these emulsions. Based on these pH-sensitive Pickering formulations, many other compounds such as graphene oxide and other nanoparticles with magnetic property and their composites have been introduced based on the evidences of their respective rheological properties (Lu et al., 2014; Crossley et al., 2010).

5. Conclusion Delivery of insoluble and solid particles in various products that include the pharmaceutical, chemical, and food industry was problematic. Pickering emulsion system not only solved this problem but also facilitated delivery of many components. Specific advantage of superior stability against flocculation or adhesion opens the scope of formation of primary coarse microsized droplets of the emulsion, double and finally multiple emulsions. Solid particles form a rigid stable barrier against interphase interference. This unique property has taken advantageous position in various fields that include drug delivery and hollow materials.

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Further Reading Eskandar, N.G., Simovic, S., Prestidge, C.A., 2009. Chemical stability and phase distribution of all-trans-retinol in nanoparticle-coated emulsions. International Journal of Pharmaceutics 376 (1e2), 186e194.

CHAPTER

Microencapsulation of bioactive compounds and enzymes for therapeutic applications

17

Ragini G Bodade1, Anand G Bodade2 1

Department of Microbiology, Savitribai Phule Pune University, Pune, Maharashtra, India; 2Department of Transfusion Medicine, Seth G S Medical College and KEM Hospital, Mumbai, Maharashtra, India

1. Introduction Microencapsulation, a rapidly expanding technology, deals with the covering of tiny solid, liquid, and gaseous particles by a continuous film of synthetic or natural polymer, thereby protecting them from the adverse environment and enabling a controlled release of particles at the desired time, rate, dose, and site of action. The resulted structure is termed as microparticles (microcapsules, microspheres, and microemulsions). Microparticles exist in a variety of size, compositions, and functions. The size diameter 1e1000 mm is termed as microparticles and more than 1 mm as macroparticles. The particle size less than 1 mm is termed as nanoparticles, nanocapsules, and nanospheres (Peanparkdee et al., 2016). Structurally each of the microparticles or microcapsules consists of an active inner core material and outer coat or shell material that covers or protects the core material. Microsphere has only a homogeneous structure (Fig. 17.1). The core or inner phase contains solid, liquid, or gaseous material, whereas the outer soft or hard film called as shell/coating or membrane made by coating materials like ethyl cellulose, sodium carboxyl methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, gelatin (GE), poly(lacticco-glycolic acid) or (PLGA), chitosan, and polyesters. the liquid core is either dispersed or dissolved form, while solid core is a mixture of active constitutes like stabilizers, diluent, or excipients. Different core materials like active pharmaceutical ingredients, proteins and peptides, food materials, dyes, pigments, volatile oils, catalysts, and pesticides encapsulated within the coat or shell materials (Jyothi et al., 2010). Likewise, different bacterial and animal cells also immobilized and applied in the field of cell and tissue engineering to treat different diseases (Tomaro-Duchesneau et al., 2013). A number of objectives have been achieved by microencapsulation techniques including protection of the core material, material structuration, and controlled release of the encapsulated product. The microsize of the particles makes them efficient in distribution throughout the system and thus improves the drug absorption. The compounds that are difficult to administer because of insolubility, volatility, reactivity, hygroscopicity, and physical state are at the higher priority for microencapsulation. It also protects labile compounds from external environments, viz., oxygen, light, heat, humidity, gastric pH, and host’s immune system, thus improves product quality (Gholse and Yeole, 2013 and Tomaro-Duchesneau et al., 2013). Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00017-5 Copyright © 2020 Elsevier Inc. All rights reserved.

381

382

Chapter 17 Microencapsulation of bioactive

FIGURE 17.1 Structures of microparticles (left: microcapsule and right: microsphere).

In the current chapter, microencapsulation techniques for therapeutic applications of bioactive compounds and enzymes in disease treatments are described in detail.

2. Types of microencapsulation Historically the microencapsulation procedure was first introduced by Bungen burg de Jong and Kan in 1931 using GE sphere and is still widely used in the food, pharmaceutical, and cosmetic industries to maintain the stability, efficiency, and bioactivity of the compounds (Suganya and Anuradha, 2017). Although controlled drug delivery concept using microencapsulation was introduced in 1970s, still it is under study (Gupta and Day, 2012). Morphology of the microcapsules can be affected by selection of the techniques and nature of core and wall material. The synthesized different forms of the microcapsules may be mononuclear, poly/ multinuclear, matrix, multiwall, and irregular type (Das et al., 2011). Principle materials used for microencapsulation are different types of carbohydrate polymers, proteins, and lipids as per Table 17.1. Selection of coating material for microencapsulation depends on its inertness toward core material, availability, chemical compatibility, and stability. It also possesses some physicalechemical properties including cohesiveness, permeability, sorption, solubility, clarity, and stability for target-selected delivery (Bansode et al., 2010; Wazarkar et al., 2016). Different mechanisms have been achieved for controlled and sustained release of core material. Under a specific environmental condition like temperature, the coating material dissolves in water or solvent and results in gradual release of the core material by simple dissolution mechanism. High pressure or cracks on the wall results in release of the material during rupture mechanism. While in diffusion mechanism, a concentration gradient is achieved in core and surrounding environment and causes release of core material (Hu et al., 2017). However, a single type of microencapsulation method cannot be universally applied for a variety of drug materials. Therefore, development of a microcapsule method for a given drug requires understanding of drug physiochemical characteristics, compatible polymer, and encapsulation. The encapsulation techniques for drugs are emerging widely as ideal method due to its wide applications in food, cosmetic, and pharmaceutical industry. Microencapsulation drug delivery system is a promising option for developing oral drug formulation. Major of the drugs studied for encapsulation are from BCS class II group, which comprises mainly low solubility and high permeability drugs. Therefore, bioavailability, stability, and controlled release of drug could be possible by varied encapsulated methods. This will eliminate the drug easily from absorption site and for those having reduced bioavailability (Sachan et al., 2014). Microencapsulation is classified as chemical (emulsification, polymerization, and liposomes), physical (freeze-drying/lyophilization, spray drying, cocrystallization, fluidized-bed coating, or

3. Microencapsulation of bioactive compounds and bioactive extracts

383

Table 17.1 Materials used for microencapsulation. Sr. No.

Source

Material

1

Plant and plant exudates

2 3

Marine extracts Animal and microbial

4

Other materials

Starch and its derivatives (amylose, amylopectin, dextrin, maltodextrins, polydextrose), celluloses (ethyl cellulose, carboxymethyl cellulose, methyl cellulose, cellulose acetate phthalate) and their derivatives (gum arabica, galactomannans), pectin, and soybean-soluble polysaccharides Carrageenan, alginate Dextran, chitosan, xanthan and gellan, caseins, albumin, gelatin and gluten, fatty acids and fatty alcohols, waxes (beeswax, carnauba wax, candelilla wax), steric acids, glycerides, and phospholipids PVP, paraffin, PEG, inorganic materials

extrusion), and physiochemical/biological method type (coacervation and solgel encapsulation), and each of these gives different morphology of the particles (Table 17.2) (Madene et al., 2006, Nedovic et al., 2011, Laouini et al., 2012 and Lam and Gambari 2014).

3. Microencapsulation of bioactive compounds and bioactive extracts Bioactive compounds extracted from different parts of the plants have been used in food and pharmaceutical industries as health-promoting agents. They are obtained easily from the plant extracts prepared from plant parts by solvent extraction method. In pharmaceutical industry, bioactive compounds revealed to be effective in treatment of cancer, obesity, infection, and cardiovascular diseases. Moreover, they are also investigated for their health-promoting effects on animals (Pangestuti and Kim, 2011 and He and Giusti, 2010). Bioactive extracts and individual bioactive compounds are microencapsulated by immobilization methods using different wall materials as per Table 17.3 and Fig. 17.2. Moreover, effect of oral delivery of microcapsules prepared by alginate, polylysine, and pectin revealed no direct effect on microbial load of the gastrointestinal tract (Afkhami et al., 2007). Natural polyphenols and polyherbal formulations (PHFs) possess dynamic medicinal and therapeutic properties. Plants contain large quantities of pigments, especially chlorophylls, carotenoids, and anthocyanin are responsible for colourization of plant flowers, fruits, and leaves. In food industry these pigments are used as colorant, flavoring agent, and taste enhancer. Anthocyanin is a water-soluble nontoxic polyphenol used widely as colorants in foods and drinks. However, several factors such as pH, light, storage temperature, free radicals, chemical structure, concentration, oxygen, and solvents affect its stability and bioavailability and thus require encapsulation (Carocho et al., 2015; Zhao et al., 2015; Joana Gil-Cha´vez et al., 2013). Recently its importance in treatment of cardiovascular, neurological, cancer, and diabetes diseases has been investigated (Yousuf et al., 2016). Encapsulation of anthocyanin by spray drying technique using maltodextrin (MD) and gum arabic (GA) has been studied for its stability (Burin et al., 2011 and Mazuco et al., 2018). Jaboticaba extract (Myrciaria cauliflora and Myrciaria jaboticaba), a rich source of anthocyanin, can be microencapsulated with MD and GA by spray drying method to improve its physicalechemical characteristics as well as minimal color loss (Silva et al., 2013). Santos et al. (2013) assessed encapsulation using polyethylene glycol (PEG), supercritical CO2 as solvents, and ethanol as cosolvent to retain its stability and protection from

384

Table 17.2 Common techniques for microencapsulation in food and pharmaceutical industry. Type of encapsulation

1

Spray drying

2

Freeze-drying/ lyophilization

3

Coacervation

Principle

Material used

Homogenized core and wall material in suitable solvents is fed into a spray dryer and atomized with a nozzle. Water gets evaporated due to high temperature with simultaneous precipitation of capsules at the bottom.

Gum arabica, maltodextrins, saccharose, cellulose, gelatin, lipids, soy proteins

The homogenized material is freezed by reducing the surrounding pressure and allowing the frozen water to sublimate directly from solid phase to gas phase by providing enough heat. Phase separation of one or more hydrocolloids from a polymeric solutions layer around the core material is suspended in the same reaction mixture under the specific conditions, viz., pH, temperature, ionic strength.

Dextran, chitosan, polyvinyl alcohol, gelatin, carrageenan, gum arabica, soy protein, guar gum

Chitosan, heparin/gelatin, gum arabica/gelatin, gliadin, carrageenan, polyvinyl alcohol, soy protein, guar gum/dextran, gelatin/carboxymethyl cellulose

Advantages

Disadvantages

for · Used hydrophobic and

of product · Loss · Degradation of

· · · · ·

hydrophilic polymers Suitable for heat labile and highly viscous solutions (300mpa) Economically feasible Inexpensive, simple, rapid High drug-loading efficiency For thermosensitive compounds (water-soluble natural aromas and essence, drugs)

· For thermosensitive ·

compounds (flavor compounds) High encapsulation efficiency and targeted and controlled release

· ·

heat-sensitive products For limited wall materials Fiber formation sometimes achieved

Expensive and timeconsuming

Expensive

Chapter 17 Microencapsulation of bioactive

Sr. No.

4

Liposomes

Phospholipid bilayers are dispersed in aqueous environment, encapsulation, and core.

targeted and · Site efficient

· ·

5

6

Emulsification

Polymerization

Mixture of emulsion is prepared by two immiscible liquids, which are either used directly as liquids or dried in the powder form. The reactive monomers get polymerized on the surface of droplet or particle to form a capsule shell.

Oil and Water system

Ethyl cellulose, carboxymethyl cellulose, methyl cellulose, cellulose acetate phthalate, gelatin

controlled drug delivery Both hydrophilic and hydrophobic compounds can be encapsulated Stable and easy production

Both hydrophilic and hydrophobic food compounds can be encapsulated

Micro- to nanosized particles will be formed

low · Expensive, reproducibility, low drug entrapment, difficulties in particle size control, and short circulation halflife of vesicles Issues with sterilization and stability Droplets are bigger in size and need separation Always coupled with other encapsulation method Difficult to control the polymerization

· · ·

3. Microencapsulation of bioactive compounds and bioactive extracts

Cholesterol and natural and/ or synthetic phospholipids (phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylinositol)

385

Table 17.3 Microencapsulated plant extracts and bioactive compounds. Technique

Material

Application

1

Anthocyanin

Spray drying method

Maltodextrin Gum arabic

Nutraceutics

2 3

Spray drying method Encapsulation

Maltodextrin and gum arabic Margarine

Nutraceutics Nutraceutics

Silva et al. (2013) Zaidel et al. (2014)

4 5

Jaboticaba extract Roselle and red cabbage extract Bilberry extract Jamun extract

With whey protein and citrus pectin Gum arabica and maltodextrin

Nutraceutics Nutraceutics

6

Black carrot extract

Encapsulation Spray and freezedried method Spray drying method

Maltodextrin 20e21 DE

Nutraceutics

7

Spray drying method

Maltodextrin/soybean protein isolates

Antioxidant

Spray drying method

Maltodextrin/dextrose and gum acacia and tricalcium phosphate Maltodextrin/dextrose and gum acacia

Antioxidant

Nayak & Rastogi (2010)

Nutraceutics

Tonon et al. (2010)

10

Pomegranate extract Garcinia indica Choisy fruit pulp Euterpe oleracea Mart. juice fruit pulp Jussara pulp

Mueller et al. (2018) Preetha and Preetha (2017) Ersus and Yurdagel (2007) Robert et al. (2010)

Spray drying method

Santana et al. (2016)

11

Betacyanin

Spray drying method

Cosmetics Antioxidant Antioxidant

12 13

Beetroot extract Cactus pear extract

Spray drying method Spray drying method

Antioxidant Antioxidant

Pitalua et al. (2010) Robert et al. (2015)

14 15

Spray drying method Coacervation

Antioxidant Nutraceutics

Tupuna et al. (2018) Aditya et al. (2015)

16

Norbixin Curcumin and catechin b-Carotene

· ·

Antiangiogenic activity

· Spada et al. (2012) · Aissa et al. (2012)

17

Lycopene

Spray drying method

· Modified starch with trehalose and · Alginate galactomannans

Antioxidant Nutraceutics

· Rocha et al. (2012) · Calvo et al. (2017)

8 9

Spray drying method

· Spray drying method · Freeze-drying method

Gum arabic and modified starch, whey protein concentrate, soy protein isolate Maltodextrin (25 and 10 DE) and starch b-Cyclodextrin and maltodextrin Gum arabic Maltodextrin, inulin, and soybean protein isolate Maltodextrin and gum arabic Olive oil, soybean oil, and sunflower oil Starch Gum arabic

· ·

Reference et al. (2011) · Burin and Wrolstad · Giusti (2003)

and Corke (2000) · Cai · Chong et al. (2014)

Chapter 17 Microencapsulation of bioactive

Core material

386

Sr No

18

21

Grape waste

19

Freeze-drying method

Gum arabic, gelatin, and maltodextrin

Spray drying method drying · Spray method · Freeze-drying method drying · Spray method · Freeze drying

Hussain et al. (2018)

Maltodextrin

Antioxidant Antidiabetic Antioxidant

Gum arabic and maltodextrin

Antioxidant

Rezende et al. (2018)

· Alginateechitosan · Guar gum and 5% polydextrose

method

22 23

24 25

Chlorogenic acid

· Paeonia rockii roots sidoides · Lippia Cham. hystrix · Citrus fruit extract

Spray drying method Spray dry method

· Chitosan arabic · MD/gum · Glucomannan/gum arabic

26

Gallic acid Quercetin Vanillin Tea extract

Spray dry method

27

Punicalagins

Spray dry method

28

Capsaicin

Coacervation

Gelatin and gum acacia

· ·

Spray dry method Spray dry method

b-Cyclodextrin

Acetylated starch and inulin Inulin

· b-Cyclodextrin · Chitosan · Maltodextrin

Antioxidant, antibacterial, antiinflammatory, and antiplatelet activity Antimicrobial Antioxidant Antimicrobial Antioxidant

Antioxidant Antioxidant Improvement of bone quality Cytotoxicity Antioxidant a-Glucosidase activity Antimicrobial Antioxidant

Da Costa et al. (2018)

et al. · Moschona (2018) · Kuck and Noren˜a (2016)

Zhao et al. (2010)

· Sansone et al. (2014) et al. · Fernandes (2012) · Adamiec et al. (2012) Robert et al. (2012) Sun-waterhouse et al. (2013) Haidong et al. (2011) Liang et al. (2011)

· ·

Cam et al. (2014)

Xing et al. (2004)

Plant Oil Drumstick oil

Spray drying method

Maltodextrin/gum arabic

30

Plant and animal oil

coating · Fluid-bed method

b-cyclodextrin · Hydroxypropyl · Maltodextrin

Cosmetics and anticholesterol Nutraceutics

Premi and Sharma (2017) Anwar et al. (2010) Encina et al. (2016) Continued

387

29

3. Microencapsulation of bioactive compounds and bioactive extracts

20

Polyherbal formulation (PHF) Cupuassu seed extract Acerola pulp

Table 17.3 Microencapsulated plant extracts and bioactive compounds.dcont’d Technique

Material

Application

Reference

31

Flaxseed oil

Spray drying method

Nutraceutics

32

Olive oil

Spray drying method

Antioxidant

Tonon et al. (2011); Carneiro et al. (2013) Calvo et al. (2012)

33

Linseed oil

Spray drying method

Nutraceutics

Gallardo et al. (2013)

34 35

Garlic oil Ginger oil

Coacervation Spray drying method

Maltodextrin/whey protein concentrate Maltodextrin/carboxymethyl cellulose and lecithin Gum arabic/maltodextrin and whey protein isolate Gelatinegum acacia Arabic/maltodextrin and inulin

Antioxidant Antioxidant

36 37 38

Lemon oil Walnut oil Green coffee oil

Spray drying method Spray drying method Spray drying method

Siow and Ong (2013) De Barros Fernandes et al. (2016) Kausadikar et al. (2015) Shamaei et al. (2017) Carvalho et al. (2014)

39

Macadamia nut oil

Spray drying method

40

Brucea javanica oil

41

Nigella seed oil

Coacervation method Spray drying method Spray drying method

42

Eugenol oil

Coacervation

Gelatinesodium alginate

Antioxidant

43

Menthol

Spray drying method

Gum arabic and modified starch

44

Omega-3 fatty acids

Spray dry method, coacervation, spray chilling, extrusion coating, liposomes

Maltodextrin/gum arabic, casein/ methyl cellulose, starch/whey protein

Antimicrobial and anticancer Nutraceutics

Encapsulation

Poly(lactic-co-glycolic acid)-catalase

drying · Spray method

Maltodextrin Skim milk powder and Tween 80 Starch Hi-Cap 100 and Corn syrup/HiCap 100 Sodium caseinate (NaCas) and maltodextrin (MD) Arabic gum and gelatin

· Sodium caseinate and maltodextrin DE 10 arabic/maltodextrin · Gum · Gum arabic/sorghum starch

Nutraceutics Nutraceutics Cosmetics and antioxidant Antioxidant Anticancer Antimicrobial, antioxidant, and nutraceutics

Laohasongkram et al. (2011) Hu et al. (2016)

· Mohammed et al. (2017) et al. (2016) · Edris · Arshad et al. (2018)

Shinde and Nagarsenker (2011) Soottitantawat et al. (2005) Kaushik et al. (2015)

Enzymes 45

Catalase (EC 1.11.1.6)

Treatment of acatalasaemic and oxidative stress

Singhal et al. (2013)

Chapter 17 Microencapsulation of bioactive

Core material

388

Sr No

46

Encapsulation

Artificial cell preparation

Treatment of phenylketonuria

Bourget and Chang (1986)

Encapsulation

Artificial cell preparation

Palmour et al. (1989)

Entrapment method

Liposomes

50

Urease (EC 3.5.1.5)

· Encapsulation · Encapsulation · Solgel method

Liquid-air nozzle method

· Carrageenan · Alginate/bentonite · Silica matrix

Hexamethylenediamine/milk casein

51

Asperginase (EC 3.5.1.1)

Entrapment method

Liposomes [poly(lactic-co-glycolic acid)/polyvinyl alcohol]

52

Superoxide dismutase (EC 1.15.1.1) Papain (EC 3.4.22.2.) and protein hydrolysate

Entrapment method

Liposomes [poly(lactic-co-glycolic acid)/polyvinyl alcohol]

Encapsulation method

Carboxymethylated flamboyant seed gum/sodium alginate

LescheNyhan disease Treatment of hyperuricemia Treatment of lactose intolerance In urea removal from artificial kidney dialysate Treatment of acute lymphoblastic leukemia Treatment of acute and chronic inflammation Nutraceutics

· Spray dry method · Coacervation

· Maltodextrin/modified starch HiCap 100 complex · Gelatin/acacia · Cyclodextrin

47 48 49

53

Micronutrients 54

Vitamin A palmitate

Ascorbic acid

56 57

Squalene Vitamin B12 Vitamin B2 Vitamin B9

· · ·

Watereoil double emulsion method Spray dry method Encapsulation method

Corn oil, polyglycerol polyricinoleate (emulsifier), and gelatin Chitosan Chitosan/alginate Gelatin Lactoferrin/b-lactoglobulin

· · ·

et al. (2017), · Zhang (1998), · Dashevsky · Nichele et al. (2011)

Miyawaki et al. (1979)

De Brito et al. (2019)

Shaheen et al., 2017

Betancur-Ancona et al. (2011) and Ruiz Ruiz et al., 2013

· Gangurde and Amin (2017) et al. · Junyaprasert (2001) and Albertini et al. (2010)

Nutraceutics and antioxidant Antioxidant Antioxidant

· Zhang et al. (2018)

Comunian et al. (2014) Kumar et al. (2017) Estevinho et al. (2016) Azevedo et al. (2014) Chapeau et al. (2016)

· · ·

389

55

Nutraceutics and antioxidant

Zhou et al. (2016)

3. Microencapsulation of bioactive compounds and bioactive extracts

Phenylalanine ammonia-lyase (EC 4.3.1.24) Xanthine oxidase (EC 1.17.3.2) Uricase (EC 1.7.3.3) Lactase (EC 3.2.1.108)

390

Chapter 17 Microencapsulation of bioactive

FIGURE 17.2 Microencapsulated bioactive compounds.

the environmental condition. The result revealed that the supercritical solvent technology and conventional Caealginate encapsulation system protected the anthocyanin pigment from light and temperature significantly. Encapsulation with Caealginate showed less release profile than the PEG (Santos et al., 2013). Zaidel et al. revealed the improved storage and stability characteristics of margarine containing encapsulated anthocyanin from roselle and red cabbage (Zaidel et al., 2014). Bilberry extract has been reported as a rich source of anthocyanins and so investigated for encapsulation with whey protein and citrus pectin (PC) for its bioavailability and intestinal accessibility in humans (Mueller et al., 2018). A comparable result has been observed with the use of spray dried and freeze-dried method for anthocyanin pigment encapsulation from Jamun (Syzygium cumini) extracts. Spray dried powder with GA and MD as wall material gave the homogeneous particle size, low moisture content, and highest encapsulation efficiency (EE) up to 8 weeks of storage (Preetha and Preetha, 2017). Six different anthocyanin pigments have been reported in black carrot (Daucus carota L.). Effects of MD (20e21 DE) as carrier agent by spray dried encapsulation method revealed the highest anthocyanin content and improved shelf life under light illumination and storage temperature (Ersus and Yurdagel, 2007). Bioactive compounds like polyphenols and anthocyanins from pomegranate juice (PJ) (Punica granatum) and ethanolic extracts (PE) were encapsulated with MD or soybean protein isolates (SPI) by spray drying method. MD microcapsules attained significant protection to the polyphenols and anthocyanin’s than SPI during storage conditions (at 60
C for 56 days). Commercial PJ has highest antioxidant activities as compared to other fruit juices, green tea, and red wine. Thus has health benefits and commercial value (Robert et al., 2010). Many of the fruit juice powders obtained by spray drying are sticky, hydroscopic, and less soluble. Use of fruit pulps encapsulated with carrier agents revealed beneficial to handle, package, and transport. Microencapsulation of anthocyanin from Garcinia indica Choisy fruit pulp using MD/dextrose and GA and tricalcium phosphate helps to enhance their antioxidant activity (69.90%) and stability (Nayak and Rastogi, 2010). Similar result was described for anthocyanin from Euterpe oleracea Mart. juice fruit pulp (Tonon et al., 2010). Therefore, encapsulated anthocyanins as food additives protect them in gastrointestinal site and induce safe release for its beneficial health effect (Robert and Fredes, 2015).

3. Microencapsulation of bioactive compounds and bioactive extracts

391

Microencapsulation by spray drying of jussara pulp (Euterpe edulis) using ternary mixtures of GA and modified starch (MS) together with either whey protein concentrate (WPC) or SPI as the carrier agents revealed to give maximum yield (PY), solubility (S), retention of total anthocyanins (RTA), EE, and minimum moisture content. Thereby, the jussara pulp product can be applicable to cosmetics, as colorants, and flavoring agents (Santana et al., 2016). Betacyanin, a close relative of anthocyanin natural pigment, is used as food colorant. Betacyanin from the Amaranthus gangeticus plant extracted and encapsulated using MD as carrier (25 and 10 DE) and starch as coating agent by spray dried method revealed highest pigment retention 97.3% and 88.7%, respectively. The 25 DE/10 DE mixed powders can be stored up to 63.6 weeks without any pigment loss (Cai and Corke, 2000). Furthermore, microencapsulation using b-cyclodextrin and MD was explored by Chong et al. (2014). The result revealed that b-cyclodextrin-encapsulated pigment had shorter range of droplet size and drying time during spray drying method (Chong et al., 2014). Betalains are naturally occurring plant pigments classified as betacyanins and betaxanthins. Betalains-containing fruits are known for their beneficial actions in maintaining the body’s redox balance, decreasing oxidative damage, and improving antioxidant status in humans. Moreover, in humans after ingestion they promote their incorporation into the LDL and red cells, thereby protecting from oxidative damage and hemolysis. Betalains and carotenes from the cactus pear have known for their colorant and health beneficial properties. Beta vulgaris (beetroot) is known for its antioxidant properties due to betacyanins and betaxanthins content. Many of the reported studies revealed its significant role in cancer, coronary diseases, and other degenerative illnesses. Encapsulation by spray drying using GA revealed no significant differences in color, antioxidant activity, betalains concentration, and redox potential up to 45 days. Water adsorption influences the stability of the product during storage (aw < 0.521 for greatest stability) and antioxidant activity (aw > 0.748 for greatest stability). Therefore, betacyanin microcapsules could be interesting food additives for their antioxidant health benefits (Pitalua et al., 2010). Also, indicaxanthin from cactus pear (Opuntia ficus-indica) improved drastically by encapsulation in an MD matrix when stored at 20
C for months (Gandia-Herrero et al., 2010). In another study, pulp and ethanolic extract of cactus pear encapsulated with MD, inulin, and cladode mucilage revealed promising thermal stability and thereby its application as food additives (Saenz et al., 2009; Ota´lora et al., 2015). Moreover, encapsulation within an SPI and polysaccharide (MD or inulin) revealed improved betalains and polyphenol EE, and stability during storage at 60
C (Robert et al., 2015). Carotenoids are natural pigments found in fruits, vegetables, some fungi, and algae. Various carotenoids from different sources are reported for oxygen radicals scavenging activity and thereby reduce oxidative stress in biological system. Moreover, it has potential to reduce cancer, cardiovascular disease, cataracts, and as immune response booster (Gul et al., 2015). Microencapsulation by spray drying and supercritical micronization method using different carrier materials, viz., MD and calciumealginate, revealed its stability against light, oxygen, and increased shelf life (JaniszewskaTurak, 2017; Fratiann et al., 2017). Norbixin, an apocarotenoid, has food additive and antioxidant applications. It effectively deactivates reactive oxygen species (ROS)-induced DNA damages from the cell. Microencapsulation using MD: GA by spray drying improved its thermal stability and shelf life (Tupuna et al., 2018). Encapsulation of curcumin and catechin by water-in-oil-in-water system was study by Aditya et al. (2015). They have been used in food and drink products as supplement to prevent several diseases such as cancer, obesity, infection, and cardiovascular ailments (Aditya et al., 2015). Bcarotene microencapsulation was studied for antiangiogenic activity using starch and GA (Spada et al.,

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Chapter 17 Microencapsulation of bioactive

2012, Aissa et al., 2012, Thakur et al., 2017 and Correˆa-Filho et al., 2019). Lycopene encapsulated in MS by spray drying was studied by Rocha et al. (2012). The plant pigment is used as colorant in food and can be prevented by oxidation reaction during storage process (Rocha et al., 2012). Release profile of lycopene from pink grapefruit was strongly influenced by encapsulating it with alginate with trehalose and galactomannans. Lycopene is used as food additive, colorant, and flavor modifiers. It is effective in reducing risk of oxidative stress, cancer, cardiovascular, and hepatic fibrogenesis diseases (Calvo et al., 2017). Microencapsulation of PHF made by the root of Chlorophytum borivilianum, Astragalus membranaceus, Eurycoma longifolia, and seeds of Hygrophila spinosa T. Anders was carried out by freezedrying method using GA, GE, and MD polymers. The result revealed excellent antioxidant activity and antidiabetic activity. Microencapsulation of PHF further proved improved bioavailability under intestinal acidic pH (Hussain et al., 2018). Cupuassu (Theobroma grandiflorum Schum.) fruits are known for their intense aroma and nutritional value due to their phenolic contents. Microencapsulation using MD for cupuassu seed extract by spray drying method was investigated for antiradical power and stability at low temperature or under simulated gastrointestinal conditions (Da Costa et al., 2018). Bioactive compounds from acerola pulp (Malpighia emarginata DC), viz., carotenoids (CA), ascorbic acid (AA), phenolic compounds (PC), anthocyanins (A), and residual extracts, retain good antioxidant activity by spray- and freeze-drying encapsulation methods, using GA and MD (Rezende et al., 2018). Phenolic compounds, viz., quercetin, kaempferol, transferulic acid, ellagic acid, and a derivative of caffeic acid, are found in many of the fruit extracts. Grape waste (Vitis vinifera) from wine industry contains high amount of these phenolic compounds and has potential to use in food and pharmaceutical industries after immobilization. Encapsulation of this waste extracts revealed high antioxidant activity, antibacterial activity, antiinflammatory, and antiplatelet activity using alginateechitosan polymer due to its controlled release property (Moschona et al., 2018). Moreover, 5% partially hydrolyzed guar gum and 5% polydextrose has proven to be the best treatment for retention of phenolics and anthocyanins (>80%) and antioxidant activities (45.4%e83.7%) by spray drying encapsulation technique (Kuck and Noren˜a, 2016). Antimicrobial activity of microencapsulated Vitis labrusca solvent extract against Staphylococcus aureus and Listeria monocytogenes and Leishmania arginase enzyme inhibition was reported by (de Souza et al., 2014). Microencapsulated chlorogenic acid from Nicotiana tabacum leaves was developed as food products with antimicrobial properties (Zhao et al., 2010). Moreover, enhanced antifungal activity of Paeonia rockii roots and Lippia sidoides Cham. leaves and antibacterial activity of Citrus hystrix DC fruit skin were reported using chitosan, MD/GA, and glucomannan/GA, respectively (Sansone et al., 2014, Fernandes et al., 2012 and Adamiec et al., 2012). Gallic acid, a natural antioxidant from plant, was investigated for microencapsulation using acetylated starch and inulin revealed higher EE and antioxidant activity (Robert et al., 2012). Health benefits of other polyphenols like quercetin and vanillin revealed significant with inulin as wall material by spray drying microencapsulation method (Sun-waterhouse et al., 2013). Microencapsulation using b-cyclodextrin of Camellia sinensis tea extract has been reported for improvement of bone quality in rat and reduction in blood Ca2þ contents due to its content of catechins, gallic acid, and caffeine (Haidong et al., 2011). Improved cytotoxicity of the tea polyphenols has also been reported using microencapsulation in chitosan and can be used as antitumor agent (Liang et al., 2011). Punica granatum L. peel extract containing polyphenol punicalagins was microencapsulated in MD and revealed enhanced antioxidant and a-glucosidase activity (Cam et al., 2014).

3. Microencapsulation of bioactive compounds and bioactive extracts

393

Enhanced antiinflammatory activity of polyphenols such as caffeic acid, carvacrol (derivatives), thymol, pterostilbene (derivatives), and N-(3-oxo-dodecanoyl)-L-homoserine lactone was studied by Coimbra et al. (2011). Capsaicin from chill has application as pharmaceutical, neuroscience, and antimicrobial drugs in medicine. Microencapsulation by coacervation process using GE and GA along with tannins revealed a high drug-loading content (19.84%) and a high EE (88.21%) (Xing et al., 2004). Food-derived micronutrients like Vitamin A from natural foods have been always a growing interest for the health promotion activities. Vitamin A palmitate (VAP) is widely used in cosmetic industry to prepare skin care products. Microencapsulation using MD and MS Hi-Cap 100 by spray dry method significantly increased its antioxidant activity and stability up to 3 months with promising EE (53%e63%) and drug release (80.18%e83.43%) (Gangurde & Amin, 2017). In another study microencapsulation with gelatineacacia complex by coacervation and double-coated alginatee chitosan microcapsule along with stabilizer butylated hydroxytoluene (BHT) improved the VAP stability (Junyaprasert et al., 2001 and Albertini et al., 2010). Microencapsulation of VAP using gcyclodextrin-based metal organic frameworks (g-CD-MOFs) enhances its stability by 1.6-fold than the current market reference product (BASF VAP). Thus, the microencapsulation method revealed useful for its food and pharmaceutical applications (Zhang et al., 2018). Other micronutrient AA (Vitamin C) is a natural antioxidant from fruits and vegetables and was investigated for physicochemical and sensory stability of chicken frankfurters and thereby its health-promoting activity. Encapsulation by watereoil double emulsion method using corn oil, polyglycerol polyricinoleate (emulsifier), and gelatin could allow the incorporation of an antioxidant with vitamin functionality and improved stability (Comunian et al., 2014). One another micronutrient of marine origin squalene has a wide range of health-promoting properties like cardio-protective, antioxidant, chemopreventive, anticancerous, etc. Therefore, squalene has applications in pharmaceutical, cosmetic, and biomedical industries. Kumar et al. studied oxidative stability of squalene by encapsulating in chitosan by spray drying method (Kumar et al., 2017). Vitamin B12 (cobalamin), Vitamin B2 (riboflavin), and Vitamin B9 (folic acid) also known for their health benefits are microencapsulated by chitosanealginate, GE, and lactoferrin/b-lactoglobulin, respectively, for their stability, bioavailability, and shelf life (Estevinho et al., 2016; Azevedo et al., 2014 and Le-gang et al., 2012, Chapeau et al., 2016). Many of the plant oils have nutritional value but are unstable due to their oxidative deterioration nature and consequent production of undesirable flavor. Encapsulation of such oils to minimize the oxidative reaction and to improve the physiochemical characteristics, viz., viscosity, stability, droplet size, and dry powder characteristics, are mostly affected by the type of carrier agent and techniques. Carrier agents used for encapsulation of these oil-based components include mostly GA, MD, WPC, skim milk powder (SMP), and sodium caseinate (NaCas). Drumstick oil or ben from Moringa oleifera (MOO) resembles to olive oil in fatty acid composition and contains high amount of useful behenic acid and oleic acid. High oleic contents of MOO attributed an increase in HDL cholesterol and decrease in serum cholesterol and triglycerides level. Therefore, are applied in cosmetics, skincare formulations, and folk medicines (Nadeem and Imran, 2016). Encapsulation of drumstick oil with MD: GA revealed better thermal and oxidative stability, thus has therapeutic values (Premi and Sharma, 2017). Other reported oils encapsulated by spray drying include fish oil (Anwar et al., 2010; Encina et al., 2016), flaxseed oil (Tonon et al., 2011; Carneiro et al., 2013), olive oil (Calvo et al., 2012), linseed oil (Gallardo et al., 2013), garlic oil (Siow and Ong 2013), and ginger oil (De Barros Fernandes

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et al., 2016). Thus, encapsulated oils revealed applications as food additive and therapeutics. Lemon oil, a flavoring agent in the tea and beverages encapsulated by spray drying techniques using MD, was developed by Kausadikar et al. (2015). Walnut oil contains health-beneficial essential fatty acids, oleic acid, linoleic acid, tocopherols, and phytosterols. Walnut oil has been effective in reducing the risk of cardiovascular and cancer diseases. Shamaei et al. (2017) evaluated the emulsion prepared using SMP and Tween 80 as wall material, with 91% encapsulated efficiency and its possible nutraceutical application (Shamaei et al., 2017). Green coffee oil from Coffea arabica and Coffea canephora contains high amount of diterpenes (cafestol and kahweol), palmitic acid, oleic acid, stearic acid, and linoleic acid. Due to its emollient properties including antioxidant and solar UV light absorption property it can be applied as active ingredients in cosmetics products. Green coffee oil microparticles of MS Hi-Cap 100 and corn syrup/Hi-Cap 100 has stabilized by lecithinechitosan through electrostatic layer-by-layer deposition technique to exhibit the highest oxidative stability and controlled release of sun protective factor (Carvalho et al., 2014). The macadamia nut oil (Macadamia integrifolia) contains high amount of monounsaturated fatty oils (70%) and is widely used in treatment of cardiovascular diseases by reducing the blood cholesterol level. Encapsulation by NaCas and MD in 1: 4 proportions improves quality and shelf life of macadamia by preventing oxidative reaction (Laohasongkram et al., 2011). Brucea javanica fruit possesses anticancer, antiinflammatory, antimalarial, and detoxification activities. Brucea javanica oil (BJO) extracted from dried ripe fruit contains stearic, oleic, and linoleic acids. Encapsulation of BJO extract prevents its off flavors, volatilization, and degradation effectively. Improved anticancer activity of encapsulated BJO microcapsules by complex coacervation method and spray drying method was evaluated by Hu et al. (2016). At the optimal conditions, the EE (82.9%) and stability against oxidation was remarkably achieved for cancer treatment using arabic gum and gelatin as coating materials (Hu et al., 2016). Nigella sativa seed is used traditionally to treat fever, headache, diarrhea, anxiety, asthma, and stroke. The Nigella seed oil (NSO) shows excellent source of essential fatty acids and lipid-soluble bioactive compounds. Thymoquinone is the active compound from NSO and possesses excellent antioxidant and antiinflammatory properties. Thymoquinone encapsulation using NaCas and MD DE 10 by spray drying method was assessed for its oxidative stability, morphology, and bioavailability by Mohammed et al. (2017). A similar study on oleoresin from N. sativa L. emulsified using GA/MD (1:1 w/w) by spray drying method was carried for its application in processed food and nutraceutical (Edris et al., 2016). In another study, microencapsulated oleoresin by GA and sorghum starches were studied for antioxidant activity and antimicrobial activity against Escherichia coli and Bacillus cereus (Arshad et al., 2018). Eugenol is a phenylpropanoid containing oil derived from clove and cinnamon and used in medicine as local antiseptic and analgesic agent. Its derivatives are also used in perfumeries, flavorings, and as essential oils. Microencapsulation of eugenol with GEesodium alginate complex by coacervation is expected to offer protection against oxidization and volatilization (Shinde and Nagarsenker, 2011). Menthol, a cyclic monoterpene alcohol from peppermint, possesses biological activities, viz., antibacterial, antifungal, antipruritic, anticancer, and analgesic. Microencapsulation of menthol with GA and MS by spray drying method affects its handling and storage conditions (Kamatou et al., 2013 and Soottitantawat et al., 2005). Omega-3 fatty acids, polyunsaturated fatty acids, implicated in the prevention of diabetes, coronary artery disease, arthritis, cancer, hypertension, other inflammatory, and autoimmune disorders. Many studies encourage the involvement of omega-3 fatty acids for healthy retina and brain development of fetus in pregnant and lactating women. Microencapsulation of omega-3 fatty acids using spray dry

4. Microencapsulation of therapeutic enzymes

395

method, coacervation, spray chilling, extrusion coating, and liposomes was also successfully studied for their oxidative deterioration and stability (Kaushik et al., 2015).

4. Microencapsulation of therapeutic enzymes Enzymes are biocatalyst responsible for conversion of substrate to product at varied biological conditions. However, enzymes become nonfunctional under stressed environmental factors like temperature, pH, mechanical process, and others due to change in conformation. Enzymes possess varied applications in detergent, pharmaceutical, and food industry. Microencapsulating the enzyme in order to improve its use in industrial process and commercial products is today’s need. Their encapsulation can be achieved by two strategies: surface immobilization by chemical or physical interaction on a solid support and by encapsulation inside the matrices such as hydrogels or within solid membranes (Trusek-Holownia and Noworyta 2015). Both Spray drying and liposome methods have been revealed advantageous for encapsulation of therapeutic enzymes and for their controlled release, using different wall materials and carrier agents (Andrea et al., 2016). Moreover, it can prolong the enzyme stability and in vivo half-life when injected intramuscularly, subcutaneously, or intraperitonially, or administered orally as an ingestible product to carry out its intended functions. Therefore, encapsulation gains importance in treatment of type 1 diabetes, Alzheimer’s disease, Parkinson’s disease, renal disease, cancers, and other disorders (Karamitros et al., 2013, Tomaro-Duchesneau et al., 2013 and Szilasi et al., 2012). Catalase (E.C. 1.11.1.6) an antioxidant enzyme plays important role in detoxifying the hydrogen peroxide production during stress-induced diseases, viz., Zellweger syndrome, acatalasemia, or Wilms tumoreaniridia syndrome (WAGR syndrome). Moreover, it is responsible for cell proliferation by inducing genetic instability and oncogenes activation. Therapeutic application of catalase as future therapeutic target in the context of cancer by using prooxidant approaches has a significance (Glorieux and Calderon, 2017). Chang and Poznansky (1968) reported microencapsulation of catalase in treatment of acatalasaemic mice. This prevents enzyme leakage and its involvement in immunological reactions (Chang and Poznansky, 1968). Poly(lactic-coglycolic acid)ecatalase-loaded nanoparticle protects human neurons from oxidative stress and UV-induced epidermal damage (Singhal et al., 2013 and Hammiller et al., 2017). Microencapsulation of the phenylalanine ammonia-lyase enzyme (EC 4.3.1.24) effectively depleted phenylalanine in phenylketonuric rats within 6 days in vivo study. Here the phenylketonurictreated rats showed no signs of abnormal behavior and weight loss as compared to phenylketonuricnontreated rats as enzyme is protected by low gastrointestinal pH and other proteolytic enzymes action (Bourget and Chang, 1986). Deficiency of the enzyme hypoxanthine phosphoribosyltransferase (HPRT) gives rise a disease called LescheNyhan syndrome. It is also associated with hyperuricemic condition. Encapsulation of xanthine oxidase (EC 1.17.3.2) enzyme protects the condition effectively (Palmour et al., 1989). Moreover, uricase (EC 1.7.3.3) entrapment inside the lipid vesicle membrane along with catalase in bicine buffer as alkaline enzymosomes provides enhanced thermal, hypothermal, acidebase, and proteolytic stabilities, in vitro and in vivo kinetic characteristics, and uric acid lowering properties (Zhou et al., 2016 and Tan et al., 2010). In adult human milk, tolerance caused due to lactose is treated by lactase enzyme/b-galactosidase (E.C.3.2.1.108) that catalyzes conversion of milk lactose to glucose and galactose. Microencapsulation

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protects lactase from highly acidic gastric fluids and allows it to reach in small intestine effectively. Oral delivery of lactase fabricated in carrageenan-based hydrogel beads revealed greater encapsulation, protection, and targeted enzyme delivery (Zhang et al., 2017). Study on microencapsulation of lactase in alginate and bentonite at low pH (4.61) revealed reduction in the protein loss without lowering the enzyme activity (Dashevsky, 1998). While lactase entrapment in a silica matrix by the sol-gel method prolongs its therapeutic action and stability in extreme pH and temperature conditions (Nichele et al., 2011). Microbial urease (EC 3.5.1.5) has been identified as emerging pathogenic factor in bacterial and fungal infection (Mora and Arioli, 2014), and induction of kidney struvite stone formation (Schwaderer and Wolfe., 2017). It is also used in urea removal from artificial kidney dialysate. Oral microcapsule containing urease and an adsorbent, zirconium phosphate, was evaluated for urea removal in stirred batch by Wolf and Chan (1987) and found effective in reducing dialysis treatment times (Do
gac¸ et al., 2014; Wolfe and Chang, 1987). In vivo study of microcapsule containing urease as intraperitoneal injection into dogs of 0.25 mL/kg resulted in rise of the arterial blood ammonia level due to enzyme activity (Chang, 2013). In another study, alginate microparticles cross-linked with Baþþ ions has been revealed as an alternative matrix for urease nanoencapsulation and its possible biomedical applications (Saxena et al., 2017). Urease microencapsulation by liquid-air nozzle method using hexamethylenediamine and a protective protein provides 78% retained initial activity (Miyawaki et al., 1979). Acute lymphoblastic leukemia (ALL) is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells. Asperginase (EC 3.5.1.1), an antitumor enzyme, hydrolyzes asparagine into aspartic acid and ammonia, thus inhibiting the protein synthesis in leukemic cells (Batool et al., 2016). However, treatment with asperginase is hampered by anaphylactic toxic reactions and development of resistance to the enzyme. Liposome-entrapped Erwiniae asparaginase investigation using C3H mice bearing 6C3HED asparagine-sensitive lymphoma cells achieved complete cure (Chang, 2013). Currently nanoencapsulation of asperginase using poly(lacticco-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) as a stabilizer gives maximum EE (87  2%) and reduced immunogenic effects using double emulsification method (De Brito et al., 2019). Superoxide dismutase (EC 1.15.1.1) enzyme is prescribed to treat acute and chronic inflammation by inhibiting the generation of molecular oxygen or hydrogen peroxide from superoxide ions produced by proinflammatory cytokine signaling pathway. PEGylated SOD liposomes can decrease inflammation effectively in a rat model of lipopolysaccharide-induced peritonitis by reducing TNF-a and oxidative species. Moreover, liposomes containing catalase and SOD found effective for skin inflammation reduction in a murine ear edema model (Farhadi et al., 2018). Papain (E.C. 3.4.22.2.) and Phaseolus lunatus protein hydrolysate were encapsulated by carboxymethylated flamboyant seed (Delonix regia) gum (CFG) for better protection in a gastric pH and controlled drug release. Both of them can thus be used as a nutraceutical and therapeutic agents (Betancur-Ancona et al., 2011 and Ruiz Ruiz et al., 2013).

5. Challenges and future outlooks Microencapsulation of bioactive compounds, plant extracts, and enzyme as drug has definitely a remarkable therapeutic potential for a wide range of diseases, due to achievement of polymer chemistry and advancement in encapsulation techniques. A large number of the bioactive compounds and plant extracts are encapsulated for their therapeutic applications but found limited for enzymes.

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However, most of the current methods have several limitations related with the complexity in procedure, selection of material, low EE, toxicity with organic solvent on exposure, maintenance of aseptic conditions, burst effect and controlled drug release, targeted delivery, effective cost, ensured product quality, stability during manufacturing process, reproducibility, and scale-up at commercial level. Therefore, still require great efforts to minimize these issues.

Acknowledgments Authors are thankful to Head, Department of Microbiology, SPPU, and Head, Department of Transfusion Medicine, Seth G S Medical College and KEM Hospital for their help in smoothing out the whole manuscript.

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Hammiller, B., Karuturi, B.V.K., Miller, C., Holmes, M., Labhasetwar, V., Madsen, G., Hansen, L.A., 2017. Delivery of antioxidant enzymes for prevention of ultraviolet irradiation-induced epidermal damage. Journal of Dermatological Science 88 (3), 373e375. He, J., Giusti, M.M., 2010. Anthocyanins: natural colorants with health-promoting properties. Annual Review of Food Science and Technology 1, 163e187. Hu, L., Zhang, J., Hu, Q., Gao, N., Wang, S., Sun, Y., Yang, X., 2016. Microencapsulation of Brucea javanica oil: characterization, stability and optimization of spray drying conditions. Journal of Drug Delivery Science and Technology 36, 46e54. Hu, M., Guo, J., Yu, Y., Cao, L., Xu, Y., 2017. Research advances of microencapsulation and its prospects in the petroleum industry. Materials 10 (4), 369e378. Hussain, S., Hameed, A., Nazir, Y., Naz, T., Wu, Y., Suleria, H., Song, Y., 2018. Microencapsulation and the characterization of Polyherbal Formulation (PHF) rich in natural polyphenolic compounds. Nutrients 10 (7), 843e868. Janiszewska-Turak, E., 2017. Carotenoids microencapsulation by spray drying method and supercritical micronization. Food Research International 99, 891e901. Joana Gil-Cha´vez, G., Villa, J.A., Fernando Ayala-Zavala, J., Basilio Heredia, J., Sepulveda, D., Yahia, E.M., Gonza´lez-Aguilar, G.A., 2013. Technologies for extraction and production of bioactive compounds to be used as nutraceuticals and food ingredients: an overview. Comprehensive Reviews in Food Science and Food Safety 12 (1), 5e23. Junyaprasert, V.B., Mitrevej, A., Sinchaipanid, N., Boonme, P., Wurster, D.E., 2001. Effect of process variables on the microencapsulation of vitamin A palmitate by gelatin-acacia coacervation. Drug Development and Industrial Pharmacy 27 (6), 561e566. Jyothi, N.V.N., Prasanna, P.M., Sakarkar, S.N., Prabha, K.S., Ramaiah, P.S., Srawan, G.Y., 2010. Microencapsulation techniques, factors influencing encapsulation efficiency. Journal of Microencapsulation 27 (3), 187e197. Kamatou, G.P., Vermaak, I., Viljoen, A.M., Lawrence, B.M., 2013. Menthol: a simple monoterpene with remarkable biological properties. Phytochemistry 96, 15e25. Karamitros, C.S., Yashchenok, A.M., Mo¨hwald, H., Skirtach, A.G., Konrad, M., 2013. Preserving catalytic activity and enhancing biochemical stability of the therapeutic enzyme asparaginase by biocompatible multilayered polyelectrolyte microcapsules. Biomacromolecules 14 (12), 4398e4406. Kaushik, P., Dowling, K., Barrow, C.J., Adhikari, B., 2015. Microencapsulation of omega-3 fatty acids: a review of microencapsulation and characterization methods. Journal of Functional Foods 19, 868e881. Kausadikar, S., Gadhave, A.D., Waghmare, J., 2015. Microencapsulation of lemon oil by spray drying and its application in flavour tea. Advances in Applied Science Research 6, 69e78. Kuck, L.S., Noren˜a, C.P.Z., 2016. Microencapsulation of grape (Vitis labrusca var. Bordo) skin phenolic extract using gum Arabic, polydextrose, and partially hydrolyzed guar gum as encapsulating agents. Food Chemistry 194, 569e576. Kumar, L.R., Chatterjee, N.S., Tejpal, C.S., Vishnu, K.V., Anas, K., Asha, K., Anandan, R., Mathew, S., 2017. Evaluation of chitosan as a wall material for microencapsulation of squalene by spray drying: characterization and oxidative stability studies. International Journal of Biological Macromolecules 104, 1986e1995. Lam, P.L., Gambari, R., 2014. Advanced progress of microencapsulation technologies: in vivo and in vitro models for studying oral and transdermal drug deliveries. Journal of Controlled Release 178, 25e45. Laohasongkram, K., Mahamaktudsanee, T., Chaiwanichsiri, S., 2011. Microencapsulation of Macadamia oil by spray drying. Procedia Food Science 1, 1660e1665. Laouini, A., Jaafar-Maalej, C., Limayem-Blouza, I., Sfar, S., Charcosset, C., Fessi, H., 2012. Preparation, characterization and applications of liposomes: state of the art. Journal of Colloid Science and Biotechnology 1 (2), 147e168.

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CHAPTER

Rice husk silica for the stabilization of food-grade oil-in-water (O/W) emulsions

18 Lanny Sapei

Department of Chemical Engineering, University of Surabaya, Raya Kalirungkut, Surabaya, East Java, Indonesia

1. Introduction Food is a basic essential for human beings. Therefore, the overall product quality such as nutrition, texture, appearance, taste, and stability are of fundamental considerations. To provide food with good stability along with desirable texture and appearance, stabilizers have been usually added into processed food products. Stabilizers are generally added into processed foods containing mixtures of aqueous and oil phases. It functions as an enhancement to food texture and appearance, since it helps produce a stable emulsion. Emulsifiers are among the stabilizers which promptly enhance the stability of water-in-oil (W/O) or oil-in-water (O/W) emulsionebased food products. Emulsifiers consist of polar heads and nonpolar tails, and they reorient themselves on the interface of oil and water emulsion to lower the surface tension arising between those two immiscible phases, thus enabling the stable dispersion of one phase into another. Natural emulsifiers have been replaced by synthetic ones for a long time since they can be mass produced and are therefore lower in price. However, there has in recent times been a growing popularity in natural emulsifiers due to the undesirable health effects of synthetic emulsifiers over the longterm such as obesity and related issues (Simmons et al., 2014). On the other hand, natural emulsifiers have low toxicity, are easily degradable, biodegradable, are effective in extreme conditions, and are able to be reused via regeneration, so that they enjoy preference in the food industry despite their higher price (Koglin et al., 2010). Solid particles have existed in emulsion formulation for many years, such as those used in the food, oil, pharmaceutical, and agrochemical industries (Binks, 2002). They are believed to enhance stability to some extent. In many food and foam emulsions stabilized mainly through proteins and phospholipids, solid particles play a significant role for necessary stabilization, such as ice crystals in ice cream and the particles of fat in whipping cream (Binks, 2002). The use of colloidal particles to stabilize emulsions and foams known as the Pickering emulsion has been known for at least a century (Aveyard et al., 2003). Ramsden (1903) concluded that the existence of viscous material at the two immiscible liquids’ interfaces contributed, in part, to the stability of many emulsions. Solid and stabilized emulsion was formed, thanks to finely divided solids dispersed between the interfaces of oil and water. Pickering (1907) observed that colloid particles dampened more easily by Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00018-7 Copyright © 2020 Elsevier Inc. All rights reserved.

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Chapter 18 Rice husk silica for the stabilization

water than by oil could more likely stabilize O/W emulsions. However, the Pickering emulsion has been slowed down, a slow development until the 1980s (Dyab, 2012). Since then, along with progress in the characterization techniques of colloidal particles, there has been renewed interest in Pickering emulsions. Solid particles like metal sulfates, hydroxides, alumina, carbon, and iron oxide have been used as stabilizers of O/W or W/O emulsions (Binks, 2002; Wang et al., 2004; Dyab, 2012; Xie et al., 2018). Recently, some other solids such as hydroxyapatite (Hap), magnetic nanoparticles, chitosan (CS), nanotube, cyclodextrin (CD), and some food-grade stabilizers and organic particles can effectively serve as Pickering emulsifiers (Yang et al., 2017; Dai et al., 2018a, 2018b). Therefore, the applications of Pickering emulsions have become very widespread, for example, as porous scaffolds, catalysts, stimulieresponsive materials, delivery vehicles, and so on (Yang et al., 2017). Pickering emulsions have drawn significant research interest as templates in many fields due to the following advantages: (i) solid particles minimize droplet coalescences and bring about higher emulsion stability; (ii) many solid particles bestow materials with preferred traits such as porosity, responsiveness, etc; and (iii) some solid food-grade particles possess reduced toxicity, making them safer to use in vivo (Yang et al., 2017). There are many behavior similarities of small solid particles and emulsifier molecules at the liquids’ interfaces. Colloidal particles show similar behavior like surfactant molecules, especially when accumulated at the interfaces due to their partitioning between the immiscible liquids, rendering the stability to the emulsion. Solid particles likewise surfactants or emulsifiers tend to be adsorbed on the interface between two phases since they possess hydrophilic portions as well as hydrophobic portions. The tendency of an emulsifier toward oil or water liking is reflected by the term hydrophileelipophile balance number, which is equivalent with the term wettability and contact angle for a spherical particle (Binks, 2002). It is well known that low molar mass emulsifiers and surface-active polymers can help diffuse powdered materials in a liquid by forming aggregated structures in liquid media. In contrast to emulsifiers, individual solid particles do not form aggregates such as micelle, therefore solubilization phenomena is not seen in the particle case. Spherical particles just simply adsorb to oilewater interfaces depending on the contact angle q which governs the particles’ wettability. The wetting efficiency of the solid by one liquid is likely greater than that of the other, leading to the weaker wetting liquid becomes the dispersed phase. For hydrophilic particles, e.g., metal oxides and silica, the particle surface stays more in water than in the oil phase and q measured is normally less than 90
. For hydrophobic particles such as modified silica and carbon, the particle stays more in oil than in water and q measured is generally greater than 90
(Binks, 2002; Aveyard et al., 2003). Colloidal particles could effectively stabilize emulsions if they were wetted in partial by both aqueous and oil phases, thereby considered to be an important group of emulsifying agents (Tambe & Sharma, 1993). Water-wet particles or hydrophilic solid particles tend to stabilize O/W emulsions, while oil-wet particles or hydrophobic solid particles tend to stabilize W/O emulsions (Binks, 2002; Aveyard et al., 2003). The emulsion-stabilizing efficiency of these solids depends on such things as interparticle interactions, particle size, and particle wettability. Furthermore, the stability and type of emulsions formed depends on particle concentration, wettability, pH of the aqueous phase, the presence of ions in the aqueous phase, and the concentration and type of emulsifier present in the system (Tambe & Sharma, 1993). Particles used for emulsion stabilization are normally submicrometer to a few micrometers in size (Tambe & Sharma, 1993). Each particle size should be smaller than the emulsion droplets themselves for the particles to properly cover the droplets. The particle interaction at some degree especially between particles at the interface is necessary for an effective stabilization. From gathered evidence, in

1. Introduction

407

some systems it seems that weak particle flocculation improves stability of the emulsion (Aveyard et al., 2003). Adjusting the pH, salt concentration, and temperature can modify the wettability of the particle (Binks et al., 2006). The adsorption of certain surfactants can also change the particles’ wettability and may occasionally lead to emulsion-phase inversion (Binks, 2002). The solid particles’ ability to steady the emulsions relates to the formation of the interfacial barrier which provides the steric hindrance to dropletedroplet fusion and the modification of the interfacial region’s rheological properties due to the particulate presence. The particles can arrest the Ostwald ripening process in emulsions due to the resulting capillary effects and high desorption energy of the particles (Tcholakova et al., 2008). The formation of highly viscous and rigid films at the interface layer induced by colloidal particles such as clays, resins, asphaltenes, wax, and biopolymers apparently promote emulsion stability (Urdahl & Sjo¨blom, 1995; Al-Sahhaf et al., 2009; Hong & Fischer, 2016; Dai et al., 2018a; Lu et al., 2018; Li et al., 2019). The capacity for solid particles to diffuse and remain in the interfacial region in a state of mechanical equilibrium would also dictate its emulsionstabilizing capacity. The adsorbed solid particles at the interface would then form rigid layer that can sterically curb the fusion of dispersed droplets. For obtaining a stable emulsion, there should be a minimum concentration of particles to sufficiently form a dense layer of particles around the droplets and to modify the degree of interparticle interaction. A heavy layer of solid particles at interfacial layer would influence the capacity of the colloidal particles to stabilize emulsions. The interfacial rheological properties change as the surface concentration of particles increase, resulting in full surface coverages. The interfacial region will tend to demonstrate viscoelastic behavior when the concentration of particles becomes sufficiently high (Tambe & Sharma, 1993). The displacement of the colloidal particles along the interface results in droplet coalescences leading to emulsion instability. The forming of a fixed protective interfacial film, with particular interfacial rheological characteristics, is of great value to the stability of these Pickering emulsions. Moreover, the huge free energy of adsorption for particles of moderate wettability (50
< q < 130
) leads to incredible stability for certain emulsions due to irreversible adsorption, in contrast to emulsifier molecules which are typically in rapid dynamic equilibrium between the bulk phases and the oilewater interface (Aveyard et al., 2003). These properties offer several possible advantages of particulate emulsifiers over conventional surfactant in providing greater stability against coalescence and a reduced rate and extent of creaming/ sedimentation due to the improved viscosity of the continuous phase (Binks, 2002; Aveyard et al., 2003; Binks et al., 2006). The high-level stability found in solid-stabilized emulsions greatly benefits the shelf life of formulas that include those (Binks, 2002). The stability of the formed emulsions increases as emulsifiers are added to the system (Tambe & Sharma, 1993). The wettability of the solids is modified by the presence of emulsifier in the system and most likely enhances the interparticle interaction too. Along with extra surfactant molecules comes an enhanced emulsification capacity, which may be due to an alteration in particle wettability that accompanies surfactant adsorption, thereby modifying the contact angle (Binks & Whitby, 2005). The incorporation of cationic surfactant also enhanced emulsion stability. This is predominantly due to the adsorption of emulsifier molecules both at the interfaces between solideliquid and liquideliquid (Binks & Whitby, 2005). The added emulsifier molecules are partitioned to the interface, and in most cases, colloidal particles would be adsorbed at the oilewater interface, rendering steric hindrance against droplet coalescences. The proceeding interfacial structure is therefore a combination of emulsifiers associated with colloidal particles which leads to long-term stable O/W emulsion due to the very rigid film surrounding the oil droplets (Reger et al., 2011) and viscoelastic interfaces that increase

408

Chapter 18 Rice husk silica for the stabilization

the magnitude of steric hindrance and decrease the rate of film thinning between coalescing droplets (Tambe & Sharma, 1995; Reger et al., 2012).

2. Silica for stabilization of O/W emulsion Silica or silicon dioxide (SiO2) is omnipotent in nature consisting of 26% of the earth’s crust (Martin, 2007) and is present in a crystalline form in nearly all the minerals rocks, clays, and sands on the earth. It belongs to the only inorganic polymer which is most often colorless to white and insoluble in water. The term “biosilica” used in this context referred to silica biopolymer which was accumulated in the living tissues such as rice husk and was isolated thereof. Biosilica is also found in silica-rich plants such as horsetail (Sapei et al., 2007, 2008), bamboo, and grass. In lower creatures such as diatoms and radiolarian, silica was mainly deposited in their cell walls. Biosilica is amorphous in nature and tends to be hydrophilic due to the presence of hydroxyl group on their surface as depicted in Fig. 18.1. Silica has widespread industrial applications as an antifoaming agent, as an excipient in drugs and vitamins, as dough modifier, and as adsorbent to clarify beverage and as viscosity modifier (Martin, 2007). Silica is also among the additive for foods (E 551) and is typically used as an anticaking agent. Silica was reported to have many potential health benefits. Many evidences supported a beneficial role of silicon in collagen formation, improving bone, cartilage, and connective tissue structure, prevention of toxicity to the brain, and maintenance of blood vessel integrity (Martin, 2007). Silica is common in the typical human diet and largely considered safe. There is no observed adverse effect level of 50,000 ppm (mg/L) for dietary silica (Martin, 2007). Biosilica accumulated in plants consisted of colloidal primary particles of about 2 nm (Sapei et al., 2007), and therefore it has a great potential to be used for stabilizing O/W emulsion by diffusing into the oilewater interfaces forming a rigid and densely packed silica particle layer. There were few publications related to biosilica for the stabilization of food-grade O/W emulsions. Sapei et al. (2017a; 2017b) has reported the kinetics of destabilization of O/W emulsion stabilized by rice husk silica

FIGURE 18.1 Schematic illustration of the nature of amorphous hydrated biosilica

2. Silica for stabilization of O/W emulsion

409

combined with lecithin and Tween-20. The destabilization rate of emulsions stabilized with biosilica and lecithin was reduced by w10% in comparison with the emulsions prepared without added emulsifiers (Sapei et al., 2017a). It seemed that the varying concentrations of both lecithin and silica slightly affected the destabilization rates of O/W emulsions. The emulsion destabilization rate constant was reduced by w20% when the emulsion was stored at refrigerated temperatures (Sapei et al., 2017a). In the O/W emulsion stabilized with rice husk silica/Tween-20, it was demonstrated that the varying concentration of Tween-20 significantly influences the overall emulsion stability. The emulsion stability increased with the increase of Tween-20 concentration (Sapei et al., 2017b). The addition of Tween-20 could help increase the wettability of biosilica to be firmly attached on the interfaces, rendering the stability of the resulting Pickering emulsion. The instability of O/W emulsion process due to mainly oil droplet coalescences followed the first-order kinetic model. Recent study demonstrates the influence of pH on the stability of W/O/W emulsion stabilized with rice husk silica/Tween20 on the outer emulsion (Sapei et al., 2018). The acidic environment of the outer aqueous phase (pH < 4) enhanced the stabilizing action of rice husk silica particles on the interfacial layer. The silica particles formed a closely congested layer due to the biosilica particle aggregations, thereby improving the barrier properties against droplet coalescences. The resulting double emulsions had in turn higher stabilities of about 97% after a storage period of 7 days. Pichot et al. (2009, 2010) investigated the effect of surfactant addition on the “food-grade” O/W emulsion stabilized by colloidal hydrophilic silica particles (Aerosil 200). Pichot et al. (2009) found out that the concentration of both monoolein and silica particles influenced the stability of O/W emulsion. Initially, monoolein decelerated the coalescence phenomena and further induced droplet break-up, during emulsification, while reducing interfacial tension by rapidly covering the newly created interfaces, thus allowing the deposition of the silica particles at the interfaces to render longterm stability. Pichot et al. (2010) investigated the effect of different emulsifiers’ types and concentrations on the O/W stability stabilized by colloidal particles. Colloidal silica particles were combined with lecithin and Tween-60/Sodium Caseinate. Emulsion stabilized by mixed emulsifiers and silica particles contained smaller droplets in comparison with the emulsion stabilized by either emulsifiers or silica particles. As the emulsifier concentration increased, dispersed droplets began to increase, followed by the detachment of silica particles from the surfaces. Pichot et al. (2012) investigated the effect of surfactants’ concentrations and types (Tween-60, Na-caseinate, and lecithin) on the contact angles and interfacial tensions in the presence of hydrophilic silica particles. The contact angle profile was governed by both emulsifiers and particles at low emulsifier concentration regardless of the surfactant’s type, whereas it was influenced by the emulsifier only at high concentration. Eskandar et al. (2011) investigated the interfacial structure along with the formation and stability of MCT (medium chain triglyceride) or paraffin-based O/W emulsions which were stabilized with either lecithin or oleylamine and nanoparticles of hydrophilic silica (Aerosil 380). A synergism between emulsifiers and hydrophilic silica nanoparticles in stabilizing O/W emulsion was observed. Dyab (2012) investigated the destabilization of Pickering emulsions in the presence of hydrophobic silica fumed nanoparticles by dictating the pH. The results suggested that pH strongly affected the silica particles’ wettability, hence their contact angles. Frelichowska et al. (2010) investigated the stability of O/W emulsions stabilized by hydrophobic fumed silica. Silica particle aggregation became an important parameter for the emulsion stability. A supplementary mechanism of oil adsorption and capillary condensation of oil within the silica aggregates contributed to the stability of emulsion as well. Binks and Whitby (2005) studied the use of hydrophilic nanoparticle silica in stabilizing O/W emulsion. The oil

410

Chapter 18 Rice husk silica for the stabilization

used included toluene, heptanes, isopropyl myristate, and methyl myristate. Changes in pH and the addition of divalent electrolyte altered the particle charge and flocculation causing temporary enhancements in emulsion stability. In addition, the incorporation of cationic type surfactant improved the emulsion stability. The particle layer formed around the droplets sterically hindered the flocculation, thus stabilizing the droplets against coalescences (Binks & Whitby, 2005). Midmore (1998) observed the synergism action between silica (Ludox) and polyoxyethylene emulsifier during the formation of O/W emulsions. There were three main roles of the surfactant, i.e., promoting solid particle flocculation, modifying the particle wettability, and lowering the interfacial tension. Surfactants played a significant role in modifying the silica surface properties (Lebdioua et al., 2018). Tambe and Sharma (1993) investigated several solid particles including hydrophilic silica in a nonefood-grade O/W emulsion with n-decane as oil phase. It turned out that low pH values (4 and 6) favored the formation of highly stable O/W emulsions stabilized by silica particles and an emulsifier, vice versa a high pH value (8 and 10) was suitable for achieving W/O emulsion with high stability. W/O emulsions are favored as Dai et al. (2019) observed the formation of solid-like films due to the self-assembly of nanosilica/surfactant at the oile water interface. Under weakly acidic conditions, higher surface coverage and lower interfacial tension was observed. Rı´os et al. (2018) found that silica particles increased the surface activity of anionic surfactant by considerably reducing their critical micelle concentration, while the effects were reversed in case of nonionic surfactants. Silica particles were considered as nontoxic and even reduced the toxicity of surfactant solutions. Pickering emulsion stabilized with silica nanoparticles demonstrated an increase in thermal resistance at high temperatures and remained stable in harsh conditions, thanks to a rigid nanoparticle layer at the oilewater interface (Taherpour & Hashemi, 2018).

3. Effect of emulsifier addition combined with rice husk silica in stabilizing O/W emulsion Pickering emulsions stabilized with particles exhibited a much higher stability when combined with the amphiphilic emulsifiers. It was shown that emulsions involving oil and water were less stable when only solid particles or only emulsifiers were used (Pichot et al., 2009, 2010; Eskandar et al., 2011; Sapei et al., 2017a, 2017b). There was a synergistic mechanism between solid particle and amphiphilic emulsifier in stabilizing O/W emulsion. The polymeric emulsifier was easily adsorbed onto the oile water interfaces, which lowered the interfacial tension while facilitating the further adsorption of the particles onto the droplet surface providing the steric barrier against droplet coalescences. The emulsions showed higher stability with greater long-term stability (Pichot et al., 2009; Sapei et al., 2017b) and lower destabilization rate constants (Sapei et al., 2017a, 2017b). The effect of the addition of lecithin and Tween-20 together with rice husk silica on the overall stability of O/W emulsions was described in the following Sections 3.1 and 3.2, respectively. No pH adjustment was made during the experiments. However, the slightly acidic pH of the outer aqueous phase favored the formation of stable O/W emulsion (Tambe & Sharma, 1993).

3.1 Addition of lecithin in the oil phase The O/W emulsion was prepared by dispersing soybean oil into the aqueous phase with the fraction of 20%. Emulsification was carried out using a rotorestator homogenizer. Lecithin was added into the oil

3. Effect of emulsifier addition combined with rice husk silica in stabilizing O/W emulsion

411

phase, whereas pure biosilica derived from citric acideleached rice husk (Sapei et al., 2017b) was dispersed in the aqueous phase. Concentrations of lecithin and silica were varied in order to study their effect on the overall emulsion stability as depicted in Table 18.1. The emulsion stability was measured based on the fraction of emulsion and cream indicated by their corresponding height in the glass vial (Sapei et al., 2017a). The results showed that the O/W emulsion without any emulsifiers had the lowest emulsion stability of below 60% after 2 days. The use of rice husk silica alone as a stabilizer enhanced the emulsion stability. However, when the concentration of silica particles was too low or too high, its emulsion stabilizing ability decreased. The optimum rice husk silica concentration obtained was 2% indicated by the higher short-term (after 1 h) as well as long-term (after 2 days) stability. A minimum concentration of particles is required to sufficiently form a dense layer of particles around the droplets and to modify the degree of interparticle interaction (Tambe & Sharma, 1993). The solid silica particles would diffuse and adsorb onto the interfacial layer, thus rendering a steric hindrance to inhibit the coalescence of oil droplets and increasing emulsion stability. When the silica particles increase, they tend to sediment since the interaction among particles at the oilewater interface is not strong. The silica particles tend to have negative charges over their surface, thereby increasing the electrostatic repulsion between the particles and lessening the silica flocculation at some degree which is required to form a rigid film barrier. Stabilization of O/W emulsion using bare silica particles resulted in an emulsion with low stability since creaming and coalescence occurred rather quickly (Frelichowska et al., 2009). The addition of lecithin ranged from 0.1% to 1.5% did not significantly improve the emulsion stability. In general, an increase in lecithin concentration slightly improved both short-term and longterm emulsion stability. The optimum emulsifier’s mixture was found to be 2% rice husk silica and 1.5% lecithin which demonstrated the highest short-term emulsion stability. The influence of W/O surfactant on the stability of O/W emulsion was almost negligible according to previous investigation (Pichot et al., 2010).

Table 18.1 Stability of oil-in-water (O/W) emulsion stabilized by Rice Husk Silica and Lecithin. Emulsifiers

Emulsion stability after 1 h (%)

Emulsion stability after 2 days (%)

No emulsifiers 1% S 2% S 3% S 2% S þ 0.1% L 2% S þ 0.6% L 2% S þ 1% L 2% S þ 1.5% L

71.15 69.23 73.72 71.15 71.79 73.08 73.72 74.36

59.97 62.18 64.38 61.54 64.10 65.10 64.46 63.82

Oil phase of 20% consisted of soybean oil and lecithin was dispersed in aqueous phase containing rice husk silica. The resulting O/W emulsions were stored at room temperature (w28
C). S and L denoted to silica and lecithin, respectively.

412

Chapter 18 Rice husk silica for the stabilization

3.2 Addition of Tween-20 in the aqueous phase The O/W emulsion with palm oil fraction of 20% was dispersed in the aqueous phase containing pure biosilica derived from citric acideleached rice husk (Sapei et al., 2017b) and Tween-20 using a rotorestator homogenizer. It was obvious as seen in Table 18.2 that O/W emulsion prepared without any emulsifiers had the lowest stability of below 50%. The addition of bare rice husk silica particles up to 2% increased the emulsion short-term stability of about 33%. Furthermore, the incorporation of 1% Tween-20 with 2.5% silica drastically increased the short-term emulsion stability of about 90% and 43% compared to those without emulsifiers and with bare rice husk silica, respectively. The long-term emulsion stability was also increased upon the addition of rice husk silica to about 58% and further increased to about 73% with the incorporation of Tween-20. The emulsion stability decreased with time for the first 1 h. after the emulsion preparation, as the function of various rice husk silica concentration could be seen in Fig. 18.2. Similar trends with emulsions prepared with rice husk silica/lecithin showed that the emulsion stability escalated with the increase of rice husk silica concentration until the optimum concentration was reached and then decreased. A minimum amount of silica particles seemed to be required to cover the oilewater interface, thus providing a rigid barrier against oil droplet coalescences. As silica concentrations were further increased, some particles would be more suspended into the aqueous phase prior to sedimentation due to weak particle interaction in the interface region. The particles on the interfacial layer would probably detach and leach into the aqueous phase due to the van der Waals attraction forces among the suspended silica particles and hydrogen bonding among hydroxyls from both silanols and water. It was obvious that at the optimum rice husk silica concentration of 2%, the emulsion stability exhibited higher value compared to others within the timeframe of 10e60 min. This could be due to a higher interaction between silica particles on the interfacial region that made the formation of rigid film possible. In Fig. 18.3, the emulsion stability profiles all emulsions stabilized with different emulsifiers and without emulsifiers. It was observed that the emulsion stability increased with the sequences as Table 18.2 Stability of oil-in-water (O/W) emulsion stabilized by Rice Husk Silica and Tween-20. Emulsifiers

Emulsion stability after 1 h (%)

Emulsion stability after 2 days (%)

No emulsifiers 1% S 2% S 3% S 2.5% S þ 0.1% T 2.5% S þ 0.3% T 2.5% S þ 0.7% T 2.5% S þ 1% T

46.67 60.00 62.22 44.44 51.11 82.22 86.67 88.89

42.22 57.78 57.78 40.00 47.78 71.11 73.33 71.11

Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica/Tween-20. The resulting O/W emulsions were stored at room temperature (w28
C). S and T denoted to silica and Tween-20, respectively.

3. Effect of emulsifier addition combined with rice husk silica in stabilizing O/W emulsion

413

FIGURE 18.2 Effect of rice husk silica particle concentrations on the oil-in-water (O/W) emulsion stability. Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica. The resulting O/W emulsions were stored at room temperature (w28
C).

follows: mixture of 2.5% rice husk silica and 1% Tween-20 > 1% Tween-20 > 2% silica > without emulsifier. The results suggested a noticeable enhancement of emulsion stability upon the addition of polymeric surfactant, compared to the use of polymeric surfactant only, or bare silica. This is in line with the earlier tests (Pichot et al., 2009; Sapei et al., 2017b). It seemed plausible that there was a synergism between polymeric surfactant and silica particles in stabilizing the oilewater interfacial region. Besides lowering the interfacial tension, polymeric surfactant could also modify the wettability of rice husk silica particles and induce the flocculation of silica particles, thus facilitating the formation of a rigid barrier against coalescences of oil droplets (Midmore, 1998). The use of polymeric surfactant only seemed to be more effective in increasing the emulsion stability compared to the use of bare rice husk silica. This was due to the higher diffusivity rate of Tween-20 as polymeric surfactant molecules into the interfacial region and their surface activity after being adsorbed onto the interfaces. The use of FIGURE 18.3 Effect of various concentrations of rice husk silica/Tween-20 concentrations on the oil-inwater (O/W) emulsion stability. Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica/ Tween-20. The resulting O/W emulsions were stored at room temperature (w28
C).

414

Chapter 18 Rice husk silica for the stabilization

bare silica particles to improve the emulsion stability has become less effective without the addition of other polymeric surfactant. The use of emulsifier mixtures of rice husk silica/Tween-20 slightly increases the overall emulsion stability compared to that stabilized with Tween-20 only. This might indicate the importance of O/W type polymeric surfactant as the predominant factor in enhancing the overall stability of O/W emulsion.

4. Effect of pH of outer continuous phase on the stability of O/W emulsion stabilized with rice husk silica The stability of O/W emulsion stabilized by silica was influenced by the pH of the outer aqueous phase. The O/W emulsion using 20% palm oil and stabilized using 1.5% rice husk silica combined with Tween-20 1% was dispersed in the outer aqueous phase with varying pH of 4.6 (no pH adjustment), 2, 7, and 10. It could be seen from Fig. 18.4 that the emulsion stability reached its maximum of 100% at pH 2 and 4.6 after being stored for 60 min at room temperature. The stability was reduced by approximately 40% at the higher pH (7 and 10) of outer aqueous phase. However, the effect of pH was insignificant for the long-term emulsion stability. The emulsion stabilized with silica gained stability against coalescence when its pH became acidic. (Tambe & Sharma, 1993) The emulsifying properties of silica were mostly affected by pH, whereby acidic pH of less than 4 was preferable to achieve longterm emulsion stability (Pichot et al., 2010). When pH was below 4, silica particles tended to have no charge (Pichot et al., 2010), although the isoelectric point of the silica particle was around pH 2 (Dyab, 2012). Therefore, the silica particles likely to form closely packed aggregates surrounding the interfacial oil and water surface are more resistant to coalescence (Pichot et al., 2009). On the other hand, at higher pH (7 and 10), silica would undergo dissociation into SiO4  with the mechanism as follows (Dyab, 2012): SiOH 4 SiO þ Hþ

FIGURE 18.4 Effect of pH of the outer aqueous phase on the stability of oil-in-water emulsion stabilized using 1.5% silica and 1% Tween-20 after 60 and 120 min storage at room temperature (w28
C). Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica/Tween-20.

5. Effect of storage temperature on the stability of O/W emulsion stabilized with rice husk silica

415

The negatively charged silica particles tended to be hydrophilic. (Dyab, 2012) As silica particles were becoming more hydrophilic, they tended to diffuse into the outer aqueous phase and were less likely to remain within the oilewater interfaces. The negative silica charges favored the electrostatic repulsion among the particles, which hindered the formation of the closely packed layer of silica particles on the interfacial layer. The emulsion stability was therefore decreased as a result of the oil globule coalescences or flocculations due to the weak barrier properties of silica particles on the interfaces. Vice versa, low pH would result in more hydrophobic and chargeless silica particles. If particles were either very hydrophilic (q > 90
), they were easily removed from the interface (Binks & Rodrigues, 2009). Low pH of outer continuous phase proved to produce higher stability of W/O/W emulsion stabilized by rice husk silica according to our previous investigation (Sapei et al., 2018). The proposed mechanisms of rice husk silica stabilization upon basic and alkaline pH has also been described (Sapei et al., 2018). The pH of the system seemed to play a significant part in influencing the wettability of silica particles and in establishing a stable O/W emulsion.

5. Effect of storage temperature on the stability of O/W emulsion stabilized with rice husk silica The emulsion stability was highly dependent on temperature. The effect of storage temperatures on the stability of O/W emulsion stabilized by rice husk silica/lecithin and rice husk silica/Tween-20 could be seen in Figs. 18.5 and 18.6, respectively. It was obvious that the stability of emulsion stored at the refrigerated temperature (w8
C) was higher than that of emulsion stored at the room temperature (w28
C). The increased stability at 8
C was obviously seen after 40 min in the emulsion stabilized using silica/Tween 20. Furthermore, in all O/W emulsions stabilized with silica and varying amount of lecithin showed a slight increase in the overall stability when emulsions were stored at 8
C. The gradual and slight increase in emulsion stability was due to the gradual cooling of the emulsions upon storage in the refrigerator. In general, the emulsion was becoming more stable at the lower temperature. As temperatures lowered, the viscosity of the emulsion increased, thus decreasing the rate of creaming according to Eq. (18.1) (McClements, 2007). The decrease in creaming velocity was due to the slowing down of oil globule flocculations or coalescences at decreasing temperatures. vStokes ¼

2gr 2 ðr2  r1 Þ 9h1

(18.1)

where vStokes is creaming velocity, g is gravitational acceleration, r is droplet radius, r is density, h is viscosity, and subscripts of 1 and 2 are denoted to continuous phase and dispersed phase, respectively. The increased stability of O/W emulsions stored at low temperatures could be observed microscopically as seen in Figs. 18.7 and 18.8. It was observed that both O/W emulsion stabilization using 2% silica/1.5% lecithin and 2.5% silica/1% Tween-20 demonstrated smaller oil droplets dispersed in the aqueous phase at refrigerated temperatures (Fig. 18.7B vs. 18.7C and Fig. 18.8B vs. 18.8C). The smaller the oil globules, the more stable the emulsion. This again inferred the increasing stability of O/W emulsions upon storage at low temperatures. The rate of coalescence and creaming was decreased as emulsion viscosity tended to be increased with the lower temperature.

416

Chapter 18 Rice husk silica for the stabilization

FIGURE 18.5 Effect of storage temperature on the stability of oil-in-water emulsion stabilized using 2% silica combined with various concentration of lecithin after 60 min and 2 day storage. Oil phase of 20% consisted of soybean oil and lecithin was dispersed in aqueous phase containing rice husk silica.

FIGURE 18.6 Effect of storage temperature on the stability of oil-in-water emulsion stabilized using 2.5% silica and 1% Tween-20. Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica and Tween-20.

6. Kinetics study on the stability of O/W emulsion stabilized with rice husk silica Research has shown that a O/W emulsion tended toward instability with time, since it was an inherently thermodynamically unstable system. However, the emulsion could be presumed to be stable when it demonstrated a high kinetic stability. The kinetic stability of the emulsion was able to be increased by many means including the choice of emulsifiers, ratio of all components added into the formulation, presence of additives, emulsification techniques, and condition during emulsification. The instability of emulsions was caused by several phenomena such as flocculation, coalescences,

6. Kinetics study on the stability of O/W emulsion stabilized with rice husk silica

417

FIGURE 18.7 The microscopic images of oil-in-water emulsions stabilized with 2% rice husk silica and 1.5% lecithin. Oil phase of 20% consisted of soybean oil and lecithin was dispersed in aqueous phase containing rice husk silica. (A) After emulsification, (B) after 3 h storage at room temperature (w28
C), and (C) after 3 h storage at refrigerated temperature (w8
C).

Ostwald ripening, creaming, sedimentation, phase inversion, and phase separation (McClements, 2007). In O/W emulsions systems, creaming generally occurred as the result of oil globule flocculation followed by coalescences. The oil globules became larger upon coalescences, thereby increasing the creaming rate leading to emulsion instability. The kinetics of emulsion destabilization was studied in some of our experiments in order to determine the effect of different emulsifier mixtures involving the use of rice husk silica upon the Pickering emulsion stabilization. It has been known that the coalescence rate of the dispersed phase globules followed the first kinetic model (Wanli et al., 2000). The data within the first 20e40 min and between 0 and 120 min were selected for the determination of the destabilization kinetics of O/W emulsion stabilized by rice husk silica/lecithin and rice

(A)

(B)

(C)

FIGURE 18.8 The microscopic images of oil-in-water emulsions stabilized with 2.5% rice husk silica and 1% Tween-20. Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica and Tween20. (A) After emulsification, (B) after 2 h storage at room temperature (w28
C), and (C) after 2 h storage at refrigerated temperature (w8
C).

418

Chapter 18 Rice husk silica for the stabilization

husk silica/Tween-20, respectively. The emulsion stability data remained quite constant after those specified timeframe. The stability data of O/W emulsion were evaluated in time using the zero-order or first-order kinetic models to determine the constant destabilization rate. The least square procedure was applied in order to determine the most appropriate kinetic order based on the best obtained correlation coefficient (R2). The emulsion destabilization rate was represented by Eq. (18.2), while the zero-order and first-order kinetics models were represented by Eqs. (18.3) and (18.4), respectively (Levenspiel, 1999): r ¼ 

dZ dt the

dZ ¼ kZ a dt

(18.2)

Z ¼ Z0  k0 t

(18.3)

lnðZÞ ¼ lnðZ0 Þ  k1 t

(18.4)

where emulsion destabilization rate, a the emulsion destabilization rate order, k0 and k1 the destabilization rate constants of emulsions for the zero order (% stability/minute) and first order (per minute), respectively, t the storage time (minute), Z the emulsion stability percentage after time t, and Z0 the initial emulsion stability percentage. The kinetics data of O/W emulsion stabilized by rice husk silica/lecithin and rice husk silica/ Tween-20 were depicted in Tables 18.3 and 18.4 respectively. Soybean oil was used for the emulsions stabilized with silica/lecithin, whereas palm oil was used for the emulsions stabilized with silica/Tween-20. Generally, the destabilization process of all O/W emulsions was fitted more closely to the first-order kinetic models compared to the zero-order kinetic models. This was reflected by the higher R2 obtained from the fitting of the experimental data with the first-order kinetic model. Silica of different concentrations ranged from 1% to 3% was investigated in a soybean/water system. The lowest destabilization rate (k) of 1.3  103 min1 was found in the O/W emulsion stabilized with 2% silica as could be seen in Table 18.3. When the concentration of silica was decreased into 1% or increased into 3%, the k values were lowered by around 3 and 7 times, respectively. This implied that there was an optimum concentration of rice husk silica that was needed to help stabilize the O/W emulsion. When the silica particle concentration was too low, there would not be sufficient coverage of silica on the interfacial layer. In contrast, when the silica particle concentration was too high, silica particles tended to be leached out to the outer aqueous phase due to the hydrophilicity of biosilica particles. The huge decrease in emulsion stability occurred with time since the particle silica on the interfacial layer was probably detached from the interface due to the hydrogen bonding interaction between the hydroxyl groups of silanol present in biosilica and water. As particle density at the oilewater interface decreased, the emulsion stability also decreased (Wang et al., 2010). Furthermore, there was an increase in O/W emulsion stability as concentrations of lecithin were increased, combined with the consistent concentration of rice husk silica. This was reflected by the decreased k with the increased lecithin concentration. This may indicate the role of lecithin in assisting the deposition of silica particles onto the interfacial layer, thus improving the barrier properties of the emulsifier layer against flocculations or coalescences. However, the synergistic action between silica and lecithin was not obvious, since the addition of lecithin did not decrease the k value by much and was comparable with that obtained when no emulsifier at all was used with k of 2.5  103 min1 (Sapei et al., 2017a). The lowest k was achieved by the use of 2% silica only and

6. Kinetics study on the stability of O/W emulsion stabilized with rice husk silica

419

Table 18.3 Kinetic oil-in-water emulsionedestabilizing rate constants and R2 values according to zero-order and first-order kinetic models for emulsions stabilized with rice husk silica/ lecithin. Storage temperature Emulsifiers

Order 1 R

k1 3 10 (min-1)

R2

28 28 28 28

0.3669 0.1293 0.8107 0.382

0.9541 0.8044 0.9082 0.9651

3.9 1.3 9.6 4.0

0.9586 0.8071 0.9064 0.9688

28 28

0.3573 0.2185

0.8048 0.8723

3.7 2.3

0.8103 0.8789

8

0.315

0.9844

3.3

0.9863

8 8

0.1523 0.1869

0.8055 0.8954

1.6 1.9

0.8118 0.9001

T ( C)

S 1% S 2% S 3% S 2% þ L 0.1% S 2% þ L 1% S 2% þ L 1.5% S 2% þ L 0.1% S 2% þ L 1% S 2% þ L 1.5%

Order 0 k0 (% stability/min)

o

2

3

Oil phase of 20% consisted of soybean oil/lecithin was dispersed in aqueous phase containing rice husk silica. S and L denoted as silica and lecithin, respectively.

Table 18.4 Kinetic oil-in-water (O/W) emulsionedestabilizing rate constants and R2 values according to zero-order and first-order kinetic models for emulsions stabilized with rice husk silica/Tween-20. Order 0

Order 1

Emulsifiers

k0 (% stability/min)

R2

k1 3 103 (minL1)

R2

Without emulsifiers S 2.5% 1% T 2.5% S þ 0.1% T 2.5% S þ 0.3% T 2.5% S þ 0.7% T 2.5% S þ 1% T

4.9621 5.7525 0.3524 1.7681 0.2326 0.1824 0.1390

0.9235 0.7749 0.9460 0.9119 0.9634 0.9447 0.9349

72.5 72.4 4.0 23.1 2.7 2.0 1.5

0.9434 0.7937 0.9540 0.9321 0.9769 0.9568 0.9422

Oil phase of 20% consisted of palm oil was dispersed in aqueous phase containing rice husk silica/Tween-20. The resulting O/W emulsions were stored at room temperature (w28
C). S and T denoted as silica and Tween-20, respectively.

420

Chapter 18 Rice husk silica for the stabilization

was comparable with the use of 2% silica/1% lecithin upon storage at 8
C. This implied that the addition of lecithin did not significantly influence the overall O/W emulsion. This was also corroborated by the previous investigation that the concentration of W/O surfactant within the emulsifier mixture did not significantly affect the stability of O/W emulsion system (Pichot et al., 2010). The silica particle seemed to predominantly determine the emulsion stability. However, the effectiveness of the silica particle as an emulsifier was influenced by pH which modulated its wettability on the interfacial layer. The emulsion stability could have been maximized by changing the pH into acidic which helps increase the performance of the silica particle on the interfaces as having been described in the previous section. The rate of emulsion destabilization was up to 2.3 times lower when the emulsion was stored at the refrigerated temperature. This confirmed that low storage temperature increases the emulsion stability. When rice husk silica was mingled with various Tween-20 as the O/W surfactant, it was obvious that the constant destabilization rate decreased as the concentration of Tween-20 increased, as seen in Table 18.4. This demonstrated the synergistic action between rice husk silica and Tween-20 in stabilizing the O/W emulsion. The Tween-20 surfactant would easily adhere to the oilewater interface and facilitate the attachment of rice husk silica on the interface. Therefore, a rigid layer of silica on the interface would prevent the oil globule coalescences. The lowest k of 1.5  103 min1 was attained with the use of 2.5% rice husk silica/1% Tween-20 as mixed emulsifiers. In contrast to silica/lecithinstabilized O/W emulsion whereby lecithin showed no effect on the emulsion stability, Tween-20 seemed to play a significant role in achieving an emulsion with high stability. A tremendous decrease in emulsion stability was observed upon the use of silica alone or without emulsifiers at all reflected by the very high k value of about 72  103 min1. Based on these experiments, the presence of Tween-20 was crucial in producing a highly stable O/W emulsion stabilized by rice husk silica. The presence of surfactant could modify the wetting properties of the solids and likely enhance the interparticle interactions (Tambe & Sharma, 1993). In summary, the stabilizing capability of rice husk silica in an oilewater interface was governed by pH, temperature, and added surfactant. The improved stability of Pickering emulsions with respect to polymeric emulsifiers stabilized classical emulsions especially for food applications would be a great advantage. The development of emulsions without the use of emulsifiers have attracted particular attention since emulsifiers may cause some adverse effects, such as air entrapment, foaming, irritancy, and detrimental interaction with living matter (Frelichowska et al., 2010). Rice husk silica seems promising to be used as the stabilizing agent of oil droplets while reducing the use of emulsifiers for the formulations of various O/W emulsionebased processed food products. Rice husk biosilicae stabilizing Pickering emulsions would also demonstrate the great potential as an encapsulation vehicle combined with the controlled release mechanism of bioactive ingredients for developing highly nutritious and healthful food products.

Acknowledgments The research was partially funded by Ministry of Research, Technology and Higher Education of the Republic of Indonesia under the research grant scheme of “Fundamental Research” 2019 under the contract number: 004/ SP2H/LT/MULTI/L7/2019.

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CHAPTER

Oil-entrapped films

19

Saumya Agarwal1, Monjurul Hoque1, Nupur Mohapatra1, Irshaan Syed1, Chanda Vilas Dhumal1, Subhadeep Bose2, Prasanta Kumar Biswas2, Padmaja Kar3, Nisarani Bishoyi3, Preetam Sarkar1 1

Department of Food Process Engineering, NIT Rourkela, Rourkela, Odisha, India; 2Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata, West Bengal, India; 3Department of Chemistry, NIT Rourkela, Rourkela, Odisha, India

1. Introduction Recent advances in the minimally processed and the ready-to-eat “fresh food” domain have given rise to the discovery of edible films which are proving to be suitable replacements to the commercially available synthetic polymer packaging. Edible films can be defined as “preformed thin layer or solid sheets of edible components placed on or between food components” (Fasihi et al., 2019). Edible coatings on food material help in enhancing the shelf life of food, maintaining the stability, and posing an advantage of serving as nondisposable packages which reduce the environmental influence to a great extent (Shit & Shah, 2014). These edible polymers were made from renewable and edible ingredients and thus are supposed to degrade more conveniently than polymeric materials. Various characterizations of the developed edible films need to be studied carefully before applying them in food systems. They mainly include barrier properties like water vapor permeability and oxygen permeability, mechanical properties like tensile strength, Young’s modulus, and elongation at break (Fig. 19.1). Incorporation of edible oils tends to produce bioactive films with antimicrobial and antioxidant activities which further needs to be assessed carefully. Cost-effectiveness, flavor, color, and simple technology for production and improvisation of their recyclability by simplifying their structure are other important parameters to be considered (Debeaufort et al., 1998). The various other functional properties of oil-entrapped films are listed in Fig. 19.2. Edible films on the incorporation of essential oils provide microbiological stability to the food by serving as additive carriers, which have the ability to extend the shelf life of the product, thereby reducing the growth risk of pathogenic bacteria and other undesirable flora on the surface of the product (Avila-Sosa et al., 2012). The high vapor pressure attributes of the plant-derived essential oils facilitates its way through the liquid and gas phase for reaching the pathogens. They also possess the potential to serve as flavoring content in food and beverages. The addition of essential oils in edible films encompasses the consumer demands to provide foods with long shelf life which are natural or mildly processed but with no added chemical preservatives. This chapter deals with different polymers like polysaccharides, proteins, and composites that are used to form edible films assimilating essential oils as a part of their matrix for improvising the overall properties of the edible films and broadening the scope of their usage (Fig. 19.3). Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00019-9 Copyright © 2020 Elsevier Inc. All rights reserved.

425

426

Chapter 19 Oil-entrapped films

FIGURE 19.1 Functional properties of oil-entrapped films.

FIGURE 19.2 Flowchart for development of oil-entrapped films.

2. Hydrocolloid-based films incorporated with essential oils Hydrocolloids include naturally occurring macromolecular proteins and carbohydrates which form the primary basis of food structure. Hydrocolloids are responsible for imparting thickness and gelling properties and in stabilizing the foams, emulsion, and dispersions consequently assisting in regulation and modification of the rheology and structure of the food systems. They interact with the molecular

2. Hydrocolloid-based films incorporated with essential oils

427

FIGURE 19.3 Schematic representation of barrier properties of films.

assemblies in the food formulations and subsequently affect the texture and nutritional and functional properties of foods including edible films (Ai et al., 2015). The matrices formed are embedded with essential oils like oregano oil, lemon oil, clove oil, and cinnamon oil to aid their functional and barrier properties. The choice of essential oils depends on the specific purpose and the type of system on which they are being implemented. Hydrocolloids like starch, chitosan, carrageenan, pectin, soy protein isolate, zein protein, etc., in association with oils are discussed in sections below.

2.1 Polysaccharide-based edible films 2.1.1 Starch-based edible films Starch is composed of amylose and amylopectin. The major sources of starch are cereal grains and tubers like corn wheat potato, tapioca, and rice. The film-forming capacity of starch is attributed to amylose (Romero-Bastida et al., 2015). Amylose is the linear fraction of the starch molecule which produces coherent and strong films as opposed to the amylopectin which results in brittle and noncontinuous films (Zobel, 1988). Starch-based films are not at par when compared to synthetic films due to their moderate gas barrier and mechanical properties (Cha & Chinnan, 2004). As previously  mentioned, this section will deal with essential oil incorporated into starch-based active films (Suput et al., 2016) (Table 19.1).

2.1.1.1 Cassava starch Cassava (Manihot esculenta Crantz) is a starch-based root crop suitable for human consumption and industrial purpose in tropical and subtropical regions of Asia, Africa, and Latin America. Cassava is enriched with major nutrient sources (carbohydrates, riboflavin, thiamin, and nicotinic acid) except proteins. Harvesting time and season of planting play an important role in ensuring high amylose

428

Biopolymer

Serial no.

Film category

Protein

Polysaccharide

Oil

Properties

References

1

Monophasic

e

Corn starch

Oregano essential oil(1%)

Suput et al. (2016)

2

Monophasic

e

Alginate

Cinnamon essential oil nanoemulsions(4%)

3

Biphasic

e

Carrageenan Carboxy methyl cellulose

4

Monophasic

Soy protein isolate

e

Oregano essential oil (0.02%) Thyme essential oil (0.03%) Rapeseed oil(3%)

5

Monophasic

Whey protein

e

Tensile strengthd2.3 Mpa Inhibition zone against Listeria monocytogenesd37 mm Moisture contentd12.3% Tensile strengthd15.63 Mpa Inhibition zone against Salmonella typhimuriumd65.67 mm Inhibition zone against Bacillus cereusd62.67 mm Moisture contente10%e12% Tensile strengthd0.31
0.04 kg/cm2 Moisture contentd38.88%
1.24 Water vapor permeabilityd3.38
0.10 gm/m2sPa Tensile strengthd0.91 Mpa Elongation at breakd4.19
0.16% Water vapor permeabilityd2.23
0.03 1010 gm1Pa1s1 Tensile strengthd109
7 Mpa Inhibition zone against Penicillium communed1.3
0.8 cm Water vapor permeabilityd0.0011
0.0001 gm m m m2 day1 kPa1

Oregano essential oil (1%)

Frank et al. (2018)

Soni et al. (2016)

Galus (2018)

Oliveira et al. (2017)

Chapter 19 Oil-entrapped films

Table 19.1 An overview of different oil-loaded films along with their biopolymeric matrix, oil added, and important properties.

Biphasic

e

Chitosan Cassava starch

Cinnamon leaf essential oil (0.25%)

7

Biphasic

e

Potato starch Zedo gum

Salvia officinalis Essential oil (250 mL)

8

Biphasic

Whey protein isolate

Cellulose nanofibres

Rosemary essential oil (2%)

9

Biphasic

Corn zein

Chitosan

Cinnamon essential oil (2%)

10

Triphasic

e

Potato starch Furcellaran Gelatin

Lavender essential oil (2%)

Tensile strengthd18
2 Mpa Total coliform countd6.5 log cfu/g Water vapor permeabilityd7.87
0.14 g mm kPa1 h1 m2 Tensile strengthd2.50
0.058 Mpa Strain to breakd38.812
0.1153% Water vapor permeabilityd6.70
0.03 1011 g/m s Pa Antioxidant propertyd64.67
0.24% Shelf life extensiond9 days Minimum inhibitory concentrationd1mg/mL Inhibition zone against L. monocytogenesd19.4
0.35 mm Tensile strengthd2 Mpa Elongation at breakd21% Water vapor permeabilityd2.058
0.281 g Pa1 h1 m1 Inhibitory area against Staphylococcus aureusd27.33
1.93 mm2 Tensile strengthd62.8
1.9 Mpa Elongation at breakd19.5
2.6% Water contentd72.28
9.75% Inhibition zone against S. aureus, e8.9
0.9 mm

ValenciaSullca et al. (2018) Pirouzifard, Yorghanlu et al. (2019)

Sani et al. (2017)

Vahedikia et al. (2019)

Jamro´z et al. (2018)

2. Hydrocolloid-based films incorporated with essential oils

6

429

430

Chapter 19 Oil-entrapped films

content from the extracted starch. Cassava has proved to be a reliable crop for such applications due to its year-end availability and draught-tolerant traits. Small concentrations of the essential oils showed inhibitory characteristics keeping in check the overall flavor profile of the product being manufactured. In the following section, properties of films obtained from cassava starch in association with cinnamon and clove essential oil, lemongrass oil, and oregano oil are discussed. Cassava starch films incorporated with cinnamon and clove essential oil demonstrated considerable antibacterial activity against microorganisms like Penicillium commune and Eurotium amstelodami. The cinnamon essential oil is more effective against the latter with a destruction rate of almost 91% of the microbial population. However, the increase in the antimicrobial oil concentration leads to a reduction in the mechanical strength including tensile strength and elongation at break of the cassava films due to lesser intermolecular strength between the polymeric chains. The water vapor permeability of the obtained films is mostly affected by the concentration of glycerol and emulsifier implying a negligible role for the essential oil to play (Souza et al., 2013). In another study, the addition of lemongrass oil to cassava starch films resulted in inhibition of microorganisms namely Penicillium and Trichoderma when applied to meat during its storage but the inhibitory action lasted only for 3 days. The addition of oil furthermore decreased the tensile strength and roughened the texture (Supardan et al., 2016). The inclusion of oregano oil in the films demonstrated a negative impact on the tensile strength alongside having a positive impact on the film elongation. Higher oregano oil concentrations were effective against Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. They also succeeded in preventing lipids oxidation for 3 days when tested in meat (dos Santos Caetano et al., 2018).

2.1.1.2 Corn starch Maize or Zea mays commonly referred to as corn has roughly 75% amylopectin and 25% amylose content. Corn starch is generally modified for applications in the food industry. The addition of black cumin and oregano essential oil (OEO) to a corn starch film considerably decreased the swelling of the film because of the hydrophobic nature of the oil. The tensile strength of the control film was 14.43 MPa, whereas the tensile strength of the films decreased from 10.02 to 2.3 MPa in case of black cumin oil and 10.65e2.12 MPa in case of OEO. These large reductions in tensile strength upon addition of the essential oils could be accounted to the plasticizing effect of the essential oils. The elongation at break values was reported to increase in similar studies after addition of essential oils in biopolymer matrices. The water vapor transfer (WVT) of the film decreased upon the addition of essential oil as WVT takes place through the hydrophilic structures of the film and addition of essential oil led to a decrease in the same. Also, the addition of black cumin oil narrows the diffraction peaks as seen from the XRD measurements. The L value of the films decreased with the addition of essential oils indicating a transition from completely transparent control films. Black cumin essential oils were not successful in providing any kind of microbial inhibitory characteristics, whereas oregano oil was highly effective against gram-positive bacteria and a larger inhibition zone was created (Suput et al., 2016). Addition of nutmeg oil nanoemulsions to corn starchebased films increased the film thickness but had no effect on the tensile strength and elongation of the film thus contradicting to the observations recorded earlier during the addition of cumin and oregano oil. Furthermore, addition of 1% nutmeg oil proved to be effective in destroying bacteria like E. coli and S. aureus (dos Santos Caetano et al., 2018). The shelf life of raw beef was extended by casing them with corn starch film incorporated with cinnamon and clove essential oil which subdued the microbial activity of Lactococcus lactis and

2. Hydrocolloid-based films incorporated with essential oils

431

Salmonella typhimurium and simultaneously reduced the rate of lipid oxidation. Eugenol in clove essential is again responsible for aiding antimicrobial activity. Eugenol in clove essential oil mainly aids the antimicrobial activity (Radha krishnan et al., 2015). The detailed mechanism of essential oil activity on microorganisms is illustrated in Fig. 19.4.

2.1.1.3 Banana starch Banana starch has proved its potential in industry for processed foods owing to its digestion and functional property, and commercial viability. Films made out of banana starch when incorporated with lemongrass and rosemary essential oils nanoemulsions improved the state of the edible film and made it suitable to serve packaging purposes. The factors that promote the usage of newly formed banana starch film in packaging were its increase in the transparency and elongation of the films. As mentioned in the previous section, water vapor permeability of the obtained films increases due to the addition of EO but the water solubility of the film does not increase because of the hydrophobic property of the essential oil which counteracts the increase in WVP (Villa, 2018).

2.1.2 Chitosan-based edible films Chitosan is a deacetylated derivative of chitin, a naturally occurring polymer present in shells of crustaceans. Films based on chitosan are obtained using the solvent casting method. As mentioned earlier, the packaging attributes of chitosan-based films can also be improvised by the addition of essential oils. Apricot kernel essential oil (AKEO) obtained from seed kernels of the apricot was incorporated in chitosan films which marked an increase in water resistance and enhancement of water vapor barrier permeability by 41% was reported when equal concentration of AKEO and chitosan was taken. The elongation showed a sharp increase up to an oil ratio of 0.125 with respect to chitosan after which on further increase in the oil ratio, a decrement in elongation was observed. The FEM image analysis reflected that by increasing the AKEO concentration, the heterogeneity increased due to the entrapped oil particles which deviate the microstructure from that of smooth surfaced pure chitosan

FIGURE 19.4 Mechanism of essential oil activity on microorganisms.

432

Chapter 19 Oil-entrapped films

films. The AKEO in the chitosan films serves as a natural antioxidant which halts oxidative degradation of the food material that is packed in it. AKEO has antimicrobial properties which damage the bacterial membrane and cause leakage of the intercellular fluid leading to the death of the microorganism. It is effective against both the positive and negative strains of bacteria (Priyadarshi et al., 2018). Chitosan films containing Eucalyptus globulus essential oil (EGO) have the ability to be served as active packaging material for sliced sausage. EGO has a special characteristic of showing antimicrobial property when present in even the vapor phase. The increase in the concentration of EGO marked a decrease in the moisture content and the water solubility, thus increasing the hydrophobicity of the films. The FE-SEM images showcased that the film was devoid of cracks and was smooth except for slight changes which were observed when there was a further increase in EGO oil concentration. The tensile strength of the film was inversely proportional to the concentration of EGO. Upon addition of 0.5% EGO, log reduction values of approximately 2 are seen against microorganisms like E. coli, S. aureus, and Bacillus cereus (Azadbakht et al., 2018). EGO incorporated films show a clear inhibitory zone without any bacterial/fungal growth and effective in vitro antioxidant property of the films, thereby extending the shelf life (Hafsa et al., 2016). Chitosan films incorporated with basil and thyme oil were tested against Aspergillus niger and Rhizopus stolonifer, wherein the added essential oils proved ineffective in destroying the fungal population. The retention of these essential oils in the films is greatly influenced by the stability of the film forming an emulsion. This stability can be increased by adding oleic acid which aids in retaining a large amount of essential oil in the film (Perdones et al., 2016). The edible film quality on antibacterial activity is improved by adding red ginger oil to chitosan films due to their active components namely shogaol and gingerol. The addition of 1.2% of purified ginger oil inhibited the growth of E. coli (Irawan et al., 2017). Essential oil from cumin and clove were also tested against a range of bacteria. Clove essential oil seemed the most likely to be put into the food packaging application on account of the minimum inhibitory concentration of the two oils (Herna´ndez-Ochoa et al., 2012).

2.1.3 Carrageenan-based edible films Carrageenan which is readily obtained from seaweed serves as an economical alternative for the raw material used during the preparation of edible films. Carrageenan contains a linear chain of partially sulfated galactans which are responsible for its film-forming ability all the while making it highly water soluble (Praseptiangga et al., 2016). Carrageenan was isolated from the seaweed flour before the preparation of edible film (Jabar et al., 2013). The addition of palm oil as a plasticizer during carrageenan film preparation showed considerable bifurcations from the control film. The general flowchart for development of biopolymer-based edible films with incorporation of essential oil is depicted in Fig. 19.5. The tensile strength and elongation were increased up to the extent of 1.5% of carrageenan concentration after which the films started becoming brittle and stiff. In another similar study, oxygen permeability started getting compromised after increase in palm oil quantities. The experimentation model to study these films was sliced apples. They were successful in decreasing the vitamin C loss from the surface and fruit shrinkage of the cut apples, preventing a weight loss of 30.7% (Jabar et al., 2013). The cinnamon essential oil along with kappa carrageenan was used to produce edible films. Meanwhile, the thickness of the film showed a positive proportionality with the increase in the cinnamon oil content. Following a similar trend, the cinnamon essential oil addition led to an increase in the overall tensile strength of the film owing to the strong cross-link effect between the polymer and

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FIGURE 19.5 Classification of biopolymer-based films.

the essential oil. The essential oil incorporation decreases the chain interactions causing the biopolymeric network to become heterogeneous leading to discontinuities in the polymer matrix, consequently decreasing the elongation at break values. As observed earlier, the water vapor permeability rate decreased with the incorporation of oil. Summing this up, 1% concentration of cinnamon oil was recommended for creating edible films (Praseptiangga et al., 2016). The concentration of 1.5% carrageenan along with concentrations of oregano oil (0.02%) and thyme oil (0.03%) was found suitable for acting as a biopreservative for chicken meat products (chicken patties). The addition of essential oils did not show any considerable thickness alteration of the edible films. In accordance with the aforementioned studies, the moisture absorbing capacity, film solubility, and the WVP of the films were decreased due to the hydrophobic oil molecules. The oil molecules increased the intermolecular attractions between the polysaccharide molecules, thereby decreasing the tensile strength as a result of oil incorporation. The elongation values did not vary much compared to the control films (Kumar et al., 2016).

2.1.4 Pectin-based edible films Pectin is a suitable material for the formation of edible films due to its biocompatibility, versatile chemical/physical properties, and biodegradability. With an aim to reduce pathogen growth and extend

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the shelf life, pectin edible films have been incorporated with antimicrobials compounds. These postprocessing foodborne pathogens and the spoilage microorganisms were threatened as the antimicrobial diffuses through the film to the food surface (Espitia et al., 2014). OEO has been found to show antimicrobial properties, but the volatile nature of the oil makes it ineffective in certain applications. Pectin obtained from citrus peel was incorporated with OEO, and its effect was tested against E. coli, Salmonella choleraesuis, S. aureus, and L. monocytogenes. After performing the disk diffusion assay, the essential oil induced inactivation of all the tested bacteria at an MIC of 0.24 mg/mL. These films when used to coat shrimps and freshly sliced cucumbers negatively affected the growth of coliforms, yeasts, and molds stored at 4 C over a storage period of 15 days. They aided in shelf life extension of the tested product by reducing the microbial populace (Alvarez et al., 2014). Edible films made out of mexican lime bagasse, pomace pectic extracts, and lime essential oil showed a negative impact on the growth conditions of foodborne pathogenic bacteria (E. coli, S. typhimurium, B. cereus, and L. monocytogenes). Again, disk diffusion method was adopted for determining the extent of inhibition zones for each bacteria. The observations helped concluding that E. coli had a higher inhibition zone as compared to the others (Sa´nchez Aldana, Andrade-Ochoa, Contreras-Esquivel, & Nevarez-Moorillon, 2014). Coatings were developed using pectin enriched with citral and eugenol essential oils for the enhancement of shelf life of strawberry. Strawberries generally possess a storage period of 7 days and after a maximum of 14 days studies indicate their deteriorate making them unfit for human consumption. Addition of the essential oils had an overall improving effect on the quality of the stored strawberry. An optimum concentration of 2% pectin and 0.1% eugenol or 2% pectin and 0.15% citral was found to be most effective (Guerreiro et al., 2015).

2.1.5 Gum-based 2.1.5.1 Guar gum Mangoes have a short shelf life and are prone to spoilage during postharvest. Edible films were made out of guar gum mixed with essential oils for postharvest preservation of mangoes. When ethanolic essential oil was used alongside guar gum, the bacterial count reduced appreciably. Also, more retention in quality of mango was observed. Without supplementing the guar gum with essential oils, spoilage of mangoes occurred after 6e10 days. Whereas incorporation of ethanolic or methanolic EO to the guar gum was able to extend the shelf life to even 24 days (Naeem et al., 2018).

2.1.5.2 Gum tragacanth For inhibiting the aging of button mushroom during cold storage, tragacanth gum (TG) treated with Zataria multiflora Boiss Essential oil (ZEO) was used and a comparison was drawn with sodium metasulfite (Hafsa et al.). After observation for a period of 16 days, it was observed that there was a significant cut down in the number of microorganisms when TG-ZEO was used. Also TG-ZEO accounted for the retention of 93.47% of mushroom tissue firmness, 86% of phenolic compounds, and 72.8% of ascorbic acid. However, there was a drop down in the BI value up to 52.23%. TG-ZEO was able to retain mushroom quality and also improved its shelf life. The results indicated that the combined effect of TG and ZEO had greater influence in preservation of button mushroom than TG or SM alone (Nasiri et al., 2017).

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2.1.5.3 Gum Arabic For the prevention of fungal diseases arising in tropical fruits such as banana and papaya, composites of gum arabic (GA), cinnamon oil (CM), and lemongrass (LG) were used. When used alone, GA treatment resulted in firmer fruit because it delayed fruit ripening, but it had no antifungal properties. Making a composite of 10% GA and 0.4% CM resulted in increased shelf life and better quality of fruit during its storage. Also, a complex of 10% GA and 0.05% LG showed antifungal properties. The most suitable composition for prevention of postharvest anthracnose in banana and papaya (tropical fruits) was found to be a composite of potato dextrose agar medium combined with 10% GA and 0.4% CM. It was observed that the fruits were more firm and had high water loss and low values of titratable acidity and soluble solid concentration (Maqbool et al., 2011).

2.2 Protein-based edible films Proteins are complex macromolecules composed of one or more long chains of amino acids forming a specific molecular structure. The varied distribution of charged, polar, and nonpolar amino acids along the protein chain provides it with the required chemical potential (Dangaran et al., 2009). The characteristics of these protein films and coating are dependent on its molecular characteristics like molecular weight, conformations, electrical property, flexibility, and thermal stabilities (Vargas et al., 2008). Multiple types of physical and chemical treatment can be applied on the protein structure (primary, secondary, or tertiary) to obtain the desired physical and functional properties so that they can be suitably modified to be used in the making of edible films (Chiralt et al., 2018). Among the various edible films prepared from polysaccharides, proteins, or lipids, the most preferred one is protein edible film because it offers not only an explicit range of functional and physical properties but also high binding potential on the intermolecular level. Studies are being conducted to improvise the moisture barrier properties of these edible protein films and increase its mechanical strength in comparison to the other synthetic packaging materials available (Bourtoom, 2009). Essential oils and their components have showcased interesting antimicrobial and antioxidant properties for obtaining natural minimally processed protein edible films and coating formulations (Barba et al., 2015). If used as an individual component in food, the essential oil can come in clash with the original aroma and flavor of food, hence it is incorporated in the film formulation to provide the developed films with antibacterial and antimicrobial properties and thereby increasing the shelf life of the food wrapped up in a protein-based edible film (Chiralt et al., 2018). Recently, studies have been done to aim the delivery of antimicrobials into food through a gradual release of essential oil from the protein edible films (Barba et al., 2015). The prime factor considered in the selection of antimicrobials used is its interaction with the film-forming biopolymer, i.e., protein along with secondary interactions with the food components of the system. Below is the discussion of resultant properties of the films obtained on incorporation of oil and proteins like soy protein isolate, whey protein isolate (WPI), and zein protein:

2.2.1 Soy protein isolate Soy protein is extricated from soya bean seeds from which soy oil is derived. It is composed of albumin and globulin (7S (b conglycinin) and 11S (glycinin) globulins). Globulin subunits are responsible for the hydrophobic hydrogen-bonding and disulphide bonds of the soy protein (Shan et al., 2015). Soy protein isolate is the most refined form of soy protein derived from defatted soy flour which is devoid of carbohydrates, fats, and all other nonprotein components (Kalman, 2014).

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Rapeseed oil homogenized with soy protein at a varying concentration of 0%, 1%, 2%, and 3% was formulated to produce film-forming emulsions using the casting method (Galus, 2018). In accordance with the past findings, rapeseed oil led to an obvious reduction in water vapor permeability when tested against three RH differentials of 0%e50%, 50%e75%, and 75%e100% due to high hydrophobicity. The values ranged from 2.84 to 5.12 ∙ 1010 gm1 Pa1 s1 for control films and from 2.36 to 3.87∙ 1010 gm1 Pa1 s1 for films containing 2% oil at 0%e50% and 50%e100% relative humidity differentials, respectively. The highlights of mechanical studies concluded that oil-entrapped film is weaker than the control films. The tensile strength, Young’s modulus, and elongation at break of soy protein marked a significant decrease with increase in the concentration of oil from 0% to 3%, attributed to weaker lipid-polar molecule interactions in comparison to the polarepolar interactions occurring in control films. The oil-entrapped films obtained were visually yellow in color due to yellow commercial soy protein isolate powder. The lightness (L parameter) values varied from 88.7 for control films to 82.8 for 3% oil film, implying the darkening of films with an increase in oil concentration. Also, the addition of rapeseed oil concentration increased the total color differences (DE) with respect to control films. Edible films from carvacrol and cinnamaldehyde are derived from essential oils namely oregano and cinnamon essential oils, respectively, and hence provide the antimicrobial characteristics to the films against disease-causing pathogens in the food system (Du et al., 2009; Friedman et al., 2004). Antimicrobial films have the capability to control illness outbreaks which are increasing due to the massive intake of freshly cut fruits and vegetables. In addition, Rojas-Grau¨ et al. (2007) reported the antimicrobial activity of these constituents in the following order: carvacrol > oregano oil > cinnamaldehyde > cinnamon oil. In the above study, edible films were developed from direct oil-inwater micro- and nanoemulsions which were obtained by mixing vegetable glycerin (plasticizer) and surfactant for the required time (30 min for microemulsions and 5 h for nanoemulsions). Attempts were made to maintain 0% RH for ensuring constant water vapor driving forces. Essential oils have been known to plasticize the films by weakening the bond in isolated soy protein films and hence allowing greater water vapor permeability across the films (Zivanovic et al., 2005). The increased hydrophobicity in the system resulted due to the incorporation of carvacrol and cinnamaldehyde balancing with the plasticizing effect of both the compounds resulting in similar WVP values. When the droplet size was decreased from micro to nano, WVP of the ISP films marked a reduction which can be owed to the porosity of the resulting films (Otoni et al., 2016). Irrespective of the particle size or how it was formulated, all the emulsified films were tested to have reduced tensile strength and elastic modulus and more elongation at break when compared to the control samples. Moreover, edible films prepared from essential oils containing smaller droplet size were observed to produce films having higher elongation at break. Thus, the plasticizing effect got increased with the increase in the surface area of smaller droplets in nanoemulsions. Furthermore, enhancement of the delivery system efficiency was increased due to surface area. Otoni et al. (2014) highlighted similar results in their study conducted separately on cinnamaldehyde and OEO (Otoni et al., 2014). In another study, microfibrillated cellulose (MFC) was introduced to improve the functionality besides clove essential oil for its antioxidant and antimicrobial properties. MFC enhanced the mechanical and barrier properties by reinforcing the protein matrix. In general, with the addition of MFC, water vapor permeability decreased with an increase in the content of MFC. This phenomenon can be explained well with the zigzag path encountered by water vapor molecules due to the addition of MFC (Tunc et al., 2007). The water vapor permeability values ranged from 13.90 to 9.77  1011 (g H2O/m Pa s) in the control sample with MFC added but decreased significantly in case of adding clove

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essential oil on whose addition, the WVP ranged from 9.66 to 7.12  1011 (g H2O/m Pa s). A contradictory result was recorded in a study conducted by Echeverrı´a et al. (2016), where no change in WVP values was seen for activated nanocomposite matrices when clove essential oil was added to SPIMMT films. The oxygen transmission rate decreased with the increase in MFC content, but similar effects were not visible in case of oxygen permeability, due to a simultaneous increase in the obtained thickness of the film. The addition of nanofiber MFC, with or without CEO enhanced the tensile strength and Young’s modulus. A significant decrease in the tensile strength and Young’s modulus can be seen in case of 8 and 12 (g/100 g SPI) MFC, with almost no change in elongation at break (%) when CEO is incorporated in the protein matrix. These results confirm the plasticizing effect of essential oil in soy protein isolate films as stated by Monedero et al. (2009). Protein nanocomposite films despite the addition of MFC (without CEO) recorded a very low antioxidant activity, although, on the incorporation of clove essential oil, the protein films showed an obvious increase in the antioxidant capacity attributing the presence of its own phenolic compound primarily eugenol, gallic acid, and caffeic acid (Dudonne´ et al., 2009). Highest antioxidant property in the case of 12 (g/100 g SPI) implies greater dispersion of CEO through the nanocomposite matrix. No antimicrobial activity was observed without CEO, with whatsoever content of MFC added. Essential oils have been proved to be more effective against Gram-positive bacteria such as B. cereus and S. aureus. According to the data recorded, B. cereus came out to be the most delicate strain against clove oil’s antimicrobial activity followed by S. aureus and E. coli (Burt, 2004). Multiple studies have been performed by Ayoola et al. (2008), (Fu et al., 2007) to study the antimicrobial properties of clove oil with and without the combination of other essential oils. The Hunter-Lab color parameter L was greater in SPI film alone compared to the values obtained after addition of clove essential oil. Note that with an increase in MFC, L value decreased in both the cases. DE in SPI and SPI þ CEO films ranged from 15.7 to 19.8 and 30.1 to 34.3, respectively, while increasing the MFC content from 0 to 12 (g/100 g SPI) MFC (Ortiz et al., 2018).

2.2.2 Whey protein isolate Zone of inhibition areas was studied against different microorganisms for OEO. Studies demonstrated that the minimum amount of oil required to show any activity against microorganism is 2% of oregano oil in the formulations. With the increase in the oil concentration, the zone of inhibition of S. aureus, Salmonella enteritidis, and L. monocytogenes increased unlike in case of Lactobacillus plantarum, where no significant change in the inhibition zone was seen after incorporating 2% of the OEO (Seydim & Sarikus, 2006). Almond oil has a high amount of monounsaturated fatty acids (linolenic and oleic acid) with a comparatively lower amount of saturated fatty acids (palmitic, palmitoleic, and stearic) (Kodad et al., 2014). Meanwhile, walnut oil is rich in triacylglycerols, with oleic acid and PUFAs and linoleic and a-linoleic acids as the major components (Martı´nez et al., 2010). Be it 0.5 or 1% oil content, the water permeability decreased with an increase in oil content. This study resulted in a considerable increase in the hydrophobicity, on the addition of even low concentrations of added walnut and almond oil. Similar values implying the reduction of WVP on the addition of lipid content have been recorded in research conducted by Han et al. (2006) (Valenzuela et al., 2013). The values of oxygen permeability ranged from 112.5 cm3 mm m2 d1 kPa1 in control films to 131.5 cm3 mm m2 d1 kPa1 for films with 1% walnut oil in formulations. At both 0.5% and 1.0%, almond oil was observed to have higher oxygen permeability compared to walnut oil. The tensile strength recorded a significant increase with incorporation of oil at 0.5% but on further increase in oil percentage to 1%, it dropped to the level

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lower than even the control. Same type of data was recorded for values of Young’s modulus. The research proved to be justified because of the homogenization process and presence of high amount of lipid phase consequently disrupting the organization of the protein matrix. The research deduced that almond oil showcased greater plasticizing effect in changing the functional properties of whey protein films (Galus & Kadzi nska, 2016). WPI films were made through Grammosciadium ptrocarpum Bioss. essential oileloaded emulsions and nanoemulsions. Nanoscale delivery system and encapsulation of essential oils had now been increasingly adapted due to its higher bioavailability (Severino et al., 2015). Water vapor permeability increased with increase in essential oil concentration varying from 0% to 1.5% of oil due to elaborately explained reasons in the above text. On the other hand, in case of nanoemulsions of GEO, the WVP decreased with increase in oil content with an exception for 1.5% oil content which was found to be higher than the control sample. This anomaly might be explained with the presence of uniformly distributed nanodroplets in the film which provided the water with a curvilinear path, henceforth decreasing the water migration rate. The tensile strength values were increased in 0.5% of essential oil composition but decreased subsequently in case of higher oil concentrations. The Young’s modulus decreased from 233.229 to 12.627 MPa on increasing the essential oil content of the emulsion from 0% to 1%. In brief, the nanoemulsion films had higher YM compared to the previous ones. These data were in accordance with the possibility that presence of essential oil in the protein matrix weakened the intermolecular forces and reduced the mechanical strength of the WPI film. Data collected by Bahram et al. (2014) resembled with this study. GEO showcases effective antimicrobial properties (Rajabi et al., 2014; Sonboli et al., 2005). Its antimicrobial activity was tested against four different microorganisms namely L. monocytogenes, S. typhimurium, E. coli, and Pseudomonas aeruginosa. Highest antimicrobial activity was observed in gram-positive bacteria, L. monocytogenes because of more complexity in cell wall of gram-negative bacteria. Nanoemulsified EO decreased the lightness values of produced films. Note that the differences in color parameters (L, a, and b) are not’ significant which concludes that incorporation of GEO in either emulsion or nanoemulsion form can cause many variations in the color of the obtained WPI films.

2.2.3 Zein proteins Zein is another by-product of corn, exhibiting protein storage properties (Guo et al., 2005). Altan et al. (2018) incorporated carvacrol in electrospun zein and poly (lactic acid) (PLA) at different concentrations (5%, 10%, and 20%) using the electrospinning process. An increasing percentage of carvacrol marked an increase in the antioxidant activity of the zein fiber films, whereas no significant difference was shown when oil concentration varied from 5% to 10% carvacrol. The research suggested that the zein fiber with 20% carvacrol exhibited the highest antioxidant activity. Zein film was made with the addition of 5% and 10% (g essential oil/g dry zein powder) where essential oil used is Z. multiflora Boiss (ZEO). Also, monolaurin was added in the emulsion as another active compound. Antimicrobial activity against L. monocytogenes was studied in model sample of minced beef using zein-based films obtained by casting method. The antimicrobial and antioxidant activity simultaneously increased with the concentration of ZEO and monolaurin. In another study, thymol was added at 35% concentration to obtain mono- and multilayer films. Corn zein was the main ingredient in all films, whereas spelt bran was loaded in outer layers in case of multilayer films. The release rate of thymol was dependent on the quantity of bran added because of the presence of microvoids in its microstructure. Subsequently, monolayer with the highest concentration of bran

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439

released the highest quantity of thymol as illustrated from its antimicrobial and antioxidant activity (Del Nobile et al., 2008).

3. Composite films incorporated with essential oils Bionanocomposites based on chitosan were incorporated with rosemary essential oil (REO). Commercial clays like Cloisite Naþ and Cloisite Ca2þ were used to form the nanocomposite films. The hydrophilic nature of Cloisite Naþ made it suitable for incorporating REO. The oil incorporated films showed decreased tensile strength and more opacity. The films also exhibited improved UV light blockage and water solubility. All these characteristics along with decreased swelling made the prepared films a contender to serve the purpose of active packaging in the food industry (Souza et al., 2018). The shelf life of foods having a high oxidation potential can be increased by coating them with films incorporated with antioxidants like ginger and eugenol essential oil. Gelatin and chitosan-based films were incorporated with eugenol and cinnamon essential oils which showed an increase in the antioxidant properties as well as the elasticity of the films. The roughness of the films also increased as seen by the SEM analysis which was due to the nonhomogeneity of the oil incorporation in the films. The water vapor permeability of the blend showed no significant changes (Bonilla et al., 2018). Starch-gellan films containing thyme essential oil were prepared. The prepared films showed great antifungal activities against Alternaria alternata (AA) and Botryotinia fuckeliana (BF). The blends were showed more inhibition of BF as compared to AA. Lecithin was also incorporated into the films in order to control the loss of the essential oil. As a consequence, its incorporation increased the water barrier properties but decreased the stiffness and resistance to break at the same time. The lecithinencapsulated EO films exhibited all the properties required to be used as packaging materials (Sapper et al., 2018). WPI and zein in different w/w ratios of 2:1, 1:1, and 1:2 were used to develop edible films. Deionized water and ethanol varying from 25%, 50%, 75%, and 95% concentration were used as solvents for the formation of films. For ethanol as solvent, as the concentration was increased, the white index and elongation of films decreased. Also, low water vapor permeability was observed for concentration 25% and 75%. For deionized water as a solvent, higher transparency and brightness in films was observed. Higher WPI concentration corresponded to more elongation and higher breaking elongation in films. All the three composite films had good solubility in hot water and could be heat sealed, and there was no appreciable effect of solvent on moisture content (Tsai and Weng, 2019). Edible films were developed using gum arabic, sodium caseinate, lemongrass, and cinnamon essential oils. They were tested in guava where they served as an edible coating. The coating led to better retention of flavonoid contents as well as phenol and ascorbic acid content. The amount of sugar loss by the guavas over the storage period was also decreased. On an overall basis, the shelf life of the guava samples was extended by 40 days (Murmu and Mishra, 2018).

4. Conclusion The applications of oil-loaded edible films have found increasing importance during the past decade. In addition to the formulation of oil-loaded films from single biopolymers, the current focus has been on formulating films from multiple biopolymeric systems. Combination of multiple biopolymers such

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as proteins and polysaccharides provide improved functionality such as water vapor permeability and tensile strength to the films. Addition of essential oils to these systems further improves the film properties by providing antimicrobial functionality. Such films with multitude of functions have found significant value within the food-processing industry. New research needs to be performed in order to evaluate their applicability on real food systems.

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CHAPTER

Tamarind seed polysaccharide: unique profile of properties and applications

20

Kazuhiko Yamatoya, Akira Tabuchi, Yumewo Suzuki, Hiroyuki Yamada DSP Gokyo Food & Chemical Co., Ltd., Osaka, Japan

1. Introduction Tamarind seed polysaccharide (TSP) is a neutral hydrocolloid obtained from the seeds of the tamarind tree, Tamarindus indica L. The tamarind tree is an evergreen tree distributed widely in Asia, in countries such as Thailand and India, and in many other parts of the world, such as Africa (e.g., Egypt) and the Americas (e.g., Florida, USA) (Glicksman, 1986). Its range is vast, but it has spread along with human habitation, indicating that the tamarind tree is a familiar part of life for much of humankind. The main component of TSP is xyloglucan, which consists of a b-1,4-linked D-glucan backbone with a-D-xylose at O-6, some of which are further linked with b-D-galactose at O-2 (York et al., 1990) (Fig. 20.1). Xyloglucans occur universally in the primary cell walls of higher plants. Cell growth and

Number of Gal residue per oligosaccharide unit : 0–2

(Notes) Glc, glucose; Xyl, Xylose; Gal, galactose FIGURE 20.1 Chemical structure of TSP. Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00020-5 Copyright © 2020 Elsevier Inc. All rights reserved.

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enlargement are controlled by the looseness of a thin net of microfibrils made of cellulose tethered by chains of xyloglucan (Hayashi, 1989). In aqueous solution, TSP has unique physicochemical properties: -

Newtonian flow in practical conditions, synergism with sugars, alcohol, and polyphenol (thickening, gelling), stability (against heat, acid, salt, mechanical impact), and water retention effect.

Based on these properties, TSP has been used mainly as a food thickener, and also as a stabilizer and gelling agent (Yamatoya and Shirakawa, 2003). TSP is used in foods such as sauces, dressings and mayonnaise, ice cream, pickles, seaweed products, flour products and jelly, and also cosmetic and personal care products. In Japan, TSP products were first marketed as food additives in 1964. Having also been called as existing food additives and placed on the List of Existing Food Additives by The Ministry of Health, Labor and Welfare in Japan, they have been safely used for many decades. These products are also used as food additives in China, South Korea, and Canada and as food ingredients in Taiwan. In the United States, TSP products are marketed with Generally Recognized As Safe (GRAS) status, granted by the US Food and Drug Administration (FDA) in 2014. Confirming the safety of TSP by assessing toxicity in long-term rodent studies and lack of concern regarding genotoxicity, reproductive toxicity, and developmental toxicity, the FAO/WHO Joint Expert Committee on Food Additives (JECFA) Committee established an acceptable daily intake (ADI) “not specified” for TSP in 2017. In this review, the properties of TSP, in structure, rheological behavior, and gelling, and emulsification and stabilizing effects are described, and furthermore some food applications that take advantage of the properties and effects of TSP are highlighted.

2. Fundamental properties 2.1 Molecular weight and structure The structure of TSP in aqueous solution has been investigated using several analysis methods, such as small angle X-ray scattering (SAXS), light scattering, and small-angle neutron scattering (SANS) (Dentini et al., 2001; Gidley et al., 1991; Lang and Burchard, 1993; Lang and Kajiwara, 1993; Muller et al., 2011; Picout et al., 2003; Yamanaka et al., 1999a). As detailed information of these analysis methods is discussed in each of the reference documents above and some reviews, such as Nishinari et al. (2009), the qualitative structural properties of TSP are described briefly here. TSP consists of high-molecular-weight polysaccharides. The reported range of the molecular mass of TSP is wide: 470 kDa (Kato, 2000; Muller et al., 2011), 880 kDa (Gidley et al., 1991), 1160 kDa (Dentini et al., 2001), 2500 kDa (Lang and Kajiwara, 1993), and 400e5870 kDa (Lang and Burchard, 1993). Several explanations have been offered for this broad range, including variations in sample preparation procedures and the tendency of TSP to self-aggregation (Nishinari et al., 2009). Based on a comparison of structure analysis parameters of other polymers, TSP molecules in aqueous solution are relatively stiff. Furthermore, multistranded lateral self-aggregation was observed between molecules. TSP, however, does not show any of the conformational changes, such as coilhelix transition, seen in xanthan gum (Nishinari et al., 2009; Nitta et al., 2003). The contour length

2. Fundamental properties

447

of TSP is likely to be slightly longer than that of galactomannan but shorter than that of xanthan gum or schizophyllan, both of which have a helix configuration. In accordance with that fact, the structural properties of TSP are intermediate between single chain polysaccharides such as galactomannan and helix-form polysaccharides such as xanthan gum. These structural profiles relate to the fact that TSP exhibits characteristic behavior and effects in solution, as described in more detail in the following sections.

2.2 Rheological properties The flow behavior of TSP is significantly different from that of xanthan gum, which exhibits a substantial decrease in shear viscosity with increasing shear rate (Fig. 20.2). Shear-thinning arises from the shear-induced orientation of the polymer against rotational diffusion, and is much more conspicuous in xanthan gum than in TSP. Viscometric analysis of TSP solutions (Nishinari et al., 2009; Sims et al., 1998) indicates that the solutions exhibit close to constant steady shear viscosity over a wide range of shear rates. The steady shear viscosity is conjectured to be due to the fact that TSP does not show any of the conformational changes, such as coil-helix transition, seen in xanthan gum. These results indicate that the flow properties of TSP aqueous solutions are near Newtonian, which is regarded as Newtonian in practical conditions. This flow behavior is important from a practical viewpoint because Newtonian flow in fluidic or paste food products permits smooth flow and easy serving. Furthermore, as TSP exhibits relatively higher viscosity compared to polymers with the same

Temperature: 25

η[Pa ・s]

TSP 5.0% (w/w) TSP 2.0% (w/w) TSP 1.0% (w/w) Xanthan 1.0% (w/w) Xanthan 0.5% (w/w)

dy/dt[s-1] FIGURE 20.2 Steady shear viscosity of TSP and xanthan gum solutions (Nishinari et al., 2009).

C

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Chapter 20 Tamarind seed polysaccharide

contour length can be expected in connection with the multistranded self-aggregation of TSP (Nishinari et al., 2009). The viscosity of TSP significantly contributes to a number of its effects, such as thickening, stabilizing, and so on. Fig. 20.3 shows the frequency and temperature dependence of the storage shear modulus G0 and loss shear modulus G00 of TSP solutions of various concentrations (Yoshimura et al., 1999). G0 and G00 are moduli representing properties of gel and sol, respectively. In regard to frequency dependence, at a lower concentration of TSP G00 is constantly larger than G0 , while at higher concentrations of TSP each modulus approaches similar values with increases of frequency to around 102 rad/s (Fig. 20.3A), indicating that G0 increases more sharply than G00 at each concentration of TSP. On the other hand, it is well known that in a xanthan gum solution, G0 is constantly higher than G00 and these moduli increase in parallel, indicating that the solution exhibits apparent gel behavior. The frequency dependence of TSP solution is reflected by its unique behavior, unlike in the case of xanthan gum. In regard to temperature dependence, both moduli decrease monotonically with increasing temperature and G00 is always larger than G0 , as observed for many polymer solutions and colloidal suspensions (Fig. 20.3B). The results indicate that TSP solution does not form a gel, but remains liquid throughout the working temperature range.

2.3 Gelling properties TSP alone does not form a gel in aqueous solution even at high concentrations, instead remaining in sol form. TSP has the ability to form gels in the presence of sugars (e.g., sucrose), alcohols (e.g., ethyl alcohol) (Glicksman, 1986), polyphenols (e.g., epigallocatechin gallate), or iodine (Yuguchi et al., 2001). The gelation of TSP solution mixed with sugar or alcohol is speculated to involve cross-linking of TSP molecule aggregation domains due to a dehydrating action by these additives. These gels can become sols by heating and revert to gel again by cooling, indicating that the transition between sol

FIGURE 20.3 Frequency and temperature dependence of G0 and G00 for TSP dispersions (Yoshimura et al., 1999), (A) Frequency dependence of G0 and G00 of TSP dispersions, (B) Temperature dependence of G0 and G00 for TSP dispersions.

2. Fundamental properties

449

and gel is thermoreversible. These gels show high elasticity and low water release. A freeze-thawing process makes these gels harder and more elastic (Nishinari et al., 2009). Although native TSP alone does not form gels in response to heat (see Fig. 20.2), it has been demonstrated that partial degalactosylation of TSP leads to the formation of a hydrogel that is thermally responsive in aqueous solutions (Shirakawa et al., 1998; Yuguchi et al., 1997). The gel strength can be increased with an increase in degalactosylation as the (1 / 2) b-D-galactose side groups interfere with the interactions between the main chains. TSP gels created by degalactosylation or mixing with some additives, such as ethanol and epigallocatechin gallate, have been investigated by structural analysis mainly using SAXS. The degalactosylated TSP molecules form a gel by cross-linking of TSP molecules aggregated laterally in the shape of a flat plate (Yamanaka et al., 1999b). In contrast, it is reported that gelation of TSP solution by alcohol is caused by the cross-linking domains formed due to aggregation of nonordered structure without any specific direction. The gelation of TSP solution with polyphenols is conjectured to involve cross-linking domains formed by the random aggregation of xyloglucan chains, the mechanism of which is similar to that of TSPealcohol gel (Yamanaka et al., 2000; Yuguchi et al., 2004). TSP also forms a gel in the presence of about 40%e70% sugar (i.e., sucrose) over a wide pH range (Fig. 20.4). In regard to TSPesugar gels, there are no structural analysis data and thus there still remains an issue to be uncovered for understanding how TSP molecules interact with sugars to form a gel. Further findings are awaited to elucidate this subject. TSP has also synergistic thickening or gelling properties in combination with gellan gum or xanthan gum even under conditions where these polysaccharides cannot form a gel alone (Kim et al., 2006; Nitta et al., 2003). These synergistic effects will involve ordered structure formation due to the intermolecular binding between TSP and either gellan gum or xanthan gum, both of which are double helix-forming polysaccharides.

Hardness[N/m2]

TSP concentration 1.5% 1.0% 0.5%

FIGURE 20.4 Hardness of TSP containing sucrose of various concentrations (Nishinari et al., 2009).

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Chapter 20 Tamarind seed polysaccharide

carrageenan gel

carrageenan + 0.5% hydrolyzed TSP

Magnification: x100. FIGURE 20.5 Comparison of SEM images of carrageenan gel images (Nagashima et al., 1999).

There have been studies on effects of TSP or Hydrolyzed TSP, whose main chains are enzymatically hydrolyzed, for other gelling polysaccharides. Addition of TSP or hydrolyzed TSP contribute a reduction of water separation from gels containing carrageenan, gellan gum, or agar, when TSP are supplemented in these gelling polysaccharides. These effects are more conspicuous after frozenthawed process, during which period the gels retain appropriate texture without any effects on their own properties. In particular, hydrolyzed TSP ameliorates decrease in hardness of carrageenan gel after freeze-thawing (Nagashima et al., 1999). Based on observation of the gel structure using SEM, the gel network structure of carrageenan, containing hydrolyzed TSP, appears to be a thicker and denser grid compared to that of carrageenan alone (Fig. 20.5). Similarly, in agar gel, TSP exhibits suppressive effect for water release from the gel and deterioration of gel strength through freeze-thaw process (Senda et al., 2008). These findings suggest that native and hydrolyzed TSPs interact with polysaccharides during the freeze-thaw process, and resulting complexes ameliorate the damage during the process.

2.4 Emulsion stabilizing and emulsification property Polysaccharides are known as emulsion stabilizers, which contribute to the prevention of solid particle precipitation in dilute aqueous solutions, limitation of creaming, flocculation, and retarding coalescence. These characteristics are attributed to the ability of the polysaccharides to modify the rheological properties of the aqueous continuous phase, by acting as a thickening or gelling agent without adsorbing onto the particle/droplet interface (Dickinson, 1987). In practice, as the emulsified state in foods often seems to be very unstable, polysaccharides are often used as an “emulsion stabilizer” in combination with an emulsifier to further increase its stability. TSP has been used for emulsion films intended for biopolymer-based food packaging materials. The films are prepared by adding TSP and glycerol to sesame oil-in-water emulsion that were already emulsified in advance using surfactants, followed by homogenization by various methods, and drying (Rodrigues et al., 2018). The results indicate that TSP can be used as an emulsion stabilizer for emulsion films, and is could be used as a biobased film-forming material.

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451

As TSP is used commercially for emulsion stability, xyloglucans from other sources may have potential for emulsion stabilizing. For example, xyloglucan from Hymenaea courbaril seeds was examined for stabilization of oil-in-water cosmetic emulsion (Vianna-Filho et al., 2013). Based on the rheological results, the authors conjecture that long-term stability of the emulsions was improved by natural polysaccharides including Hymenaea xyloglucan. It has been also reported that Maillard-type protein-TSP conjugates enhanced emulsifying properties compared to the protein alone (Kato, 2000). The results show that TSP conjugate with soy protein has a greater emulsifying activity and emulsion stability than conjugates with other polysaccharides, indicating that some properties of TSP will contribute to increases of emulsifying activity or emulsion stability. In contrast to emulsion stabilizer, an emulsifier is a single chemical or mixture of components having the capacity for promoting emulsion formation and short-term stabilization by interfacial action (Dickinson, 1987). Some polysaccharides such as gum arabic and PGA have been used as emulsifying agents. It has been shown that TSP solution (0.5%) can form an o/w emulsion without any emulsifiers and stabilize the emulsion for 15 days at 25 C (Fig. 20.6), suggesting that TSP can be used as an emulsifier as well as an emulsion stabilizer. TSP with different purification and composition in particular carbohydrate/protein ratios showed emulsion stability and produced initial oil droplets to some extent (Crispı´n-Isidro et al., 2019). As these effects were different from purification methods, it was concluded that emulsion stability was

FIGURE 20.6 Oil in water emulsion composed of vegetable oil, water, and TSP (DSP Gokyo Food & Chemical, 2014a), Oil in water emulsions were prepared using vegetable oil (50%) and water (the rest) with added TSP (0% (control), right; 0.5%, left) by homogenizing at 15 MPa.

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Chapter 20 Tamarind seed polysaccharide

Table 20.1 Surface tensions of TSP and alkylated derivatives (0.1% solutions) measured using DuNouy ring apparatus (Lang et al., 1992).

Sample

% Substitution at galactose

Surface tensiona (air/ water) dyne cmL1

Interfacial tensiona (decane/water) dyne cmL1

H2O 0.1 M NaCl TSP/H2O Ethyl TSP/H2O Octyl TSP/H2O Octyl TSP/0.1 M NaCl Nonyl TSP/H2O

e e e 19 7 7 23

71.5 69.2 61.3 65.1 55.6 59.3 59.3

43.1 42.5 34.9 34.2 32.7 30.6 29.6

2 dyne/cm, Lang et al., 1992.

a

due to a complex interplay between several factors, including chemical composition, apparent viscosity, contact angle, interfacial tension dynamics, surface charge, all of which had bearing on the electrosteric repulsion terms and adsorbed layer mechanical properties around the oil droplets. In a study using a DuNouy ring apparatus to examine surface and interfacial activities of purified TSP, 0.1% TSP aqueous solution lowered the interfacial and surface tensions (Lang et al., 1992) (Table 20.1). Although the surface tension of the TSP solution was modest (61.3 dyne/cm), compared with those for other polysaccharides such as methyl cellulose (48 dyne/cm), gum tragacanth (43 dyne/ cm), sugar beet pectin (53 dyne/cm), guar, and locust bean gum (55 dyne/cm) (Garti and Reichman, 1994), TSP did exhibit surface activity. When surface or interfacial activities are detected in a polysaccharide solution even after purification, there is a possibility that samples contain a residual and strongly bound protein/peptide fraction (Dickinson, 2003). However, Lang et al. (1992) mention that nitrogen was not detected in the purified TSP by elemental analysis, which excludes the possibility that proteins contributed to the activity. Therefore, these surface or interfacial activities are at least partly based on the structure of TSP molecules in solution. In the study, in addition to native TSP, a series of derivatives of TSP was examined for surface, interfacial, and emulsifying activities (Lang et al., 1992). The results are that alkylaminated TSP shows only a limited decrease in surface and interfacial tensions compared with native TSP whereas nonylaminated TSP shows significant activities for formation and stabilization of a foam consisting of sunflower oil and water. Furthermore, enzymatic depolymerization of the nonylaminated TSP substantially decreases surface and interfacial tensions, suggesting that access of the hydrophobic groups to the interface is increased after the depolymerization, as the overall hydrophilic/lipophilic balance is not changed by depolymerization. The decrease in surface and interfacial activities is more likely to be due to decreased solution viscosity rather than the loss of specific group interactions. Conversely, the volume of the foam using the depolymerized nonylaminated TSP is reduced compared to the nondepolymerized one, suggesting that the stiff polymer backbone affecting the solution viscosity might be required for the foam stability and air cell size.

3. Food applications

453

From these results, it is concluded that TSP has not only emulsion-stabilizing capabilities, but also emulsifying activity. Although TSP molecule has the same backbone as cellulose, which is insoluble in water, its high solubility in water is attributable to the side chains of TSP. There is a possibility that the substitution patterns of the glucan backbone and/or molecular aggregation (see the previous sections) create partial hydrophobic regions, which allow for contribution to emulsifying activity as potential sites of adsorption on the oil surface. Further study of the emulsifying activities of TSP should broaden our understanding of the functions and mechanisms of TSP in an emulsion system.

3. Food applications 3.1 Functions The fundamental properties of TSP described earlier are the basis for its wide use in food products. This section will describe how the functions of TSP are applied in these food products, and give specific use examples of TSP.

3.1.1 Thickening TSP is used as a thickener because of its smooth flow and lack of sticky texture or trailing threads, which are sometimes shortcomings for other thickeners. In addition, it can also provide body and a denser feeling in the mouth. It can be especially used as a replacement for or in combination with starch. TSP has been used in sauces for pork cutlets and meat requiring high viscosity and high stability in low pH over a long period. In low-fat milk, fruit beverages, and cocoa, TSP can provide body or improve texture. As TSP stabilizes small particles in suspension, it can be used in fruit-pulp beverages or shiruko (traditional Japanese soup containing pureed sweet red bean with rice cake). In batter mixes for fried products, it is important to maintain a stable viscosity during the battering process. Even though enzymes in wheat flour often reduce the viscosity of batter mix, TSP can stabilize the viscosity throughout the battering process by its protective effect against the enzymes.

3.1.2 Gelling and water-retaining TSP provides an elastic gel with concentrated sugar solutions over a wide pH range. Because of its heat stability, TSP is not affected by boiling in aqueous solutions. The gel is water-release free and resistant to freeze-thawing. It can be used as a substitute for pectin in fruit jelly or jam. Some Japanese traditional desserts such as yokan, and kudzu mochi, which are gelatinized principally by agar or kudzu, gelling agents unique to Japan, can also be produced partially using TSP (Nussinovitch and Hirashima, 2014). This not only helps gelatinization, but also is effective at preventing syneresis. TSP is also used for various jellies, puddings, and Japanese traditional seaweed preserve products such as tsukudani, for the purpose of suppressing water release and making the product look bright without changing texture and physical properties. The effect of preventing water release is also useful for foods for people who have trouble in swallowing.

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Chapter 20 Tamarind seed polysaccharide

3.1.3 Starch modification TSP can suppress the aging of starch under particular conditions. It can also protect starch by conferring heat stability and mechanical strength. TSP improves the texture of starch. Based on the effects of TSP on the gelatinization and retrogradation of corn starch, it was suggested that TSP entangles with corn starch molecules and prevents the structure reordering, hence retarding retrogradation during long-term storage, even though it promotes retrogradation during short-term storage (Yoshimura et al., 1999). To supplement, the result showed the promotion of retrogradation by TSP during short-term storage from only rheological properties on the specific model condition, but TSP is practically used in many commercial food products for both short- and long-term antiretrogradation, as mentioned later. Furthermore, in mixture model with tapioca starch, it was indicated that retrogradation was masked by TSP in the early stage of storage immediately after gelatinization, and TSP improved the thermal stability from the results that the mixture showed a lower activation energy in thermodynamic and more freeze-thaw stability than that for tapioca starch alone (Pongsawatmanit et al., 2006).

3.1.4 Emulsification and emulsion stabilization It was indicated in the preceding chapter that TSP has its own emulsification activity and stabilizing effect. Food applications used in the form of oil-in-water emulsions vary widely in their consumption, texture and flavor. Food manufacturers have been using polysaccharides as stabilizers to confer substantial improvements in shelf-life (long-term emulsion stability). The main stabilizing action of food polysaccharides is said to be via viscosity modification or gelation in the aqueous continuous phase (Dickinson, 2003). TSP has also been used especially as an emulsion stabilizer for various foods requiring emulsion or its stability, such as salad dressings, mayonnaise, and ice cream.

3.2 Applications TSP has been used not only for thickening, but also for gelling, starch modification, and emulsion stabilizing or a combination of these. As there has been considerable previous information describing applications using TSP simply as a thickener, major applications utilizing more beneficial effects of TSP are explained with the use of examples in this section.

3.2.1 Bakery products There are many applications using TSP in combination with starch (or a substitute) for bakery products, custard cream, flour paste, stew, noodles, and traditional Japanese confections (e.g., dango or rice cake). In bread applications, hydrocolloids can be used to improve dough performance, act as antiretrogradation agents for starches, and preserve dough and product quality (Ferrero, 2017). It is reported that TSP can also improve bread volume and storage properties (Maeda et al., 2007; Morimoto et al., 2015). In the study performed by Maeda et al. (2007), water-soluble fractions containing TSP and its hydrolysates, fractionated by gel filtration chromatography, improved the quality of dough by increasing its stability, as noted by observations of loaf volume, storage properties, and good appearance with the distribution of small size gas bubbles in bread samples. Fig. 20.7 shows the SEM

3. Food applications

455

FIGURE 20.7 SEM images of dough containing TSP (Maeda et al., 2007). (A) Control (B) TSP hydrolysate (polymerization degree: 17) (C) TSP hydrolysate (polymerization degree: 223) (D) TSP (native).

images containing TSP and its hydrolysates, indicating that they exhibited thicker matrix structure than that of the control. Thus, it was suggested that these thicker gluten matrices lead to extensible dough and improved final products. Moreover, in sponge cakes, hydrolyzed TSP increased cake volume and suppressed hardening (Fig. 20.8A and B, Nagashima, 2005). The effect can also be explained by suppressed aging of gelatinized starch as hydrolyzed TSP preserved gelatinization status (Fig. 20.8C). These effects can be explained with respect to the suppressive action on retrogradation of starches and assistance to dough matrix by addition of TSP, indicating that TSP is a versatile and effective additive suitable for bakery products. TSP can also improve the storage properties and texture of gluten-free rice bread. As rice flour does not contain gluten, which has an impact on bread staling, it is noted that rice bread is labile to retrogradation compared to wheat flour bread. Meanwhile, research is being conducted to assess the effect of polysaccharides on gluten-free rice bread. Results have shown that addition of TSP led to

(B)

Volume of sponge cakes

Hardness during storage Control branched dextrin hydrolyzed guar gum hydrolyzed TSP

branched dextrin hydrolyzed guar gum hydrolyzed TSP 250 Hardness (g/cm2)

1350 Volume (ml)

1300 1250 1200 1150 1100

addition rate:2% 200 150 100 50 0

1050 0

1

2

Hydrolyzed polysaccharides (%)

0

2

4

Storage period (day)

(C)

Degree of gelatinization (%)

(A)

Degree of gelatinization change during storage Control branched dextrin hydrolyzed guar gum hydrolyzed TSP

120 100 80 60 40 20 0 0

2

5

Storage period (day)

FIGURE 20.8 The effects of hydrolyzed polysaccharides in sponge cakes (Nagashima, 2005): (A) volume of sponge cakes, (B) hardness during storage, (C) degree of gelatinization change during storage.

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Chapter 20 Tamarind seed polysaccharide

significant improvement of appearance, volume, and storage properties of gluten-free rice bread (Jang et al., 2018). TSP has a similar effect to gluten. Thus, there is potential for the use of TSP in the production of gluten-free products, as “Gluten-Free” is an area currently experiencing a global boom. Based on the earlier findings, there seems to be further potential for TSP use in the production of several types of baked goods.

3.2.2 Dressing and mayonnaise In oil-in-water-type dressings, xanthan gum has been routinely used because it has good emulsion stability in acidic conditions and strong pseudoplastic properties. Dressings containing xanthan gum alone as an emulsion stabilizer, however, are often difficult to pour from the product container due to their characteristic flow behavior. If the container is tilted excessively, the viscosity rapidly decreases and the dressing flows in large quantities because of its characteristic fluidity, showing high viscosity at lower shear and low viscosity at higher shear. On the other hand, TSP exhibits good emulsion stability in acidic conditions, but, different from xanthan, is a near-Newtonian fluid flowing from the bottle mouth with constant fluidity. TSP therefore compensates for the shortcomings of xanthan. Taking this benefit, TSP is used for the adjustment of texture of dressings in combination with xanthan gum. There is also a report that the combination synergistically exhibits high viscosity caused by thermoreversible physical gel-like properties (Kim et al., 2006). From these characteristics, it is evident that TSP can provide benefits in dressing applications. In addition, TSP is also useful for low- or no-fat dressings and mayonnaise, which are in increasing demand due to recently widespread health-conscious diet trends. Mayonnaise is intrinsically an emulsion food prepared by emulsifying oil and vinegar with egg yolk, but it is said that reduction of the fat cuts the body texture and shape retention property caused by the fat content. In this case, the emulsion stability of TSP can make up the deficits. In low-fat mayonnaise applications, TSP exhibits higher adhesiveness than xanthan gum, while xanthan gum exhibits better shape retention (Fig. 20.9). As a result, it is thought that TSP can make dressing/mayonnaise stay in the mouth longer and thus improve flavor release by adding adherence.

Adherence(ratio%)

TSP 2.0%

Xanthan gum 0.5% TSP 1.0%

Xanthan gum 1.0%

100

70

23

Adherence(appearance)

Shape retention FIGURE 20.9 Comparison of mayonnaise type dressings (DSP Gokyo Food & Chemical, 2014a).

3. Food applications

457

Therefore, the combination of TSP and xanthan gum provides both adherence and shape retention, demonstrating why TSP has been used in this way in combination with other polysaccharides such as xanthan gum. From these findings, it is concluded that TSP possesses further potential as an effective stabilizer, and as a part of fat replacement or mimetic in dressings and mayonnaise.

3.2.3 Frozen desserts Frozen dessert (e.g., ice cream) is another important food class using oil-in-water emulsion. Ice cream is arguably one of the most complex food products, with multiple phases that can influence product quality and attributes (Goff et al., 1999). The mix ingredients include fat globules, air bubbles, and ice crystals dispersed in a high-viscosity freeze-concentrated matrix phase containing a solution of proteins, salts, polysaccharides, and sugars. Polysaccharides such as locust bean gum (LBG), guar gum, and carboxymethyl cellulose (CMC) are generally included in frozen dessert formulations where they provide many functions including increasing viscosity, aiding aeration and meltdown control (Goff and Hartel, 2013; Sutton et al., 1996). Melting rate and shape retention have been two of the main factors influencing the quality and processing of frozen dessert. Although TSP has been used as a stabilizer for frozen products, to date there have been few reports regarding ice cream using TSP as a stabilizer. This section will describe possible functions of TSP in stabilizing frozen desserts, giving practical use cases as examples. Our studies on frozen desserts indicate that TSP can give an over run and suppress ice crystal growth and sugar crystallization after prolonged storage. As described earlier, TSP has water-retention capabilities, indicating that TSP holds more free-water in freezing mixes around the ice crystals and prevents their growth. In addition, as described in section:2.3, TSP-sugar gel becomes harder and more elastic after freeze-thaw, thus it is suggested that freeze-thaw processes make the gel-like network stronger and TSP suppresses ice crystal growth (Yamatoya and Shirakawa, 2003). TSP or hydrolyzed TSP can also reduce water separation from gels composed of other gelling agents, especially after freeze-thaw (Nagashima et al., 1999; Senda et al., 2008). The thicker and denser gel network is observed in the SEM image of hydrolyzed TSP with added carrageenan gel. From these findings TSP alone or in combination with other ingredients such as sugar or polysaccharides forms a gel network in the freezing mixture, and so it can be considered that the network captures a variety of particles, including emulsion globules, and stabilizes the state in the frozen environment. Ice cream formulations containing both TSP and LBG clearly exhibit an improvement of meltdown rate and shape retention during storage at room temperature compared to those with LBG or TSP alone (Fig. 20.10). In practice, this ability has also been used for shape retention of soft-serve ice cream, where it is very important to make a pointed top when extruded. These characteristics also contribute to melting in the mouth during consumption. These effects of TSP may at least partly account for the gel-like network in combination with LBG as a structural support, although TSP does not exhibit any synergism with LBG in aqueous solution in viscosity and gelling property. The gel-like network of LBG involves trapping melting water in the freezing mixture and the network is responsible for heat-shock stability. However, such a network is not evident with guar gum (Goff et al., 1999). It is further indicated that these stabilizers, including polysaccharides, sugars and proteins, are freeze-concentrated in the unfrozen continuous phase of ice cream where they interact with each other and significantly affect the rheological and textural properties of the products (Goff and Hartel, 2013).

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Chapter 20 Tamarind seed polysaccharide

(A)

1

2

3

4

(B)

Dissolved amount [g]

0min.

30min.

:Locust bean gum(LBG) 0.3% :LBG 0.2%/TSP 0.1% :LBG 0.1%/TSP 0.2% :TSP 0.3%

60min. 1: Locust bean gum(LBG) 0.3%, 2: LBG 0.2% + TSP0.1%, 3: LBG 0.1% + TSP 0.2%, 4: TSP 0.3%

Shape-retaining properties

Stroge time[min.] at room temperature

Amounts of dissolved ice cream over time

FIGURE 20.10 Comparison of ice creams prepared with polysaccharides (DSP Gokyo Food & Chemical, 2014b).

Therefore, regarding the synergic effect of TSP and LBG during the freezing procedure, both of them with higher relative concentrations form a cryptic gel-like network together with both concentrated sugars and each other, and it has been suggested that the network is at least partly responsible for higher heat-shock stability. Furthermore, it is presumed that the network conformation is caused by a hydrophobic network formed by some interaction of both of the rod-like molecules. That is, aggregation occurs due to hydrophobic interaction not at the side chain, but at the main chain section of both TSP and LBG, because guar gum, which has a similar main chain but with more side residues compared to LBG, has no synergistic effect with TSP. These interactions are compared with thickening or gel formation due to the interaction between TSP and xanthan gum or TSP and gellan gum (Nishinari et al., 2009). In commonly known molecule models of the synergic gelation of xanthan gum/galactomannan, the binding region of galactomannan side chains exists in a continuous unsubstituted mannose main chain region, the so-called “smooth region” (Brownsey et al., 1988; Cairns et al., 1987; Dea et al., 1972; Takemasa and Nishinari, 2016), and the binding region with TSP is also expected to be on such a main chain region. In summary, it is assumed that the gel-like intermolecular interactions in the frozen product may be prerequisites for their effectiveness in heat-shock stability. The possible hypothesis of the mechanism described earlier needs to be proven experimentally. Recently, manufacturers have started using TSP in vegan products, including ice cream, because TSP is a plant-based polymer and is therefore compatible with these products. TSP is expected to play an important role in the stream of current dietary trends, such as “Vegan” or “Gluten-Free”.

4. Conclusion In this review, the structural makeup, rheological behavior and mechanism of gelation of TSP have been reviewed, and not only the emulsion stability, but also the emulsification activity of TSP has been discussed more deeply than ever before. Based on its fundamental properties, TSP has a beneficial

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effect on thickening, water retention, gelation, antiretrogradation of starch, and combinations of these functions, in applications such as bakery products, dressings, mayonnaise, and frozen desserts, which consist of complex elements. Although the mechanisms of the effects of TSP are described in reference to recent studies and current use, to elucidate more detailed mechanisms, further experimental and theoretical proof is required in the future.

Acknowledgments We thank Dr. Stuart Clegg, Fuerst Day Lawson Ltd., for his great contribution. We thank Dr. Hiroshi Egawa, Kenji Takasaka, and Akiko Miyamoto, DSP Gokyo Food & Chemical Co., Ltd., for their helpful advice.

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Sims, I.M., Gane, A.M., Dunstan, D., Allan, G.C., Boger, D.V., Melton, L.D., et al., 1998. Rheological properties of xyloglucans from different plant species. Carbohydrate Polymers 37 (1), 61e69. Sutton, R.L., Lips, A., Piccirillo, G., 1996. Recrystallization in aqueous fructose solutions as affected by locust bean gum. Journal of Food Science 61 (4), 746e748. Takemasa, M., Nishinari, K., 2016. Solution structure of molecular associations investigated using NMR for polysaccharides: xanthan/galactomannan mixtures. The Journal of Physical Chemistry B 120 (12), 3027e3037. Vianna-Filho, R.P., Petkowicz, C.L.O., Silveira, J.L.M., 2013. Rheological characterization of O/W emulsions incorporated with neutral and charged polysaccharides. Carbohydrate Polymers 93 (1), 266e272. Yamanaka, S., Mimura, M., Urakawa, H., Kajiwara, K., Shirakawa, M., Yamatoya, K., 1999. Conformation of tamarind seed xyloglucan oligomers. Journal of Fiber Science and Technology 55 (12), 590e596. Yamanaka, S., Yuguchi, Y., Urakawa, H., Kajiwara, K., Shirakawa, M., Yamatoya, K., 1999b. Gelation of enzymatically degraded xyloglucan extracted from tamarind seed. Journal of Fiber Science and Technology 55 (11), 528e532. Yamanaka, S., Yuguchi, Y., Urakawa, H., Kajiwara, K., Shirakawa, M., Yamatoya, K., 2000. Gelation of tamarind seed polysaccharide xyloglucan in the presence of ethanol. Food Hydrocolloids 14 (2), 125e128. Yamatoya, K., Shirakawa, M., 2003. Xyloglucan: structure, rheological properties, biological functions and enzymatic modification. Current Trends in Polymer Science 8, 27e72. York, W.S., van Halbeek, H., Darvill, A.G., Albersheim, P., 1990. Structural analysis of xyloglucan oligosaccharides by 1H-n.m.r. spectroscopy and fast-atom-bombardment mass spectrometry. Carbohydrate Research 200, 9e31. Yoshimura, M., Takaya, T., Nishinari, K., 1999. Effects of xyloglucan on the gelatinization and retrogradation of corn starch as studied by rheology and differential scanning calorimetry. Food Hydrocolloids 13 (2), 101e111. Yuguchi, Y., Mimura, M., Urakawa, H., Kajiwara, K., Shirakawa, M., Yamatoya, K., et al., 1997. Cross-linking structure formation of some polysaccharides in aqueous solution. In: Adisesha, H.T., Sudirjo, S.T., Panggabean, P.R., Arda, J., Soetrono, C.W. (Eds.), Proceedings of the International Workshop on Green Polymers, Reevaluation of Natural Polymers. Indonesian Polymer Association, Bogor, Indonesia, pp. 306e329. Yuguchi, Y., Urakawa, H., Kajiwara, K., Shirakawa, M., Yamatoya, K., 2001. Gelation of xyloglucan polysaccharide extracted from tamarind seed. In: Radiman, C.L., Iguchi, M., Yatabe, T., Fischer, S. (Eds.), Proceedings of the Second International Workshop on Green Polymers. Indonesian Polymer Association, Bogor, Indonesia, pp. 253e263. Yuguch, Y., Kumagai, T., Wu, M., Hirotsu, T., Hosokawa, J., 2004. Gelation of xyloglucan in water/alcohol systems. Cellulose 11, 203e208.

CHAPTER

Thermomechanical and surface morphology of biopolymere nanoparticle composite films

21 Jasim Ahmed

Food and Nutrition Program, Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Safat, Kuwait

1. Introduction The negative impact of fossil-derived polymers on the environment and human health has led to the development of the market, use, and production of polymers from natural resources including starch, cellulose, hydrocolloids, gelatin, and chitosan (CS), which are termed biopolymers. The use of biopolymer-based composites has increased significantly because of their high biocompatibility, biodegradability, optimal mechanical properties, wide availability from natural sources, low immunogenicity, and nonmutagenic, noncarcinogenic, and nonirritating properties (Jacob et al., 2018). Biopolymer-derived composites have attracted considerable attention for their use in packaging applications. However, biopolymer films have poor mechanical, thermal, and barrier properties, which limits their use as a potential food packaging material. To overcome those limitations, various approaches have been taken so that biopolymers can be successfully used in packaging applications. The first approach is plasticization of the biopolymer by incorporating lowemolecular weight plasticizers (e.g., polyethylene glycol [PEG], polypropylene glycol, monomer) or blending with a miscible polymer so that the glass transition temperature (Tg) of the resultant polymers is lowered (Ahmed et al., 2016). Although the resultant biopolymers exhibit increased elongation at break (EAB), it still needs further improvement in the mechanical, thermal, and barrier properties. The addition of suitable nanoparticles (NPs) could bring a desired change in the properties of biopolymer composites, known as bionanocomposites. Nanofillers offer enormous advantages over microparticles owing to their interfacial interactions on polymer branches resulting from their increased surface area and high surface energy, which significantly improve the thermal, mechanical, and water barrier properties of polymers (Kovacevic et al., 2008). Therefore, significant efforts have been made to advance food-packaging films using bio-nanocomposites with the improved mechanical, barrier, rheological, and thermal properties. The focus of the chapter is on elucidating the interaction and miscibility between biopolymers and NPs, which eventually produce the best nanocomposites (NCs) with desired properties for packaging applications. The list of biopolymers is long, and it is impossible to put into a single chapter all Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00021-7 Copyright © 2020 Elsevier Inc. All rights reserved.

463

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information related to biopolymers. The chapter includes only selected biopolymers and their composites that have commercial potential and are widely studied.

2. Nanoparticles Based on size, particles are classified as NPs with a size of approximately 1e100 nm. According to the ASTM International 2456-06 standard, for three-dimensional (3D) particles, two or three dimensions must be 1e100 nm. Nanomaterials have some properties that make them unique from that of the source materials. Metal and metal oxide NPs just a few nanometers in diameter and sizes somewhere between single atoms or molecules and bulk materials have pronounced size-dependent properties. The strongly size-related properties of NPs offer numerous opportunities for composites impregnated with NPs with superior performance. NPs consist of three layers: (1) surface, which may be functionalized with a variety of small molecules, metal ions, surfactants, and polymers; (2) shell, a material chemically different from the core in all aspects; and (3) core, essentially the central portion of the NP, and which usually refers to the NP itself. NPs are derived from different sources and their properties may vary according to the preparation method and their aspect ratios. NPs are classified into four major categories, as illustrated in Fig. 21.1. The most common NPs employed for reinforcement into biopolymers are metal-based (silver [Ag], titanium dioxide [TiO2], copper [Cu], and zinc oxide [ZnO]) and their alloys (silver-copper [AgeCu]), carbon nanotubes, clays, silicates, cellulose, starch nanocrystals, and many more (Alcaˆntara et al., 2014; Koriche et al., 2014; Kontou et al., 2011; Laredo et al., 2010; Angellier et al., 2006; Wawro et al., 2012; Villanueva et al., 2016; Ahmed et al., 2010a,b; Arfat et al., 2017). Among polysaccharides, cellulose is the most frequently used natural polymer; it is obtained from different sources. Cellulose nanostructures are available in two forms: cellulose nanocrystals and nanofibers (Zhao et al., 2014). Cellulose nanostructures have been used as an alternative in the production of biodegradable films because of their biodegradability and biocompatibility (Pelissari et al., 2014; Vilarinho et al., 2018). Various analytical techniques have been employed to characterize NPs. These include particle size analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic

FIGURE 21.1 Classification of nanoparticles (NPs).

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465

force microscopy (AFM), x-ray diffraction (XRD), x-ray photoelectron spectroscopy, and the BrunauereEmmetteTeller equation. The fabrication of polymer NC has been facilitated by the use of ultrasonication to disperse nanofillers. However, the controlled amount of the weight percent and size of the nanomaterial is carefully considered. The key challenge in NCs is to eradicate agglomerate formation when a nanofiller comes into contact with the host polymer. Different methods are adopted to functionalize the surface of nanofillers so that uniform dispersion can be obtained.

3. Biopolymers Most biopolymers are naturally derived and biodegradable; therefore, they have no negative impact on the environment. Polysaccharides are a major source of biopolymers. Some are derived from proteins, lipids, and microbial sources. A list of biopolymers is illustrated in Fig. 21.2. The biomass has been selectively converted into biopolymer by chemical modification, which further turns into a bionanocomposite by reinforcing selective NPs. The composites have active applications in packaging and coatings. The interfacial interactions via functional groups between the biopolymer matrix and NPs control the formation of a network that improves the homogeneity in dispersion. In addition, biodegradability makes the composite a wondrous material.

4. Biopolymerenanoparticle fabrication techniques Many synthetic methods are depicted in the literature for bio-based NCs. The major challenge in synthesizing NCs is the adequate distribution of NPs at a critical concentration in the polymer matrix, which finally controls the properties of NCs. The major techniques employed for synthesizing NCs are discussed next.

FIGURE 21.2 Biopolymers produced from various sources. PLA, polylactic acid; PHA, polyhydroxyalkanoate.

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Chapter 21 Thermomechanical and surface morphology

5. In situ polymerization Synthesis of the confined polymer is performed by adsorption of a guest molecule (monomer) into a host compound containing intraplanar spaces, channels, or other cavities, followed by in situ polymerization of the monomer (Messersmith and Giannelis, 1993). In this type of polymerization, the nanofiller is dispersed into the monomer of the polymer matrix before the polymerization reaction (Fig. 21.3). The resulting polymers are expected to be intercalated or occluded. The polymere inorganic NCs can be obtained by intercalative polymerization of suitable monomers in the galleries of layered solids and the resulting hybrids often exhibit unique properties. However, the limitation of the process is to obtain inter-gallery polymerization; hence, there are not many applications.

6. Melt intercalation Melt intercalation has been considered a promising technique because it can be carried out with conventional polymer mixing or extrusion and the process is environmentally benign. NPs and polymer matrix form an intercalated network in the molten state within polymeric chains to produce an NC. Polylactic acid (PLA)-based NCs are produced by this method. The interaction between the organic modifiers and the molecules of the polymer matrix favor the intercalation process of polymer molecules into the interlayers of NPs. However, the process is unsuitable for most biopolymers because of heat degradation.

7. Solution cast intercalation Solution cast is the most common technique used in the laboratory for developing biopolymer-based NCs because of its simplicity and easy in producing the film. NPs are dispersed in a suitable solvent for a predefined time with constant shearing on a magnetic stirrer or ultrasonication followed by evaporating the solvent to obtain the film. Common solvents for the process are dicholoromethane and chloroform for PLA and 1% acetic acid for CS. The major limitation is in the handling of solvents and a significant loss of solvents with environmental concerns.

FIGURE 21.3 In situ intercalative polymerization. From Messersmith, P. B. and Giannelis, E. P. (1993). Polymer-layered silicate nanocomposites: in situ intercalative polymerization of ε-caprolactone in layered silicates. Chemistry of Materials, 5, 1064e1066.

9. Rheology of polylactic acidebased nanocomposites

467

The following sections discuss NC films obtained from some important biopolymers with potential commercial values and their properties.

8. Polylactic acidebased nanocomposites PLA is one of the most well-studied biopolymers. It has been approved as having generally regarded as safe status for food packaging. Although PLA possesses a high Young’s modulus (YM) of around 3e4 GPa and a tensile strength (TS) of 50e70 MPa, major limitations of PLA are its inherent brittleness and low toughness. PLA is generally blended with lowemolecular weight and low-Tg polymers such as PEG, polypropylene glycol, and polyhydroxy butyrate to improve their flexibility. It has been observed that incorporating 10% PEG drastically reduced the mechanical rigidity of the blend with a significant drop in the Tg. Further addition of PEG to PLA resulted in a blend with significantly low Tg that may not be suitable for processing and molding. In addition, there may be a chance of migration of the monomer to the film’s surface, which may be unsuitable for food packaging (Ahmed et al., 2010a). Therefore, incorporating nanofiller is a novel way to improve the thermomechanical and mechanical properties of plasticized PLA. Ahmed and his group (2010e19) conducted a series of works on the synthesis of NCs of plasticized PLA using a range of nanofillers including nanoclay, ZnO, AgeCu, graphene oxide (GO), and evaluated the rheological, thermal, tensile, and antimicrobial properties of the developed films.

9. Rheology of polylactic acidebased nanocomposites Study of the rheological characteristics of polymeric blends and NCs in the molten state is critical to obtaining a fundamental understanding of the nature of the processability and structureeproperty correlation of NCs. Ahmed et al. (2010b) prepared PLAeNCs by blending L-PLA, PEG, and montmorillonite (MMT) clay by solvent cast technique. The elastic modulus, G0 , of the PLAeclay blend showed a significant improvement in the magnitude in the melt, whereas the clay concentration was at 6% wt. or higher. However, the addition of PEG reduced the dynamic moduli and the complex viscosity (h) of PLAePEG blend significantly as a function of concentration. The PLAePEGeclay (74/ 20/6) NC exhibited intermediate values of G0 and h with excellent flexibility compared with the neat PLA and PLAePEG blend. The miscibility of clay into the PLA blend was further assessed by the ColeeCole plot of the rheological data representing the relation between real (h0 ) and imaginary (h00 ) parts of complex viscosity. A smooth, semicircular shape of the curve indicates a good dispersibility or phase homogeneity in the melt; any deviation from the shape indicates nonhomogeneous dispersion or immiscibility. The h0 and h00 are calculated from the equations (Eqs. 21.1 and 21.2): h0 ¼ G0=u

(21.1)

h00 ¼ G0=u

(21.2)

The smooth semicircular shape of the ColeeCole plot for the PLAeclay blend at 196 C indicates a smooth distribution between the PLA and the nanoclay at loading concentrations of 3% and 6%, whereas the plot failed at the highest loading concentration of 9%, indicating nonmiscibility (Fig. 21.4).

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Chapter 21 Thermomechanical and surface morphology

15000

η" (Pa.s)

10000

5000

3% 6% 9%

0 0

5000

10000

15000

20000

η' (Pa.s)

FIGURE 21.4 Cole-Cole plot for polylactic acideclay blend at 196 C at selected clay loading concentrations. Adapted from Ahmed, J., Varshney, S. K., Auras, R., & Hwang, S. W. (2010b). Thermal and rheological properties of L-polylactide/ polyethylene glycol/silicate nanocomposites films. Journal of Food Science, 75(8), N97eN108.

Reinforcing selected NPs (AgeCu alloy [97%wt of liquid oil. Corresponding cryo-SEM images of these samples are provided in the bottom panel (from left to right: image width ¼ 40, 40, and 200 mm, respectively). Modified from Patel, A.R., Rajarethinem, P.S., Cludts, N., Lewille, B., De Vos, W.H., Lesaffer, A., Dewettinck, K., 2015. Biopolymer-Based Structuring of Liquid Oil into Soft Solids and Oleogels Using Water-Continuous Emulsions as Templates. Langmuir 31, 2065e2073.

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Chapter 24 Biopolymer-based oleocolloids

steps. However, it does suffer from the drawback of long drying time that can lead to oxidation of liquid oil, especially when elevated temperature is used for drying the emulsions. The long drying time is due to the presence of unabsorbed nonsurface-active polymer in the bulk phase that binds and structures the water. In recent work by Wijaya et al., this drawback was circumvented by (a) using high internal phase emulsions (HIPEs) as the templates to decrease the overall water content, and (b) using complexes of proteinepolysaccharides as stabilizers that accumulate at the interface so the bulk phase is relatively unstructured (Wijaya et al., 2018b). In this work, HIPEs with oil volume fraction >0.74 (more than the close packing of spherical oil droplets) were first formulated using preformed complexes of proteins (whey protein isolate or sodium alginate) with low methoxy pectin. Owing to the relatively lower content of water in the bulk phase, these emulsions could be efficiently dried to obtain oil-in polymer gels.

2.3 Gels prepared from protein hydrogels via stepwise solvent exchange route A more sophisticated technique called solvent exchange route has also been tried to make biopolymer suitable for oil structuring (de Vries et al., 2015). A hydrogel is first created by dispersing the hydrophilic protein in water, followed by heat treatment to expose the hydrophobic groups of protein and create basic building blocks. Once the network is established in the aqueous solvent, a stepwise exchange from water to oil is carried out with the use of solvents with intermediate polarity to prevent any agglomeration-induced disruption of the protein network. Specifically, authors of this work have found that heat-set network of whey protein isolates formed in water could be successfully used as a framework for supporting the structure of oleogels containing more than 90 %wt of liquid oil. Transparent to translucent-turbid oleogels with negligible residual water (1 mm) ensures a rapid creaming of the coated capsules, which are then separated and dried. This powdered hard stock can then be used to deliver hydrated polymer strands into the liquid oil (Fig. 24.4).

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Chapter 24 Biopolymer-based oleocolloids

3. Biopolymer-based O/W/O emulsions In the last few years, research on emulsions with complex microstructures such as double emulsions have received a considerable interest especially from industrial scientists working in the field of colloid structuring. They can be used for a number of applications including compartmentalization of incompatible bioactives, controlled delivery of micronutrients, and fat and salt reduction in reformulated food products. For fat reduction, mainly water-continuous double emulsions (water-in-oil-in-water, W/O/W) have been explored where the goal is to decrease the energy density of the formulation by replacing a part of oil with water for applications in products such as mayonnaise, salad dressings, sauces, and dips. Oil-continuous double emulsions (oil-in-water-in-oil, O/W/O) have not received the same level of attention as WOW emulsions, and this is mainly because of the processing difficulties encountered in the preparation of oil-continuous double emulsions and/or requirement of high level of surfactant(s). Although W/O/W emulsions are applicable for reduction in fat content of food products, O/W/O emulsions may find use in reduction of the amount of solid fat required for structuring of food products that are based on indirect emulsions (W/O) (such as table spreads, baking margarines, and cooking fats). The lowering of solid fats in indirect emulsions through use of O/W/O emulsions is explained with the help of a hypothesis shown in Fig. 24.5A (Patel, 2018b). Basically, the partitioning of a portion of liquid oil in the inner water phase results in the lowering of the amount of liquid oil left to be structured in the external oil phase, and thus a better texture may be obtained at a relatively lower mass fraction of crystalline fat. With this hypothesis, O/W/O were first prepared and the rheological and textural properties of double emulsion were compared with indirect emulsion containing same level of hard stock (Fig. 24.5B and C) as a proof of concept (Patel, 2018b). As seen from the figure, by distributing the same phases differently, a higher firmness is achieved in double emulsion compared to the indirect emulsion. O/W/O double emulsions were prepared using a two-step emulsification approach by first creating a primary emulsion (O1/W) stabilized by biopolymers (proteinegelatin and polysaccharideexanthan gum) followed by subsequent emulsification with oil phase (O2) containing different proportion of crystallizing solid fat of vegetable origin (palm oil) (Patel, 2017e). The homogenization and crystallization was carried out simultaneously to stabilize primary emulsion droplets (O1/W) in double emulsion through interfacial accumulation of fat crystals as well as physical entrapment in solidified crystalline network in the bulk oil phase (Fig. 24.5D). The microscopy images of representative primary emulsion and O1/W/O2 emulsion are shown in Fig. 24.6AeD. Stabilization of concentrated primary emulsion (4oil ¼ 0.6) was achieved by using an optimized combination of gelatin:xanthan gum (GL:XG) with a total biopolymer concentration ranging between 1.4 and 2.2 %wt. The average droplet size (d4,3) was below 10 mm for emulsions prepared at different ratios of GL:XG with zeta potential values well above 60 mV (pH z 6). The droplet size distribution curves shown in Fig. 24.6E also confirmed that variation in the concentration of the surface-active component (GL) had a stronger influence on the fineness of droplets compared to the viscosity building component (XG) as seen in one of the previously reported work (Patel et al., 2015). Although resulting in a decrease in the mean droplet size, the increased GL levels also led to a bimodal distribution of droplets suggesting the formation of significant proportion of finer droplets in the submicron range. On the other hand, XG while not contributing strictly to the colloidal stability of primary emulsion, was required for improving the viscoelastic properties of the primary emulsion thereby stabilizing it against gravitational separation of the phases (creaming). Moreover, primary emulsions stabilized only with GL (in the absence of XG) resulted in the formation of watercontinuous emulsions (O/W) when mixed with molten oil phase before showing complete phase

3. Biopolymer-based O/W/O emulsions

597

FIGURE 24.5 (A) Schematic representation of hypothesis to explain the use of O/W/O for structuring indirect emulsionbased products. As a part of liquid oil is partitioned into the inner water phase, the amount of crystalline mass (solid fat) required for structuring the external oil phase can be decreased; (B) and (C) Amplitude stress sweep and load-extension curves comparing the firmness and deformation properties of DE and corresponding indirect emulsion (Ref) containing same level of components. Both DE and Ref contained 30%wt sunflower oil, 20%wt water (with 1.6%wt biopolymers), and 50%wt. palm stearine. However, the phase distribution in DE was 30/20/50 (sunflower oil/water/palm stearine) whereas in Ref it was 20/80 (water/ sunflower oil þ palm stearine). This is a proof of concept to prove the hypothesis that by encapsulating liquid oil in the inner water droplet, we can get an increase in the firmness and consistency of DE over Ref due to relatively higher proportion of crystalline mass in the external oil phase; and (D) Representation of stages involved in the formation of O/W/O, the microstructure changes are explained with the help of microscopy images (image widths ¼ 400 mm). Reproduced from Patel, A.R., 2018b. Innovative dispersion strategies for creating structured oil systems. In: Edible Oil Structuring: Concepts, Methods and Applications. The Royal Society of Chemistry, pp. 308e330.

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Chapter 24 Biopolymer-based oleocolloids

FIGURE 24.6 (A and B) Cryo-SEM image of primary emulsion (O1/W) and freeze-fractured double emulsion droplet showing a high encapsulation of inner oil droplets; (C and D) PLM images confirming that the emulsion droplets are stabilized by interfacial crystallization and physical entrapment in the network formed by bulk crystallization respectively; (E) Droplet size distribution curves for primary emulsions prepared at different concentrations of XG (at GL ¼ 1 %wt) and GL (at XG ¼ 0.6 %wt); and (F) Solid fat content profiles of different fat phases used for the preparation of double emulsions. Reproduced with permission from Patel, A.R., 2017d. Surfactant-free oil-in-water-in-oil emulsions stabilized solely by natural components-biopolymers and vegetable fat crystals. MRS Advances 2(19e20), 1095e1102.

separation. Hence, the presence of XG was necessary both for stability of the primary emulsion as well as for ensuring the formation of oil-continuous double emulsions. As seen from the schematic representation in Fig. 24.5D, the viscoelastic property of primary emulsion is responsible for the initial formation of islands of macrophase consisting of emulsion droplets that are broken down into discrete double emulsion droplets under homogenization at high temperature. With subsequent cooling, crystals are first formed to stabilize the interfaces followed by bulk crystallization that together contributes to the stabilization of double emulsion droplets through physical immobilization (Fig. 24.6C and D, PLM images) in fat crystal network. Stabilization of droplets via combination of interfacial crystallization and network development in the bulk oil phase has been a subject of investigation for quite a few years. As confirmed by several authors, stabilization of interfaces by in situ crystallization gives better stabilization compared to preformed crystals. Similarly, in our work it was found that the stabilization of primary emulsion droplets (O1/W) in the external oil phase (O2) could only be obtained when crystallization was carried

4. Biopolymer-based oleofilms

599

out along with homogenization. As seen from Fig. 24.6B, a high oil encapsulation (100% yield of inner oil phase) that is expected due to the process involved in the double emulsion formation (that simply involves breakdown of the macrophase of primary emulsion into discrete droplets) was obtained. Possibility of preparing double emulsions with such high loading of inner oil phase could be very useful for delivering oil soluble components in controlled release formulations. Another commercial application of such a system could be exploited in spreads-like products (Patel, 2017e, 2018b). Spreads constitute a range of products differing in the level of water phase, fat phase as well as the fat types that together influences the bulk properties of the systems such as texture and rheology. To explore the flexibility of our formulation for applications in spreads-like products we varied the amount of solid fat content in O2 as well as the ratio of (O1/W): O2. The solid fat content was altered (Fig. 24.6F) by replacing semisolid palm oil (PO) rich in saturated fatty acids (z49 %wt) with liquid sunflower oil (SFO) containing high amount of unsaturated fatty acids (saturated fatty acids < 10% wt). Stable double emulsions could be prepared by replacing as much as 50% of PO with SFO, further replacement of PO resulted in phase separation (within 24 h) suggesting that a minimum 10 %wt of crystalline fat (at 5 C) in O2 phase is required to stabilize double emulsion against coalescence and eventual phase separation. For possible applications in low-fat spreads, the ratio of (O1/W):O2 was also changed to investigate the possibility of formulating double emulsions with high water content. Double emulsions with up to 45%wt water (and minimum of 15% inner oil phase) could be prepared without addition of any emulsifier. The levels of all three phases were altered to obtain emulsions with high water content while making sure that the inner oil phase is at a level sufficient for future encapsulation-related applications. Based on the data from diffusive NMR, it was found that the inner oil droplet size showed a slight increase over 30 days of storage; however, the inner oil yield remained more or less unaffected. This indicates that although the inner oil droplets did show some degree of coalescence, the diffusion of oil droplets into the continuous oil phase was found to be insignificant. The relative stability of encapsulated oil droplets to diffusion could be viewed as a positive attribute of these surfactant-free double emulsions that could find interesting applications in compartmentalizing and controlling the release of hydrophobic bioactives loaded in the inner oil phase (Patel, 2017e).

4. Biopolymer-based oleofilms Biopolymers such as proteins and polysaccharides are known to display synergistic interactions that can be exploited to structure a range of soft matter systems, such as hydrogels, gel emulsions, oleogels, and films (Eghbal et al., 2016; Wijaya et al., 2017; Wijaya et al., 2018a,b). Under appropriate conditions, associative interactions among the biopolymers can create an interpolymeric matrix that can form a structure framework for bulk systems such as gels and films. As mentioned in Section 2.2.2, indirect method is generally employed to enable the interpolymeric matrix to entrap the hydrophobic solvents, that is, liquid oils. Using concentrated emulsions as templates that are stabilized by biopolymers, oleogels with a tightly packed arrangement of oil droplets into a structured interpolymeric matrix are obtained by simply drying the emulsions at mild temperatures. However, the structured oil obtained by drying the emulsions (which are stabilized by both native biopolymers) usually ends up being rigid and brittle due to extensive intermolecular interactions. The use of plasticizers such as polyols (e.g., glycerol) could improve the flexibility and processability of the dried products by increasing the interchain spacing and reducing interchain interactions as is usually seen in protein-

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Chapter 24 Biopolymer-based oleocolloids

based films. Working on this hypothesis, a novel colloidal system called as oleofilm was successfully fabricated using a simple modification of emulsion-templated approach (Wijaya et al., 2018a). As shown in Fig. 24.7, thin layered elastic oleofilms containing an ultrahigh concentration of liquid oil (>97 wt.% sunflower oil) was prepared by simply drying o/w HIPEs that are stabilized by proteine polysaccharide complexes with glycerol incorporated in the continuous phase as a plasticizer. In our previous research, oleogels (containing >97 wt.% liquid oil) were successfully prepared from emulsion templates stabilized by uncomplexed food polymers (gelatin or methylcellulose and xanthan gum) (Patel et al., 2014a; Patel et al., 2015). However, due to the relatively high proportion of the bulk water phase (oil volume fraction, foil z 0.60) that was structured with the uncomplexed xanthan gum, a higher temperature (i.e., 50 C) and longer drying time (i.e., 72 h) were required to completely evaporate the aqueous phase (Patel et al., 2014b). This drying process led to undesired oil oxidation as indicated by an increase of peroxide and p-anisidine values. In this study, the issue with the drying process was rectified by (i) using emulsion templates with a high oil volume fraction (foil ¼ 0.82) and (ii) using biopolymeric complexes for stabilization of the oilewater interfaces. Together, these modifications led to a shortening of the drying time and a lowering of the drying temperature.

FIGURE 24.7 A) Schematic representation of the process where liquid oil is first mixed with sodium caseinate (SC):alginate (ALG) complexes to prepare an 80 wt.% o/w emulsion, followed by the casting and removal of water (blue background) through drying, which results in the formation of an oleofilm. Furthermore, the interface of the yellow emulsion droplets shows an adsorbed layer of SC:ALG complexes: SC particles (dots) embedded in ALG (chains); (B and C) Representative photographs of HIPE and oleofilm with their corresponding microstructures seen under optical microscope and cryo-SEM, respectively. Modified from Wijaya, W., Van der Meeren, P., Dewettinck, K., and Patel, A. R., 2018. High internal phase emulsion (HIPE)templated biopolymeric oleofilms containing an ultrahigh concentration of edible liquid oil. Food and Function 9(4), 1993e97 with permission from The Royal Society of Chemistry.

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SC:ALG colloidal complexes were preformed at pH 7.0 by mixing the stock solutions of SC and ALG with the help of sonication. The aqueous dispersions containing complexes of SC:ALG were then used as the aqueous phase and homogenized together with sunflower oil (oil/aqueous phase mass ratio ¼ 4: 1) to obtain HIPEs. Homogenization was carried out at low and high shear at 11,500 and 13,500 rpm (using an UltraTurrax T25 Basic, Germany) to distribute slowly the oil phase within the aqueous phase (for efficient coverage of the oilewater interfaces) and to force adsorption of complexes at the surface of the oil droplets. Glycerol was added after the low shear emulsification. The resultant emulsion was then casted and dried to form elastic films. The representative appearance of HIPE sample is shown in Fig. 24.8B. After subjecting the HIPEs to casting and drying, the complete removal of water resulted in the formation of solid films (Fig. 24.8C). The mass-weighted average diameter D [4,3] of droplets in HIPEs ranged from 4.70 to

FIGURE 24.8 Infographic providing an overview of the field of oleocolloids.

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Chapter 24 Biopolymer-based oleocolloids

6.80 mm. The microstructure of oleofilms showed a unique structure where distinct oil droplets are seen to be tightly packed together in an interpolymeric matrix. The possibility of obtaining such a microstructure could be attributed to the prevention of coalescence during drying, owing to the wellstructured interfaces formed around the oil droplets in the emulsion (Fig. 24.8A). These interfaces were occupied by SC:ALG complexes, where ALG served as building blocks and SC particles were embedded along the ALG networks. This is in line with our previous studies where combinations of proteins and polysaccharides have been used to prevent dehydration-induced coalescence in emulsions. These films showed resistance to rupture on application of shear and thermostability at elevated temperatures. Thus, making them suitable for applications in bakery products (especially, laminated products) where they can improve the fatty acid profile (by replacing saturated fats contributed by commercial plastic shortening) and also improve the baking process by providing a bake-stable shortening option.

5. Conclusion and future trends A concise overview of the field of oleocolloids is provided in Fig. 24.8. Although the academic research has proliferated in the last few years, there is still a significant lack of knowledge in the understanding of soluteesolvent interactions in hydrophobic mediums such as edible oils. Hence, there is an urgent need to investigate the links between molecular features and parameters such as solubility, self-assembly (the process by which individual molecules form defined aggregates), and self-organization (the process by which the aggregates create higher-ordered structures) of different categories of solutes in liquid oil (Patel, 2017a). On one hand, this will help us unravel new materials in processed or unprocessed form that could be used to structure the oil phase. Moreover, on the other hand, an in-depth study of coassembly and self-sorting behavior of building blocks could unravel new synergistic combinations of gelling agents for bulk phase structuring of oleocolloids (Patel, 2019). Furthermore, innovation in dispersion techniques could assist in the formulation of novel complex colloids, such as high internal phase emulsions (W/O type), double emulsions (O/W/O), oleofoams, and oleofilms, which could be used to reformulate existing food products or to develop new product formats. It is also important to keep in mind that reformulated products may lose some functionality and not be able to match the characteristics of conventionally formulated food products. Such loss of functionality may include a decreased plasticity of intermediate-use products (such as bakery fats) and less-desirable organoleptic properties of final products (such as texture and mouthfeel of spreads). Moreover, as most of these studies are mainly exploratory in nature, extensive developmental work in collaborations with industry will be required to commercially exploit some of these potential systems for food product formulation. Some anticipated industry actions (in collaboration with academia) may include: (i) rework/optimize formulations to suit the processing conditions (shear, temperature modulation) encountered in large-scale production; (ii) understand the behavior of oleocolloids in food product matrixes; and (iii) explore innovative approaches in liaison with ingredient suppliers to improve the functionality of commonly used ingredients. To summarize, the estimated benefits of replacing saturated fats with healthy unsaturated fats (especially with PUFAs), replacing palm oil, and developing clean-labeled products with unconventional textures, far outweighs the costs associated with innovating and reworking oleocolloids for better scalability.

References

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Patel, A.R., 2018b. Innovative dispersion Strategies for creating structured oil systems. In: Edible Oil Structuring: Concepts, Methods and Applications. The Royal Society of Chemistry, pp. 308e330. Patel, A.R., 2018c. Structuring edible oils with hydrocolloids: where do we stand? Food Biophysics 13 (2), 113e115. https://doi.org/10.1007/s11483-018-9527-6. Patel, A.R., 2019. Oleogelation for food structuring based on synergistic interactions among food components. In: Melton, L., Shahidi, F., Varelis, P. (Eds.), Encyclopedia of Food Chemistry. Academic Press, Oxford, pp. 715e718. Patel, A.R., 2018d. Hydrocolloid-based structuring of edible oil. Inform 29, 6e10. Patel, A.R., Cludts, N., Bin Sintang, M.D., Lewille, B., Lesaffer, A., Dewettinck, K., 2014a. Polysaccharide-based oleogels prepared with an emulsion-templated approach. ChemPhysChem 15 (16), 3435e3439. https:// doi.org/10.1002/cphc.201402473. Patel, A.R., Cludts, N., Sintang, M.D.B., Lesaffer, A., Dewettinck, K., 2014b. Edible oleogels based on water soluble food polymers: preparation, characterization and potential application. Food & Function 5 (11), 2833e2841. https://doi.org/10.1039/C4FO00624K. Patel, A.R., Rajarethinem, P.S., Cludts, N., Lewille, B., De Vos, W.H., Lesaffer, A., Dewettinck, K., 2015. Biopolymer-based structuring of liquid oil into soft solids and oleogels using water-continuous emulsions as templates. Langmuir 31 (7), 2065e2073. https://doi.org/10.1021/la502829u. Qiu, C., Huang, Y., Li, A., Ma, D., Wang, Y., 2018. Fabrication and characterization of oleogel stabilized by gelatin-polyphenol-polysaccharides nanocomplexes. Journal of Agricultural and Food Chemistry 66 (50), 13243e13252. https://doi.org/10.1021/acs.jafc.8b02039. Romoscanu, A.I., Mezzenga, R., 2006. Emulsion-templated fully reversible protein-in-oil gels. Langmuir 22 (18), 7812e7818. https://doi.org/10.1021/la060878p. Sa´nchez, R., Stringari, G.B., Franco, J.M., Valencia, C., Gallegos, C., 2011. Use of chitin, chitosan and acylated derivatives as thickener agents of vegetable oils for bio-lubricant applications. Carbohydrate Polymers 85 (3), 705e714. https://doi.org/10.1016/j.carbpol.2011.03.049. Tanti, R., Barbut, S., Marangoni, A.G., 2016a. Hydroxypropyl methylcellulose and methylcellulose structured oil as a replacement for shortening in sandwich cookie creams. Food Hydrocolloids 61, 329e337. https://doi.org/ 10.1016/j.foodhyd.2016.05.032. Tanti, R., Barbut, S., Marangoni, A.G., 2016b. Oil stabilization of natural peanut butter using food grade polymers. Food Hydrocolloids 61, 399e408. https://doi.org/10.1016/j.foodhyd.2016.05.034. Tharanathan, R.N., Kittur, F.S., 2003. Chitin d the undisputed biomolecule of great potential. Critical Reviews in Food Science and Nutrition 43 (1), 61e87. https://doi.org/10.1080/10408690390826455. Wijaya, W., Van der Meeren, P., Dewettinck, K., Patel, A.R., 2018a. High internal phase emulsion (HIPE)templated biopolymeric oleofilms containing an ultra-high concentration of edible liquid oil. Food & Function 9 (4), 1993e1997. https://doi.org/10.1039/C7FO01945A. Wijaya, W., Van der Meeren, P., Patel, A.R., 2017. Cold-set gelation of whey protein isolate and low-methoxyl pectin at low pH. Food Hydrocolloids 65, 35e45. https://doi.org/10.1016/j.foodhyd.2016.10.037. Wijaya, W., Van der Meeren, P., Patel, A.R., 2018b. Oleogels from emulsion (HIPE) templates stabilized by proteinepolysaccharide complexes. In: Edible Oil Structuring: Concepts, Methods and Applications. The Royal Society of Chemistry, pp. 175e197.

CHAPTER

Gum-based hydrogels in drug delivery

25

Amit Kumar Nayak1, Md Saquib Hasnain2, Kunal Pal3, Indranil Banerjee3, Dilipkumar Pal4 1

Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj, Odisha, India; 2 Department of Pharmacy, Shri Venkateshwara University, Amroha, Uttar Pradesh, India; 3Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha, India; 4Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Koni, Bilaspur, Chhattisgarh, India

1. Introduction Hydrogels are three-dimensional cross-linked network structures generally composed of hydrophilic polymeric systems, which are capable of holding high volume of aqueous fluids devoid of altering the structure (Hoffman, 2002). In swollen condition, hydrogels are accountable for the rubbery and soft characteristics. However, these are not able to dissolve swiftly in the aqueous solutions resembling with the living tissues and demonstrating exceptional mechanical capabilities (Hoffman, 2002; Nayak and Pal, 2016a). In 1894, the term “hydrogel” was used first time as hydrogel was used to elucidate a colloidal gel (Bemmelen, 1894). Usually, hydrogels swell due to absorptions of large amount of water, which make these as valuable materials for various biomedical uses (Nayak and Pal, 2016a; Pal et al., 2018). Due to the exceptional mechanical strength, chemically cross-linked hydrogels are identified as the biomedical material of great demand (Mishra and Mishra, 2016; Nayak and Pal, 2016a). The hydrogel structure comprises the covalent junction among the polymeric structures that can be attained via the cross-linking methodologies such as physical method, chemical method, enzymatic method, high-energy irradiation, photochemical reaction, and grafting (Singhal and Gupta, 2016). During past few decades, different kinds of hydrogels get the technical values well in various important commercial healthcare as well as biomedical applications employing tablets, tissue expanders, contact lenses, osmotic devices, implantable devices, inserts, etc. (Mawad et al., 2012; Mishra and Mishra, 2016). Currently, hydrogels have gained the exceptional attention in the controlled release drug delivery applications where the drugs are dispersed all through the hydrogel matrices and are capable of delivering the drug candidates at the steady rate over a longer time (Hua et al., 2010; Pal and Nayak, 2015b). Recently, different types of hydrogels are prepared by employing several natural, semisynthetic and synthetic polymers for utilizations in the applications in drug delivery area (Mishra and Mishra, 2016). Among these, hydrogels made of natural gums have been considered as more advantageous in terms of biodegradability, biocompatibility, material cost, ease of production, wide array of applications, etc. (Pal et al., 2018; Rana et al., 2011). Considering these advantages, in the recent years, Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00025-4 Copyright © 2020 Elsevier Inc. All rights reserved.

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Chapter 25 Gum-based hydrogels in drug delivery

numerous gum-based hydrogels made of chitosan (Pal et al., 2018), alginate (Pal and Nayak, 2015a,b), gellan gum (Agnihotri et al., 2006; Jana et al., 2013), pectin (Munarin et al., 2012; Nayak et al., 2013b), tamarind gum (Nayak and Pal, 2011, 2015), sterculia gum (Nayak and Pal, 2016a; Singh and Pal, 2011, 2012), guar gum (Soppimath and Aminabhavi, 2002; Soppimath et al., 2000), locust bean gum (Maiti et al., 2010), etc., have already been investigated as potential drug delivery carriers, and researchers have found some considerable interest in applications and quality. This chapter deals with an inclusive discussion of a number of gum-based hydrogel systems for the application in releasing of different drugs. Besides this, sources and classifications of gum have been explained, in detail.

2. Gums and their classifications Natural gums are the polysaccharides that are made up of multiple units of sugar that linked each other to form large molecules (Bashir et al., 2016; Nayak et al., 2015; Nayak et al., 2018a,b,c; Prajapati et al., 2013a,b). These are the pathological yields made up of calcium, magnesium, potassium, etc., salts of complex materials acknowledged as “polyuronides” (Prajapati et al., 2013a,b). These are available in great amount in a number of plants, animals, algal and microbes, where these gums execute a number of structural as well as metabolic functions (Nayak and Hasnain, 2019a,b). These are heterogeneous natured in their compositions. These produce simple sugar residues such as arabinose, galactose, glucose, mannose, xylose, and uronic acids when hydrolyzed. Naturally derived gums are extensively employed in the pharmaceuticals nowadays for varied characteristics and uses in the development of liquid oral and topical products (Bashir et al., 2016; Prajapati et al., 2013a,b). These are nontoxic, economical, and readily available, and thereby favored over comparable synthetic materials (Nayak and Pal, 2018). Majority of them are adequately safe for oral use, for example, in food additives or carriers of drug (Prajapati et al., 2013a,b). The intestinal microflora metabolized them and finally degraded to their respective sugars units. The classifications of different available gums are listed in Table 25.1.

3. Chitosan-based hydrogels used in drug delivery Chitosan is obtained from the chitin by deacetylation (Nayak and Pal, 2015). Chitin is the major structural constituent of in vertebrates’ exoskeletons, for example, crustaceans, insects’ cuticles and most fungi and algae cell walls (Hasnain and Nayak, 2018). It is a biocompatible cationic polysaccharide extensively employed as biopolymeric excipients (Jana et al., 2013, 2014; 2015b; Ray et al., 2018). Chitosan molecules comprise amino groups (free), and it is insoluble in the aqueous medium. The occurrences of amino groups in the chitosan molecules experience the protonation in acidic pH and, therefore, make them water soluble (Verma et al., 2017). Its aqueous solubility is reported to be dependent upon the presence of free amino groups as well as N-acetyl groups in the chitosan molecular structure (Sonia and Sharma, 2011). It is fervently available for cross-linking due to a number of free amino groups in their molecular structure (Hasnain and Nayak, 2018b; Ray et al., 2018). It produces hydrogels in attendance of multivalent anions, for example, tripolyphosphate by ionic interaction among positive charged eNH2 groups occurred in chitosan and negative charged tripolyphosphate counter ions (Shu and Zhu, 2000). These ionically gelled beads of chitosan have already been employed as drug delivery carriers. During past few decades, numerous chitosan-based

3. Chitosan-based hydrogels used in drug delivery

607

Table 25.1 Classifications of gums. Basis

Classes

Examples

Sources

Plant gums

Tamarind gum Guar gum Locust bean gum Sterculia gum Okra gum Konjac glucomannan Cashew exudate gum Moringa exudate gum Gum kondagogu Dillenia fruit gum Abelmoschus gum Albizia gum Terminalia gum Gum odina Gum cordia Chitin and chitosan Chondroitin sulfate Xanthan gum Gellan gum Pullulan Agareagar Alginate Carrageenan Tamarind gum Guar gum Xanthan gum Locust bean gum Gum Arabic Sterculia gum Sodium alginate Low methoxy pectin Gellan gum, Carrageenans Chitin Chitosan Xanthan gum Guar gum Gum Arabic Gum tragacanth

Animal gums Microbial gums

Algal gums

Charges

Nonionic gums

Anionic gums

Cationic gums Shape

Short branched gums Branch on branch gums

hydrogels for the effective delivery of drugs have been developed and evaluated by several researchers and scientists (Aycan and Alemdar, 2018; Pella´ et al., 2018). In an investigation, Sun et al. (2011) developed a kind of chitosan-tripolyphosphate hydrogel beads for the pH-sensitive controlled release of an antidiabetic agent (glipizide). These hydrogel beads were synthesized by ionically gelation methodology utilizing an ionically cross-linking agent,

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Chapter 25 Gum-based hydrogels in drug delivery

tripolyphosphate. In this research, different issues (such as, concentrations of chitosan and tripolyphosphate, drug to polymer ratio, chitosan to tripolyphosphate ratio, and cross-linking time) influencing the in vitro swelling performance of these glipizide-encapsulated chitosan-based hydrogel beads were estimated and analyzed. The swelling behavior of these chitosan-based hydrogel beads was found increased as the pH value of swelling media was increased. In vitro swelling pattern of glipizideencapsulated chitosan-based hydrogel beads at the acidic pH (1.5) milieu was found comparatively higher than that of at alkaline pH (6.8) milieu. Additionally, in vitro delivery profile of encapsulated glipizide of ionically gelled chitosan-based hydrogel beads were investigated. About 90% of the encapsulated glipizide released, in vitro, from the ionically gelled chitosan-based hydrogel beads at pH 1.5 was estimated, while this was found comparatively lower (about 36%) at pH 6.8. The overall outcome of this study clearly demonstrated that the glipizide-encapsulated chitosan-tripolyphosphate hydrogel beads can be employed as a pH-responsive controlled releasing carrier to enhance the bioavailability and patient compliances by reducing drawbacks of multiple dosing. Jana et al. (2013b) prepared and assessed novel chitosan-tamarind polysaccharide-made interpenetrating polymeric network (IPN) microparticles for sustained releasing of aceclofenac. The aceclofenac entrapment efficiency of these microparticles was observed within 85.84
1.75 to 91.97
1.30%; whereas, the average particle size was reported in the range of 490.55
23.24 to 621.60
53.57 mm. Scanning electron microscopy (SEM) images indicated nearly spherical particles devoid of agglomeration in the micrometer size with rough surface morphology having some wrinkles (Fig. 25.1). Fourier-transform infrared (FTIR) analyses suggested the development of IPN structure among chitosan and tamarind polysaccharide, suggesting the aceclofenac stability in the IPN hydrogel microparticles. Differential scanning calorimetry (DSC) analyses showed the absence of any interaction (chemical and/or physical) among the encapsulated aceclofenac and the IPN hydrogel matrix

(A)

(B)

FIGURE 25.1 SEM photographs of chitosan-tamarind polysaccharide IPN microparticles containing aceclofenac. Jana, S., Saha, A., Nayak, AK., Sen, KK., Basu, SK., 2013b. Aceclofenac-loaded chitosan-tamarind seed polysaccharide interpenetrating polymeric network microparticles. Colloids Surf B: Biointerf. 105, 303e309; Copyright @ 2013, with permission from Elsevier (B)V.

3. Chitosan-based hydrogels used in drug delivery

609

FIGURE 25.2 In vitro drug releasing from various chitosan-tamarind polysaccharide IPN microparticles containing aceclofenac. Jana, S., Saha, A., Nayak, A.K., Sen, K.K., Basu, S.K., 2013b. Aceclofenac-loaded chitosan-tamarind seed polysaccharide interpenetrating polymeric network microparticles. Colloids and Surfaces B: Biointerfaces 105, 303e309; Copyright @ 2013, with permission from Elsevier B.V.

made of chitosan and tamarind polysaccharide. Outcome of aceclofenac-releasing analysis illustrated sustained releasing of aceclofenac over 8 h (Fig. 25.2), which also found to be obeyed the KorsmeyerePeppas kinetic modeling with anomalous (non-Fickian) diffusional mechanism of in vitro aceclofenac releasing. In vivo antiinflammatory studies illustrated the sustained antiinflammatory action in the carrageenan-induced rats after oral intake of aceclofenac-encapsulated novel chitosanbased IPN microparticles. Angadi et al. (2010) formulated glutaraldehyde induced cross-linked IPN hydrogel microspheres made of hydroxyethyl cellulose and chitosan for controlled releasing of isoniazid (an antitubercular agent) via the emulsion cross-link methodology. The utmost drug (here isoniazid) entrapment efficiency was reported 75% and this was significantly found reliant upon the characteristics of IPN matrix structure. SEM images of these isoniazide-loaded chitosan-hydroxyethyl cellulose IPN hydrogel microspheres demonstrated the spherical shaped microspheres with even surface morphology (Fig. 25.3). FTIR spectroscopic analyses results indicated the chemical cross-linking in-between chitosan and hydroxyethyl cellulose to produce the IPN structure by the action of glutaraldehyde (covalent cross-linker). However, the results of X-ray diffraction (XRD) illustrated the homogeneous occurrence of isoniazid (drug) and nonappearance of crystals of isoniazid in these chitosanhydroxyethyl cellulose IPN hydrogel microspheres. DSC analysis and thermogravimetric analysis suggested good quality thermal stabilities of encapsulated isoniazid within the isoniazid-loaded IPN hydrogel microspheres. These prepared microspheres demonstrated sustained in vitro releasing of isoniazid over 16 h showing the non-Fickian mechanism and the isoniazid-releasing patterns were

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Chapter 25 Gum-based hydrogels in drug delivery

FIGURE 25.3 SEM photographs of chitosan-hydroxyethyl cellulose IPN hydrogel microspheres containing isoniazid. Angadi, S.C., Manjeswar, L.S., Aminabhavi, T.M., 2010. Interpenetrating polymer network blend microspheres of chitosan and hydroxyethyl cellulose for controlled release of isoniazid. International Journal of Biological Macromolecules 47, 171e179; Copyright @ 2010, with permission from Elsevier B.V.

affected by the cross-linking extents by glutaraldehyde, compositions of IPN blending and isoniazid loading. Some other chitosan-based hydrogels used in drug delivery are listed in Table 25.2.

4. Alginate-based hydrogels used in drug delivery Alginates are a group of naturally derived marine polysaccharidic gums containing various salts of alginic acid (Repka and Singh, 2009). These alginates are produced from different brown marine algae, and recently, these are also being extracted from the bacterial resources (bacterial alginate) (Pal and Nayak, 2015a). In the natural marine resources, alginates exist as the mixture of alginic acid salts of Mg2þ, Sr2þ, Ba2þ, Naþ, etc. (Goh et al., 2012). These possess excellent biodegradability as well as biocompatibility with the anionic nature (Nayak and Pal, 2016b). Among various salt forms of alginic acid, the sodium salt-sodium alginate has been extensively exploited in drug delivery. It is well known as the copolymer of a-L-guluronic acid (G) and b-D-mannuronic acid (M), with the 1,4-glycoside linking these (Malakar et al., 2014). Being the anionic natured biopolysaccharide, sodium alginate

4. Alginate-based hydrogels used in drug delivery

611

Table 25.2 Various chitosan-based hydrogels used in drug delivery. Chitosan-based hydrogels

Drug released

References

Hydrogels based on interpenetrating network of chitosan and polyvinyl pyrrolidone Intelligent semi-IPN chitosanePEGepolyacrylamide hydrogel Hybrid pH-sensitive hydrogels of chitosanco-acrylic acid IPN hydrogels of chitosan and poly(2hydroxyethyl methacrylate) Chitosan-based composite hydrogels

Repaglinide Insulin

Subhash and Madhabhai (2011) Bahman et al. (2016)

Verapamil

Nazar et al. (2010)

Quetiapine

Garcia et al. (2017)

Tinidazole and theophylline Thyroxin Isoniazid

Himadri and Ray (2014) Abdur et al. (2017) Angadi et al. (2013)

Indomethacin

Kumbar et al. (2003)

Diclofenac sodium

Kurkuri and Aminabhavi (2004)

Chlorpheniramine maleate Theophylline

Kumari and Kundu (2008) Rokhade et al. (2007b)

Bovine serum albumin

Abureesh et al. (2016)

Mesalamine, curcumin, and progesterone Tenofovir

Neufeld and BiancoPeled (2017) Yang et al. (2017)

Cefadroxil Amoxicillin

Reddy et al. (2006) Aycan and Alemdar (2018)

Chitosan/collagen-based smart hydrogels Coated interpenetrating blend microparticles of chitosan and guar gum Chitosan-grafted-polyacrylamide hydrogel microspheres Poly(vinyl alcohol) and poly(acrylic acid) sequential interpenetrating network pHsensitive microspheres Semi-IPN hydrogel composed of chitosan and glutamic acid IPN microspheres of chitosan and methylcellulose Glucose-sensitive chitosan-poly(vinyl alcohol) hydrogel Pectin-chitosan physical hydrogels Thermosensitive chitosan hydrogels containing polymeric microspheres Chitosan-guar gum semi-IPN microspheres pH-responsive chitosan-based hydrogel modified with bone ash

is cable of forming hydrogel microparticles/beads via the ionic cross-linking gelation methodology by some divalent/trivalent cations (e.g., Zn2þ, Ba2þ, Ca2þ, Fe3þ, Al3þ, etc.) because of the intermolecular interaction involving the carboxylic acid groups of alginate molecules and metal cations (Hasnain et al., 2018; Racovita et al., 2009). Actually, these metal cations are positioned into the electronegative cavities of sodium alginate molecules resembling the eggs in the “egg-box” modeling for the development of ionic cross-linking gelled alginate matrices as a result of the intermolecular interaction (Hasnain and Nayak, 2018a; Pal and Nayak, 2015a, 2017). The ionically gelled alginate-based hydrogels have already been researched and employed for the encapsulation of numerous drugs and other bioactive materials (Jana et al., 2015a; Pal and Nayak, 2017; Tønnesen and Karlsen, 2002). Unfortunately, these ionically gelled alginate-based hydrogels experience some severe weaknesses

612

Chapter 25 Gum-based hydrogels in drug delivery

such as lower drug encapsulation, premature releasing of encapsulated drugs, and lower stability in the alkaline pH (Hasnain et al., 2016). To overcome these severe weaknesses, various modifications have already been explored to fabricate efficient hydrogels for the use in drug releasing (Giri et al., 2012; Nanda et al., 2019; Pal and Nayak, 2017). In a research, Al-Kassas et al. (2007) manufacturing gliclazide-encapsulated calcium alginate beads via ionically cross-linked gelation technique for the enhancement of oral bioavailability. They assessed the influences of polymer concentrations, volume of internal phase, types of surfactants and stirring speed on the particle sizing, flow characteristics, and gliclazide entrapment of ionically gelled gliclazide-encapsulated calcium alginate beads. The morphology of gliclazide-encapsulated calcium alginate beads was visualized via the SEM analyses, and these beads were of spherically shaped (Fig. 25.4). In vitro swelling of these beads was found highly dependent on the polymer concentrations in the bead formula and the pH of swelling media. The in vitro drug-releasing result demonstrated that the releasing of encapsulated gliclazide was mainly controlled by the pH of dissolution media (Fig. 25.5). In addition, these ionically gelled gliclazide-encapsulated calcium alginate beads demonstrated a pH-dependent profile of gliclazide releasing over a longer time. In vivo effects of these gliclazide-encapsulated calcium alginate beads on the diabetic rabbits demonstrated that the hypoglycemic action induced by the gliclazide-encapsulated calcium alginate beads was found greater, significantly. This was also found more prolonged in comparison with that induced by the commercial gliclazide tablet (conventional).

FIGURE 25.4 SEM photograph of gliclazide-encapsulated calcium alginate beads. Al-Kassas, R., Al-Gohary, O.M.N., Al-Fadhel, M.M., 2007. Controlling of systemic absorption of gliclazide through incorporation into alginate beads. International Journal of Pharmaceutics 341, 230e237; Copyright @ 2007, with permission from Elsevier B.V.

5. Pectin-based hydrogels used in drug delivery

613

FIGURE 25.5 In vitro drug-releasing pattern of gliclazide-encapsulated calcium alginate beads in various pHs. Al-Kassas, R., Al-Gohary, O.M.N., Al-Fadhel, M.M., 2007. Controlling of systemic absorption of gliclazide through incorporation into alginate beads. International Journal of Pharmaceutics 341, 230e237; Copyright @ 2007, with permission from Elsevier B.V.

Nayak et al. (2012) investigated the formulation development of calcium alginateegum Arabic hydrogel beads for sustained glibenclamide release via the ionically cross-linking gelation technique. In this research, when the aqueous solutions of sodium alginate and gum Arabic were extruded drop by drop to the aqueous solution of calcium chloride, glibenclamide-encapsulated calcium alginateegum Arabic hydrogel beads were instantaneously produced owing to the electrostatically ionically crosslinking interaction among carboxylate groups of sodium alginateegum Arabic (negatively charged) and divalent calcium cations (positively charged). The effects of the sodium alginate as well as gum Arabic contents in bead formula on glibenclamide encapsulations and in vitro releasing of glibenclamide were optimized and evaluated. These optimized glibenclamide-encapsulated calcium alginateegum Arabic hydrogel beads demonstrated high glibenclamide encapsulation efficiency (i.e., 86.02
2.97%). The increasing of glibenclamide encapsulations and the decreasing of the rate of in vitro drug releasing were noticed with the increment of both the polymer contents. The average bead size range was measured as 1.15
0.11 to 1.55
0.19 mm. SEM photographs of glibenclamideencapsulated optimized hydrogel beads demonstrated a spherically shaped morphology having rough surface, onto which, some debrises were seen (Fig. 25.6). The in vitro swelling of these glibenclamide-encapsulated optimized hydrogel beads was dependent by pH of swelling media. In vitro drug releasing of these beads exhibited a sustained glibenclamide releasing over 7 h (Fig. 25.7), which obeyed first-order model with anomalous (non-Fickian) diffusion mechanism. Some other alginate-based hydrogels used in drug delivery are enlisted in Table 25.3.

5. Pectin-based hydrogels used in drug delivery Pectin is a water-soluble natural biopolysaccharide possessing good biocompatibility and biodegradability nature (Nayak et al., 2013f). It is a low-priced material and industrially, extracted from

614

Chapter 25 Gum-based hydrogels in drug delivery

(A)

(B)

FIGURE 25.6 SEM photographs of optimized glibenclamide-encapsulated calcium alginate-gum Arabic hydrogel beads. Nayak, A.K., Das, B., Maji, R., 2012. Calcium alginate/gum Arabic beads containing glibenclamide: development and in vitro characterization. International Journal of Biological Macromolecules 51, 1070e1078; Copyright @ 2012, with permission from Elsevier (B)V.

FIGURE 25.7 In vitro drug release from various glibenclamide-encapsulated calcium alginate-gum Arabic hydrogel beads. Nayak, A.K., Das, B., Maji, R., 2012. Calcium alginate/gum Arabic beads containing glibenclamide: development and in vitro characterization. International Journal of Biological Macromolecules 51, 1070e1078; Copyright @ 2012, with permission from Elsevier B.V.

5. Pectin-based hydrogels used in drug delivery

615

Table 25.3 Various alginate-based hydrogels used in drug delivery. Alginate-based hydrogels

Drug released

References

Sodium alginate-polyacrylamide graft-copolymer-based stomach targeted hydrogels pH-sensitive IPN hydrogel beads of carboxymethyl cellulose-(PAAm-graftedalginate) Novel alginate hydrogel coreeshell systems

Famotidine

Tripathi and Mishra (2012) Kulkarni and Sa (2008)

pH-sensitive sodium alginate/poly(vinyl alcohol) hydrogel beads prepared by combined Ca2þ cross-linking and freezethawing cycles pH-sensitive barium alginate/carboxymethyl guar gum hydrogel beads Barium ions cross-linked alginate and sterculia gum-based gastroretentive floating beads Esterified alginate/gellan gum microspheres Novel alginate-sterculia IPN microparticles pH-sensitive starch-g-poly(acrylic acid)/ sodium alginate hydrogels Zinc alginate-carboxymethyl cashew gum microbeads Oil entrapped alginate buoyant beads Oil entrapped alginate-magnesium stearate buoyant beads Oil-entrapped sterculia gum-alginate buoyant beads Alginate gel-coated oil-entrapped alginateetamarind gumemagnesium stearate buoyant beads Alginate-sterculia gum gel-coated oilentrapped alginate beads Alginate-PVP K 30 microbeads Alginate-methyl cellulose mucoadhesive microcapsules Soluble starch-blended Ca2þ-Zn2þ-alginate composites-based microparticles pH-sensitive tamarind seed polysaccharidealginate composite beads Tamarind seed polysaccharide-alginate mucoadhesive microspheres

Ketoprofen

Ranitidine HCl and aceclofenac Diclofenac sodium

Jana et al. (2015b) Hua et al. (2010)

Vitamin B6

Bajpai and Sharma (2006) Singh and Chauhan (2011)

Pantoprazole

Aceclofenac Repaglinide Diclofenac sodium

Jana et al. (2013) Kulkarni et al. (2014) Chang (2015)

Isoxsuprine HCl

Das et al. (2014)

Cloxacillin

Malakar et al. (2012) Malakar and Nayak (2012) Guru et al. (2013)

Ibuprofen Aceclofenac Risperidone

Bera et al. (2015a)

Risperidone

Bera et al. (2015c) Nayak et al. (2011) Pal and Nayak (2011) Nayak et al. (2018b) Nayak and Pal (2011) Pal and Nayak (2012)

Diclofenac sodium Gliclazide Aceclofenac Diclofenac sodium Gliclazide

Continued

616

Chapter 25 Gum-based hydrogels in drug delivery

Table 25.3 Various alginate-based hydrogels used in drug delivery.dcont’d Alginate-based hydrogels

Drug released

References

Mucoadhesive alginate-ispaghula husk mucilage beads Ispaghula husk mucilage-alginate mucoadhesive beads Jackfruit seed starch-alginate beads

Gliclazide

Nayak et al. (2010) Nayak et al. (2013c) Nayak et al. (2013d) Nayak and Pal (2013a) Malakar et al. (2013a) Malakar et al. (2013b) Hasnain et al. (2018) Nayak and Pal (2013b); Nayak et al. (2016a,b) Nayak et al. (2013e) Sinha et al. (2015a) Sinha et al. (2015b) Nayak et al. (2013f)

Glibenclamide Pioglitazone

Jackfruit seed starch-alginate mucoadhesive beads Modified starch (cationized)-alginate beads

Aceclofenac

Potato starch-blended alginate beads

Tolbutamide

Alginate-linseed polysaccharide beads

Diclofenac sodium

Tamarind seed polysaccharide-alginate mucoadhesive beads

Metformin HCl

Fenugreek seed mucilage-alginate mucoadhesive beads Zinc alginate-okra gum blend beads

Metformin HCl

Calcium alginate-okra gum mucoadhesive beads Oil entrapped tamarind seed polysaccharidealginate emulsion-gelled floating beads

Metformin HCl

Diclofenac sodium Glibenclamide Diclofenac sodium

citrous peels, apple pomaces, etc. (Sharma and Ahuja, 2011). As a biopolysaccharide, pectin molecules comprise linearly attached (1e4)-linked N-D-galacturonic acid residues, which are interrupted by means of rhamono-galacturonic acid residue and N-L-rhamnopyranose via N-1e2 linkage (Nayak et al., 2013f; Sriamornsak et al., 2010). The galacturonic acid residue of pectin molecular structure is found esterified, in part. Based on the methoxylation degree, pectins are classified as low methoxyl and high methoxyl pectins having 25%e50% and 50%e80% methoxylation degrees, respectively (Sharma and Ahuja, 2011). In general, low methoxy pectins undergo the ionically cross-linked gelation by the divalent cations (e.g., Ca2þ, Zn2þ, etc.) and these ionically cross-linked pectinate hydrogels have already been exploited as sustained drug-releasing carriers (El-Gibaly, 2002; Sriamornsak et al., 2010). The ionically cross-linked gelation of carboxyl groups of low methoxy pectin molecular structure with metal cations (such as Ca2þ, Zn2þ, etc.) persuade the structure of “egg-box” modeling. However, this kind of ionically cross-linking of low methoxy pectin differs to some extent from the “egg-box” modeling explained for ionically cross-linking of alginates (Munarin et al., 2012). During past few years, many ionically cross-linked pectinate hydrogels have already been researched for the use as drug-releasing matrices (Das et al., 2010; El-Gibaly, 2002; Sriamornsak et al., 2010).

5. Pectin-based hydrogels used in drug delivery

617

In a research, El-Gibaly, (2002) designed zinc pectinate hydrogel microparticles for orally administrable colonic delivery of ketoprofen. These ketoprofen-encapsulated zinc pectinate hydrogel microparticles were processed together with the polymeric mixtures of pectin-dextran to prepare tablets. These tablets made of ketoprofen-encapsulated zinc pectinate hydrogel microparticles have been studied, with the conditions selected to influence pH and time during the transit to the colonic area. With the purpose of searching suitable ketoprofen-encapsulated zinc pectinate hydrogel microparticles, the formulation optimization was done via the 23 factorial design model. The influences of different variables on in vitro drug releasing and surface properties of the zinc pectinate hydrogel were analyzed. The in vitro drug-releasing results demonstrated that the releasing of encapsulated ketoprofen from these zinc pectinate hydrogel microparticles was found to be significantly prolonged. The maximum prolonged ketoprofen releasing was noticed by zinc pectinate hydrogel microparticles formulated using 2.5% w/v of ketoprofen, 2.5%e3% w/v of pectin and 2.75% w/v of zinc acetate. In addition, it was found that the in vitro ketoprofen releasing in the simulated intestinal fluid (pH 7.4) was highly influenced by concentrations of ionic cross-linker and initial ketoprofen quantity, but not predominantly influenced by the pectin amounts used during the microparticle production. The ketoprofen quantity as a formulation variable increased the ketoprofen entrapment efficiency of these zinc pectinate hydrogel microparticles, significantly. The optimal colonic delivery of ketoprofen by these formulated tablets facilitated the desired delayed ketoprofen releasing of sigmoidal profiles having a lag period of 4.125e4.85 h and the time for 50% of ketoprofen released within a period of 7.45e8.70 h (Fig. 25.8), which were found dependent on the ratio of pectin/dextran utilized. The results of this research also revealed that the untableted ketoprofen-encapsulated zinc pectinate hydrogel microparticles displayed the delayed releasing of encapsulated ketoprofen (5.28e37.82 times) in the simulated intestinal fluid (pH 7.4). Hence, ketoprofen-encapsulated zinc pectinate hydrogel microparticles and the modified-releasing tablets made of these microparticles can be employed in the colon-targeted delivery. In another investigation, the ionically gelled zinc pectinate beads demonstrated a retarded drug-releasing profile as compared to that of the conventional ionically gelled calcium pectinate beads (Assifoui et al., 2011). In addition, Hagesaether et al. (2008) revealed the outstanding ex vivo biomucoadhesivity of the zinc pectinate hydrogel beads. Nayak et al., (2013f) developed a new kind of mucoadhesive hydrogel beads made of calcium pectinate-fenugreek seed mucilage. These mucoadhesive hydrogel beads were loaded with metformin HCl by the ionically cross-linked gelation. The influences of fenugreek seed mucilage and low methoxy pectin quantities on metformin HCl encapsulations and in vitro drug releasing were optimized via the 32 factorial design. The encapsulation efficiencies of drug (metformin HCl) within these pectinate-based hydrogel beads within 63.16
2.88% to 96.03
4.67% w/w; whereas, bead sizes were measured within 1.47
0.14 to 2.08
0.18 mm. The morphological observation of these polymeric hydrogel beads of metformin HCl was done via SEM analysis, showing discrete and spherical morphology with a rough surface containing typical wrinkles and cracks (Fig. 25.9). The in vitro metformin HCl releasing from these calcium pectinate-fenugreek seed mucilage hydrogel beads demonstrated sustained-releasing phenomena over 10 h (Fig. 25.10) and obeyed a controlled-releasing (zero order) kinetic profile along with the releasing mechanism of super case-II transport. In vitro swelling as well as ex vivo biomucoadhesivity of these pectinate-based hydrogel beads of metformin HCl were found to be influenced by pH of swelling medium employed in testing. The optimized pectinate-based mucoadhesive hydrogel beads of metformin HCl also demonstrated the prolonged significant antidiabetic action in the alloxaninduced diabetic rats after the oral intake (Fig. 25.11).

618

Chapter 25 Gum-based hydrogels in drug delivery

FIGURE 25.8 The in vitro release of ketoprofen from matrix tablets containing ketoprofen-encapsulated zinc pectinate hydrogel microparticles under conditions simulating gastrointestinal transit times and pH. Dissolution media: simulated gastric fluid, pH 1.2 (2 h); simulated intestinal fluid, pH 7.4 (3 h) and simulated intestinal fluid, pH 6.5 (7 h). El-Gibaly, I., 2002. Oral delayed-release system based on Zn pectinate gel (ZPG) microparticles as an alternative carrier to calcium pectinate beads for colonic drug delivery. International Journal of Pharmaceutics 232, 199e211; Copyright @ 2002, with permission from Elsevier Science B.V.

Sutar et al. (2008) developed pH-responsive hydrogel matrices made of polyacrylamide-grafted pectin for controlled releasing of salicylic acid. In this work, the grafting of polyacrylamide was carried out onto the pectin. The grafted pectin was chemically cross-linked with various amounts of glutaraldehyde and the hydrogel membranes were prepared via the conventional solution casting methodology. Within these pectin-based hydrogels, salicylic acid was incorporated as a model drug. The polyacrylamide-grafted pectin-based hydrogel matrices demonstrated a better film producing characteristics and gelling capability than pure pectin-based matrices. The relative rheological characteristics of grafted pectin and pure pectin demonstrated the transformation in the character of the product tested. polyacrylamide-grafted pectin-based hydrogel was characterized by FTIR, DSC, and XRD analyses. FTIR results demonstrated the incorporation of amide group within the synthesized hydrogel. Both the DSC and XRD results indicated the development of a new polymeric-structure in the form of grafted pectin-based cross-linked hydrogel. In vitro swelling evaluation suggested the pHdependent swelling pattern of the grafted pectin-based cross-linked hydrogel. In vitro drug releasing from the polyacrylamide-grafted pectin-based hydrogel matrices was carried out in a modified Franz’s diffusion cell. In vitro releasing of salicylic acid from hydrogel demonstrated a pH-dependent

5. Pectin-based hydrogels used in drug delivery

619

FIGURE 25.9 SEM photograph of optimized calcium pectinate-fenugreek seed mucilage mucoadhesive hydrogel beads of metformin HCl. Nayak, A.K., Pal, D., Malakar, J., 2013f. Development, optimization and evaluation of emulsion-gelled floating beads using natural polysaccharide-blend for controlled drug release. Polymer Engineering & Science 53, 338e350; Copyright @ 2012, with permission from Elsevier Ltd.

FIGURE 25.10 In vitro drug release from various calcium pectinate-fenugreek seed mucilage mucoadhesive hydrogel beads of metformin HCl. Nayak, A.K., Pal, D., Malakar, J., 2013f. Development, optimization and evaluation of emulsion-gelled floating beads using natural polysaccharide-blend for controlled drug release. Polymer Engineering & Science 53, 338e350; Copyright @ 2012, with permission from Elsevier Ltd.

620

Chapter 25 Gum-based hydrogels in drug delivery

FIGURE 25.11 (A) Comparative in vivo blood glucose level in alloxan-induced diabetic rats after oral administration of pure metformin HCl and optimized calcium pectinate-fenugreek seed mucilage mucoadhesive hydrogel beads of metformin HCl. The data were analyzed for significant differences (P < .05) by paired samples t-test; (B) comparative in vivo mean percentage reduction in blood glucose level in alloxan-induced diabetic rats after oral administration of pure metformin HCl and optimized calcium pectinate-fenugreek seed mucilage mucoadhesive hydrogel beads of metformin HCl. Nayak, A.K., Pal, D., Malakar, J., 2013f. Development, optimization and evaluation of emulsion-gelled floating beads using natural polysaccharide-blend for controlled drug release. Polymer Engineering & Science 53, 338e350; Copyright @ 2012, with permission from Elsevier Ltd.

releasing pattern. These pH-responsive polyacrylamide-grafted pectin-based hydrogel was found hematocompatible and biocompatible, when tested. Some other pectin-based hydrogels used in drug delivery are listed in Table 25.4.

6. Gellan gum-based hydrogels used in drug delivery Gellan gum is a naturally derived heteropolysaccharide of the microbial origin possessing an anionic nature (Maiti et al., 2011). It is extracted from Pseudomonas eloda (Nayak and Pal, 2014). It is aqueous soluble and has two different chemical varieties: (i) the native or natural gellan gum possessing high acyl contents, and (ii) the low or deacetylated gellan gum. Both the gellan gum varieties have a linear molecular structure comprising the repeating tetrasaccharide units with b-D-glucose, b-D-glucuronic acid, and a-L rhamnose sugar residues in the molar ratio of 2:1:1 (Nayak and Pal, 2014). The gelation of gellan gum entails the arrangement of double helical junctional zones and afterward, aggregations of double helical segments to shape a three-dimensional networking via complexation by the influence of divalent or trivalent cations (such as Zn2þ, Ca2þ, Al3þ, Fe3þ, etc.) and the formation of hydrogen bonds with water molecules (Babu et al., 2010; Grasdalen and Smidsroed, 1987; Maiti et al., 2011). In the previous research reports by numerous research groups, several gellan gum-based hydrogels for the use in drug releasing have already been researched and reported (Maiti et al., 2011). In a research by Kedzierewicz et al. (1999), the characteristics of gellan gum-based hydrogels were found

6. Gellan gum-based hydrogels used in drug delivery

621

Table 25.4 Various pectin-based hydrogels used in drug delivery. Pectin-based hydrogels

Drug released

References

Pectin-poly(sodium acrylate-co-acrylamide) hydrogel Pectinate-poly(vinyl pyrrolidone) beads Mucoadhesive-floating zinc-pectinate-sterculia gum IPN beads Calcium pectinate gel beads

Ibuprofen Aceclofenac Ziprasidone HCl

Sadeghi (2011) Nayak et al. (2013a) Bera et al. (2015b)

Metronidazole

Zinc-pectinate beads Calcium pectinate-fenugreek seed mucilage mucoadhesive beads Jackfruit seed starch- pectinate mucoadhesive beads Calcium pectinate-tamarind seed polysaccharide mucoadhesive beads Calcium pectinate-fenugreek seed mucilage mucoadhesive beads Alginate-pectinate bioadhesive microspheres

Resveratrol Metformin HCl

Sriamornsak et al. (2010) Das et al. (2010) Nayak et al. (2013b)

Metformin HCl Metformin HCl

Nayak and Pal (2013c) Nayak et al. (2014d)

Metformin HCl

Nayak et al. (2013b)

Aceclofenac Diltiazem HCl

Chakraborty et al. (2010) Giri et al. (2013a)

Diclofenac sodium

Giri et al. (2013b)

Indomethacin

Jung et al. (2013)

Cross-linked biodegradable IPN hydrogel beads of pectin and modified xanthan gum Biodegradable IPN hydrogel beads of pectin and grafted alginate Pectin and charge modified pectin hydrogel beads as a colon-targeted drug delivery carrier

to be influenced by the formulation variables such as concentrations of gellan gum, concentrations of the drug, concentrations of cross-linker, pH of cross-linking solutions, cross-linking time, stirring time, etc. (Kedzierewicz et al., 1999). Agnihotri et al. (2006) investigated the formulation of cephalexinencapsulated gellan gum hydrogel beads via the ionically cross-linked gelation by the cross-linker solutions contained mixtures of zinc and calcium cations. The encapsulation of cephalexin within these gellan gum beads achieved was 69.24%. The cephalexin-encapsulated gellan gum hydrogel beads were of spherical morphology, which was visualized in SEM (Fig. 25.12). In vitro drug-releasing results clearly demonstrated that the drug release was dependent by pH of cross-linker containing solutions and amount of cephalexin loading. Maiti et al. (2011) developed a novel kind of glipizide-loaded acetylated gellan gum hydrogel networking beads via the ionically cross-linking gelation via trivalent aluminum cations and covalent cross-linking (using glutaraldehyde). Following treatment by glutaraldehyde, spherically shaped trivalent aluminum cation-induced gellan gum beads were shrinked with showing characteristic wrinkles on the bead surface. Highest glipizide entrapment efficiency of 97.67% was attained; whereas, treatment by glutaraldehyde reduced the glipizide entrapment efficiency of these gellan gum hydrogel beads by 11.89%. All the gellan gum hydrogel beads released, in vitro, less than 10% of glipizide in the acidic pH for the initial 2 h; however, it was measured 38%e47% of glipizide releasing by trivalent aluminum cation-induced gellan gum beads and only 15% of glipizide releasing by

622

Chapter 25 Gum-based hydrogels in drug delivery

(A)

(B)

FIGURE 25.12 SEM photographs of cephalexin-encapsulated gellan gum hydrogel beads produced in pH 9 (A) and pH 5 (B) media. Agnihotri, S.A., Jawalkar, S.S., Aminabhavi, T.M., 2006. Controlled release of cephalexin through gellan gum beads: effect of formulation parameters on entrapment efficiency, size, and drug release. European Journal of Pharmaceutics and Biopharmaceutics 63, 249e261; Copyright @ 2006, with permission from Elsevier Ltd.

7. Tamarind gum-based hydrogels used in drug delivery

623

FIGURE 25.13 In vitro drug release profiles of glipizide-loaded gellan gum beads in pH 1.2 KCl/HCl buffer solutions for 2 h and subsequently in pH 7.4 phosphate buffer solution for 6 h. Maiti, S., Ranjit, S., Mondol, R., Ray, S., Sa, B., 2011. Alþ3 ion cross-linked and acetylated gellan hydrogel network beads for prolonged release of glipizide. Carbohydrate Polymers 85, 164e172; Copyright @ 2011, with permission from Elsevier Ltd.

glutaraldehyde-treated hydrogel beads in the alkaline pH (Fig. 25.13). In vitro glipizide releasing from these hydrogel networking beads was not in correlation with the in vitro swelling results. For the glutaraldehyde-treated gellan gum hydrogel beads, the anomalous (non-Fickian) drug transport mechanism was found to be changed to the super case II transport mechanism, where the trend of polymer relaxation was found prevailing. FTIR, DSC, and XRD analyses demonstrated that the encapsulated glipizide present within these gellan gum hydrogel beads was relatively stable and amorphous. Therefore, the glutaraldehyde-treated gellan gum hydrogel beads and the untreated aluminum cation-induced gellan gum hydrogel beads can be used as controlled drug delivery carriers. Some other gellan gum-based hydrogels used in drug delivery are listed in Table 25.5.

7. Tamarind gum-based hydrogels used in drug delivery Tamarind gum is obtained from the seed endosperm of the plant, Tamarindus indica L. (family: Fabaceae) (Nayak and Hasnain, 2019c). It is a biopolysaccharide of neutral character having no-charge with branched structure (Nayak, 2016). Tamarind gum is made up of (1 / 4)-b-D-glucan skeleton, which is substituted with a-D-xylopyranose and b-D-galactopyranosyl (1 / 2)-a-D-xylopyranose side chains connected to glucose-moieties at (1 / 6). Glucose, xylose, and galactose units are approximately 55.4%, 28.4%, and 16.2%, equivalent to a molar ratio of 2.8:2.25:1.0 for glucose, xylose, and galactose, correspondingly (Nayak et al., 2011). As a result, tamarind gum is considered as galactoxyloglucan (Nayak, 2016). It is water soluble, hydrophilic in nature and has the capability of gel formation as well as mucoadhesive properties (Nayak and Pal, 2017). It is also well established that tamarind gum is nontoxic, biodegradable, biocompatible, noncarcinogenic, and nonirritant (Nayak and

624

Chapter 25 Gum-based hydrogels in drug delivery

Table 25.5 Various gellan gum-based hydrogels used in drug delivery. Gellan gum-based hydrogels

Drug released

References

Gellan gum-poly(vinyl alcohol) hydrogel microspheres

Carvedilol

IPN microcapsules of gellan gum and egg albumin Acrylamide-grafted gellan gum system Methacrylamide-grafted gellan gum system Esterified alginate/gellan gum microspheres Ionotropically gelled gum cordial/gellan beads Tamarind seed polysaccharide-gellan mucoadhesive beads Ispaghula mucilage-gellan mucoadhesive beads Fenugreek seed mucilage-gellan mucoadhesive beads Jackfruit seed starch-blended gellan gum mucoadhesive beads Hydrogel polyspheres of gellan gum and carrageenan Gellan-xanthan pH-responsive hydrogels

Diltiazem Metformin HCl Diclofenac sodium Aceclofenac Metformin HCl Metformin HCl Metformin HCl Metformin HCl Metformin HCl

Agnihotri and Aminabhavi (2005) Kulkarni et al. (2011) Vijan et al. (2012) Nandi et al. (2015) Jana et al. (2013) Ahuja et al. (2010) Nayak et al. (2014a) Nayak et al. (2014b) Nayak and Pal (2014) Nayak et al. (2014c)

Cross-linked gellan gum and retrograded starch blend hydrogels pH-dependent gellan beads Gellan gum macro beads Gellan gum beads

Simvastatin Bovine serum albumin and diclofenac sodium Ketoprofen Naproxen Amoxicillin Meloxicam

Kulkarni et al. (2013) Ramburrun et al. (2017) Oliveira Cardoso et al. (2017) Osmalek et al. (2018) Babu et al. (2010) Osmalek et al. (2017)

Hasnain, 2019c). In pharmaceutical, cosmetic, and food industries, tamarind gum is exploited as the promising biopolymer (Nayak, 2016). It is extensively investigated and utilized in a number of drug delivery systems (such as oral, ocular, nasal, buccal, and colon drug delivery) as the valuable pharmaceutical excipients in current years (Nayak, 2016; Nayak and Hasnain, 2019c; Nayak and Pal, 2017). Recent years, numerous drug delivery hydrogels made of tamarind gum have already been developed by various drug delivery research endeavors. Ghosh and Pal (2013) developed pH-responsive hydrogels made of grafted tamarind gum as the matrix former for the use in controlled drug delivery application. Synthesis of these grafted tamarind gum was done by grafting of polyacrylamide chains onto tamarind gum molecular skeleton by microwave-irradiation involving the use of ceric ammonium nitrate as a reaction initiator. This prepared tamarind gum-graft polyacrylamide was then assessed as the matrix-forming materials for controlled releasing of aspirin (as a model drug). Guar gum was utilized as the tablet binder to develop the matrix tablets of tamarind gum-graft polyacrylamide containing aspirin in a ratio of 10:1:0.3. The formulated matrix tablets containing aspirin was characterized with the help of SEM and FTIR analyses. SEM photographs demonstrated the changes in morphology, suggesting the physical (but, not chemical) interaction among aspirin and tamarind gum-graft polyacrylamide (which was used as table matrix former) (Fig. 25.14). FTIR data revealed that there was not any kind of chemical interaction(s) among matrix of tamarind gum-graft polyacrylamide and aspirin (drug), advocating the compatibility in-between drug and excipients within these formulated matrix tablets containing aspirin. In vitro

7. Tamarind gum-based hydrogels used in drug delivery

(A)

(B)

625

(C)

FIGURE 25.14 SEM photographs of (A) tamarind gum-graft polyacrylamide, (B) aspirin, and (C) matrix tablet of aspirin made of tamarind gum-graft polyacrylamide. Ghosh, S., Pal, S. 2013. Modified tamarind kernel polysaccharide: a novel matrix for control release of aspirin. International Journal of Biological Macromolecules 58, 296e300; Copyright @ 2013, with permission from Elsevier B.V.

aspirin-releasing performance of tamarind gum-graft polyacrylamide-made matrix tablets containing aspirin was assessed in the dissolution medium of various pHs (1.2, 6.8, and 7.4). It was observed that after 24 h, aspirin was totally released from these formulated aspirin matrix tablets made of tamarind gum-graft polyacrylamide. The current study also demonstrated that in vitro aspirin-releasing rate from these formulated matrix tablets was lesser in the acidic pH than that of the neutral and alkaline pHs (Fig. 25.15). Furthermore, the higher percentage of grafting reduced the aspirin-releasing rate. In addition, it was observed that as the percent grafting augmented, the swelling rate of these table

FIGURE 25.15 Cumulative in vitro aspirin-releasing profiles of matrix tablets of aspirin made of tamarind gum-graft polyacrylamide at (A) pH 1.2, (B) pH 6.8, and (C) pH 7.4. Ghosh, S, Pal, S., 2013. Modified tamarind kernel polysaccharide: a novel matrix for control release of aspirin. International Journal of Biological Macromolecules 58, 296e300; Copyright @ 2013, with permission from Elsevier B.V.

626

Chapter 25 Gum-based hydrogels in drug delivery

matrices increased, while a reduction in the rate of aspirin releasing and the erosion of tablet matrices were reported. These tamarind gum-graft-polyacrylamide matrix tablets of aspirin obeyed the zeroorder model kinetics as well as non-Fickian diffusion mechanism, advocating the controlled releasing of aspirin from matrix tables. The swelling performance evaluations of these formulated matrix tablets containing aspirin was performed for 24 h in different pHs (1.2, 6.8, and 7.4). Upon the contact with the swelling media, the dry polymeric tablet matrices made of tamarind gum-graft polyacrylamide could possibly be hydrated and swelled. After that this produced viscous gelledlike barrier layer that hindered the in vitro releasing of aspirin from these grafted matrices, in vitro. This also noticed that the in vitro swelling performances of tablets were elevated as the percentage grafting of tamarind gum-graft polyacrylamide was increased. In addition, the matrix erosion rate of tamarind gum-graft polyacrylamide-made matrix tablet matrices was noticed lesser with the rise in percent grafting. In a research by Sharma et al. (2018), novel tamarind gum-based physical hydrogels containing moxifloxacin HCl were developed and investigated. Tamarind gum-based hydrogels containing moxifloxacin HCl were prepared via the physical gelation methodology employing different concentrations of tamarind gum. These hydrogels containing moxifloxacin HCl were instrumentally characterized by microscopy, FTIR, and XRD analyses. Microscopical evaluations demonstrated the development of globular polymeric structures throughout the hydrogel matrix. The results of FTIR and XRD analyses recommended the increment of the associative interactions when tamarind gum amounts were augmented in the hydrogel formula that sequentially resulted in the amendment in the mechanical characteristics of these tamarind gum-based hydrogels. The mechanical as well as electrical characteristics of these tamarind gum-based hydrogels containing moxifloxacin HCl were evaluated. From the results of the electrical evaluations, an enhancement in the intrinsic ionic conductivity characteristics of these hydrogels was envisaged. This occurrence might be due to the fact of modulation of the drug releasing from the tamarind gum-based hydrogels, which was released (drug) from the hydrogels in its active state. The overall results of this research clearly demonstrated that these tamarind gum-based physical hydrogels can be utilized as the drug-releasing carriers. Some other tamarind gum-based hydrogels used in drug delivery are listed in Table 25.6.

8. Sterculia gum-based hydrogels used in drug delivery Sterculia gum (also known as karaya) is extracted from the exudates of Sterculia urens tree (family: sterculiaceae) (Nayak and Hasnain, 2019d). It is partially acetylated polysaccharides having high molecular weight (Cerf et al., 1990; Leung, 1980). It is water soluble and consists of three different kinds of polysaccharidic chains. Among these three chains, one of them (50% of the total polysaccharide) has four galacturonic acid moieties recurring units, which contains b-D-galactose branches with L-rhamnose moieties at the reducing end. On the other hand, the second one (17%) has an oligorhamnan unit, which contains branch residues of D-galacturonic acid with the interrupted intermittently by D-galactose moieties; whereas, the third one (33%) has D-glucuronic acid moieties that constitute from galactose, rhamnose, and uronic acid in a quantity of 13%e26%, 15%e30% and 40%, respectively (Nayak and Hasnain, 2019d). Sterculia gum displays a number of exceptional characteristics such as biocompatibility, biodegradability, nonallergic, nonmutagenic, nonteratogenic, acidic stability, excellent viscosity, elevated swelling ability, and water retention capability (Nayak and

8. Sterculia gum-based hydrogels used in drug delivery

627

Table 25.6 Various tamarind gum-based hydrogels used in drug delivery. Tamarind gum-based hydrogels

Drug released

References

IPN hydrogels of O-carboxymethyl Tamarind gum and alginate Chitosan-tamarind seed polysaccharide IPN microparticles Alginate gel-coated oil-entrapped alginateetamarind gumemagnesium stearate buoyant beads Oil entrapped tamarind seed polysaccharide-alginate emulsiongelled floating beads pH-sensitive tamarind seed polysaccharide-alginate composite beads Tamarind seed polysaccharide-gellan mucoadhesive beads Tamarind seed polysaccharide-alginate mucoadhesive microspheres Tamarind seed polysaccharide-alginate mucoadhesive beads

Acyclovir Aceclofenac Risperidone

Jana et al. (2016) Jana et al. (2013b) Bera et al. (2015a)

Diclofenac sodium Diclofenac sodium Metformin HCl Gliclazide

Nayak et al. (2013f)

Calcium pectinate-tamarind seed polysaccharide mucoadhesive beads Tablets containing hydrogel polysaccharides of tamarind gum and xanthan gum Hydrogel films of carboxymethyl tamarind gum using citric acid

Metformin HCl

Nayak and Pal (2011) Nayak et al. (2014a) Pal and Nayak (2012)

Metformin HCl

Nayak and Pal (2013b); Nayak et al. (2016a,b) Nayak et al. (2014d)

Famotidine

Razavi et al. (2014)

Moxifloxacin HCl

Mali et al. (2017)

Hasnain, 2019d; Nayak and Pal, 2016a). It is also reported to have antimicrobial activity (Nayak and Hasnain, 2019d). In pharmaceutical and food industries, sterculia gum is employed as emulsifier, stabilizer as well as the thickener (Singh and Sharma, 2011; Kulkarni et al., 2014). Sterculia gum is acknowledged as one of the promising biodegradable excipients of polysaccharidic origin in numerous dosage forms (Nayak and Hasnain, 2019d). Due to the favorable hydration as well as swelling rates, a number of efforts have already been made for development of various sterculia gum-based hydrogels (Singh and Sharma, 2011). The majority of the studies by now showed that these hydrogels were found efficient for sustained release of a number of drugs. For the application of oral administration, a number of sterculia gum-alginate hydrogel beads have already been researched and developed (Guru et al., 2013; Kulkarni et al., 2014). Among these, majority of these hydrogel beads made of sterculia gum and alginate were formulated by employing ionic cross-linkers such as Ca2þ and Ba2þ. Kulkarni et al. (2014), in one of their study, developed IPN microparticles of sterculia gum-alginate loaded with repaglinide (an antidiabetic drug) by means of ionically cross-linked gelation and emulsion cross-linking methodology. Aqueous solutions of barium chloride, calcium chloride, and aluminum chloride were employed as the ionic cross-linker for the formulation of these repaglinide-encapsulated IPN microparticles. The repaglinide encapsulation efficiency of these IPN microparticles was observed as 81.10%e91.70%, and it was also observed that as the concentrations of sodium alginate was reduced, the repaglinide encapsulation efficiency was revealed to be reduced. The order of repaglinide encapsulation efficiencies of these IPN microparticles formulated with Al3þ, Ba2þ, and Ca2þ ion-induced cross-linker was observed as

628

Chapter 25 Gum-based hydrogels in drug delivery

FIGURE 25.16 In vitro release profiles of repaglinide from various repaglinide-encapsulated sterculia gum-alginate IPN microparticles. Kulkarni, R.V., Patel, F.S., Nanjappaiah, H.M., Naikawadi, A.A., 2014. In vitro and in vivo evaluation of novel interpenetrating polymer network microparticles containing repaglinide. International Journal of Biological Macromolecules 69, 514e522; Copyright @ 2014, with permission from Elsevier B.V.

Al3þ > Ba2þ > Ca2þ. The mean sizing of these IPN microparticles was reported as 19.75e61.52 mm. The results of in vitro repaglinide releasing showed sustained delivery of encapsulated repaglinide over 24 h (Fig. 25.16). The repaglinide releasing was reported slower as the sodium alginate concentrations augmented in these formulated sterculia gum-alginate IPN microparticles. The encapsulated repaglinide releasing from IPN microparticles prepared via ionically cross-linker demonstrated repaglinide releasing swiftly; whereas, the IPN microparticles prepared via dually cross-linked demonstrated an extended in vitro releasing of repaglinide over prolonged period. These repaglinide-encapsulated IPN microparticles were reported to pursue the mechanism of non-Fickian transport. In the diabetic rats (streptozotocin induced), in vivo hypoglycemic action of these repaglinide-encapsulated sterculia gum-based IPN microparticles was reported. The outcomes of the in vivo study demonstrated that an unexpected fall in blood glucose level was produced by the oral intake of pristine repaglinide in the diabetic rats up to 3 h after oral intake and subsequently, this was found to be recovered. It was also noticed that the decrease of blood glucose level in these diabetic rats was steadier by the action of repaglinide-encapsulated sterculia gum-alginate IPN microparticles in comparison to pristine repaglinide within 3 h (Fig. 25.17). However, it was steadily augmented to 81.27% up to 24 h. Singh and Sharma (2008a) developed a novel type of hydrogels, where sterculia gum was crosslinked acrylamide, for the delivery of ranitidine HCl. These sterculia gum cross-linked polyacrylamide hydrogels were prepared by employing N,N0 -methylene bisacrylamide and ammonium persulfate as the cross-linker and reaction initiator, respectively. Evaluation of the optimal reaction variables for the development of sterculia gum cross-linked polyacrylamide hydrogels was carried out via the initial trial studies by varying the concentrations of N,N0 -methylene bisacrylamide, acrylamide,

8. Sterculia gum-based hydrogels used in drug delivery

629

FIGURE 25.17 Percentage reduction of blood glucose in rats treated with pristine repaglinide and repaglinide-encapsulated sterculia gum-alginate IPN microparticles (KA8). Kulkarni, R.V., Patel, F.S., Nanjappaiah, H.M., Naikawadi, A.A., 2014. In vitro and in vivo evaluation of novel interpenetrating polymer network microparticles containing repaglinide. International Journal of Biological Macromolecules 69, 514e522; Copyright @ 2014, with permission from Elsevier B.V.

ammonium persulfate, and quantity of sterculia gum on the basis of synthesized sterculia gum-based hydrogels swelling in the distilled water over 24 h. The structural integrity, swelling, and shape of these hydrogels were maintained throughout this study. Based on the initial trials, the optimal reaction condition for the preparation of sterculia gum cross-linked polyacrylamide hydrogels was selected as N,N0 -methylene bisacrylamide, acrylamide, ammonium persulfate, and sterculia gum of 6.486 mM/L, 1.125 mM/L, 13.158 mM/L, and 0.8 g, respectively. SEM images demonstrated even and homogeneous morphology of sterculia gum; whereas, in the case of prepared sterculia gum cross-linked polyacrylamide hydrogels, the structural heterogeneity was confirmed (Fig. 25.18). FTIR images indicated the cross-linking among acrylamide and sterculia gum to develop the sterculia gum crosslinked polyacrylamide hydrogels. As the concentration of monomer (i.e., acrylamide) augmented in the polymeric hydrogel, swelling rates of these hydrogels was reduced; whereas, the swelling rate of these was augmented as the quantity of sterculia gum increased in the hydrogel-matrix composition. However, as the concentration of N,N0 -methylene bisacrylamide (cross-linker) augmented during the hydrogel preparation, the swelling rate of prepared hydrogels was reported to be decreased. The swelling behavior of optimized sterculia gum cross-linked polyacrylamide hydrogels in different swelling mediums of varying pH demonstrated that the increment of swelling as the swelling medium pH tested in swelling study was increased. Additionally, it was observed that the swelling of these sterculia gum cross-linked polyacrylamide hydrogels was faster in distilled water as compared to that in the solution containing 0.9% NaCl. The drug (ranitidine HCl) loading onto the optimized sterculia gum cross-linked polyacrylamide hydrogels was made via the swelling equilibrium technique. The in vitro ranitidine HCl release, these sterculia gum-based hydrogels was performed in distilled water, pH 2.2, and pH 7.4 buffers. In comparison to the in vitro drug (ranitidine HCl) released in the pH 7.4

630

Chapter 25 Gum-based hydrogels in drug delivery

FIGURE 25.18 SEM images showing the surface morphologies of sterculia gum (A) and sterculia gum cross-linked polyacrylamide hydrogels (B). Singh, B, Sharma, N., 2008a. Development of novel hydrogels by functionalization of sterculia gum for use in anti-ulcer drug delivery. Carbohydrate Polymers 74, 489e497; Copyright @ 2008, with permission from Elsevier Ltd.

buffer and distilled water, these sterculia gum-based hydrogel released drug in higher quantity in the pH 2.2 buffer. The fashion of ranitidine HCl releasing was not found analogous to the swelling model of these formulated hydrogels, where in vitro swelling behavior was found maximum in distilled water as well as pH 7.4 buffer. In the pH 2.2 buffer and distilled water, releasing of drug from the optimized sterculia gum cross-linked polyacrylamide hydrogels, in vitro, obeyed the Fickian diffusional mechanism; whereas, in the pH 7.4 buffer, the non-Fickian diffusion mechanism was followed. Hence, the newly developed sterculia gum cross-linked polyacrylamide hydrogels demonstrated aptness as the drug-releasing matrices. Some other sterculia gum-based hydrogels used in drug delivery are listed in Table 25.7.

9. Guar gum-based hydrogels used in drug delivery Guar gum is extracted from Cyamopsis tetragonoloba (family: Leguminosae) seeds (Prabaharan, 2011). It is a water-soluble polysaccharide with nonionic nature (Sarmah et al., 2011). Guar gum

9. Guar gum-based hydrogels used in drug delivery

631

Table 25.7 Various sterculia gum-based hydrogels used in drug delivery. Sterculia gum-based hydrogels

Drug released

References

Sterculia gum cross-linked polymethacrylic acid hydrogels

Ranitidine HCl

Sterculia gum cross-linked poly (N-vinylpyrrolidone) hydrogel Sterculia gum cross-linked polyvinyl alcohol hydrogel membrane Sterculia gum cross-linked polyvinyl alcohol and polyvinyl alcohol-polyacrylamide hydrogel films Radiation-induced graft-copolymerized of sterculia gum cross-linked polyvinyl alcohol hydrogel Radiation-induced cross-linked sterculia gum-polyvinyl alcohol-N vinylpyrrolidone based hydrogel Sterculia gum-alginate beads and floating sterculia gumalginate beads

Ornidazole

Singh and Sharma (2008b) Singh and Sharma (2011) Singh and Pal (2008)

Oil-entrapped buoyant beads made of sterculia gum-sodium alginate blends Mucoadhesive-floating zinc-pectinate-sterculia gum IPN beads Alginate-sterculia gum gel-coated oil-entrapped alginate beads

Tetracycline HCl Gentamicin sulfate and tetracycline HCl Tetracycline HCl

Singh and Pal (2012)

Doxycycline hyclate

Singh and Pal (2011)

Pentoprazole

Aceclofenac

Singh and Chauhan (2011); Singh et al. (2016) Guru et al. (2013)

Ziprasidone HCl

Bera et al. (2015b)

Risperidone

Bera et al. (2015c)

Singh and Pal (2008)

molecular structure is made up of linear polymeric chains possessing (1 / 4)-b-D- mannopyranosyl units, which is substituted with a-D-galactopyranosyl units linked (1 / 6) linkage (Prabaharan, 2011; Sarmah et al., 2011). Gastrointestinal tract microbials are capable to ferment the galactomannan moieties very well in such way that these produce fatty acids in huge amounts (having short chain) and cannot be digested by animals as well as humans (Reddy and Tammishetti, 2002; Sharma et al., 2013). Guar gum as a versatile polymer is very imperative in the biomedical field because of some encouraging characteristics such as nonallergic, nonmutagenic, biocompatibility, and biodegradability (Prabaharan, 2011; Sarmah et al., 2011). It possesses valuable gelling and enzymatic degradation properties at the colonic fluids and is exploited as a promising colon-targeted carrier material (Prabaharan, 2011). Guar gum swells in the presence of cold water and produces colloidal dispersions. The gelling networking of guar gum hinders the releasing of drug from the dosage forms made of guar gum. Therefore, both physical and chemical modifications of guar gum have already been researched to control the swelling properties of guar gum-based hydrogels in different buffer solutions (Aminabhavi et al., 2014; Reddy and Tammishetti, 2002). In a research by George and Abraham (2007), a pH-responsive guar gum-alginate hydrogel system was developed and evaluated for controlled releasing of bovine serum albumin (a protein). The guar gum-alginate hydrogel systems were formulated by covalent cross-linking using glutaraldehyde, where guar gum was incorporated into the matrix of sodium alginate. The drug (bovine serum albumin) loading procedure used for the guar gum-alginate hydrogel system was carried out in the aqueous

632

Chapter 25 Gum-based hydrogels in drug delivery

milieu. The bovine serum albumin-loaded guar gum-alginate hydrogel system prepared using alginate to guar gum ratio of 3:1 clearly demonstrated an excellent encapsulation of bovine serum albumin within the polymeric hydrogels. The in vitro release profile of loaded bovine serum albumin from the hydrogel system made of guar gum-alginate exhibited nominal amount of protein releasing (w 20%) in the simulated gastric fluid (pH 1.2); whereas, it was reported considerably higher (w 90%) in the simulated intestinal fluid (pH 7.4). It was also seen in release studies that the protein (bovine serum albumin) entrapment efficiency was augmented by the incorporation of guar gum and glutaraldehyde. This averted the releasing of bovine serum albumin in the acidic pH milieu (1.2) and also displayed a controlled-releasing profile in the alkaline pH milieu (7.4). SEM photographs of freeze-dried guar gum-alginate hydrogel system revealed a highly porous morphology; whereas, the air-dried guar gumalginate hydrogel system was found to be nonporous in nature. The cross-sectional view of freezedried guar gum-alginate hydrogel systems demonstrated many open channels and network-like structural morphological features. Bajpai and Sharma (2006) produced hydrogel beads prepared of carboxymethyl guar gum and sodium alginate by employing barium ions as the ionic cross-linker. Drug entrapment efficiency of carboxymethyl guar gum/alginate hydrogel beads was found approximately 50%, when these beads were prepared through ionically cross-linking using barium chloride solutions (5/6% w/v). These formulated carboxymethyl guar gum/alginate hydrogel beads were found to be swelled lesser (approximately 15%) in the gastric pH milieu (1.2) and higher (approximately 310%) in the intestinal pH milieu (7.4) after 3 h. The results of in vitro drug-releasing study suggested that the drug (vitamin B12) released from these prepared carboxymethyl guar gum/alginate hydrogel beads was measured 20% (approximately) after 3 h in the gastric pH milieu (1.2) and 70% (approximately) for the next 7 h in the intestinal pH milieu (7.4). Some other guar gum-based hydrogels used in drug delivery are listed in Table 25.8.

Table 25.8 Various guar gum-based hydrogels used in drug delivery. Guar gum-based hydrogels

Drug released

References

Cross-linked polyacrylamide grafted guar gum hydrogel microspheres Poly(vinyl alcohol)eguar gum IPN hydrogel microspheres Phosphated cross-linked guar gum microspheres Chitosan-guar gum semi-IPN microspheres Gastric resistant microbeads of metal ion cross-linked carboxymethyl guar gum Guar gum microspheres Cross-linked guar gum hydrogel discs Semi-IPN hydrogels made of carboxymethyl guar gum and gelatin Acryloyl guar gum hydrogels Semi-IPN hydrogels consisted of poly(methacrylic acid) and guar gum for colon-specific drug delivery Cross-linked guar gum hydrogel discs for colon-specific delivery pH-responsive guar gum hydrogels

Verapamil HCl and nifedipine Nifedipine 5-Fluorouracil Cefadroxil Bovine serum albumin

Soppimath and Aminabhavi (2002) Soppimath et al. (2000) Gowda et al. (2012) Reddy et al. (2006) Reddy and Tammishetti (2002) Patel and Amin (2011) Das et al. (2006) Ghosh et al. (2018)

Mebeverine HCl Ibuprofen Ciprofloxacin L-DOPA

and L-tyrosine 5-Aminosalicylic acid

Thakur et al. (2009) Li and Liu (2008)

Ibuprofen

Das et al. (2006)

Dexamethasone

Das and Subuddhi (2015)

10. Locust bean gum-based hydrogels used in drug delivery

633

10. Locust bean gum-based hydrogels used in drug delivery Locust (carob) bean gum is a plant derived branched nonionic biopolysaccharide (Nayak and Hasnain, 2019e). It is isolated from Ceratonia siliqua (carob tree) seeds (Mathur and Mathur, 2005). It is of galactomannan-type biopolysaccharide possessing galactose and mannose units in a ratio of 1:4 (Mathur and Mathur, 2005; Nayak and Hasnain, 2019e). Locust bean gum molecular structure comprises (1, 4)-linked b-D-mannopyranose skeleton with the branching points at the 6-positions and is also connected to the a-D-galactose residue (Prajapati et al., 2013a,b). It is less soluble in cold or normal water and required heat to obtain the aqueous solutions (Nayak and Hasnain, 2019e). Even in the smaller concentrations, the locust bean gum has the capability to form the incredibly viscous solutions and is relatively unaffected when salts added in this changes in pH or temperature (Malik et al., 2011). Locust bean gum is reported as biocompatible, biodegradable, nonteratogenic, and nonmutagenic polysaccharidic material, which is exploited as the natural polymeric pharmaceutical excipients in a number of dosage forms (Malik et al., 2011; Prajapati et al., 2013a,b). Prajapati et al. (2014) formulated aceclofenac-encapsulated mucoadhesive hydrogel beads composed of locust bean gum-alginate matrices with the plan to use locust bean gum as mucoadhesive polymer to develop mucoadhesive hydrogel beads for sustained aceclofenac releasing over a longer period. These locust bean gum-alginate mucoadhesive beads of aceclofenac were prepared via the ionically cross-linked gelation method by employing calcium chloride as the ionic cross-linking agent. For the optimization of locust bean gum-alginate mucoadhesive hydrogel beads, 32 factorial design model was used to check the influences of polymeric-blending amounts on the drug entrapments, mucoadhesivity, and in vitro cumulative drug release. The percentage yields of these aceclofenacencapsulated mucoadhesive beads were found within 93.19%e96.65%; whereas, aceclofenac entrapment efficiencies were within 56.37%e68.54%. Average sizes of these hydrogel beads were found within 1.32
0.11 to 1.42
0.13 mm. SEM photographs indicated that formulated beads were of spherically shaped; whereas, FTIR and DSC analyses indicated that the encapsulated drug (aceclofenac) and excipient were compatible with each other. In the in vitro swelling study at pH 1.2 and pH 6.8, it was seen that the swelling media pH influenced the in vitro swelling behavior of these beads. This did not show the polymer matrix dissolution over 10 h. These formulated locust bean gum-based beads in gastric pH (1.2) illustrated distinctly poorer swelling ratio in comparison to that in the intestinal pH (6.8). These locust bean gum-based hydrogel beads demonstrated excellent mucoadhesiveness onto the goat intestinal mucosal tissue over a longer period. In vitro aceclofenac release study was reported a sustained drug-releasing pattern over 12 h, and it was found depended on compositions of polymers used. The in vitro drug releasing obeyed the first-order kinetic modeling and super case-II transport mechanism. In another research, Maiti et al. (2010) formulated glipizide-encapsulated ionically cross-linked hydrogel beads using synthesized carboxymethyl locust bean gum by varying cross-linker (aluminum chloride) concentrations. These developed glipizide-encapsulated hydrogel beads were of spherically shaped and glipizide encapsulation efficiencies were within the range, 85.14%e97.68%. As the aluminum chloride concentration was reduced in the hydrogel preparation, the drug entrapment efficiency was found to be augmented. In acidic pH (1.2), in vitro releasing of encapsulated glipizide was observed lower from these locust bean gum-based hydrogel beads containing glipizide in comparison to that of alkaline pH milieu. The beads prepared by higher concentration of cross-linker

634

Chapter 25 Gum-based hydrogels in drug delivery

Table 25.9 Hydrogels made of miscellaneous gums for the use in drug delivery. Hydrogels made of miscellaneous gums

Drug released

References

Isoxsuprine HCl

Das et al. (2014)

Moxifloxacin Amoxicillin

Singh et al. (2016) Gupta et al. (2018)

Glibenclamide Mitsugumin-53 Bromelain

Nayak et al. (2012) Li et al. (2017) Ataide et al. (2017)

Xanthan gum-based hydrogel for topical delivery Xanthan-O-carboxymethyl hydrogel particles

Liranaftate Glibenclamide

Gellan-xanthan pH-responsive hydrogels

Bovine serum albumin and diclofenac sodium

Mishra et al. (2018) Maiti and Mukherjee (2014) Ramburrun et al. (2017)

Cashew gum Zinc alginate-carboxymethyl cashew gum microbeads Tragacanth gum Tragacanth gum-based sterile hydrogel by radiation method Tragacanth gum cross-linked poly (lactic acid-co-itaconic acid) hydrogel Gum Arabica Calcium alginate/gum Arabic beads Bioinspired alginate-gum Arabic hydrogel Hydrogel formulated using alginate and Arabic gum. Xanthan gum

Moringa gum Hydrogel network of Moringa gum by radiation induced cross-linking Hydrogel prepared by radiation induced cross-linked copolymerization of acrylamide onto moringa gum

Ciprofloxacin HCl Levofloxacin

Singh and Kumar (2018a) Singh and Kumar (2018b)

Dextran Semi-IPN microspheres of acrylamide grafted dextran and chitosan Hydrogel loaded with self-assembled dextran sulfate

Acyclovir

Rokhade et al. (2007a)

Doxorubicin

Niu et al. (2017)

5-Fluorouracil

Pal et al. (2018)

Diclofenac sodium Glibenclamide

Sinha et al. (2015a) Sinha et al. (2015b)

Ropinirole HCl

Ray et al. (2017)

Sesbania gum Sesbania gum based hydrogel Okra gum Zinc alginate-okra gum blend beads Calcium alginate-okra gum mucoadhesive beads Gum ghatti Smart hydrogels of etherified gum ghatti

References

635

(aluminum chloride) solution showed slower releasing of encapsulated glipizide. Additionally, a prolonged hypoglycemic effect was observed by these glipizide-encapsulated carboxymethyl locust bean gum beads.

11. Miscellaneous Besides the hydrogels made of chitosan, alginate, gellan gum, pectin, tamarind gum, sterculia gum, guar gum, and locust bean gum, other natural gums such as gum Arabica, tragacanth gum, cashew gum, xanthan gum, sesbania gum, moringa gum, gum ghatti, dextran, and okra gum have been used to prepare hydrogels for the investigation of their drug-releasing capabilities. These hydrogels also displayed excellent controls of drug releasing, when loaded with various drugs. These hydrogels for the uses in deliveries of various drugs are listed in Table 25.9.

12. Conclusion With the advancements in the field of biopolymer sciences, many multifunctional biopolymeric hydrogel systems have been researched by various research groups with the special spotlight for their prospective uses in the biomedical applications including drug delivery. During past few decades, different kinds of hydrogels achieved the technical values well in terms of biodegradability, biocompatibility, material cost, ease of production, wide array of applications, etc. Considering these advantages, hydrogels made of natural gums such as chitosan, alginate, gellan gum, pectin, tamarind gum, sterculia gum, guar gum, and locust bean gum have already been proved the effectiveness as drug delivery carrier systems to control the releasing of numerous drugs in an effective way.

References Abdur, R.A., Lubna, S., Farah, A., Khan, A.F., Chaudhry, A.A., Rehman, I., Yar, M., 2017. Thyroxin releasing chitosan/collagen based smart hydrogels to stimulate neovascularization. Materials and Design 133, 416e425. Abureesh, M.A., Oladipo, A.A., Gazi, M., 2016. Facile synthesis of glucose-sensitive chitosan-poly(vinyl alcohol) hydrogel: drug release optimization and swelling properties. International Journal of Biological Macromolecules 90, 75e80. Agnihotri, S.A., Aminabhavi, T.M., 2005. Development of novel interpenetrating network Gellan gum-poly(vinyl alcohol) hydrogel microspheres for the controlled release of carvedilol. Drug Development and Industrial Pharmacy 31, 491e503. Agnihotri, S.A., Jawalkar, S.S., Aminabhavi, T.M., 2006. Controlled release of cephalexin through gellan gum beads: effect of formulation parameters on entrapment efficiency, size, and drug release. European Journal of Pharmaceutics and Biopharmaceutics 63, 249e261. Ahuja, M., Yadav, M., Kumar, S., 2010. Application of response surface methodology to formulation of ionotropically gelled gum cordial/gellan beads. Carbohydrate Polymers 80, 161e167. Al-Kassas, R., Al-Gohary, O.M.N., Al-Fadhel, M.M., 2007. Controlling of systemic absorption of gliclazide through incorporation into alginate beads. International Journal of Pharmaceutics 341, 230e237. Aminabhavi, T.M., Nadagouda, M.N., Joshi, S.D., More, U.A., 2014. Guar gum as platform for the oral controlled release of therapeutics. Expert Opinion on Drug Delivery 11, 753e766.

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Osmałek, T., Milanowski, B., Froelich, A., Szybowicz, M., Białowąs, W., Kapela, M., Gadzi nski, P., Ancukiewicz, K., 2017. Design and characteristics of gellan gum beads for modified release of meloxicam. Drug Development and Industrial Pharmacy 43, 1314e1329. Pal, D., Nayak, A.K., 2011. Development, optimization and anti-diabetic activity of gliclazide-loaded alginatemethyl cellulose mucoadhesive microcapsules. AAPS PharmSciTech 12, 1431e1441. Pal, D., Nayak, A.K., 2012. Novel tamarind seed polysaccharide-alginate mucoadhesive microspheres for oral gliclazide delivery. Drug Delivery 19, 123e131. Pal, D., Nayak, A.K., 2015a. Alginates, blends and microspheres: controlled drug delivery. In: Mishra, M. (Ed.), Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, vol. I. Taylor & Francis Group, New York, NY 10017, U.S.A, pp. 89e98. Pal, D., Nayak, A.K., 2015b. Interpenetrating polymer networks (IPNs): natural polymeric blends for drug delivery. In: Mishra, M. (Ed.), Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, vol. VI. Taylor & Francis Group, New York, NY 10017, U.S.A, pp. 4120e4130. Pal, D., Nayak, A.K., 2017. Plant polysaccharides-blended ionotropically-gelled alginate multiple-unit systems for sustained drug release. In: Thakur, V.K., Thakur, M.K., Kessler, M.R. (Eds.), Handbook of Composites from Renewable Materials, Polymeric Composites, vol. 6. WILEY-Scrivener, USA, pp. 399e400. Pal, D., Nayak, A.K., Saha, S., 2018. Interpenetrating polymer network hydrogels of chitosan: applications in controlling drug release. In: Mondal, I.H. (Ed.), Cellulose-Based Superabsorbent Hydrogels, Polymers and Polymeric Composites: A Reference Series. Springer, Cham, pp. 1e41. Pal, P., Pandey, J.P., Sen, G., 2018. Sesbania gum based hydrogel as platform for sustained drug delivery: an ’in vitro’ study of 5-Fu release. International Journal of Biological Macromolecules 113, 1116e1124. Patel, M.M., Amin, A.F., 2011. Process, optimization and characterization of mebeverine hydrochloride loaded guar gum microspheres for irritable bowel syndrome. Carbohydrate Polymers 86, 536e545. Pella´, M.C.G., Lima-Teno´rio, M.K., Teno´rio-Neto, E.T., Marcos, R., Guilherme, M.R., Muniz, EC,c, Rubira, A.F., 2018. Chitosan-based hydrogels: from preparation to biomedical applications. Carbohydrate Polymers 196, 233e245. Prabaharan, M., 2011. Prospective of guar gum and its derivatives as controlled drug delivery systems. International Journal of Biological Macromolecules 49, 117e124. Prajapati, V.D., Jani, G.K., Moradiya, N.G., Randeria, N.P., 2013a. Pharmaceutical applications of various natural gums, mucilages and their modified forms. Carbohydrate Polymers 92, 1685e1699. Prajapati, V.D., Jani, G.K., Moradiya, N.G., Randeria, N.P., Nagar, B.J., 2013b. Locust bean gum: a versatile biopolymer. Carbohydrate Polymers 94, 814e821. Prajapati, V.D., Jani, G.K., Moradiya, N.G., Randeria, N.P., Maheriya, P.M., Nagar, B.J., 2014. Locust bean gum in the development of sustained release mucoadhesive macromolecules of aceclofenac. Carbohydrate Polymers 113, 138e148. Racovita, S., Vasilu, S., Popa, M., Luca, C., 2009. Polysaccharides based micro- and nanoparticles obtained by ionic gelation and their applications as drug delivery systems. Revue Roumaine de Chimie 54, 709e718. Ramburrun, P., Kumar, P., Choonara, Y.E., du Toit, L.C., Pillay, V., 2017. Design and characterization of neurodurable gellan-xanthan pH-responsive hydrogels for controlled drug delivery. Expert Opinion on Drug Delivery 14, 291e306. Rana, V., Rai, P., Tiwary, A.K., Singh, R.S., Kennedy, J.F., Knill, C.J., 2011. Modified gums: approaches and applications in drug delivery. Carbohydrate Polymers 83, 1031e1047. Ray, S., Roy, G., Maiti, S., Bhattacharyya, U.K., Sil, A., Mitra, R., 2017. Development of smart hydrogels of etherified gum ghatti for sustained oral delivery of ropinirole hydrochloride. International Journal of Biological Macromolecules 103, 347e354. Ray, S., Sinha, P., Laha, B., Maiti, S., Bhattacharyya, U.K., Nayak, A.K., 2018. Polysorbate 80 coated crosslinked chitosan nanoparticles of ropinirole hydrochloride for brain targeting. Journal of Drug Delivery Science and Technology 48, 21e29.

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Razavi, M., Nyamathulla, S., Karimian, H., Moghadamtousi, S.Z., Noordin, M.I., 2014. Hydrogel polysaccharides of tamarind and xanthan to formulate hydrodynamically balanced matrix tablets of famotidine. Molecules 19, 13909e13931. Reddy, K.M., Babu, V.R., Sairam, M., Subha, M.C.S., Mallikarjuna, N.N., Kulkarni, P.V., Aminabhavi, T.M., 2006. Development of chitosan-guar gum semi-interpenetrating polymer network microspheres for controlled release of cefadroxil. Design. Monomers Polymer 9, 491e501. Reddy, T., Tammishetti, S., 2002. Gastric resistant microbeads of metal ion cross-linked carboxymethyl guar gum for oral drug delivery. Journal of Microencapsulation 19, 311e318. Repka, M.A., Singh, A., 2009. Alginic acid. In: Handbook of Pharmaceutical Excipients, sixth ed. Pharmaceutical Press, London, UK, pp. 20e22. Rokhade, A.P., Patil, S.A., Aminabhavi, T.M., 2007a. Synthesis and characterization of semi-interpenetrating polymer network microspheres of acrylamide grafted dextran and chitosan for controlled release of acyclovir. Carbohydrate Polymers 67, 605e613. Rokhade, A.P., Shelke, N.B., Patil, S.A., Aminabhavi, T.M., 2007b. Novel interpenetrating polymer network microspheres of chitosan and methylcellulose for controlled release of theophylline. Carbohydrate Polymers 69, 678e687. Sadeghi, M., 2011. Pectin-based biodegradable hydrogels with potential biomedical applications as drug delivery systems. Journal of Biomaterials and Nanobiotechnology 2, 36e40. Sarmah, J.K., Mahanta, R., Bhattacharjee, S.K., et al., 2011. Controlled release of tamoxifen citrate encapsulated in cross-linked guar gum nanoparticles. International Journal of Biological Macromolecules 49, 390e396. Sharma, P.K., Taneja, S., Singh, Y., 2018. Hydrazone-linkage-based self-healing and injectable xanthanpoly(ethylene glycol) hydrogels for controlled drug release and 3D cell culture. ACS Applied Materials and Interfaces 10, 30936e30945. Sharma, R., Ahuja, M., 2011. Thiolated pectin: synthesis, characterization and evaluation as a mucoadhesive polymer. Carbohydrate Polymers 85, 658e663. Sharma, S., Kaur, J., Sharma, G., et al., 2013. Preparation and characterization of pH-responsive guar gum microspheres. International Journal of Biological Macromolecules 62, 636e641. Sharma, V., Patnaik, P., Senthilguru, K., Nayak, S.K., Syed, I., Singh, V.K., Sarkar, P., Thakur, G., Pal, K., 2018. Preparation and characterization of novel tamarind gum-based hydrogels for antimicrobial drug delivery applications. Chemical Papers 72, 2101e2113. Shu, X.Z., Zhu, K.J., 2000. A Novel Approach to prepare tripolyphosphate/chitosan complex beads for controlled release drug delivery. International Journal of Pharmaceutics 201, 51e58. Singh, B., Chauhan, D., 2011. Barium ions crosslinked alginate and sterculia gum-based gastrortentive floating drug delivery system for use in peptic ulcers. International Journal of Polymeric Materials 60, 684e705. Singh, B., Kumar, A., 2018a. Network formation of Moringa oleifera gum by radiation induced crosslinking: evaluation of drug delivery, network parameters and biomedical properties. International Journal of Biological Macromolecules 108, 477e488. Singh, B., Kumar, A., 2018b. Hydrogel formation by radiation induced crosslinked copolymerization of acrylamide onto moringa gum for use in drug delivery applications. Carbohydrate Polymers 200, 262e270. Singh, B., Pal, L., 2008. Development of sterculia gum based wound dressings for use in drug delivery. European Polymer Journal 44, 3222e3230. Singh, B., Pal, L., 2011. Radiation crosslinking polymerization of sterculia polysaccharideePVAePVP for making hydrogel wound dressings. International Journal of Biological Macromolecules 48, 501e510. Singh, B., Pal, L., 2012. Sterculia crosslinked PVA and PVA-poly(AAm)hydrogel wound dressings for slow drug delivery: mechanical, mucoadhesive, biocompatible and permeability properties. Journal of the Mechanical Behavior of Biomedical Materials 9, 9e21.

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Singh, B., Sharma, N., 2008a. Development of novel hydrogels by functionalization of sterculia gum for use in anti-ulcer drug delivery. Carbohydrate Polymers 74, 489e497. Singh, B., Sharma, N., 2008b. Modification of sterculia gum with methacrylic acid to prepare a novel drug delivery system. International Journal of Biological Macromolecules 43, 142e150. Singh, B., Sharma, N., 2011. Design of sterculia gum based double potential antidiarrheal drug delivery system. Colloids and Surfaces B: Biointerfaces 82, 325e332. Singh, B., Varshney, L., Francis, S., Rajneesh, 2016. Designing tragacanth gum based sterile hydrogel by radiation method for use in drug delivery and wound dressing applications. International Journal of Biological Macromolecules 88, 586e602. Singhal, R., Gupta, K., 2016. A review tailor-made hydrogel structures (classifications and synthesis parameters). Polymer - Plastics Technology & Engineering 55, 54e70. Sinha, P., Ubaidulla, U., Hasnain, M.S., Nayak, A.K., Rama, B., 2015a. Alginate-okra gum blend beads of diclofenac sodium from aqueous template using ZnSO4 as a cross-linker. International Journal of Biological Macromolecules 79, 555e563. Sinha, P., Ubaidulla, U., Nayak, A.K., 2015b. Okra (Hibiscus esculentus) gum-alginate blend mucoadhesive beads for controlled glibenclamide release. International Journal of Biological Macromolecules 72, 1069e1075. Sonia, T.A., Sharma, C.P., 2011. Chitosan and its derivatives for drug delivery perspective. In: Jayakumar, R., Prabaharan, M., Muzzarelli, R.A.A. (Eds.), Chitosan for Biomaterials I. Springer, Berlin, Heidelberg, pp. 23e53. Soppimath, K.S., Aminabhavi, T.M., 2002. Water transport and drug release study from cross-linked polyacrylamide grafted guar gum hydrogel microspheres for the controlled release application. European Journal of Pharmaceutics and Biopharmaceutics 53, 87e98. Soppimath, K.S., Kulkarni, A.R., Aminabhavi, T.M., 2000. Controlled release of antihypertensive drug from the interpenetrating network poly(vinyl alcohol)eguar gum hydrogel microspheres. Journal of Biomaterials Science, Polymer Edition 11, 27e34. Sriamornsak, P., Nunthanid, J., Cheewatanakornkool, K., Manchun, S., 2010. Effect of drug loading method on drug content and drug release from calcium pectinate gel beads. AAPS PharmSciTech 11, 1315e1319. Subhash, S.V., Madhabhai, M., 2011. Hydrogels based on interpenetrating network of chitosan and polyvinyl pyrrolidone for pH-sensitive delivery of repaglinide. Current Drug Discovery Technologies 8, 126e135. Sun, P., Li, P., Li, Y.M., Wei, Q., Tian, L.H., 2011. A pH-sensitive chitosan-tripolyphosphate hydrogel beads for controlled glipizide delivery. Journal of Biomedical Materials Research Part B: Applied Biomaterials 97, 175e183. Sutar, P.B., Mishra, R.K., Pal, K., Banthia, A.K., 2008. Development of pH sensitive polyacrylamide grafted pectin hydrogel for controlled drug delivery system. Journal of Materials Science: Materials in Medicine 19, 2247e2253. Thakur, S., Chauhan, G.S., Ahn, J.H., 2009. Synthesis of acryloyl guar gum and its hydrogel materials for use in the slow release of l-DOPA and l-tyrosine. Carbohydrate Polymers 76, 513e520. Tønnesen, H., Karlsen, J., 2002. Alginate in drug delivery systems. Drug Development and Industrial Pharmacy 28, 621e630. Tripathi, R., Mishra, B., 2012. Development and evaluation of sodium alginate-polyacrylamide graft-co-polymerbased stomach targeted hydrogels of famotidine. AAPS PharmSciTech 13, 1091e1102. Verma, A., Dubey, J., Verma, N., Nayak, A.K., 2017. Chitosan-hydroxypropyl methylcellulose matrices as carriers for hydrodynamically balanced capsules of moxifloxacin HCl. Current Drug Delivery 14, 83e90. Vijan, V., Kaity, S., Biswas, S., Isaac, J., Ghosh, A., 2012. Microwave-assisted synthesis and characterization of acrylamide grafted gellan, application in drug delivery. Carbohydrate Polymers 90, 496e506. Yang, T.T., Cheng, Y.Z., Qin, M., Wang, Y.H., Yu, H.L., Wang, A.L., Zhang, W.F., 2017. Thermosensitive chitosan hydrogels containing polymeric microspheres for vaginal drug delivery. BioMed Research International 2017, 356406.

Further reading

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Further reading Bueno, V.B., Petri, D.F., 2014. Xanthan hydrogel films: molecular conformation, charge density and protein carriers. Carbohydrate Polymers 101, 897e904. Hanna, D.H., Saad, G.R., 2019. Encapsulation of ciprofloxacin within modified xanthan gum- chitosan based hydrogel for drug delivery. Bioorganic Chemistry 84, 115e124. Liu, J., Qi, C., Tao, K., Zhang, J., Zhang, J., Xu, L., Jiang, X., Zhang, Y., Huang, L., Li, Q., Xie, H., Gao, J., Shuai, X., Wang, G., Wang, Z., Wang, L., 2016. Sericin/dextran injectable hydrogel as an optically trackable drug delivery system for malignant melanoma treatment. ACS Applied Materials and Interfaces 8, 6411e6422. Nayak, A.K., Pal, D., Santra, K., 2013g. Plantago ovata F. Mucilage-alginate mucoadhesive beads for controlled release of glibenclamide: development, optimization, and in vitro-in vivo evaluation. International Journal of Pharmaceutics, 151035. Singh, S., Sharma, V., Pal, L., 2011. Formation of sterculia polysaccharide networks by gamma rays induced graft copolymerization for biomedical applications. Carbohydrate Polymers 86, 1371e1380. Wu, C., Zhao, J., Hu, F., Zheng, Y., Yang, H., Pan, S., Shi, S., Chen, X., Wang, S., 2018. Design of injectable agarbased composite hydrogel for multi-mode tumor therapy. Carbohydrate Polymers 180, 112e121.

CHAPTER

Implant surface modification strategies through antibacterial and bioactive components

26

Agustin Wulan Suci Dharmayanti1, Rajni Dubey2, Navneet Kumar Dubey3, 4, Win-Ping Deng3, 4, 5 1

University of Jember, Jember, East Java, Indonesia; 2Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan; 3School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 4 Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 5Graduate Institute of Basic Medicine, Fu Jen Catholic University, New Taipei City, Taiwan

1. Introduction Traumatic injury, degenerative diseases, congenital, and developmental disorders present the primary etiology of human organ deficiency leading to body deformities, or anatomy structural alterations, called as defect. These defects need appropriate treatment to prevent advance failure and complications, which further interferes vital organ or tissue around the damage. Prosthetic surgery is one of the treatment measures to manage human structural deformities. The prosthesis is a medical device, such as implants, which issued to restore the defect and replace the damaged organ or tissue. This treatment uses autograft, allograft, or artificial materials to replace and repair the damaged tissues. However, the clinician prefers to apply artificial materials or prosthesis/implants to manage the damaged tissues or organs due to their ease of availability, biocompatibility, less immunogenicity, and infection. The implant is made up of organic or inorganic materials, which confers it biocompatible characteristics. These include ability of material to release leachable products without inflammatory reactions, cellular apoptosis, and tissues function, and rejection response to implant materials (Muddugangadhar et al., 2011; Bauer et al., 2013). This characteristic is influenced by bulk biomaterials and surface properties of the implant (Muddugangadhar et al., 2011); both of which determine biodynamics (behavior and performance) of implant including bioinertness, bioactivity, and biotolerance (Bhasin et al., 2015). The two cells type that are implicated in implantation process are the cells involved in the integration process of implantetissue interfaces and immune cells. Specifically, the cells in the integration process are those surrounding tissue defects, such as fibroblast, bone, and progenitor cells; whereas, the immune cells identify whether implant material is foreign or not (Conserva, Lanuti dan Menini, 2010; Morais, Papadimitrakopoulos dan Burgess, 2010; Se´rgio et al., 2016; Al-maawi et al., 2017). Furthermore, the microenvironment surrounding implant and tissue specifies implant behavior and endurance in the human body. Tissue environment is an unpredictable factor of materials implantation due to the possibility to induce undesirable effects. The environment influences bacterial adherence, ion Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00026-6 Copyright © 2020 Elsevier Inc. All rights reserved.

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release from materials, and corrosivity level, leading to triggering of inflammation, toxicity, and rejection reaction (Anderson, Rodriguez dan Chang, 2008; Love et al., 2013; Zhao et al., 2013; Saini et al., 2015). Besides, biomaterials could be categorized based on the type of biologic response they elicit when implanted and the long-term interaction that develops with host tissue. Three major types of biodynamic activity have been reported biotolerant, bioinert, and bioactive (Navarro et al., 2008). Bioinert materials does not release toxic agents and enhance osseointegration, and therefore are the primary choice. Bioinert materials include titanium, titanium alloys, and ceramic represent bioinert material due to their surface oxide (Saini et al., 2015). These biomaterials allow osteogenic cells to migrate on the implantetissue interface and promote osteogenesis directly (Muddugangadhar et al., 2011; Bhasin et al., 2015). Biotolerant represents unrejected materials after being implanted in tissues. They may release toxic agents and stimulate host response by fibrous encapsulation within connective tissue. Several metals (gold, steel, alloy, cobalt-chromium, tantalum, and niobium) and polymers exhibit tolerant materials properties (Bhasin et al., 2015; Yamada dan Egusa, 2018). Bioactive is a kind of implant such as titanium and ceramic implant, which is modified to improve the biomechanical anchorage and induce osseointegration, usually utilized in Garg et al. (2012), Bauer et al. (2013), Osman and dan Swain (2015), Fouziya et al. (2016), Mandracci et al. (2016). These modifications will be described in the later section of this chapter.

2. Response of cells and tissues to implant materials The implant as bone restoration treatment is aimed to restore bone function with pain relief. However, during initial stages of implantation, the implant devices are recognized as a foreign body. Consequently, the clinicians must consider the interaction between bone cell, tissue microenvironment, and immune system. The bone cells represent osteoblast, lining cell, osteoclast, and osteocytes. The environment involves physical, chemical and biological; whereas, the immune cells play a role in inflammatory reactions mediated by dendritic cells, neutrophils, basophils, monocytes, macrophages, and lymphocytes (Chen et al., 2016; Al-maawi et al., 2017). In particular, immune cells interact with bone cells to undertake bone formation by regulating the balance between osteoclastogenesis and osteogenesis. The three major cytokines regulate involved in osteoclastogenesis are macrophagecolony stimulating factor (M-CSF), receptor activator of NF-kB ligand (RANKL), and osteoprotegerin (OSP). Immune cells also release cytokines inducing osteogenesis and mineralization, inflammatory cytokines (TNF-a, TGF-b, IFN-g, and IL-17), bone morphogenetic protein 2 (BMP-2), transforming growth factor b (TGF-b), and vascular endothelial growth factor (VEGF) (Chen et al., 2016), which provides an adequate environment facilitating osseointegration, an early principal indicator of the successful implant.

2.1 Bone tissue: the most fundamental unit supporting implant Bone is the primary tissue supporting implant existence for a long time in the human body. To achieve the integration, implant materials while interacting with bone induce bone remodeling and osseointegration, and form an entity with tissue surrounding the implanted site. The bone cells involved in bone-implant interaction are osteoblast, osteoclast, osteocytes, and bone lining cells (Fig. 26.1).

2. Response of cells and tissues to implant materials

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FIGURE 26.1 Bone cells and their function.

Osteoblasts are derived from multipotent bone progenitor cells and are the functional bone forming unit located along the bone surface (Insua et al., 2017). However, in the remodeling process, osteoblasts not only produce bone matrix proteins, but also express osteoclastogenic factors (IL-6, TNF-a, M-CSF) (Clarke, 2008; Florencio-silva et al., 2015). Bone lining cell represents cells implicated in bone formation such as preosteoblasts, osteoblast, and osteoclast. They are called latent or rest osteoblast that do not participate in apoptosis and differentiation (Insua et al., 2017). These cells are also involved in the resorption and apposition phase. Osteocytes are derived from osteoblast and are embedded in the bony matrix. These cells regulate activities between osteoclast and osteoblast, and thereby control bone mass and structure (Insua et al., 2017). Contrary to osteoblast, the osteoclast played a role in the breakdown of the bone matrix. Osteoclast presents a multinucleated cell that is derived from monocytic lineage of hematopoietic stem cell. Moreover, it produces cytokines to maintain cellular homeostasis (Insua et al., 2017). In their activity, the M-CSF and the RANKL are critical factors that control proliferation, differentiation, and maturation of osteoclasts (Mori et al., 2013; Florencio-silva et al., 2015). Mesenchymal stem cells (MSCs) are originated from various sources and act as an immunosuppressant. Bone MSCs are multipotent cells that play a role in bone regeneration and repair through their differentiation ability into various cells including bone cells, particularly osteoblast. The boneMSCs cells are a pivotal role in bone implant treatment, as they migrate along implant surface during initial period of the prosthesis implanted. Thereafter, they differentiate into preosteoblast and osteoblast (Hosseini et al., 2015; Bai et al., 2018). All the cells participate in the osseointegration process, which consists of four stages, hemostasis (primary stability), inflammatory, proliferative

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FIGURE 26.2 The stages of osseointegration.

(temporary loosening), and remodeling phase (secondary stability). These stages undertake chronologically after the prosthesis is implanted in tissue, particularly bone tissue, although this process sometimes overlaps (Fig. 26.2) (Hosseini et al., 2015; Wang et al., 2016; Vaidya et al., 2017). The hemostasis or injury phase occurs immediately after bone drilling and implant insertion. This process causes the rupture of blood vessels and bleeding, which further results in ruptured platelets pool around the implant wound area (Hosseini et al., 2015). Subsequently, platelet and tissue protein adhere and aggregate to form a blood clot. When platelets and tissue protein contact to the implant surface, platelets release growth factor and vasoactive agents, such as leukocytes, complement system, and anti-inflammatory cytokines and enhance the healing process. This phase takes place in an hour to a few days. On the first day, pluripotent mesenchymal stem cells migrate to interface implant surfacetissue to stimulate bone regeneration. Besides, this process presents to prevent bacterial adherence and infection (Hosseini et al., 2015; Vaidya et al., 2017). During inflammatory and proliferative stage (third day), the osteoblasts of bone surrounding implant activate Runx2 and OSP transcription factors. Subsequently, on the fourth day, the macrophages and monocytes are differentiated to be osteoclasts, which resorb the necrotic bone around the wound site and release cytokine to stimulate new bone tissue formation. This process takes place about a week to 4 weeks (Hosseini et al., 2015; Vaidya et al., 2017; Chen et al., 2018). Almost a week after surgery, the neovascularization occurs surrounding the implantetissue interface that provides nutrition and oxygen around the wound site. Furthermore, the fibroblasts in connective tissue migrate to the wound site and produce collagen to form the extracellular matrix. Mesenchymal cells also migrate to

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wound site to differentiate into fibroblasts, osteoblasts, and chondroblasts. The extracellular matrix and cells transform to initial immature and woven bone. During this period, there is a cohesion between bone matrix and implant to form woven bone, which is important for implant stability and represent the initiation of mineralization and remodeling (Qin et al., 2014; Hosseini et al., 2015). In this stage, although the osteogenesis and osteoclastogenesis are balanced, the ossification is more dominant than resorption (Wang et al., 2016; Shah etal., 2018).

2.2 Effect of environmental factors to implant performance To withstand a longer time in the human body, an implant as foreign body in the human body must be adapted with tissue environment. Although the tissue environment tends to be stable, the implant stability might be influenced. After being implanted within the human body, the implant is exposed to internal tissue environment such as temperature, biofilm, bacteria, protein, tissue fluid, and pH. In response to surgical bone drilling for implantation as well as implant materials and healing process, the inflammatory changes occurs. These unfavorable conditions might lead to implant failure and loosening. After the implant is inserted into the human body, implant surface is surrounded by film containing tissue fluid and blood. This film facilitates the adsorption of fibronectin and fibrinogen proteins from interstitial fluid and tissue to implant surface. Furthermore, although the biofilm establishes adherence between these growth factor proteins and bone cells, these proteins allow bacterial attachment on the implant surface. The other tissue environment (pH and oxygen level) combined with implant characteristics and design also determine biofilm formation and bacterial growth. If biofilm and bacterial growth is uncontrolled, it induces implant failure (Xu et al., 2005; Gasik, 2017). Implant employed for replacing joint may be exposed to joint fluid containing organic and inorganic compounds. The organic compounds, such as fat, mucin, albumin, leukocytes, and cytokines, trigger kinetic corrosion. Specifically, the inorganic compounds such as CoCrMo-based implants have been reported to stimulate corrosion (Heakal et al., 2014; Royhman et al., 2014). In this chapter, we have later discussed in detail about the role of inorganic compound, implant properties, and designs of implant. It is known that tissue fluid is also an active solution that experiences micromotion and flows along boneeimplant interface during the osseointegration process. The motion serves ion reactive movement and provides oxygen supply. The excessive motion disturbs the osseointegration process and triggers aseptic loosening and induces corrosion. The acidity and ion charge within tissue also determine implant performance, particularly corrosion resistance, as pH of tissue is associated with tissue protein level and hyaluronic acid, which makes it acidic. In normal condition, the pH of tissue is about 7.3e7.4, and the inflammatory reaction after implanted materials alters pH in the range of 6.6e7.4, to even less than 5.5 (Royhman et al., 2014; Yu et al., 2015; Dong et al., 2017). However, the pH deprivation is utilized as an effective stimulant to release antimicrobial agent coated on the implant surface, like silver nanoparticles (AgNPs) (Dong et al., 2017). Besides, the low pH leads to electronic movement and molecular solubility, which finally generates corrosion. Furthermore, implant generates reactive oxygen species (ROS), especially hydrogen peroxide, by inflammatory response. These species stimulate electrochemical changes among ion-containing in tissue as well as the implant (Xu et al., 2005; Ng and Chiu dan Cheng, 2010), which leads to pitting corrosion. Metal alloys-based implant allows ion changes, particularly from metal made up of different alloys. These ionic changes induce cytotoxicity through the reduction of enzymatic activity or biochemistry cascade. The excessive ion movement

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leads to carcinogenicity, mutagenicity, localized tissue irritation, inflammation, and several organ system disorders (Royhman et al., 2014). Further, mechanical stress is the external factor during motional function of implant in the human body. The mechanical environment such as walking, running, or chewing stimulate micromotion within the implantetissue interface, and thereby stimulate cellular adhesion and growth, which play pivotal role in osseointegration. However, the cyclic loading regularly accepts frictional load and the over loading stimulates detrimental effects, such as wear and tear that induces inflammation. These adverse activities allow physical damage, leading to reduced implant resistance (Bicudoa et al., 2016; Gasik, 2017).

3. Implant materials and their surface modification The success of implant treatment is determined by three main factors, namely biomaterial implants, host response, and bone tissue. This indicator of success can be evaluated through short-term or longterm responses. The short-term evaluation is more related to host and osseointegration responses that have been described in the previous part of this chapter. The long-term evaluation is more influenced by the characteristics of the implant and functional material. However, these three factors may not be separated, as they are associated to each other. The implant characteristic is determined by their bulk composition, materials combination, implant type, and the modification. All of these characteristics influence cells adherence and attachment, and host response by the immune system.

3.1 Classification of implant materials Before we discuss the classification of implant materials, it is imperative to know about the development of implant biomaterials. Biomaterial underwent evolution for more than 60 years. This evolution is evidenced in in vitro, in vivo, preclinical, and clinical implications, which are aimed to obtain appropriate biomaterials for a human. Moreover, the objective of representative materials is the ability to replace and restore defect and damaged tissue without any complications. Based on the evolution, the materials are divided into four generations (Fig. 26.3) (Navarro et al., 2008; Gluck, 2010). The first generation presents materials based on inertness. The development started at 1950 to early 1980 using the bulk of biomaterials. The biomaterials that are included in this generation represent metal, alloy, and ceramic. The clinicians are more concerned with mechanical properties, such as corrosion resistance, carcinogenicity, toxicity, allergy, and inflammation (Gluck, 2010). The second generation was developed to attain bioactive and biodegradable materials. Through these biomaterials, the implants are expected to control interaction between implant and physiological environment and enhance tissue-implant surface bonding. Moreover, the biodegradable characteristic is exhibited by material that degrades progressively within the tissue to stimulate regeneration and healing. The third generation is made to trigger specific cellular responses at the molecular level. The advantages of these biomaterials include tissue regeneration and repair (Hench and dan Thompson, 2010). Besides, these biomaterials not only promote the cellular activities, but also inhibit the specific cellular activities to prevent adverse effects (Gluck, 2010). The fourth generation is known as biomimetic materials. Biomimetic is tissue engineering materials that are developed to mimic specific biologic processes and natural tissue (Fig. 26.3) (Sachot et al., 2015).

3. Implant materials and their surface modification

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FIGURE 26.3 Evolution of implant biomaterial.

The implant biomaterials may be categorized into two major types of materials, that is, metals and nonmetals (polymer and ceramic). Metallic materials are titanium, titanium alloys, cobalt-chromium alloys, stainless steel, and precious metals. Although every metallic material exhibit different properties, they possess several advantages and disadvantages (Table 26.1). Titanium represents the mostly used implant materials due to its excellent mechanical properties, particularly corrosion resistance. However, this implant biomaterial is commonly modified to enhance cells/tissue-implant interaction and osseointegration (Bhasin et al., 2015; Saini et al., 2015). In contrast, the nonmetallic biomaterials are classified into polymer and ceramic. The polymer is high molecular weight compound obtained by addition or condensation of smaller molecules. This material exhibits properties that are similar to human soft tissue, though they undergo easy degradation (Babu et al., 2013; Bhasin et al., 2015). There are two types of polymers: biopolymers (natural polymer produced from a living organism) and synthetic. Biopolymer will be described later. The synthetic represents polymethyl methacrylate, polytetrafluoroethylene, polyethylene, polysulfone, polyurethane, and polyether ether ketone (Saini et al., 2015; Yadav et al., 2015). Ceramics are nonmetallic biomaterial manufactured by compacting and sintering at elevated temperatures (Muddugangadhar et al., 2011).

3.2 Properties of implant biomaterials Implant biomaterials must exhibit good biocompatibility, as they are implanted in the human body possessing a unique environment. The essential mechanical properties of implant biomaterials present modulus elasticity, strength, and others. The modulus elasticity of implant biomaterials must be similar

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Table 26.1 The advantages and disadvantages of implant biomaterials (Muddugangadhar et al., 2011; Bhasin et al., 2015; Saini et al., 2015). Types Metals

Titanium

Titanium alloys

CoCreMo

FeeCreNi

Noble metals

Stainless steel

Advantages

Disadvantages

Utilize

degree of · High biocompatibility corrosion · Excellent resistance mechanical · Excellent strength · Good osseointegration · Low risk of loosening and failure mechanical strength · Good fatigue and · Good corrosion resistance osseointegration · Good · Good oxide formation corrosive · Excellent resistant mechanical · Superior properties shielding · Stress · Low cost · Ease of fabrication mechanical strength · High ductility · High · Low cost

cost · High secure processing · Not (machining, forging

· Prostheses stem

· Excellent inertness · Available · Easy to fabricate

· Low mechanical strength · Costly · Low density · Aseptic loosening wear debris · High loosening · Rapid use for Ni · Cannot sensitive patients pit and · Susceptible crevice corrosion · Galvanic reaction

resistant · Corrosive adherent · Strongly · Self-healing · Low cost · Easy processing

or heat treatment)

ductility · Low · Difficult to cast

· Dental implant

· Poor ductility

· Prostheses stem

corrosion · Low resistance · Ni allergy · Galvanic reaction

for · Alongfixator bone shafts

· Spinal connector · Vertebral spacers · Anchoring prostheses · Staples · Orthopedic implant · Screw · Pins · Plates · Fracture plates · Screws · Hip nails

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Table 26.1 The advantages and disadvantages of implant biomaterials (Muddugangadhar et al., 2011; Bhasin et al., 2015; Saini et al., 2015).dcont’d Types Ceramic

Polymer

Composites

Advantages

Disadvantages

· High abrasion resistance friction · Low impact strength · High · Excellent toughness density · Low fabrication · Ease · Biocompatibility · Biostability

· Low mechanical strength in · Unstable temperature,

biocompatibility · Excellent · Minimal thermal and electrical conductivity esthetic · Good to origin tissue · Similar · Minimal biodegradation · Corrosion resistance

mechanical · Excellent properties · Corrosive resistant

fracture · Low toughness · High elastic modulus · Difficult to mold

· · · · ·

chemical, and environment changes Pollutant Leachable in body fluid High wear debris Costly Difficult to fabricate

Utilize

form · Root · Endosteal plate form · Dental implant · Total hip arthroplasties head · Femoral · Acetabular cups · The

·

acetabular cups liner A spacer in intervertebral artificial disc replacement

· Orthopedic implant

to bone (18 GPa). The property is aimed to ensure uniform stress distribution when the implant is functioned in the human body. The tensile, compressive, and shear strength represent properties that are mostly considered while selecting implant biomaterials. The implant materials withstand applied pressure to prevent fractures and improve functional stability. The other mechanical properties are ductility, hardness, and toughness. The ductility is about 8% or more for implant, so the materials can be conformed at the host site. The hardness and toughness also must be adjusted with the human tissue to prevent fractures and improve implant performances (Bhasin et al., 2015; Saini et al., 2015).

3.3 Technique and methods for surface modification of implant materials Besides considering of bulk properties of implant biomaterials, we must also consider the surface properties of biomaterial. If the bulk properties determine the mechanical strength of biomaterial, the surface properties specify biological response of biomaterials. The surface properties affect osseointegration and implant viability; therefore, the surface characteristics are an essential indicator of long-term success of implant in the human body. The surface properties consist of topography, wettability, and roughness. The wettability is established with surface tension and energy and is associated with tissue protein adsorption and osteoblast attachment. The topography and roughness also elicit similar characteristics; however, these are aimed to enhance surface area for implantetissue

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interaction; therefore, the bone cells attachment are expected even more than flat surfaces. Based on the surface roughness, surfaces of implant materials are classified into smooth (0.04e0.4 mm), minimal (0.5e1 mm), intermediate (1e2 mm), and rough (2e3 mm). According the texture of surface, implants are grouped into concave and convex. The texture is determined by modification on implant surface that will be discussed in the later section of the chapter. Concave texture is formed by hydroxyapatite (HA) coating and titanium plasma spraying; therefore, convex texture is configured by etching and blasting (Bhasin et al., 2015; Saini et al., 2015). Moreover, the implant designs are manufactured based on the face angel (angel between thread and plane and implant axis), called topography (Fig. 26.4). The designs determine implant topography that also affect osseointegration. To obtain the good implant surface properties such as topography, the researchers have attempted to develop many surface treatments such as chemical composition, energy level, morphology, microtopography, and roughness (Shetty et al., 2014; Koshy dan Philip, 2015). The two principal techniques to modify surface properties are additive and subtractive. Additive technique involves materials or agent’s addition on to implant surface, both in the form of coating or impregnation. In the coating method, the materials/agents of various thicknesses are added on the implant. For example, titanium plasma spray, plasma sprayed HA, alumina, and biomimetic calcium phosphate (CaP) coating. In contrast, in the impregnation method, materials/agents are completely integrated into titanium core, for instance, integration of CaP crystals within the titanium oxide layer, and fluoride ion on the surface. Subtractive techniques are purposed to remove layers of core materials, distort the superficial surface of materials, and get roughness of materials. The surface materials removal includes mechanical, physical, chemical, and other treatment methods. The mechanical methods involve shaping/ removing, grinding, machining, and grit blasting through physical force. An example of physical treatments is plasma and thermal spray. The chemical treatment may utilize either acid or alkaline solution to titanium alloy. The other techniques are ion implantation, laser treatment, sputtering, etching, and ion deposition. Every technique exhibits different characteristics (Table 26.2) (Bele´m et al., 2010; Alla et al., 2011; Dahiya, Shukla dan Gupta, 2014; Jemat et al., 2015; Koshy dan Philip, 2015; Vilardell et al., 2015; El-gammal et al., 2016; Shi et al., 2017).

FIGURE 26.4 Macro-topography of implant design. (A) Buttress, (B) Standard V-Shape, (C) Square Shape, (D) Reserve Buttress, (E) Spiral.

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Table 26.2 Implant surface modification techniques. Techniques

Advantages

Limitation

Turned/machined

· Enhance bone

· Create macroroughness imperfection of cell · The attachment · Produce osseointegration slowly bonding strength · Decrease (hydroxyapatite coatings) · Reduce tensile strength

interlocking

Plasma spray

surface area · Enhance surface · Enhance roughness · Enhance mechanical properties the healing · Enhance periods

· ·

Grit blasting (using sand, hydroxyapatite, alumina, or titanium oxide)

· · · · · ·

Acid etching (hydrofluoric acid, nitric acid, sulfuric acid)

· · · ·

(hydroxyapatitecoated titanium) Promote cell proliferation (hydroxyapatitecoated implant) Increase bond strength (zirconia addition to hydroxyapatite coatings) Less corrosion Enhance surface roughness Clean from residual blasting particle (etching combination) Enhance surface microhardness (zirconia particle) Improve osteogenesis Uniform surface roughness (microtextured titanium surface) Clean the surface Increase the roughness Enhance cell adhesion Enhance bone formation and osseointegration

· · · · ·

(zirconia addition to hydroxyapatite coatings) Enhance bacteria entrapment Increase fatigue failure Increase plaque retention Costly (coated plasma sprays) Increase contamination

bacterial adhesion · Increase (zirconia particle) · Decrease osseointegration

· Depend on bulk materials, ·

surface microstructure, acid solution and soaking time Decrease mechanical properties due to the process

Continued

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Chapter 26 Implant surface modification strategies

Table 26.2 Implant surface modification techniques.dcont’d Techniques

Dual acid etching

Sand blasting

Sandblast, large grit, and acid etching (SLA)

Advantages

bonding · Enhance surface-cement

· · · · · · · · · · · ·

Ion implantation

Laser treatment (YAG laser)

Anodized

· · ·

(hydrofluoric acid etching) Enhance implant stability Enhance microrough surface Increase osseointegration rapidly Increase removal torque Enhance boneimplant contact Create microretentive topography Increase surface area Increase roughness Increase surface roughness Increase osseointegration Increase surface area by macroroughness and micropit Improve tissue integration and cell proliferation High shear strength Prevent infection Increase osseointegration

removal · Increase torque · Enhance osseointegration hemispheric · Produce pores for bone

· ·

apposition Produce chemical bond Increase osteoconductivity

Limitation

· Decrease mechanical properties due to the process

endurance of the metal · Reduce implant on size, shape, and · Depend kinetic energy

shallow depth · Reduce · Costly secondary ion · Produce · Delamination on energy density · Depend thermal damage · Induce · Produce microcracks

· Depend on density, ·

concentration, temperature, and solution Uneasy process

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Table 26.2 Implant surface modification techniques.dcont’d Techniques

Biochemical coating

Sputtering

Advantages

· Create nanotopography · Bioadhesive · Promote cell proliferation bone · Induce formation · Bioactive · Increase antimicrobial effect · Improve adhesion cell bioactive · Preserve compound (magnetron)

antibacterial · Improve performance (pulsed

·

Limitation

· Limited commercially

· Time consuming (sputtering deposition) amorphous coating · Produce and slow deposition · Low process (sputtering deposition)

magnetron sputtering) Uniform coating thickness

3.4 Implant surface coating techniques Nowadays, the surface modification is being developed by covering implant surface with bioactive compounds, either organic or inorganic. These coating compounds that are expected can induce osteogenic process and osseointegration. In this regard, titanium and polymer are the most suitable implant materials for coating bioactive compound. The coating can assist in optimizing implant stability, improving integration of soft tissue and perimplant, and decreasing periimplantitis by bacterial adhesion disturbance. The antiresorptive drugs have been used as an implant coating, such as bisphosphonate. Besides, osteogenic drugs, the antimicrobial, protein, and growth factors are also developed for implant coatings.

3.4.1 Hydroxyapatite Hydroxylapatite and nanocomposites are the osteoconductive agents that are frequently used to cover metallic implant surface. Plasma spray is the most common technique used to produce implant-coated hydroxyapatite, which strengthens organic matrix in the mineralization process. However, this coating is undurable due to low bond strength under high temperature. To improve the characteristics of hydroxyapatite, nanocomposite hydroxyapatites are being fabricated. The advantages of this type of composite coating include improved mechanical properties similar to bone and negative shielding stress inhibition (Xie et al., 2010). These hydroxyapatite-based coatings are done on implant surface by various techniques such as plasma spray, vacuum deposition, sol, and gel dip (Garg et al., 2012).

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3.4.2 Growth factors The growth factors are soluble signaling molecules controlling various cellular responses such as differentiation, proliferation, and activation. The basis of using growth factor as implant coating of platelet aims to achieve hemostasis during the primary osseointegration phase. In this process, platelets release the growth factors that stimulate cellular differentiation, proliferation, and activation, especially of mesenchymal stem cells (Smeets et al., 2016). Moreover, growth factors have also been reported to induce osteogenesis and osseointegration (Macdonald et al., 2011; Vo et al., 2012). In a seminal report, TGF-b combined with hydroxylapatite has been evidenced to enhance bone formation (Lind et al., 1996).

3.4.3 Extracellular matrix protein Extracellular matrix in bone is composed of inorganic and organic matrix. Although both of extracellular matrix components utilize as implant coating and exhibit their potency to induce bone remodeling, the hydroxyapatite, as inorganic component, can inhibit osteoblast and induce immune response. Therefore, the organic matrices, especially protein, or substitute could be combined by hydroxyapatite for implant coating. Extracellular matrix proteins guide osteoprogenitor cells migration to implant surface, and thereby promote integrin interaction. Collagen, chondroitin, fibronectin, vitronectin, and proteoglycan represent extracellular matrix protein using implant surface coating. Titanium alloy-coated collagen and chondroitin significantly enhance osteoblastic activities and reduction of macrophage and osteoclast activities, either alone or combined with hydroxyapatite or hyaluronic acid (Bierbaum et al., 2006; Rammelt et al., 2007; Ao et al., 2016; Ao et al., 2018).

3.4.4 Peptides Peptides are biomolecules consisting of short-chain amino acids. These peptides facilitate cell adhesion during osseointegration process and also act as an antibacterial agent (Chen et al., 2017). Although the role of integrin (RGD) peptide as an implant coating is still uncertain, the in vivo studies demonstrated osseointegration induced by RGD peptide coatings. Moreover, human b-defensins, another peptide type, also indicate antibacterial effect and promote osteoblast and mesenchymal stem cell proliferation when it covers the implant surface (Smeets et al., 2016).

3.4.5 Drug coating The drugs such as statin, bisphosphonate, and tetra HCl are frequently used as implant coating. Every drugs exhibit different effects on the implant surface, although the principal purpose is to enhance osseointegration. Statin (simvastatin) triggers have been reported for their ability toward bone synthesis and osseointegration (Fouziya et al., 2016; Tao et al., 2016). Bisphosphonate is an antiresorptive agent for osteoporotic bone. This agent inhibits osteoclastic activity and increases bone mineral density in the periimplant area. Further, a combination of fibrinogen and bisphosphonate (ibandronate and pamidronate) coating are able to improve bone-implant fixation and reduce bone resorption (Abtahi et al., 2010; 2012). Plasma spray implant coated with 0.5 mg/kg/day alendronic acid demonstrated enhanced surface treatment, bone density, and bone formation marker and bone-implant contact. However, its adverse effects such as jaw osteonecrosis have also been reported (Ryabov et al., 2016). Tetracycline HCl is a potent antibiotic agent utilized as an implant coating. The purpose to employ this coating is to remove periimplantitis incidence that frequently occurred while implant treatment,

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especially titanium based, which is caused by negative bacterial infection (Blanchard et al., 2017). The tetracycline HCl inhibits biofilm formation and removes the endotoxin layer on the implant surface (Herr et al., 2008). It also stimulates osseointegration by enhancing bone cell proliferation, reduction of collagenase activity, and increasing blood clot attachment (Herr et al., 2008; Lee et al., 2011; Fouziya et al., 2016).

3.4.6 Fluoride coating Fluoride is reactive ion implanted on titanium alloy. The fluoride treatment produces hydrophilicity that facilitates protein and water adsorption, and stimulates differentiation of bone progenitor cells and activates osteogenesis. It can also induce newly synthesized extracellular matrix along implant tissue (Garg et al., 2012). In a seminal report, it has been reported that chemical interaction between Ti and fluoride forms TiF4, and thereby enhances osseointegration (Dahiya et al., 2014).

3.5 Biopolymer materials Biodegradable polymers are developed to reduce polymeric waste that endangers the environment. These biodegradable polymers are classified into two types: synthetic and natural. Synthetic polymers are petroleum-based synthetic polymers, which have been used widely for a long time. However, the source of these polymers increasingly reduced being nonrenewable, nonbiodegradable, and toxic in nature (Tanase et al., 2014). The advantages of synthetic polymers include control of their mechanical and physical properties, even they exhibit possible risk of infection, toxicity, carcinogenicity, and immunogenicity, particularly by impure polymers. The synthetic biodegradable polymers belong to wide range types. For example, saturated aliphatic polyesters includes poly(lactic acid) (PLA/ PDLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolide), polypropylene fumarate (PPF), polyhydroxyalkanoates (PHA), such as poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly-4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly-3-hydroxyoctanoate (PHO). PDLLA is one of the synthetic polymers extensively observed as a coating on orthopedic implant material. PDLLA has high mechanical stability, excellent biocompatibility, and potential osteoconductivity. Due to its low molecular weight, it could be combined with growth factor, antibiotic, or thrombin inhibitor. Another synthetic polymer is PHB that can inhibit or prevent a chronic inflammatory response and enhance bone formation (Rezwan et al., 2006). Although these materials indicate biocompatibility, their biodegradable products can induce inflammation and unwanted immunological reaction (Rebelo et al., 2017). On the other hand, the natural polymer, called biopolymer, is synthesized from living matter, such as plants, algae, animals, and microorganism. There are three groups of biopolymer: polysaccharide, protein, and polynucleotides. The polysaccharide is among the most natural polymers type and is originated from the plant, algae, animal, bacteria, fungi, and lipid/surfactants (Table 26.3) (Onar, 2014; Tanase et al., 2014; Rebelo et al., 2017). Although the natural polymers are renewable and biodegradable, their manufacturing is complex and the conventional method produces poor performance (Tanase et al., 2014). The cells incorporate biopolymer enzymatically in their cytoplasmic and other organelles. They produce polymer for their structural integrity. Moreover, these kinds of polymer play a role in essential life function, such as genetic information, energy storage, catalysis, nutrients, and protective agents against hazardous microorganism (Rao et al., 2014).

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Chapter 26 Implant surface modification strategies

Table 26.3 Classification of biopolymers. Type

Origin

Production technique

Biopolymers

Polyester

Classical chemical synthesis

Polylactic acid

Polynucleotide Protein

Corn starch rice, potatoes, and other natural resources Bacteria Bacteria Animal

Genetically modified Genetically modified Classical chemical synthesis

Polysaccharide

Plants/alga

Polyhydroxyalkanoates DNA, RNA Silk, collagen, gelatin, elastin, resilin, adhesives, polyamino acids, soy, zein, wheat gluten, casein, serum albumin Starch (amylase/ amylopectin), cellulose, agar, alginate, carrageenan, pectin, konjac, and various gums (guar) Chitin/chitosan, hyaluronic acid Xanthan, dextran, gellan, levan, polygalactosamine, Pullulan, selsinan, yeast glucans Acetoglycerides, waxes, surfactants, emulsan Lignin, tannin, humic acid Shellac, poly-gglutamic acid, natural rubber, synthetic polymers originated natural fats, and oils nylon-derived castor oil.

Classical chemical synthesis

Animal Bacterial

Genetically modified

Fungal Lipid/surfactant Polyphenol Special type

3.6 Antimicrobial activity of biopolymer implant coating Even though bulk materials of implant exhibit excellent properties and biocompatibility in the human body, failure of the implant occur due to its frequent loosening. This failure leads to many adverse consequences, such as revision surgery, inexpensive, and complications leading to death. The primary factor for loosening of implant is bacterial infection, which causes hard and soft tissue destruction near the implant site. These uncontrolled growing bacterial colonies are also supported by implant surface

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structure that provides an adequate site for bacteria attachment, leading to disturbed osseointegration and cause implant loosening. An antimicrobial is therefore used for inhibiting or killing microorganisms. The antimicrobial agents used to coat implant (silver, copper, and bismuth based) are antibiotic drugs and metals (Gosau et al., 2015; Kulkarni Aranya et al., 2017). The synthetic polymers are frequently utilized with antimicrobial agents. The action of these polymers is mediated through their interaction with bacterial cell membrane, leading to its destruction and cell lysis.

3.6.1 Alginate Alginate is composed of D-mannuronic acid (M) and L-guluronic acid (G) and is extracted from polysaccharide of brown algae. This biopolymer is generally present in the hybrid form, that is, calcium alginate/gelation (cross-link of alginate chain with calcium ion by divalent cationic cross-link) (Xiao et al., 2009). Calcium alginate is frequently utilized as coating materials owing to biocompatible and biodegradable nature. Titanium-coated composite of protamine, alginate, and BMP-2 exhibited enhanced bioactivity and increased osseointegration (Chen et al., 2018). Alginate is oftentimes utilized as an antimicrobial agent in the food industry due to its ability to reduce Escherichia coli and Staphylococcus aureus growth (Fundador et al., 2018). Composite alginate coated on titanium implant surface indicated excellent antibacterial activity (Xiao et al., 2009). Furthermore, dental implant coated layer by layer with antibiotic/chitosan/alginate exhibited the ability to inhibit biofilm synthesis and bacteria growth (Lv et al., 2014).

3.6.2 Hyaluronic acid Hyaluronic acid is an unbranched biopolymer, which is present in the extracellular matrix (ECM) and soft connective tissues, and is responsible for flexibility (Arpacay and dan Turkan, 2015). However, animal-derived hyaluronic acid represents a potent titanium coating implant material and have demonstrated osteoconductive, antiinflammatory, and antibacterial activity (Boot et al., 2017).

3.6.3 Silk fibroin Silk fibroin represents biocompatible natural polymer synthesized from several spiders and insects. It consists of hydrophobic and hydrophilic peptide chains with molecular weight about w390 kDa. The silk fibroin acts as a substance. Silk fibroin is a biodegradable material, with low inflammatory response and potent bactericidal activity. In a seminal study, the silk fibroin loaded with gentamicin exhibited inhibition of Staphylococcus aureus and stimulated osteoblast adhesion (Sharma et al., 2016). The hybrid/gelatin microsphere loaded with gentamicin sulfate showed inhibited growth of both Staphylococcus aureus and Staphylococcus epidermidis and also stimulated osteogenesis. Besides, a combination of levofloxacin and silk fibroin/hydroxyapatite inhibited the growth of Staphylococcus aureus on CoCrMo alloy implant. A hybrid of silk fibroin and hyaluronic acid arranged in multiple complex layers enhanced biocompatibility and antibacterial effect (Arpacay and dan Turkan, 2015).

3.6.4 Albumin Albumin is a protein contained in blood serum. The function of albumin on the implant surface is to enhance osseointegration by stimulating biomolecular adsorption. Bovine serum albumin is used as an implant coating that has proven to be enhanced corrosion resistant on titanium-based implant. Albumin

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Chapter 26 Implant surface modification strategies

coating by electrophoretic deposition allows to prevent corrosion and inhibit biofilm synthesis (Ho¨hn et al., 2017).

3.6.5 Tannic acid Tannic acid is a polyphenol-based water-soluble biopolymer and is extracted from plants and fruits. Polyphenol exhibits potent antibacterial and antioxidative activities. Tannic acid acts by destructing bacterial cytoplasmic membrane, intensifying its permeability and finally deteriorating lipid-protein layer. The combination of tannic acid/hydroxyapatite on implant surface has revealed an inhibited bacterial growth (Yang et al., 2017).

3.6.6 Collagen Collagen is an organic component of the ECM providing cell support, adhesion, and movement. Animal-derived collagen, a natural polymer, can be utilized as coating implant surfaces owing to the involvement in cellular proliferation, adhesion, migration, and survival. The collagen characteristics include biocompatibility, biodegradability, and high versatility. For example, titanium-coated collagen exhibited an accelerated implant fixation (Cecconi et al., 2014). The collagen for coating of the biomedical device must be combined with other materials, such as hydroxyapatite that stimulates osteoblastogenesis (Bose et al., 2018). Collagen composites of other materials has been utilized as a tissue engineering or scaffold, and indicated antibacterial and osteoconductivity (Tu et al., 2012; Bronk et al., 2014; Lu et al., 2016; Dhand et al., 2018).

3.6.7 Gelatin Gelatin is derived from collagen of animals by the hydrolysis process. Gelatin presents rich in amino and hydrophilic carboxyl groups that provide nutrients and oxygen infiltration. Although it is derived from collagen, gelatin indicates more worthwhile than collagen, being as less immunogenic, economic, and convenient. It can be applied as a dental implant coating owing to high cellular affinity and adsorbability. Moreover, gelatin is utilized as a hemostatic sponge for bone healing (bone tissue engineering) (Makita et al., 2018). Further, though gelatin demonstrates a weaker antimicrobial action, it could act as antimicrobial agent carrier, like AgNPs (Chen and dan Wang, 2018). It has also shown its antimicrobial effect against Staphylococcus aureus and Escherichia coli when combined with chitosan, hydroxyapatite, and graphene oxide (Gao et al., 2016).

3.6.8 Poly(lactic acid) PLA is a thermoplastic polymer of the a-hydroxy acid family. The lactic acid is a fermentation product of plant starch, such as corn, sugarcane, potatoes, and beets. PLA is synthesized either by direct lactic acid polycondensation or by ring opening polymerization of lactide dimer using a suitable catalyst (Saini et al., 2016). PLA demonstrates good properties, such as high strength, modulus elasticity, corrosion resistance, creep resistance, biocompatibility, and biodegradability. Therefore, it is widely used as commercial products, such as screws and pins, in an orthopedic and dental application, during reconstruction surgery, etc. Beside these properties, PLA can be easily fabricated in several structures adjusted to specific biomedical applications, including the topography, geometry, and architecture (Narayanan et al., 2016; Saini et al., 2016). Furthermore, PLA blended with other materials, either synthetic or natural polymer, has demonstrated its improved properties. Interestingly, PLA solution

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blended with chitosan and poly(ε-caprolactone) (PCL) by emulsification showed a good antimicrobial effect. Hence, this hybrid is a promising material for implant coating (Saini et al., 2016).

3.6.9 Polyhydroxyalkanoates PHAs are natural polymers, belonging to hydrophobic biopolyesters family. The polyhydroxyalkanoates, both homo- and heteropolymers, are fabricated within microorganism cytoplasm, such as Cupriavidus necator and Alcaligenes latus. They are formed when the nutrients are limited. The major characteristics of this polymer includes biodegradability, biocompatibility, and no cytotoxicity (Singh et al., 2019). However, the PHAs exhibit inadequate biocompatibility due to hydrophobic property. As a result, PHAs are frequently modified with a polymer, antibiotic or inorganic materials to improve their properties, especially cellular attachment and proliferation (Dong et al., 2010). The combination of PHAs/hydroxyapatite produces a biodegradable product for implant coating and improves their properties (Raydan Kalia, 2017).

3.6.10 Chitosan Chitosan is a natural polymer that originated from marine crustacean’s shells and fungi cell walls. Specifically, it is produced from chitin through deacetylation reaction. The chitosan presents a linear polysaccharide that is biodegradable, nontoxic, and insoluble in organic/inorganic solvents and water (Shahid et al., 2019). This biopolymer exhibits several activities, such as antimicrobial, inducer of wound healing, and osteoconduction. Nowadays, chitosan is widely used for coating implant surface materials and orthopedic devices that are combined with other polymers or inorganic compounds, for example, alginate, hydroxyapatite, hyaluronic acid, CaP, poly(methyl methacrylate) (PMMA), poly-Llactic acid (PLLA), and growth factors (Di et al., 2005; Lv et al., 2014). The gallium-based implant covered by chitosan/poly(acrylic acid) have shown a reduced the orthopedic and dental implants failure. The hybrid also exhibits antibacterial activity against Escherichia coli and Pseudomonas aeruginosa (Bonifacio et al., 2017). The antimicrobial action of chitosan occurs through several mechanisms. Firstly, chitosan acts through electrostatic interaction between the polycationic compounds of chitosan and the anionic components of microorganisms. Secondly, the antibacterial activity of chitosan is presented through hydrophobic sites interaction between chitosan and bacterial cell wall that allows an increased chain length of chitosan salt. Eventually, the physical properties of chitosan, such as low molecular weight, allow the chitosan to penetrate into the cell walls of bacteria and inhibit mRNA synthesis (Lu et al., 2016).

3.7 Methods for biopolymer processing as surface modification and implant coating Implant surface forms biofilm that must be controlled, as it may stimulate protein adsorption and induce bacterial infection to disturb osseointegration. To prevent and control biofilm formation to implanted biomaterials, various antimicrobial agents are being developed by several techniques. These techniques utilized three principal methods for coating polymeric implant. The techniques include passive surface finishing/modification (PSM), active surface finishing/modification (ASM), and perioperative antibacterial local carriers or coating. PSM employs physical and chemical modification on implant surface without releasing antibacterial agent (Romano` et al., 2015a). This strategy aims to

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reduce protein adsorption on implant surface and prevent bacterial adhesion. The PSM activities do not actively interact with bacteria and therefore unable to kill them. ASM is a technique that modifies implant surface with antibacterial agents, such as antibiotics, antiseptics, metal ions, or other organic and inorganic compounds. These agents are coated on implant surface that kills the microorganism in contact or elute to tissue environment in implant site. Antibacterial activity is indicated by cross-link between ion on coating material and cell membrane of microorganism. This bonding will interfere their cell membrane permeability and produce ROS that disturbs cellular metabolism (Bose et al., 2018). The last method is LLC. This strategy protects implant and tissue through a biodegradable or nonbiodegradable antibacterial carrier or coating. The materials may be applied during surgery on the implant or around it or during the implant manufacturing. The solegel process is another method that uses modified biopolymer to implant surface. This process usually involves the use of metal-organic precursors that are converted to inorganic materials either in water or in an organic solvent. This technique can control the surface morphology of the bioactive glass nanoparticles to provide the desired properties such as enhanced mechanical and biological properties of implant, particularly osseointegration and antibacterial activity (Boccaccini et al., 2010; Kumeria et al., 2015).

4. Conclusion and future prospects Recent trends in surface implant modification strategies indicate that the surface properties of implants could be modified with various materials/compound to improve osseointegration and reduce healing time. However, only a few organic polymers possess characteristics of implant materials, due to limitations such as poor mechanical properties, cytotoxicity, immunoreactivity, or genotoxicity, leading to their unsuitability for orthopedic/dental implants and in other clinical applications. Moreover, the biopolymer needs further studies, especially in clinical implication, such as orthopedic and dental implantology, to achieve optimized implant stability during natural cascades of osseointegration, the improvement of periimplant soft tissue integration, and reduction of periimplantitis by impairing bacterial adhesion to the implant surface. Various preclinical reports demonstrated significant differences in outcomes of surface implant modifications; notwithstanding, further clinical studies should be performed to corroborate these findings. Moreover, the aims of future implant studies should focus on synthesizing a surface enhancing clinical behavior, in particular facilitating osteogenesis and cellular growth, soft-tissue attachment, and retarded microbial colony on the implant surface.

References Abtahi, J., Tengvall, P., dan Aspenberg, P., 2010. Bisphosphonate coating might improve fixation of dental implants in the maxilla : a pilot study. International Journal of Oral & Maxillofacial Surgery. International Association of Oral and Maxillofacial Surgery 39 (7), 673e677. https://doi.org/10.1016/j.ijom.2010.04.002. Abtahi, J., Tengvallc, P., dan Aspenberg, P., 2012. A bisphosphonate-coating improves the fixation of metal implants in human bone. A randomized trial of dental implants. Bone 50 (5), 1148e1151.

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Further reading Calliess, T., et al., 2012. Antimicrobial surface coatings for a permanent percutaneous passage in the concept of osseointegrated extremity prosthesis. Biomedizinische Technik 57 (6), 467e471. https://doi.org/10.1515/bmt2011-0041. Chen, G.Q., dan Zhang, J., 2018. Microbial polyhydroxyalkanoates as medical implant biomaterials. Artificial Cells, Nanomedicine and Biotechnology. Informa UK Limited, trading as Taylor & Francis Group 46 (1), 1e18. https://doi.org/10.1080/21691401.2017.1371185. ˜ ., dan Badot, P., 2008. Application of chitosan, a natural aminopolysaccharide, for dye removal from Crini, A aqueous solutions by adsorption processes using batch studies : a review of recent literature. Progress in Polymer Science 33 (April), 399e447. https://doi.org/10.1016/j.progpolymsci.2007.11.001. Das, S., et al., 2018. Osteogenic nanofibrous coated titanium implant results in enhanced osseointegration: in vivo preliminary study in a rabbit model. Tissue Engineering and Regenerative Medicine 15 (2), 231e247. https:// doi.org/10.1007/s13770-017-0106-6. Springer Singapore. Douglas, T.E.L., et al., 2019. Pectin-bioactive glass self-gelling, injectable composites with high antibacterial activity. Carbohydrate Polymers 205, 427e436. https://doi.org/10.1016/j.carbpol.2018.10.061. Elsevier Ltd. (February). Drago, L., et al., 2014. Does implant coating with antibacterial-loaded hydrogel reduce bacterial colonization and biofilm formation in Vitro ? Clinical Orthopaedics and Related Research 472 (11), 3311e3324. https:// doi.org/10.1007/s11999-014-3558-1. Folkert, J., et al., 2017. Nanocoating with plant-derived pectins activates osteoblast response in vitro. International Journal of Nanomedicine 12, 239e249. https://doi.org/10.2147/IJN.S99020. Gregurec, D., et al., 2016. Bioinspired titanium coatings : self-assembly of collagen e alginate films for enhanced. Journal of Materials Chemistry B 4, 1978e1986. https://doi.org/10.1039/C6TB00204H. Gurzawska, K., et al., 2017. Pectin nanocoating of titanium implant surfaces - an experimental study in rabbits. Clinical Oral Implants Research 28 (3), 298e307. https://doi.org/10.1111/clr.12798. Han, B., Yang, Z., dan Nimni, M., 2005. Effects of moisture and temperature on the osteoinductivity of demineralized bone matrix. Journal of Orthopaedic Research 23 (4), 855e861. https://doi.org/10.1016/ j.orthres.2004.11.007. Huang, K., et al., 2016. Recent advances in antimicrobial polymers: a mini review. International Journal of Molecular Sciences 17 (1578). https://doi.org/10.3390/ijms17091578.

Further reading

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Kokkonen, H., et al., 2008. Differentiation of osteoblasts on pectin-coated titanium. Biomacromolecules 9 (9), 2369e2376. https://doi.org/10.1021/bm800356b. Landry, M.J., et al., 2018. Layers and multilayers of self-assembled polymers: tunable engineered extracellular matrix coatings for neural cell growth. Langmuir 34 (30), 8709e8730. https://doi.org/10.1021/ acs.langmuir.7b04108. Lopez-Esteban, S., et al., 2003. Bioactive glass coatings for orthopedic metallic implants. Journal of the European Ceramic Society 23 (15). Mistry, S., et al., 2011. Comparison of bioactive glass coated and hydroxyapatite coated titanium dental implants in the human jaw bone. Australian Dental Journal 56, 68e75. https://doi.org/10.1111/j.18347819.2010.01305.x. Nawae, S., et al., 2018. Layer-by-layer self-assembled films of silk fibroin/collagen/poly (diallyldimethylammonium chloride) as nucleating surface for osseointegration to design coated dental implant materials. Materials and Design 160, 1158e1167. https://doi.org/10.1016/j.matdes.2018.10.041. Nayak, S., et al., 2013. The promotion of osseointegration of titanium surfaces by coating with silk protein sericin. Biomaterials 34 (12), 2855e2864. https://doi.org/10.1016/j.biomaterials.2013.01.019. Elsevier Ltd. Raphel, J., Karlsson, J., Galli, S., Wennerberg, A., Lindsay, C., Haugh, M.G., et al., 2016a. Biomaterials Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants. Biomaterials 83, 269e282. https://doi.org/10.1016/j.biomaterials.2015.12.030. Elsevier Ltd. Raphel, J., Karlsson, J., Galli, S., Wennerberg, A., Lindsay, C., Haugh, M., et al., 2016b. Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants. Biomaterials 83, 269e282. https://doi.org/10.1016/j.biomaterials.2015.12.030.Engineered. Ritz, U., et al., 2017. The effect of different collagen modifications for titanium and titanium nitrite surfaces on functions of gingival fibroblasts. Clinical Oral Investigations 21 (1), 255e265. https://doi.org/10.1007/ s00784-016-1784-5. Romano`, C.L., et al., 2015b. Antibacterial coating of implants in orthopaedics and trauma: a classification proposal in an evolving panorama. Journal of Orthopaedic Surgery and Research. Journal of Orthopaedic Surgery and Research 10 (1), 1e11. https://doi.org/10.1186/s13018-015-0294-5. Sobczak, M., et al., 2013. Polymeric systems of antimicrobial peptidesdstrategies and potential applications. Molecules 18, 14122e14137. https://doi.org/10.3390/molecules181114122. Sobczuk-Szul, M., et al., 2013. Changes in the bioactive protein concentrations in the bovine colostrum of Jersey and polish holstein-friesian cows. Turkish Journal of Veterinary and Animal Sciences 37 (1), 43e49. https:// doi.org/10.3906/vet-1107-42. Stanfield, J.R., dan Bamberg, S., 2014. Durability evaluation of biopolymer coating on titanium alloy substrate. Journal of the Mechanical Behavior of Biomedical Materials 35, 9e17. https://doi.org/10.1016/ j.jmbbm.2014.03.003. Elsevier. Urgery, S., et al., 2015. Sclerostin antibody treatment improves implant fixation in a model of severe osteoporosis. Journal of Bone and Joint Surgery 97, 133e140. Virdi, A.S., et al., 2015. Sclerostin antibody treatment improves implant fixation in a model of severe osteoporosis. Journal of Bone and Joint Surgery 97 (2), 133e140. Zarrintaj, P., et al., 2018. Agarose-based biomaterials for tissue engineering. Carbohydrate Polymers 187, 66e84. https://doi.org/10.1016/j.carbpol.2018.01.060. Elsevier Ltd. Zhang, F., et al., 2008. Biomaterials Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion. Biomaterials 29 (36), 4751e4759. https://doi.org/10.1016/j.biomaterials.2008.08.043. Elsevier Ltd.

CHAPTER

Edible films and coatings: an update on recent advances

27

Navneet Kumar Dubey1, 2, Rajni Dubey3 1

School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 2Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan; 3Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan

1. Introduction Usually, the foodstuffs we buy are encased in metal foils or plastic, which is removed and tossed in garbage bins. However, the environmental concern regarding the nonrenewable and nonbiodegradable nature of the packaging materials has now encouraged researchers to develop greener alternatives (Gao et al., 2017). In this regard, the films made up of edible materials are the best alternatives because, in addition of being eco-friendly, they can be used to increase the organoleptic, nutritional, and microbiological properties of food products (Lopez-Rubio et al., 2017). Moreover, as food products are prone to progressive alterations while storage and distribution, these films act as a blockade for agents such as oxygen, water vapor, and moisture, and therefore retard the aging of coated fruits and vegetables by selective permeability to gases (Otoni et al., 2017). Edible films and coatings are the materials used to cover food products to increase their shelf life by offering barrier to moisture, oxygen, temperature, and UV (Fig. 27.1). It may be consumed together with food without removal (Otoni et al., 2017). Coatings may be either employed to or formed directly on food materials; whereas, films are self-supporting constructs that could be used for wrapping food products. These are present either on surface of food or in the form of thin layers between various parts of the food product. They are composed of only food-grade components and mostly of a biomacromolecular matrix responsible for synthesizing a cohesive structure and a solvent. Additionally, a plasticizer is generally needed to reduce brittleness and enhance flexibility. Furthermore, to promote the usability of these kinds of packaging materials at industrial level, the processing of biopolymers should be under the same conditions as petroleum-based plastics (Sharma and Ghoshal, 2018). Traditionally, edible films and coatings were designed flavorless and transparent, which do not obstruct sensory properties of food. However, novel research findings showed that particular sensory characteristics might be necessary for some applications, like sushi wraps and of pizza toppings. Moreover, the antioxidant edible films may protect from food oxidation and nutritional losses; whereas, antimicrobials could refrain from food-borne bacterial spoilage and organoleptic deterioration (Debeaufort and Karbowiak, 2018) (Fig. 27.1). Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00027-8 Copyright © 2020 Elsevier Inc. All rights reserved.

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Chapter 27 Edible films and coatings: an update

FIGURE 27.1 Major functions of edible films and in food packaging applications. The edible films and coating can save the food from temperature, moisture, UV, and gases. They may be incorporated with antimicrobials, probiotics, antioxidants, and aroma compounds to enhance their applicability.

Since the early to mid-20th century, coatings have been employed to rescue water loss and impart shine to fruits and vegetables (Pavlath, 2009). Cellulose derivatives, starches, gums, chitosan/chitin, lipids, and plant or animal-based proteins offer the possibility to fabricate edible films to elongate shelf life of fresh or processed food packaging (J., 2019). However, last decade witnessed one of the most inventive trends in the use of biopolymers with the conversion of food-waste products or agricultural commodities to edible films, for instance, cheese-derived whey protein, corn zein from ethanol production, and chitosan from crustacean shells (Zuo et al., 2019). Use of the agricultural waste in packaging edible films can be associated with various economic and environmental advantages. This can also assist in avoiding competition for food resources. These polymers provide supplementary benefits in their commercial use, such as nontoxicity, biocompatibility, barrier to moisture and gases, mechanical integrity, nonpolluting, and relatively low cost (J., 2019). However, before the commercial production of edible film or coating, the manufacturing companies should follow regulatory guidelines and current legislation of the nation. All the film-synthesizing elements as well as additives should be made of safe materials (food grade), with all processing facilities meeting high levels of hygiene (Pavli et al., 2018). Moreover, the presence of allergens (proteins) in the coatings or films such as wheat, milk (whey/casein), and soy should be clearly labeled. This chapter aims to comprehensively update the most recent advances on previously published and current studies on edible active films for their future applications.

2. Novel patterns in basic structural matrices

677

2. Novel patterns in basic structural matrices Numerous compounds could be employed in formulating edible packaging for strengthening the physical properties of food products (Sharma and Ghoshal, 2018). The decision on using specific compound will reply mainly upon target application. Components synthesizing the edible films or coating are mostly categorized into four categories. These are polysaccharides, proteins, lipids, and composites. There are numerous scientific papers focused on edible packaging; however, last 5 years have witnessed huge change in the fabrication methods, functionalities, and properties of edible films and coatings.

2.1 New trends in polysaccharides-based edible films In the recent years, the polysaccharides-derived edible films (starch, cellulose, chitosan, pectins, gums, seaweeds, and pullulan) have been widely employed in food industry. Polysaccharides are primarily potent oxygen blocker, which could be attributed to their organized network of bonded hydrogens; however, being hydrophilic in nature, their ability to behave as moisture barriers is limited (Li et al., 2017). Hence, they are blended with other materials to enhance the water evaporation permeability (WVP) properties. Few of the novel combinations of polysaccharide matrix and reinforcements in edible films for extending its functionalities in recent years are listed in Table 27.1.

2.1.1 Cellulose nanocrystal and hemicellulose Recently, among the abundant natural polysaccharides, lignocellulosic materials have been evidenced as suitable material for production of edible film. Cellulose nanocrystals (CNC) acquired from sugarcane bagasse were reported to be used as reinforcement in starch film (Slavutsky and Bertuzzi, 2014), bio-nanocomposite films of carboxymethyl cellulose (CMC)/starch (ST) polysaccharide matrix (El Miri et al., 2015a), and polyvinyl alcohol/carboxymethyl cellulose (PVA/CMC) blend (El Achaby et al., 2017). Films exhibited improved transparency, surface hydrophobicity, tensile properties, and reduced WVP, which are key characteristics for packaging applications. CNC obtained from cotton linter through acid hydrolysis have been exploited to synthesize composite films with improved mechanical and optical features, thermal stability, and water vapor barrier (Chang et al., 2013). Moreover, the work on use of lignocellulosic polysaccharides was also extended to hemicellulose. Biodegradable transparent and translucent films were developed from the hemicellulosic portions of Pinus densiflora leaves in the absence of additives (Shimokawa et al., 2015). Besides, edible coating can also be used to reduce oil uptake in the deep-fried products. The coronary heart disease has been associated with excess of fatty diet; therefore, food coatings applied before frying could assist in diminishing health problems linked to overconsumption of fat. Cellulose derivatives, such as methylcellulose and hydroxypropyl-methylcellulose, which display thermal gelation, can reduce oil absorption by film synthesis (Tavera et al., 2012).

2.1.2 Pectinepectin Apart from lignocellulosic components, pectin is the other polysaccharide gaining the attention of researchers now-a-days for fabricating edible films. Pectin obtained from various vegetable origins such as carrot, apple, and hibiscus could be utilized to synthesize active edible films with antimicrobial

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Chapter 27 Edible films and coatings: an update

Table 27.1 Recent advances in polysaccharide-based edible films. Polysaccharide component Cellulose nanocrystals (CNCs)

Source

Additive

Sugarcane bagasse

Starch (4%), glycerol Carboxymethyl cellulose (CMC)/ starch (ST) polysaccharide matrix Polyvinyl alcohol/ carboxymethyl cellulose (PVA/ CMC) blend e

Cotton linter

Hemicellulose Methylcellulose and hydroxypropylmethylcellulose Pectin

Carrageenan

Properties changed Improved transparency, surface hydrophobicity, tensile properties, and reduced WVP

Sorbitol and glycerol as plasticizers Apple, carrot, and hibiscus Mexican limes

Carvacrol and cinnamaldehyde Lime essential oil

Improved antimicrobial properties

Citrus pectin

Clove bud essential oil (CEO)

e

Silver nanoparticles

k-carrageenan, icarrageenan

Alginate

More resistant to breakage and more flexible and improved antimicrobial properties Better optical, thermal, and antimicrobial activity Improved the moisture barrier and overall tensile properties

Without additives

Slavutsky and Bertuzzi (2014) El Miri et al. (2015)

El Achaby et al. (2017)

Enhancement of optical and mechanical properties, water vapor barrier, and thermal stability High light transmission Reduce oil absorption in fried potato chips

Pinus densiflora leaves

Reference

Chang et al. (2013)

Shimokawa et al. (2015) Tavera et al. (2012)

Ravishankar et al. (2012) ContrerasEsquivel et al. (2015) Nisar et al. (2017)

Shankar et al. (2016)

Paula et al. (2015)

2. Novel patterns in basic structural matrices

679

Table 27.1 Recent advances in polysaccharide-based edible films.dcont’d Polysaccharide component

Source

Additive

Semi-refined kcarrageenan (SRC)

Glycerol Sorbitol

Hybrid carrageenan, with extracted from Mastocarpus stellatus seaweeds Chitosan

Antioxidants

Natural antioxidants from peanut skin (EPS) and pink pepper residue (EPP) extracts Grape seed extract-carvacrol microcapsules Stem, leaf, and seed extracts of Pistacia terebinthus Chitosan nanoparticles

Gums

Chitosan nanoparticles Guar gum

Quinoa protein and thymol

Basil seed gum

Guar gum

Pea starch

Properties changed Decrease in water loss, weight loss, and respiratory rate in coated fruits Enhanced UV barrier, oxygen barrier, and hydrophobic properties Enhance shelf life of meagre (Argyrosomus regius) fillets Enhance shelf life of restructured chicken product

Enhance shelf life of refrigerated Salmon Enhanced its antioxidant and antimicrobial activities Improved film mechanical strength, stiffness, and antimicrobial activity Antimicrobial and water vapor barrier Enhance physicochemical, microbial, and sensorial quality properties of roma tomato Reduced oil uptake and oxidation in shrimp Improved mechanical and

Reference Lasekan et al. (2018) Farhan and Hani (2017) Larotonda et al. (2016)

_ Izci and S¸im¸sek (2018)

Serrano-Leo´n et al. (2018)

Alves et al. (2018)

Kaya et al. (2018)

Gomes et al. (2018)

Medina et al. (2019) Ruelas-Chacon et al. (2017)

Khazaei et al. (2016) Saberi et al. (2017) Continued

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Chapter 27 Edible films and coatings: an update

Table 27.1 Recent advances in polysaccharide-based edible films.dcont’d Polysaccharide component

Source

Additive

Guar gum

Chitosan and whey protein isolate

Xanthan, pullulan, and gellan gums

Starch

Properties changed antimicrobial properties Improved mechanical and antimicrobial properties Enhancing the film strength and resistance to break and preserving starch retrogradation

Reference

Dhumal et al. (2019)

Sapper et al. (2019)

and antioxidant characteristics. The natural additives, such as cinnamaldehyde and carvacrol (Ravishankar et al., 2012) or lime essential oil (Contreras-Esquivel et al., 2015), were used to provide novel nutritional and antimicrobial advantages to the film. Antimicrobial and food-compatible edible film wraps made from apples, carrots, and hibiscus calyces have been demonstrated to inactivate Listeria monocytogenes on readymade meat products (Ravishankar et al., 2012). Some more studies on citrus pectin were also reported recently. One such study revealed the antibacterial activity in edible films derived from pectin of Mexican limes against common food-borne bacteria (Contreras-Esquivel et al., 2015). Although in another study, the influence of clove bud essential oil on characteristics of the citrus pectin films were examined and found to be more resistant, less hydrophilic and highly flexible with improved mechanical, physicochemical, antioxidant, and antimicrobial properties (Nisar et al., 2017). Moreover, an attempt on combining pectin with silver nanoparticles (AgNPs) exhibited great influence on optical and thermal properties of Pectin/AgNPs composite films and powerful antimicrobial activity against food-borne pathogenic bacteria, Escherichia coli and Listeria monocytogenes (Shankar et al., 2016).

2.1.3 Carrageenan Carrageenan is polysaccharide obtained from red seaweed (Rhodophyta). In a seminal study by Paula et al., the impact of relative proportions of i-carrageenan, k-carrageenan, and alginate on physical characteristics of glycerol-plasticized edible films were determined (Paula et al., 2015). Of them, kcarrageenan demonstrated highly improved characteristics in the terms of moisture barrier and overall tensile strength, while i-carrageenan exerted highest impact on opacity. Few similar works on the plasticizing of carrageenan with glycerol (Lasekan et al., 2018) and sorbitol (Farhan and Hani, 2017) reported preservation of fruits with minimal changes in quality and loss in quantity by using a semirefined k-carrageenan (SRC) edible films. Results demonstrated that enhanced carrageenan concentration significantly decreased water and weight loss and respiratory rate in coated fruits. The

2. Novel patterns in basic structural matrices

681

transparency and seal strength of the films were significantly increased with increase in plasticizers concentration from 20% to 30% (Farhan and Hani, 2017). In recent times, as an alternative to commercial k -carrageenan, few studies employed hybrid carrageenan, isolated from Mastocarpus stellatus seaweeds. Films formulated with hybrid carrageenan showed significantly boosted hydrophobic properties and obstruction to UV and oxygen (Larotonda et al., 2016). Thus, for such application, the hybrid carrageenan proved to be a suitable alternative to k-carrageenan.

2.1.4 Chitosan Chitosan is obtained through alkaline deacetylation of chitin, which composes the exoskeleton of crustaceans and molluscs. Recent advances in chitosan film incorporated with several antioxidants are _ reported to enhance shelf life of meagre (Argyrosomus regius) fillets (Izci and S¸im¸sek, 2018), carp fillet (Cyprinus carpio) (Morachis-Valdez et al., 2017), restructured chicken product (Serrano-Leo´n et al., 2018), and refrigerated Salmon (Alves et al., 2018). A recent study reported that edible chitosan films combined with leaf, stem, and seed extracts of Pistacia terebinthus showed an improved antimicrobial and antioxidant activities. The elasticity of chitosan-stem and chitosan-seed films was also improved compared to only chitosan film (Kaya et al., 2018). Moreover, incorporation of chitosan nanoparticles with chitosan into films has opened a new door for development of edible food package films with fine-tuned properties, without the inclusion of plasticizers (Gomes et al., 2018). Following this idea, Medina et al. reported use of chitosan thymol nanoparticles in upgrading the performance of quinoa chitosan/protein edible films to elongate postharvest life of tomato cherries and blueberries (Medina et al., 2019).

2.1.5 Polysaccharide gums The term “gum” refers to polysaccharides composed of sugars other than glucose and capable of causing an increment in a solution’s viscosity to form gels or stabilize emulsion systems. Numerous studies have suggested that natural gums can be casted as edible films owing to their biocompatible biodegradable and sustainable nature. Moreover, they are chemically inert, less expensive, nontoxic, odorless, and widely available. Guar gum composite film was also reported to favorably affect the microbial, physicochemical, and sensorial properties of Roma tomato (Ruelas-Chacon et al., 2017) while basil seed gum reduced oxidation and oil uptake in shrimp while deep-fat frying (Khazaei et al., 2016). Regarding sensory evaluation, these films showed no significant difference in taste, smell, and color. Additionally, two or more gums can be blended together or with other polysaccharides as a coating to provide synergistic effects due to polymerepolymer interaction. Recent advances in the use of gum in edible film includes biphasic film with pea starch-guar gum (Saberi et al., 2017) and triphasic film with chitosan, guar gum, and whey protein isolate (Dhumal et al., 2019) with natural antimicrobial agents for food packaging. The antimicrobial and mechanical features of films showed a greater change in a concentration-dependent fashion, and therefore could be utilized for food preservation improving their shelf life. Moreover, Sapper et al. reported that the mixture of starch and small amounts of the xanthan, pullulan, and gellan gums improves the properties of starch film (Sapper et al., 2019). Edible films prepared from natural gums obtained from some novel sources such as Alyssum homolocarpum seed gum (Mohammadi Nafchi et al., 2017), gum ghatti (Zhang et al., 2016), and Tragacanth gum (Pourmolaie et al., 2018) showed that natural gums are promising materials for the production of edible packaging films.

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Chapter 27 Edible films and coatings: an update

2.2 Protein films Owing to excellent carbon dioxide, oxygen, and lipid barrier properties, the proteins are good films and hold unique characteristics like protecting against moisture and aroma loss. These films are generally synthesized from solutions or dispersions of the protein when solvent/carrier evaporates. The composition of protein films could be tuned to restrain the decrease in intercomponent moisture as well as solute migration in foods. In addition, protein-based coatings can be done in multilayer food packaging materials together with nonedible films or between different layers of components in heterogeneous foods. The commonly used proteins in edible films and their recent advances are listed in Table 27.2.

2.2.1 Collagen Till date, the ultimate commercially successful edible protein films are made up of collagens as they offer several advantages such as biocompatibility, nontoxicity to most tissues, and well-known immunological properties. They can be easily isolated and purified in large quantities, as they constitute about one-third of total body protein in mammals. Few novel research attempts demonstrated the scale-up preparation and characterization of films of collagen/sodium alginate blend. An interesting study reported that the addition of sodium alginate effectively improved the viscosity, thermal stability, and tensile strength of the film but decreased elongation and water vapor permeability (Wang et al., 2017). However, a collagen-chitosan-based biofilm has been documented to display high UV barrier potential and antioxidant activity when compared to only collagen film. This film could be used as green bioactive films to preserve nutraceutical products (Slimane and Sadok Emna, 2018). Another approach included transglutaminase (TGase) catalyzed cross-linking between intra- and interchain lysine and glutamine peptide residues. The thickness, mechanical properties, and thermal stability of the resulting collagen film were found to be highly improved by attaining an equilibrium of denaturation temperature and TGase cross-linking (Cheng et al., 2019).

2.2.2 Zein Zein is a prolamin protein with high content of nonpolar amino acids. The excellent thermoplastic, hydrophobic behavior and high barrier properties make it an excellent biomaterial for the fabrication of biodegradable films. However, the hydrogen, disulfide, and hydrophobic bonds between zein chains result in brittle films and therefore addition of plasticizer is often required for increasing the flexibility. Several reports on zein film has demonstrated its properties as a carrier of antioxidants like plant phenolics, and natural antimicrobial enzymes, including lactoperoxidase and glucose oxidase, lysozyme (LYS), bacteriocins and essential oils. A recent study on LYS-zein films showed its high performance in inhibition of Gram-positive pathogenic bacteria, Listeria monocytogenes (Gao et al., 2017). However, in another study, the edible bilayer films with an additional zein layer casted on the corn-wheat starch films displayed increased thickness, elongation at break, water content, color, and antioxidative capacity while zein thermomodified starches contributed a higher water contact angle and tensile strength to the film (Zuo et al., 2019). A novel research reported that a chitosanezein mixture with three different essential oils added (orange, cinnamon, and anise) when casted to film exhibited excellent mechanical properties, reduced water vapor permeability, and ability to inhibit the growth of Rhizopus sp. and Penicillium sp. (EscamillaGarcı´a et al., 2017).

2. Novel patterns in basic structural matrices

683

Table 27.2 Recent advances in protein-based edible films. Protein component

Additive/modification method

Collagen

Sodium alginate

Chitosan

Zein

Transglutaminase (TGase) cross-linking between intra- and interchain glutamine and lysine peptide residues LYS-zein, starch

Corn-wheat starch films

Chitosan with three different essential oils added (anise, orange, and cinnamon)

Gelatin

Chitosan and procyanidin Chitosan and citric acid

Hydroxytyrosol (HT) and 3,4dihydroxyphenylglycol (DHPG) and pectin

Properties changed

Reference

Higher viscosity and thermal stability tensile strength. Lower elongation and water vapor permeability decreased Higher UV barrier properties, antioxidant elongation. Lower tensile strength, water solubility, and lightness Lower films thickness, higher mechanical properties, and thermal stability

Wang et al. (2017)

High performance in inhibition of Grampositive pathogenic bacteria, such as Listeria monocytogenes Increased color, thickness, water content, elongation at break and antioxidative contact angle, and tensile strength Excellent mechanical properties, reduced water vapor permeability, and ability to inhibit the growth of Penicillium sp. and Rhizopus sp. High antioxidant and antimicrobial activities Increased elongation at break of the films and UV barrier properties, reduced the E. coli growth Reduced formation of oxidation products and film’s oxygen permeability

Gao et al. (2017)

Slimane and Sadok Emna (2018)

Cheng et al. (2019)

Zuo et al. (2019)

Escamilla-Garcı´a et al. (2017)

Ramziia et al. (2017) Uranga et al. (2019)

Bermu´dez-Oria and Rodrı´guez-Gutie´rrez (2019) Continued

684

Chapter 27 Edible films and coatings: an update

Table 27.2 Recent advances in protein-based edible films.dcont’d Protein component

Additive/modification method

Soy protein

Plasticizer

Rapeseed oil

Milk

Halloysite nanotubes (HNTs), poly-vinyl alcohol (PVA), and 1,2,3-propanetrioldiglycidyl-ether (PTGE) Glycerol and sorbitol

Whey protein concentrateepullulan by application of beeswax

Properties changed

Reference

Concentrationdependent thickness and tensile strength, Young’s modulus, and elongation Decrease of water vapor permeability and tensile properties Excellent strength and flexibility

Nandane and Jain (2015)

Higher film thickness, TEs, and water vapor permeability (WVP) Concentrationdependent film thickness, TEs, and water vapor permeability

Wagh et al. (2014)

Galus (2018)

Liu et al. (2017)

Khanzadi et al. (2015)

2.2.3 Gelatin Gelatin is a hydrocolloid commonly used in edible and pharmaceutical applications it forms a thermoreversible substance with a melting point close to human body temperature. Among all the proteinbased films, gelatin film and coatings have been the most extensively studied to protect food against oxygen, light, and oil migration; however, its low water vapor barrier property limits its application. Social and health reasons related to bovine and porcine gelatin has encouraged the use of fish gelatin as an alternative in recent years. Studies on adding chitosan-fish gelatin composite film supplemented with antimicrobial agents like procyanidin (Ramziia et al., 2017) and plasticizer like citric acid (Uranga et al., 2019) showed excellent potential of gelation in food packaging. Moreover, the use of pectin-fish gelatin edible film for preservation of raw beef meat during refrigerated storage was found to be significantly improved by supplementing the film with two antioxidants naturally present in olives, hydroxytyrosol (HT), and 3,4-dihydroxyphenylglycol (DHPG). Moreover, the addition of beeswax to this film facilitated the dropping of oxygen permeability of the film (Bermu´dez-Oria and Rodrı´guez-Gutie´rrez, 2019).

2.2.4 Soy protein films Soy proteins possess high nutritional value and hence it could be a great matrix for edible film, but strong charge and polar interactions between side chains of soy protein make the film stiff. In addition, the hydrophilic properties of soy protein result in films with poor moisture barrier. Hence, substantial

2. Novel patterns in basic structural matrices

685

amount of plasticizer is added to soy protein film to balance the tensile strength and moisture barrier properties. A study on correlation of the mechanical properties of soy protein-based edible film with matrix-plasticizer composition reported that the thickness of film increases with increase in n amount of soy protein isolate while increase in plasticizer and pH reduced the film thickness (Nandane and Jain, 2015). Another approach to enhance the water vapor barrier of films is the incorporation of hydrophobic compounds such as lipids. A bimodal distribution of the oil droplets in film-forming emulsions was observed with increase in the concentration of rapeseed oil in soy protein isolate (SPI) edible films. Addition of oil altered the microstructure of the films from homogeneous and smooth to heterogeneous and rough with high opacity and total color differences (Galus, 2018). In another study, the synergistic effects of the PVA, 1,2,3-propanetriol-diglycidyl-ether (PTGE), and halloysite nanotubes (HNTs) incorporation showed excellent strength and flexibility, water resistance, and thermal stability of SPI films (Liu et al., 2017).

2.2.5 Milk films Milk proteins can be classified into two types: casein and whey protein. Milk protein films show similar moisture barriers to soy protein and wheat gluten films but less moisture barriers than corn zein films (Di Pierro et al., 2018). Caseinate films are better than soy proteins in transparency and flexibility, but have poor water barrier properties. Hence, recent attempts to enhance the moisture barrier property of casein and whey protein concentrate (WPC) films involve the use of plasticizers like glycerol and sorbitol. Increasing the plasticizer concentration, increased the film tensile strain, thickness, and WVP, but reduced the elastic modulus and tensile strength. However, adding plasticizers had no effect on organoleptic quality of Cheddar cheese (Wagh et al., 2014). Another study reported on biodegradable films of WPCepullulan supplemented with beeswax showed that adding beeswax could decrease color differences of films and improved the stressestrain curves for films with 30% beeswax (Khanzadi et al., 2015).

2.3 Lipid films The need to reduce moisture losses from packaged or nonpackaged food materials makes lipids good candidates for edible films and coatings. The apolar nature of lipids makes them excellent barriers against moisture migration (Morillon et al., 2002). Coating produced from blend of lipid with proteins and polysaccharides show higher residences to mechanical and barrier properties. Much research has been done in recent years on various lipid suspensions. The commonly used lipid-based compounds for shielding film/coating include paraffin, herbal wax, beeswax, and acetylated monoglycerides. The recent advances in lipid-based edible films are listed in Table 27.3. Incorporation of lipid in films has previously been reported to enhance tensile strength and water barrier properties. Two recent reports corroborated this finding. In the first study, the effects of oligomeric procyanidins, flaxseed gum (FG), and lauric acid were determined on the mechanical properties, peroxide value, and WVP of edible gumephenolicelipid composite film (Liu et al., 2018); whereas, the second study comprised corn starch-olive oil film with glycerol and vinegar as plasticizers (Jeevahan and Chandrasekaran, 2018). The properties of yuba edible film has been evaluated by researchers after supplementing it with various additives such as emulsifier (sodium pyrophosphate), cross-linking agent (oxidized ferulic acid), plasticizer (glycerol and sorbitol), thickening agent (sodium carboxymethyl cellulose), and lipid (beeswax), either alone or in combination. Depending on

686

Chapter 27 Edible films and coatings: an update

Table 27.3 Recent advances in lipid-based edible films. Lipid component

Additives

Properties changed

Reference

Lauric acid

Gumephenolicelipid composite (flaxseed gum, oligomeric procyanidins and lauric acid) Corn starch-olive oil with glycerol and vinegar as plasticizers Plasticizer (glycerol and sorbitol), cross-linking agent (oxidized ferulic acid), emulsifier (sodium pyrophosphate), thicker (sodium carboxymethyl cellulose) and lipid (beeswax) Gelatin

Water vapor permeability (WVP), mechanical properties, and peroxide value (POV) Enhanced tensile strength and water barrier properties Exceptional edible film properties, including water resistance, elongation, and water vapor permeability

Liu et al. (2018)

Barrier properties, thermal stability, and antioxidant properties

Zhang et al. (2018)

Olive oil

Yuba

Beeswax or carnauba wax

Jeevahan and Chandrasekaran (2018) Kim et al. (2018a,b)

types of additives, the films showed extensive range of applications, but glycerol and sodium carboxymethyl cellulose-added yuba film displayed exceptional film properties, such as elongation, WVP, and water resistance (Kim et al., 2018a). Moreover, the use of beeswax or carnauba wax in gelatin film was found to confer barrier properties, thermal stability, and antioxidant properties (Zhang et al., 2018).

2.4 Nanobiocomposite edible films Polymer nanotechnology is a vibrant area that attempts to develop polymer or copolymer matrix encumbered with specialized nanoscale particulates. Nanocomposites are being extremely investigated to add interesting characteristics in food stuffs packages, and few recent research reports have confirmed that nanobiocomposite film may be a safe alternative for packaging of edibles (Table 27.4). A recent study on the use of nanocomposite edible coating for extension of shelf life of Indian olive (Elaeocarpus floribundus Blume) examined the effects of different concentrations of guar gum matrix on shelf life of Indian olives (Ghosh et al., 2017). Among the different treatments, Guar gum concentration of 1.5% was found to be most effective in considerably delaying the physicochemical parameters of fruit during storage. Few other studies on bionanocomposite films reinforced with CNCs in carboxymethyl cellulose (CMC)/starch (ST) polysaccharide matrix (El Miri et al., 2015b), polyvinyl alcohol/carboxymethyl cellulose (PVA/CMC) blend matrix and chitosan, alginate, and k-carrageenan matrices (El Achaby et al., 2018) showed that the film remains

2. Novel patterns in basic structural matrices

687

Table 27.4 Recent advances in designing of nanobiocomposite edible films. Matrix components

Additive

Properties improved

Reference

Guar gum

e

Ghosh et al. (2017)

Carboxymethyl cellulose starch polysaccharide matrix reinforced Cellulose nanocrystals and polyvinyl alcohol/ carboxymethyl cellulose blend Chitosan, alginate, and k-carrageenan

Cellulose nanocrystals

Low loss in weight and decay percent in the chemical composition of the packed fruits Improved optical transparency, reduced WVP, and enhanced tensile properties Increased the tensile modulus and strength, same transparency level

k-carrageenan film and nanoclay

e

Cellulose microfibers and cellulose nanocrystals production from alfa fibers Zataria multiflora essential oil

Pectin and clove essential oil nanoemulsions

e

Caseinate/zein nanocomposite film

Curcumin

El Miri et al. (2015)

El Achaby et al. (2017)

Mechanically strong and flexible

El Achaby et al.(2018)

Better mechanical, antimicrobial, and WVP properties Mechanical properties, water vapor permeability (WVP), and antibacterial activity Curcumin presence give red-yellow color and antioxidant properties

Shojaee-Aliabadi et al. (2014) Sasaki et al. (2016)

Wang et al. (2019)

transparent due to CNC dispersion at the nanoscale. Furthermore, the addition of CNC resulted in significantly reduced WVP and elevated levels of elastic modulus and tensile strength. Because of the natural occurrence and antibacterial activity of essential oils, few studies on edible bionanocomposite film-made nanoemulsions are reported lately. A film composed of pectin and clove essential oil nanoemulsion has been fabricated. The film displayed interesting improvement in film mechanical properties and antibacterial activity to Escherichia coli and Staphylococcus aureus (Sasaki et al., 2016). Similar improvement in properties have also been reported in other nanobiocomposite film designed from k-carrageenan and nanoclay supplemented with Zataria multiflora essential oil (Shojaee-Aliabadi et al., 2014). Wang et al. reported the synthesis of nanocomposite films with antioxidant property from self-assembled curcumin-loaded sodium caseinate (NaCas)zein composite nanoparticles. The characterization of the film showed that under higher temperatures and long-time storage, curcumin exhibited the highest stability when the zein proportion in the formulation was 50:50 (Wang et al., 2019). Several recent investigations are going-on on nanocomposites with the aim to add new characteristics in food stuffs packages, and the area of nanobiocomposite edible film has numerous new possible combinations in future.

688

Chapter 27 Edible films and coatings: an update

3. Recent advances in the applications of edible films Active packaging is a way of packaging in which the food product, the packaging material, and the environment interact in a positive way to extend the shelf life of food. As defined in the European regulation (EC) No 450/2009, active packaging systems are designed to “deliberately incorporate components that would absorb or release substances from or into the packaged food or the surrounding environment of food” (Yildirim et al., 2018). When food products are stored by active packaging system, the chemical, physical, and biological activities of system alter the condition of food leading to an increase in its shelf life and the without effecting its quality (Pavli et al., 2018). Apart from packaging, the edible films can also be used for numerous other applications such as delivery of active substance and protection of food products from oxidation and microbial growth. The following active packaging systems are used in the food industry:

3.1 Flavor encapsulation The integration of flavoring substances in food products aims to enhance consumer satisfaction. The edible packaging films can also be used to encapsulate flavorings but the degree of encapsulation and release of these compounds largely depends upon film composition and the process used to encapsulate flavoring substance. Natural flavorings substances are molecules that can be obtained from animal or vegetal material by appropriate physical, enzymatic, or microbiological processes. Aroma compounds are usually volatile and expensive; hence, the encapsulation of these molecules in edible coatings could prevent their loss and degradative reactions, such as oxidation during food conservation. Essential oils commonly used for this purpose are obtained from sources such as cinnamon, oregano, coriander, and peppermint. The search for appropriate matrix to carry the flavor compounds showed that emulsion-based edible films can act as carriers of aroma compounds in the form of lipid globules. In addition, compared to the usual lipid-based matrices, the carrageenan films were found to be more efficient in retention of polar aroma compounds. Carrageenans, in the form of beads, gels, and films, are capable of encapsulating flavors and fragrances and reduce their volatility (Chakraborty, 2017). A carrageenan film can preserve the volatile compounds during film-process formation, and then gradually release it with time (Marcuzzo et al., 2010). The emulsified films formed by addition of lipids to iota-carrageenan-based films exhibited lower water vapor and oxygen transfer and improved mechanical properties and stabilization of emulsions (Hambleton et al., 2008).

3.2 Carrier of probiotics A recent study on edible films and coatings as carriers of living microorganisms shows that it could be a new strategy toward biopreservation and healthier foods (Guimara˜es et al., 2018). Although more than 500 probiotic food products exist in the market worldwide (Tripathi and Giri, 2014), they differ country wise as they need to fit on the legislation requisites of the nation. Species of Lactobacillus and Bifidobacterium are most commonly used as probiotics in most countries, while E. coli, some Bacillus species and the yeast Saccharomyces boulardii are also used (Pavli et al., 2018). Table 27.5 lists the recently published works on encapsulation of probiotics in edible films. Numerous studies were reported on incorporation of probiotics in edible films using the various matrices. In this context, L rhamnosus encapsulated in sodium carboxymethyl cellulose and hydroxyethyl cellulose film cross-

3. Recent advances in the applications of edible films

689

Table 27.5 Recent advances in edible films encapsulating probiotics. Probiotic

Matrix composition

Properties changed

Reference

L rhamnosus

Sodium carboxymethyl cellulose and hydroxyethyl cellulose film cross-linked with citric acid (CA) Carboxymethyl cellulose (CMC)

Tunable mechanical properties and storage stability

Singh et al. (2019)

More water vapor permeability (WVP) and opacity, and less tensile strength (TS) and elongation at break (EB) Higher antilisterial activity and improves the appearance of films Storage stability

Ebrahimi et al. (2017)

Lactobacillus acidophilus, L. casei, L. rhamnosus, and Bifidobacterium bifidum Lactobacillus acidophilus and Lactobacillus casei cells Lactobacillus rhamnosus GG

Bifidobacterium animalis and Lactobacillus

Sodium caseinate

Low and high viscosity sodium alginate, low esterified amidated pectin, k-carrageenan/ locust bean gum, whey protein concentrate, and gelatin) Whey protein isolate

Storage stability

Abdollahzade and Al. (2018) Soukoulis and Behboudi-Jobbehdar (2017)

Odila Pereira et al. (2016)

linked with citric acid confirmed that the mechanical properties and storage stability of film can be tuned by varying the composition of matrix (Singh et al., 2019). Ebrahimi et al. reported that the carboxymethyl cellulose film incorporating four probiotic strains (Lactobacillus casei, L. acidophilus, L. rhamnosus and Bifidobacterium bifidum) showed high water vapor permeability and opacity, but low tensile strength and elongation compared to the control film and cell viability was found to be stable till 42 days (Ebrahimi et al., 2017). However, in contrast to this, Abdollahzadeh et al. reported no difference in tensile strength in sodium caseinate film containing L. acidophilus and casei with stability in cell viability only up to 12 days (Abdollahzade and Al., 2018). The comparison of the ability of various films matrices such as low esterified amidated pectin, sodium alginate, gelatin, k-carrageenan/locust bean gum, and WPC to encapsulate L. rhamnosus GG revealed that the k-carrageenan/ locust bean gum showed highest storage stability while pectin showed lowest (Soukoulis and Behboudi-Jobbehdar, 2017). Moreover, the formulation of whey protein isolate edible with bioactive compounds opens new prospects as a carrier for lactic acid bacteria because the organisms, Bifidobacterium animalis and Lactobacillus were found to remain viable for a longer time without any effect on the mechanical properties of the film (Odila Pereira et al., 2016). In future, it is crucial to investigate the details of the properties of the matrix materials to effectively protect the incorporated probiotic organism. In addition, the symbiotic (combination of probiotics and prebiotics) in edible films and coatings will be a thought-provoking area to extend their use in food industry.

690

Chapter 27 Edible films and coatings: an update

3.3 Carriers of antioxidant and antimicrobial compounds In the last few years, the incorporation of antioxidants and antimicrobial compounds has led to the development of a new generation of active edible films that could improve the food quality and extend their shelf life. These compounds differ in their mechanism of actions pertaining to their structure, the targeted microorganism, the choice of biopolymer and the process of encapsulation (Benbettaı¨eb et al., 2018). Edible films with antimicrobial or antioxidative agents could reduce the need of preservatives added in the food by controlling the diffusion of active compounds at the surface of food. Methanol extracts of Pistacia terebinthus stem, leaf, and seed, was reported to provide antioxidative and antimicrobial properties to chitosan-based films as they are rich in various phenolic compounds (Kaya et al., 2018). The blend films with Pistacia terebinthus extracts were eco-friendly in nature and can be easily incorporated in food packaging materials. Another study on chitosan triphasic films with whey protein isolate and guar gum, supplemented with eugenol, citral, and carvacrol as the oil phase showed good antimicrobial activity when (Dhumal et al., 2019). When chitosan and chitosan-nanoparticles

Table 27.6 Recent advances in edible films carrying antioxidant and antimicrobial compounds.

Matrix components

Antioxidant/ antimicrobial compound

Chitosan

Methanol extracts of stem, leaf, and seed obtained from Pistacia terebinthus

Guar gum, chitosan, and whey protein isolate

Eugenol, carvacrol, and citral

Chitosan

Chitosan-nanoparticles

Apple, carrot, and hibiscus-based pectin edible films against

Carvacrol and cinnamaldehyde

Pectin

Clove essential oil

Pea starch and guar gum

Epigallocatechin-3gallate, blueberry ash fruit, and macadamia (MAC) skin extracts Montmorillonite nanoclay and zataria multiflora Boiss essential oil

k-carrageenan

Properties improved

Reference

Enhanced the antioxidant and antimicrobial activities and elasticity of chitosan-seed and chitosan-stem films Improved mechanical and antimicrobial properties Improved antimicrobial properties Improved antimicrobial properties against Listeria monocytogenes on contaminated ham and bologna Antibacterial activity against E. coli and S. aureus Antimicrobial activity against S. typhimurium and Rhizopus sp

Kaya et al. (2018)

Improved mechanical and antimicrobial properties

Shojaee-Aliabadi et al. (2014)

Dhumal et al. (2019)

Gomes et al. (2018) Ravishankar et al. (2012)

Sasaki et al. (2016)

Saberi et al. (2017)

References

691

were combined to prepare biobased and unplasticized film blends, it displayed antimicrobial activity against both gram-negative and gram-positive bacteria (Gomes et al., 2018). Moreover, a study on incorporation of the antimicrobial agents carvacrol and cinnamaldehyde, into carrot, apple, and hibiscus-based edible films showed protection against Listeria monocytogenes on contaminated ham and bologna. Moreover, the carvacrol films showed higher activity against Listeria monocytogenes than cinnamaldehyde films. This study provides a scientific basis on the development of fruit- and vegetable-based antimicrobial films for microbial food safety (Ravishankar et al., 2012). In a similar work, cocoa nibs extract imparted antioxidant property to starch extracted from adzuki bean film. The addition of cocoa nibs extract increased the elongation at break and storage stability of the adzuki bean films (Kim et al., 2018b). Other recent attempts to develop antimicrobial films include adding clove oil to pectin film (Sasaki et al., 2016), epigallocatechin-3-gallate and blueberry ash fruit and macadamia skin extracts to pea starch, and guar gum composite film (Saberi et al., 2017) and nanoclay and Zataria multiflora essential oil to nanobiocomposite k-carrageenan film (Shojaee-Aliabadi et al., 2014). Some novel antimicrobial agents have also been recently incorporated in packaging films such as allyl isothiocyanate, lauric arginate ester (Guo et al., 2017), and nisin peptide (Wu et al., 2019), which show activity against Gram-negative and Gram-positive bacteria (Table 27.6).

4. Conclusion The food grown in field or manufactured in industries takes considerable time to reach the table of consumer. As a result, the products remain with transportation distribution systems and supermarkets for long time and during which they begin to dry, decay, and lose flavor and nutritional contents. Therefore, an efficient food packaging methods is the only key to prevent food deterioration. Current approaches of packaging food with petroleum-based materials not only create environmental concerns but also restrict the function of films only for packaging. However, films and coatings synthesized from edible components (polysaccharides, protein, and lipids) has emerged as a good approach for the improvement of viability of encapsulated living microorganisms such as probiotics, during food storage and processing. The base material as well as plasticizers or other additives used may affect the mechanical and physicochemical properties of films and coatings. Recently, several new formulations such as emulsions and micro or nanoencapsulations are being developed to be casted to film, and that can enhance the distribution and stability of the encapsulated probiotics, aroma, and antioxidants; however, an extensive research is still needed. In future, the alteration in consumer needs can direct this area toward many solutions with almost infinite combinations of materials, method, and encapsulates.

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Serrano-Leo´n, J.S., Bergamaschi, K.B., Yoshida, C.M.P., Saldan˜a, E., Selani, M.M., Rios-Mera, J.D., Alencar, S.M., Contreras-Castillo, C.J., 2018. Chitosan active films containing agro-industrial residue extracts for shelf life extension of chicken restructured product. Food Research International 108, 93e100. Shankar, S., Tanomrod, N., Rawdkuen, S., Rhim, J.-W., 2016. Preparation of pectin/silver nanoparticles composite films with UV-light barrier and properties. International Journal of Biological Macromolecules 92, 842e849. Sharma, R., Ghoshal, G., 2018. Emerging Trends in Food Packaging. Shimokawa, T., Togawa, E., Kakegawa, K., Kato, A., Hayashi, N., 2015. Film formation and some structural features of hemicellulose fractions from pinus densiflora leaves. Journal of Wood Science 61, 53e59. Shojaee-Aliabadi, S., Mohammadifar, M.A., Hosseini, H., Mohammadi, A., Ghasemlou, M., Hosseini, S.M., Haghshenas, M., Khaksar, R., 2014. Characterization of nanobiocomposite kappa-carrageenan film with Zataria multiflora essential oil and nanoclay. International Journal of Biological Macromolecules 69, 282e289. Singh, P., Magalha˜es, S., Alves, L., Antunes, F., Miguel, M., Lindman, B., Medronho, B., 2019. Cellulose-based edible films for probiotic entrapment. Food Hydrocolloids 88, 68e74. Slavutsky, A.M., Bertuzzi, M.A., 2014. Water barrier properties of starch films reinforced with cellulose nanocrystals obtained from sugarcane bagasse. Carbohydrate Polymers 110, 53e61. Slimane, B.E.N., Sadok Emna, S., 2018. Collagen from cartilaginous fish by-products for a potential application in bioactive film composite. Marine Drugs 16, 211. Soukoulis, C., Behboudi-Jobbehdar, E.A., 2017. Stability of Lactobacillus rhamnosus GG incorporated in edible films: impact of anionic biopolymers and whey protein concentrate. Food Hydrocolloids 70, 345e355. Tavera, M., Urriza, M., Pinotti, A., Bertola, N., 2012. Plasticized methylcellulose coating for reducing oil uptake in potato chips. Journal of the Science of Food and Agriculture. Tripathi, M.K., Giri, S.K., 2014. Probiotic functional foods: Survival of probiotics during processing and storage. Journal of Functional Foods 9, 225e241. https://doi.org/10.1016/j.jff.2014.04.030. Uranga, J., Puertas, A.I., Etxabide, A., Duen˜as, M.T., Guerrero, P., de la Caba, K., 2019. Citric acid-incorporated fish gelatin/chitosan composite films. Food Hydrocolloids 86, 95e103. Wagh, Y.R., Pushpadass, H.A., Emerald, F.M.E., Nath, B.S., 2014. Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese. Journal of Food Science and Technology 51, 3767e3775. Wang, L., Xue, J., Zhang, Y., 2019. Preparation and characterization of curcumin loaded caseinate/zein nanocomposite film using pH-driven method. Industrial Crops and Products 130, 71e80. Wang, Z., Hu, S., Wang, H., 2017. Scale-up preparation and characterization of collagen/sodium alginate blend films. Journal of Food Quality 2017, 10. Wu, Y., Li, Q., Zhang, X., Li, Y., Li, B., Liu, S., 2019. Cellulose-based peptidopolysaccharides as cationic antimicrobial package films. International Journal of Biological Macromolecules 128, 673e680. Yildirim, S., Ro¨cker, B., Pettersen, M.K., Nilsen-Nygaard, J., Ayhan, Z., Rutkaite, R., Radusin, T., Suminska, P., Marcos, B., Coma, V., 2018. Active packaging applications for food. Comprehensive Reviews in Food Science and Food Safety 17, 165e199. Zhang, P., Zhao, Y., Shi, Q., 2016. Characterization of a novel edible film based on gum ghatti: effect of plasticizer type and concentration. Carbohydrate Polymers 153, 345e355. Zhang, Y., Simpson, B.K., Dumont, M.-J., 2018. Effect of beeswax and carnauba wax addition on properties of gelatin films: a comparative study. Food Bioscience 26, 88e95. Zuo, G., Song, X., Chen, F., Shen, Z., 2019. Physical and structural characterization of edible bilayer films made with zein and corn-wheat starch. Journal of the Saudi Society of Agricultural Sciences 18 (3), 324e331.

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Rheology and tribology assessment of foods: a food oral processing perspective

28

Rituja Upadhyay1, 2, Jianshe Chen2 1

School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, China; 2School of Food Science, University of Idaho, Moscow, ID, United States

1. Introduction Variety of food products available in the market are in the form of gels, for example, yoghurt, mayonnaise, custards, sauces, etc., making them a good representation of the food products commercially available. Hence, most of the common prototypical food systems that are used for research studies are emulsions, gels/hydrogels, fluid gels, emulsion-filled gels, etc. Since gelation mechanism of biopolymers (composed of proteins, polysaccharides, and combinations) is considerably known, biopolymer gels can be considered as a suitable model system, in this framework, to link rheological properties to food structure and texture, and therefore, in a sensorial test, samples meet the requirements for parameter variations. This chapter will have a brief discussion on a few rheological tests most relevant and of interest in food industry on gels and emulsions. Emulsions are dispersion of one liquid phase in another immiscible liquid phase in the form of fine droplets. These emulsions are broadly classified as oil-in-water or water-in-oil emulsions, depending on the phase dispersed. Ice cream, butter, milk cream, margarine, salad dressing, and meat emulsions are the most common food emulsions (Barbosa-Ca´novas et al., 1996). Gels, on the other hand, cover the gamut of viscoelastic materials that exhibit no steady-state flow. They typically contain dispersed particles and macromolecules forming a network structure that immobilizes the liquid, present in substantial quantity, and produces solid-like properties. Flory (1953) defined gel as a soft, solid, or solid-like material of two or more components, one of which is a liquid present in substantial quantity. Emulsion gel is a major type of semisolid food, in which oil/fat droplets are embedded into the gel matrix either as the so-called active fillers or inactive fillers (Chen and Dickinson, 1999). At the molecular level, gelation arises due to the network formation of polymer chains (primarily polysaccharides and proteins) either from chemical or physical cross-linking due to covalent reaction or through polymerepolymer interactions, respectively (Fig. 28.1). Depending on the gelation mechanism, food biopolymers can be divided into “cold set” and “heat set.” “Hydrocolloids” is the common term used in the food industry, but these are also referred to as “biopolymers.” They will be referred to as the “biopolymers” throughout this chapter. The major reason behind the extensive use of biopolymers in foods is their ability to modify the rheology of food Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00028-X Copyright © 2020 Elsevier Inc. All rights reserved.

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FIGURE 28.1 Classification of gelation mechanism and relevant examples. Adapted from Syed, K.H.G, Saphwan, A. & Glyn, O.P. (2011) Hydrogels: methods of preparation, characterization and applications C. Angelo Ed., Progress in Molecular and Environmental BioengineeringdFrom Analysis and Modeling to Technology Applications, InTech, Rijeka, pp. 117e150 under CC BY-NC-SA 3.0 license. Available from: https://dx.doi.org/10.5772/24553.

system viz. flow behavior (viscosity) and mechanical property (texture). The texture and/or viscosity modification in a food system helps manipulate its sensory properties and, therefore, have the desired and tailored sensory experience. Polymers have been characterized using rheological techniques and methods for many decades. This chapter will focus on introducing rheology basics and discussing the rheological aspects, lubrication behavior of foods, and the role of saliva in food oral processing. This chapter shall also describe the relevant rheological tests for characterizing foods, viz. fluid, semifluid, and soft solids along with an emphasis on oral behavior of foods.

2. Rheology: basic understanding of the tests used in the food industry On a laboratory scale, rheological measurements are done with the help of rheometer wherein a welldefined deformation either shear or extensional can be imposed and the resulting stress response of the fluid is measured. This is called strain-controlled rheometry. Alternatively, a shear stress or an extensional stress is applied on the fluid and the corresponding strain response is measured. This is called stress-controlled rheometry. The imposed flow could be a steady shear flow or a dynamic oscillatory flow. Rheology offers far more than just viscosity measurements. A formulation can be refined and scaled up for commercial use by switching from a simple viscometer to a rheometer,

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FIGURE 28.2 Behavior of fluid and semifluid foods. (A) Simple deformations and the response of materials and (B) Rheological behavior of liquid foods. Reproduced from Sala, G. & Scholten, E. (2015). Instrumental characterization of textural properties of fluid food. Modifying Food Texture, vol. 2. second ed., Elsevier Ltd, with permission from Elsevier.

providing access to a range of extended test capabilities. Exploiting the full potential of rheology has been restricted due to the lack of understanding of the choice of test and the interpretation of the resulting data. Fig. 28.2 provides an overview of the behavior of fluid and semifluid foods. Food gel behavior can be studied using stressestrain tests and generally can be categorized as two types, small-strain testing and large-strain testing. Rotational rheometers enable oscillatory testing (shear force or displacement as sinusoidal) unlike rotational viscometers (shear in one rotational direction). Food scientists and product developers commonly use two types of experimental tests to characterize the viscoelastic properties of foods: stationary and dynamic measurements. Tests with oscillatory stresses or strains are often named “dynamic” tests. Dynamic testing, i.e., either controlled rate or controlled stress, is very popular for measuring the viscoelasticity of foods, including gel strength, starch gelatinization, protein coagulation, dough formation, shelf life testing, etc. Dynamic oscillatory shear tests may be broken down into two categories: small amplitude oscillatory shear (SAOS) and large amplitude oscillatory shear (LAOS). The main difference between SAOS and LAOS is that SAOS testing is performed in the linear viscoelastic region and are supported by a strong theoretical basis, while LAOS testing is performed beyond this region of linearity (Figs. 28.3 and 28.4). Here we shall focus on small amplitude tests which are widely used by food industry, while LAOS, although becoming popular, is beyond the scope of this chapter. Distinction between “strong” and “weak” gels can be made using small deformation oscillatory measurements applicable for the gels, thickeners, and stabilizers. The viscoelasticity of gels is characterized by determining G0 and G00 in the linear viscoelastic region; no other method gives dynamic moduli values. The complex shear modulus (G ¼ G0 þ G 00 ) describes the dynamic shear rheological properties of a material, where the parameters G0 and G00 are referred to as the storage modulus and loss modulus, respectively. G0 ¼ elastic behavior of the material tested represented as the energy stored may be interpreted as the component in phase with the G00 .

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FIGURE 28.3 Schematic representation of the dependence of the dynamic (G0 and G00 ) moduli with time or temperature and the critical value given by linear viscoelasticity limits.

G00 ¼ viscous behavior of the material tested and refers to the amount of energy dissipated and is the component out of phase with the strain (viscous behavior). d ¼ phase angle corresponding to the arc tangent of the ratio G00 /G0 , and it is a measurement of the response delay of strain to the applied stress. The phase angle indicates the relative importance of the viscous and elastic elements in the emulsionfilled gels (Geremias-Andrade et al., 2016). The time scale and the type of deformation applied are the factors responsible for the degree of viscoelasticity exhibited by non-Newtonian fluid food.

FIGURE 28.4 Schematic illustration of the strain sweep test at a fixed frequency. This sweep test can be used for determining the linear and nonlinear viscoelastic region. Hyun, K., Kim, W., 2011. A new non-linear parameter Q from FT-Rheology under nonlinear dynamic oscillatory shear for polymer melts system. Korea-Australia Rheology Journal 23, 227. https://doi.org/10.1007/s13367-011-0028-0.

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2.1 Flow properties of fluid foods using rotational tests For fluids the stress depends upon the rate of change of strain with time rather than the amount of deformation. Fluid food behavior can usually be described by plotting the shear stress against shear rate having direct proportionality between the two (flow curve). The proportionality constant is equal to the viscosity (h) of the material. The viscosity curve, which is a plot of viscosity versus shear rate, will show a straight line at a concentration value equal to h. However, the presence of macromolecules and moderate concentrations of dispersed solids may give rise to non-Newtonian behavior under shear, mostly shear thinning. These materials cannot be defined by a single viscosity value at a specific temperature. Apart from being shear rate dependent, the viscosity of non-Newtonian fluids may also be time dependent, in which case the viscosity is a function not only of the magnitude of the shear rate but also the duration and, in most cases, of the frequency of successive applications of shear. The viscosity of shear thinning decreases with increasing shear rate. Most liquid food systems belong to this category of fluids. The shear rate dependency of the viscosity can differ substantially between different products, and for a given liquid, depending on temperature and concentration. The reason for shear thinning flow behavior is that an increased shear rate deforms and/or rearranges particles, resulting in lower flow resistance and consequently lower viscosity. A traditional method to assess the mouthfeel of a fluid uses a rheometer to measure viscosity as a function of shear rate (Fig. 28.5; Shama and Sherman, 1973). Shama and Sherman (1973) made an important contribution by establishing a link between rheological properties and the sensory perception. They studied sensory perceived thickness of a wide range of fluid foods and established a master curve of shear deformation of fluid foods during oral processing (Fig. 28.6). Shama and Sherman (1973) indicated that thickness perception of fluid foods is likely to correspond to the viscosity of the sample measured at a rate between 10 s1 (for highly viscous fluids) and 1000 s1 (for very thin fluids) using both subjective approach (oral sensory tests by 26 panelists) and

FIGURE 28.5 Shear stresseshear rate bounds in which fluid foods are likely to be orally evaluated. Reproduced with permission from Shama, F. & Sherman, P. (1973). Identification of stimuli controlling the sensory evaluation of viscosity II. Oral methods. Journal of Texture Studies, 4(1), 111e118.

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FIGURE 28.6 A master curve for shear stress and shear rate associated with the oral viscosity evaluation of various foods. Shama, F. & Sherman, P. (1973). Identification of stimuli controlling the sensory evaluation of viscosity II. Oral methods. Journal of Texture Studies, 4(1), 111e118.

objective approach (viscometer) (Shama and Sherman, 1973). According to this, the characteristic shear rate of a given food depends on its flow characteristics. Around the curve is a zone where shear stress has the best correlation with sensory properties. It has been proposed that dynamic small deformation measurements at an oscillatory frequency of 50 s1 had a good correlation with perceived thickness, stickiness, and sliminess for a wide range of food products including Newtonian fluids, true solutions, weak gels, flocculated emulsions, and lemon pie filling (Shama and Sherman, 1973). It has been observed that some aspects of the sensory mouthfeel, e.g., creaminess, smoothness, do not correlate with viscosity behavior of the food (Baier et al., 2009; Kokini, 1987).

2.1.1 Zero shear viscosity and yield stress point determination Zero shear viscosity is the viscosity of the material when it is effectively at rest (Fig. 28.7A). Zero shear viscosity describes a plateau value which you sometimes see when you use a powered rheometer to measure viscosity under extremely low shear conditions. Most liquid and semisolid foods spend significant amount of their resistance under these low shear conditions. Therefore, arguably, this makes zero shear viscosity one of the best rheological methods for describing how products behave in storage. Foods such as mayonnaise, yoghurt, sauces can exhibit solid- or gel-like behavior at rest but begin to flow once the shear applied to them exceeds a certain value. While designing a new product and benchmarking product performance relating to customer experience, quantifying and controlling yield stress become extremely useful. For these products, yield stress correlates with the force needed to dispense the sauce from the bottle. The transition from solid- to liquid-like behavior is marked by the yield stress. Solid-like behavior at rest often imparts consumer-appealing structure, making a product look thicker and of perceived higher quality. These examples of materials are rheologically classified as “soft solids.” These materials, when subjected to very small deformations, are characterized by a relatively low modulus, but they possess solid properties due to such an internal structure and a highly

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FIGURE 28.7 A. Zero shear viscosity and apparent yield stress.

elastic response. They, however, exhibit a complex flow behavior if larger deformations are applied. A range of methods are available to determine yield stress with a modern rotational rheometer, but such testing is limited on a typical rotational viscometer.

2.2 Small amplitude oscillatory tests Strain or stress sweep, frequency sweep, isothermal time sweep, and temperature sweep tests fall under the SAOS test category. Strain/stress sweep tests are performed to define the limits of linear viscoelasticity, where the material is nondestructive in nature as discussed in the previous sections (Fig. 28.8). Grouping materials as dilute solutions, concentrated suspensions, or gels permits the following generalizations with respect to their storage and loss moduli: • •

•

With dilute solutions, G00 is larger than G0 for the entire frequency sweep. However, at the larger frequencies, the moduli approach each other. For concentrated solutions, G00 starts greater than G0 and at some point, they cross over. This cross over point is a function of the frequency and may be an important parameter when describing the material. For gels, G0 and G00 are independent of frequency and run parallel to each other with G0 being larger than G00 . Generally, the gel point is identified when the storage and loss moduli cross over. However, gelation point should be independent of anything. At the gel point, G00 and G0 are parallel to each other regardless of frequency, u (strong gel).

Storage and loss moduli exhibit power law dependence on frequency at gel point (Cuvelier and Launay, 1990) with an exponent varying from 0.5 to 0.65. Gel point can be determined precisely by a combination of the variation of log(tan d ¼ G00 /G0 ) versus the gelation (or melting) parameter (time or temperature) at different frequencies leads. Among the range of liquids, viscoelastic liquids have the worst stability because under low frequencies the phase angle is escalating. A high phase angle signifies that the material and the suspended particles can sediment and settle if left long enough. To reduce this effect, a gel-like system will demonstrate a more solid-like behavior at low frequencies.

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FIGURE 28.8 Microstructure of colloidal dispersions and the behavior of G0 and G00 as functions of angular frequency for (A) a stable dispersion, (B) a weakly flocculated dispersion, and (C) a strongly flocculated dispersion or a gel. Adapted from Khan et al. (1997).

3. Tribology fundamentals 3.1 Background and principle Rheology is a powerful tool, used for decades now, to link food physical properties, structure, and food texture. Yield stresses, flow profiles, and fracture behavior of firm solids are relatively easily determined with standard rheometry. However, there are still aspects of food texture that cannot be measured via standard rheological testing. Food texture in the later stages of mastication, when the food is being prepared for swallowing, shows poor correlation to mechanical measurements. One of the major objectives of food tribological research is to relate food sensory texture to mechanical friction measurements. In recent time, tribology has been a potential tool to uncover and instrumentally measure physical properties of foods that relate to mouthfeel during oral processing. Although food tribology is quite recent, this approach has been used for evaluating and understanding the perceived oral texture of semisolid foods Chojnicka (2008) and (2006). It is useful to consider the basic deformation processes that occur in the mouth (Fig. 28.9) during consumption to appreciate how the rheology of food and beverages is important in oral processing. Conventional tribometers are generally only configured to determine a frictional force response of a sheared sample as a function of an applied velocity under a constant normal load. The results of tribology measurements are therefore commonly reported in terms of quantities that do not require knowledge of the size, shape, or separation of the shearing surface. Stribeck curve is used in tribology which is obtained by plotting friction coefficient against sliding speed to observe the food behavior in the oral cavity (Fig. 28.10). This curve is divided into three

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FIGURE 28.9 Schematic of the interactions between the tongue and the palate. The right-hand side shows idealized depictions of the fluid dynamics taking place as the tongue moves. Slightly modified. Reproduced from Food Oral Processing: Fundamentals of eating and Sensory perception, First edition. Edited by Jianshe Chen.

regimes: boundary layer (BL), mixed, and hydrodynamic regime. In the BL regime, there is not a much change of coefficient of friction with the increase in the sliding speed, but in the mixed region, there is a decrease in the friction coefficient with an increase in the sliding speed. However, in the hydrodynamic regime, there is again an increase in the friction coefficient with the increase in the sliding

FIGURE 28.10 Typical Stribeck curve as a function of film thickness or the parameter h/W (h is viscosity, U is entrainment speed, and W is load). Adapted from Upadhyay, R., Chen, J. & Brossard, N. (2016). Mechanisms underlying astringency: introduction to an oral tribology approach. Journal of Physics. D: Applied Physics, 49, 104003.

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speed, because to keep the normal force constant as sliding speed increases the gap size increases. When the gap increases, more lubricant fluid comes in between the gap size; therefore, there is an increase in the friction coefficient due to increase in the amount of lubricant in the gap size because the viscosity of the fluid is also a kind of friction, which adds up in the friction of system. The quantity of fluid in between the surface varies in the following order: hydrodynamic regime > mixed > BL regime. This is because with the increase in the sliding speed the gap size increases to keep the normal force constant, and thus more liquid enters the gap in between the two surfaces. Stribeck curve showing changes in friction and how the saliva film thickness could be altered depending on the film viscosity and how this is affected by load. The type of lubrication is indicated above the graph. The curve can be typically divided into three regimesdthe boundary regime, the mixed regime, and the hydrodynamic regime, representing three very different friction scenarios and, in case of oral processing, different amount of food sample between the tongue and palate (Stokes, 2012; Chen et al. 2014). Boundary lubrication regime: the shear stress is virtually independent of the relative velocity between the shearing surfaces and hence independent of the nominal shear rate. This is counterintuitive for the rheologist for whom rate in dependent stresses is normally encountered only for idealized yielding processes, but such observations are a regular tribological result of a sliding friction process for which the friction coefficient m is constant and independent of the velocity difference between the shearing surfaces. Hydrodynamic or elastohydrodynamic lubrication (EHL) regime: rheological properties of the lubricant film in the contact define the friction behavior, at the high shear rate condition. In a typical tribological experiment, the pressure that arises from hydrodynamic lubrication in this regime forces the shearing surfaces apart against the applied normal load until, at a certain fluid film thickness, the two forces balance. The stresses that arise from the sheared fluid film are larger than any direct frictional stress between the surfaces. Not surprisingly, the hydrodynamic lubrication regimes measured in these simple fluids directly match the bulk flow curves, independent of the gap setting, a feature that has been utilized to obtain the high shear rate part of the flow curve. Mixed lubrication regime: in general, the mixed lubrication regime results in a lower coefficient of friction than boundary lubrication and a nonmonotonous variation of the shear stress with the shear rate. This is again counterintuitive to the rheologist on a first glance who would demand a monotonous increase of stress with rate for simple shear. However, in tribological terms, a transition from a static friction, or “stick-slip motion,” to a sliding friction when increasing the sliding velocity goes along with a reduction of the shear stress, thus allowing a nonmonotonous variation. Gelled emulsions have been characterized for their mechanical properties by many researchers (Dickinson et al., 1985; Lorenzo et al., 2011; Sala et al. 2007, 2009) for mouthfeel. The unique sensory feature of such gel systems is the release of oil droplets during eating, which will not only lead to an enhanced oil/fat sensation but will also alter the lubrication behavior inside the oral cavity. During oral processing, the release rate of the oil droplets that are strongly associated with the gel matrix (active fillers) is predominantly determined by the melting of the gel matrix. However, in the case of unbound oil droplets (inactive fillers), the release rate appears to depend on the size of the shear-disrupted gel matrix fragments. In emulsion-filled gels, sensorial properties of the systems are impacted greatly by the gel firmness and the fracture properties (Sala et al., 2008). Gel firmness (E) and brittleness, dependent on fracture or yield stress and on apparent modulus, are the common oral processing properties in the initial phase of oral processing, measured at large deformation (van Vliet

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and Walstra, 1995; Brandt et al., 1963; Kramer and Szczesniak, 1973). de Lavergne et al. (2016) investigated how the composition of emulsion-filled gels and how their fracture properties can influence the dynamic textural perception of the systems.

3.2 Tongue movements and role of saliva Tongue movements dominate the semisolid foods’ oral processing behavior unlike mastication, which occurs for hard-solid foods. The shear stress of the tongue and lower jaw is the main component during oral processing of semisolid foods. The tongue performs compression and decompression movements while moving the semisolid foods against the hard palate along with lateral movements, a deformation dominantly shear which is very similar to that occurring between the upper and lower plates of a rheometer. Therefore, shear deformation tests are used extensively to predict oral texture sensation of fluid and semisolid foods (and sometimes it does very well). All these oral manipulations result in shear and elongational deformation of the food bolus (van Vliet, 2002). However, once bolus is swallowed, there was no longer bulk deformation but a thin layer lubrication within the oral cavity. In this case, tribology is believed to be a dominating mechanism for oral texture sensation. The exact pattern of forces and velocities caused by the oral movements is still largely unknown. Shama and Sherman (1973), in a pioneering study, showed that for different semisolid food products not only the amount but also the type of oral deformation varies. The oral movements, forces, and tongue speed result in mechanical deformation and structural breakdown of the semisolid food. Mixing with saliva further affects the breakdown. The role of saliva in oral processing is multifold. Saliva corresponds to complex physiological secretion, which performs functions such as oral health protection, lubrication of the oral tissues, and the food predigestion. The tribological properties of saliva, thus, cannot be ignored. A fair amount of research is published on the influence of load, presence of surfactants, and substrate roughness on lubrication properties of saliva (Macakova et al. 2010; Gibbins et al., 2013). The viscoelasticity and rate of secretion of saliva depend on food and beverage-related stimuli, and it is demonstrated that this may subsequently influence the sensory properties, particularly the mouthfeel and after-feel associated with the product being consumed. Thus, knowledge of the contribution of the different salivary components in the oral process is a key point in understanding the detection of fat in the mouth (Fig. 28.11).

3.3 Tribology configurations Currently, there are a relatively small number of techniques that are being used in the food application to study tribological properties of structures. These range from simple configurations sliding one surface over another at a fixed speed through rheometer geometries to custom tribology equipment, allowing a full range of parameters such as surfaces, speeds, and geometries to be controlled.

3.3.1 Mount tribological device (a ball-on-three-plates principle) The capability of mount tribological device for lubrication studies has been validated with a range of Newtonian and non-Newtonian fluid materials. Tests using fat-in-water emulsions thickened with

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FIGURE 28.11 Foodesaliva interactions and impacts on eating and sensory perception. Adapted from Aktar T., Upadhyay R., Chen J. (2019) Sensory and Oral Processing of Semisolid Foods. In: Joyner H. (eds) Rheology of Semisolid Foods. Food Engineering Series. Springer, Cham.

maltodextrin or xanthan gum showed a relationship between friction factor and human creaminess perception (Fig. 28.12). Malone et al. (2003) worked with guar gum and sunflower oil to correlate the sensory attributes like fattiness and slipperiness perception with the lubrication behavior using friction coefficient as the parameter. The slipperiness scores obtained for guar gum solutions (0.05%, 0.2%, 0.4%, and 0.6%) were compared with the friction measurements from mini-traction machine (MTM). The Stribeck curves shown in Fig. 28.13 demonstrate that the biopolymer significantly reduces the friction coefficient in the mixed region. According to them, two mechanisms could explain the reduction in friction with high viscosity fluids: (1) presence of a layer of polymer in the contact zone physically stops the solid surfaces from contacting; (2) high viscosity of the fluid leads to the suppression of turbulent flow in the contact zone, and, therefore, limits drag. At the much higher speed, the guar polymer is no longer confined within the contact zone and the film in the gap is dominated by the bulk fluid viscosity. Therefore, at higher speeds, the effect of the polymer on the lubrication properties tends to be negligible.

3. Tribology fundamentals

(A)

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Top view

Slide view Normal force

Rotating shaft

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Ball contact Rotating shaft Plate contact

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1.6 2%Reduced Fat Milk

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1.2 Whole Milk Half & Half

0.8 Heavy Cream

0.4

0.0 0.1

Fat Free Skim Milk

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FIGURE 28.12 (A) Diagram of the Anton Paar tribology equipment, a ball-on-three-plates setup within a mounted tribological device. (B) Friction factor as a function of sliding speed for five different fluid dairy samples using a mounted tribological device (Heyer and La¨uger, 2009).

3.3.2 Friction tester The friction tester consists of two interacting surfaces: a specially designed rubber band and a metal cylinder of an electric motor (Fig. 28.14). The materials of these surfaces have flexibility to change the substrate materials (friction tester). The friction between these surfaces is measured with the load cell F1. When the cylinder reverses in the direction, the load produces F2. The friction coefficient, m, is calculated using the following equation:   1 F1 m ¼ log (28.1) p F2

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FIGURE 28.13 (A) Stribeck curves obtained for guar gum solutions, T ¼ 35 C. Model for the behavior of guar; (B) in the boundary regime, the film thickness is too small for the nonadsorbed polymer to enter in the contact zone; (C) the polymer can fit in the gap. Confinement of the polymer in the contact zone increases the inlet viscosity; (D) at higher speeds the inlet viscosity is based on that of the base fluid. Adapted from Malone, M.E. Appelqvist, I. A. M. & Norton, I.T. (2003). Oral behaviour of food hydrocolloids and emulsions. Part 1. Lubrication and deposition considerations. Food Hydrocolloids, 17 (6), 763e773.

3.3.3 Optical tribological configuration Optical tribological configuration consists of two interacting surfaces and a confocal scanning light microscopy (Fig. 28.15).

3.3.3 The mini-traction machine MTM has emerged, in the past twodecades, as one of the popular technologies specializing in frictional and lubricating properties of food (Malone et al., 2003). Fig. 28.16 shows the measurement chamber where the sample can be conveniently placed on the disk surface with the machine working on a ballon-disk principle. The speed and the force of the rotating balls as well as the disk are recorded and coefficient of friction is automatically tabulated (Meyer et al., 2011). The MTM has been used extensively for a number of soft-tribological studies to evaluate the lubrication behavior for Newtonian and nonNewtonian fluids including food systems, beverages, suspensions, hydrocolloids, and multiphase

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FIGURE 28.14 Schematic of the Friction tester apparatus. Reproduced from Prakash, S., Tan, D .D. Y. & Chen, J. (2013). Applications of tribology in studying food oral processing and texture perception. Food Research International, 54, 1627e1635 with permission from Elsevier.

FIGURE 28.15 Optical tribological configuration; an emulsion (A) is confined between an upper changeable surface (B) and a glass surface (C). A force (Fz) is applied and the friction force (Fx) is measured, while surface C is oscillating. Simultaneously with a confocal scanning laser microscope (CSLM) the behavior of the emulsion is observed.

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Chapter 28 Rheology and tribology assessment of foods

FIGURE 28.16 (A) Measurement chamber with two rubbing surfaces for original MTM (a) and modified setup used to simulate the oral environment (b). (B) Schematic diagram of mini-traction machine. Both the disk and the ball rotate at a controlled speed and create a lubrication flow between the two surfaces. (A) Reproduced with permission from Chojnicka, A., de Jong, S., de Kruif, C.G., & Visschers, R.W. (2008). Lubrication properties of protein aggregate dispersions in a soft contact. Journal of Agricultural and Food Chemistry, 56(4), 1274e1282. (B) Redrawn from Bongaerts, J.H.H., Fourtouni, K., & Stokes, J.R. (2007). Soft-tribology, Lubrication in a compliant PDMS-PDMS contact. Tribology International, 40, 1531e1542.

fluids (Pradal and Stokes, 2016; Hamilton and Norton, 2016). Another advantage is the ease of replacing the surface materials of the metallic ball and disk, if desired, including PDMS (de Vicente et al. 2006a, 2006b; de Vicente et al., 2006a; Bongaerts et al., 2007; Ranc et al., 2006).

3.3.4 The double-ball-on-plate tribological apparatus This apparatus is on of the latest and has been used in studies carried out by Dr. Joyner and her group at University of Idaho. The apparatus comprises of a spindle attached to two 12.7-mm diameter balls with the help of a pivot. The unevenness of the surface of the tribological plate can thus be compensated for any vertical adjustment using the pivot point. The spindle that is attached to the rheometer allows for normal force control as well as rotational speed (Fig. 28.17).

3.3.5 Three-ball-on-plate configuration attached to a texture analyzer Chen and his group at Zhejiang Gongshang University, China, have been using this configuration laid down sideways (Morell et al., 2017; Chen et al., 2014; Brossard et al., 2016; Sanahuja et al., 2017; Morell et al., 2017; Cai et al. 2017; Upadhyay and Chen, 2019).

4. Conclusions Foods can be rheologically characterized using several different tests, but the fact that no single test can adequately provide a complete rheological description is well accepted. Therefore, one needs to be careful while selecting the test considering the available instrumentation, the type of food, and the objective. Lubrication studies using the most representative tribology apparatus can provide additional information, especially while looking at the thin-film sensory attributes such as thickness, mouth coating, creaminess, etc. The authors believe that rheology and tribology go hand-in-hand when one is characterizing foods for sensory texture.

References

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FIGURE 28.17 Schematic (A) and photo (B) of double-ball tribological system. Adapted from Joyner, H.S. Pernell, C.W. & Daubert, C.R. (2014). Impact of oil-in-water emulsion com-position and preparation: method on emulsion physical properties and friction behaviors, Tribology Letters 56,143e160.

References Akhtar, M., Stenzel, J., Murray, B.S., Dickinson, E., 2005. Factors affecting the perception of creaminess of oil-inwater emulsions. Food Hydrocolloids 19 (3), 521e526. Baier, S., Elmore, D., Guthrie, B., Lindgren, T., Smith, S., Steinbach, A., 2009. A new tribology device for assessing mouthfeel attributes of foods. In: 5th International Symposium on Food Structure and Rheology. ETH Zurich, Switzerland, p. 2009. Barbosa-Ca´novas, G.V., Kokini, J.L., Ma, L., Ibarz, A., 1996. The rheology of semiliquid foods. Advances in Food & Nutrition Research 39, 1e69. Bongaerts, J.H.H., Fourtouni, K., Stokes, J.R., 2007. Soft-tribology, Lubrication in a compliant PDMS-PDMS contact. Tribology International 40, 1531e1542. Brandt, M.A., Skinner, E.Z., Coleman, J.A., 1963. Texture profile method. Journal of Food Science 28, 404e409. Brossard, N., Cai, H., Osorio, F., Bordeu, E., Chen, J., 2016. “Oral” tribological study on the astringency sensation of red wines. Journal of Texture Studies 47 (5), 1745e4603. Cai, H., Li, Y., Chen, J., 2017. Rheology and tribology study of the sensory perception of oral care products. Biotribology 10, 17e25. Chen, J., Dickinson, E., 1999. Effect of surface character of filler particles on rheology of heat-set whey protein emulsion gels. Colloids and Surfaces B: Biointerfaces 12, 373e381. Chen, J., Stokes, J.R., 2012. Rheology and tribology: two distinguish regimes of food texture sensation. Trends in Food Science & Technology 25, 4e12.

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Chen, J., Liu, Z., Prakash, S., 2014. Lubrication studies of fluid food using simple experimental set up. Food Hydrocolloids 42, 100e104. Chojnicka, A., de Jong, S., de Kruif, C.G., Visschers, R.W., 2008. Lubrication properties of protein aggregate dispersions in a soft contact. Journal of Agricultural and Food Chemistry 56 (4), 1274e1282. Cuvelier, G., Launay, B., 1990. Frequency dependence of viscoelastic properties of some physical gels near the gel point. Makromolekulare Chemie, Macromolecular Symposia 40, 23e31. Devezeaux de Lavergne, M., Strijbosch, V.M., Van den Broek, A.W., Van de Velde, F., Stieger, M., 2016. Uncoupling the impact of fracture properties and composition on sensory perception of emulsion-filled gels. Journal of Texture Studies 47, 92e111. de Vicente, J., Stokes, J.R., Spikes, H.A., 2006a. Soft lubrication of model hydrocolloids. Food Hydrocolloids 20, 483e491. de Vicente, J., Stokes, J.R., Spikes, H.A., 2006b. Rolling and sliding friction in compliant, lubricated contact. Proceedings of the Institution of Mechanical Engineers e Part J: Journal of Engineering Tribology 220, 55e63. de Wijk, R.A., Prinz, J.F., Janssen, A.M., 2006. Explaining perceived oral texture of starch-based custard desserts from standard and novel instrumental tests. Food Hydrocolloids 20 (1), 24e34. Dickinson, E., Stainsby, G., Wilson, L., 1985. An adsorption effect on the gel strength of dilute gelatin-stabilized oil-in-water emulsions. Colloid & Polymer Science 263 (11), 933e934. Flory, P.J., 1953. Principles of Polymer Chemistry. Cornell University, Ithaca. NY. Geremias-Andrade, I.M., Souki, N., Moraes, I., Pinho, S.C., 2016. Rheology of emulsion-filled gels applied to the development of food materials. Gels (Basel, Switzerland) 2 (3), 22. Gibbins, H., Carpenter, G., 2013. Mechanisms of astringency. Journal of Texture Studies 44, 364e375. Hamilton, I.E., Norton, I.T., 2016. Modification to the lubrication properties of xanthan gum fluid gels as a result of sunflower oil and triglyceride stabilized water in oil emulsion addition. Food Hydrocolloids 55, 220e227. Heyer, P., La¨uger, J., 2009. Correlation between friction and flow of lubricating greases in a new tribometer device. Lubrication Science 21 (7), 253e268. Joyner, H.S., Pernell, C.W., Daubert, C.R., 2014. Impact of oil-in-water emulsion com-position and preparation: method on emulsion physical properties and friction behaviors. Tribology Letters 56, 143e160. Khan, S.A., Roger, J.R., Raghavan, S.R., 1997. Rheology: tools and methods. In: The National Academy of Sciences. Aviation fuels with improved fire safety, Washington DC, USA, pp. 39e46. Proceedings. Kokini, J.L., 1987. The physical basis of liquid food texture and texture-taste interactions. Journal of Food Engineering 6 (1), 51e81. Kramer, A., Szczesniak, A.S., 1973. Texture Measurements of Foods: Psychophysical Fundamentals: Sensory, Mechanical, and Chemical Procedures, and Their Interrelationships. D. Reidel Publishing Company, Dordrecht, The Netherlands. Lorenzo, G., Checmarev, G., Zaritzky, N., Califano, A., 2011. Linear viscoelastic assessment of cold gel-like emulsions stabilized with bovine gelatin. LWT e Food Science and Technology 44 (2), 457e464. Macakova, L., Yakubov, G.E., Plunkett, M.A., Stokes, J.R., 2010. Influence of ionic strength changes on the structure of pre-adsorbed salivary films. A response of a natural multi-component layer. Colloids and Surfaces B: Biointerfaces 77, 31e39. Malone, M.E., Appelqvist, I.A.M., Norton, I.T., 2003. Oral behaviour of food hydrocolloids and emulsions. Part 1. Lubrication and deposition considerations. Food Hydrocolloids 17 (6), 763e773. Meyer, D., Vermulst, J., Tromp, R.H., de Hoog, E.H.A., 2011. The effect of inulin on tribology and sensory profiles of skimmed milk. Journal of Texture Studies 42 (5), 387e393. Morell, P., Chen, J., Fiszman, S., 2017. The role of starch and saliva in tribology studies and the sensory perception of protein-added yogurts. Food and Function 8 (2), 545e553. Pradal, C., Stokes, J.R., 2016. Oral tribology: bridging the gap between physical measurements & sensory experience. Current Opinion in Food Science 9, 34e41.

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Prakash, S., Tan, D.D.Y., Chen, J., 2013. Applications of tribology in studying food oral processing and texture perception. Food Research International 54, 1627e1635. Ranc, H., Servais, C., Chauvy, P.-F., Debaud, S., Mischler, S., 2006. Effect of surface structure on frictional behaviour of a tongue/palate tribological system. Tribology International 39 (12), 1518e1526. Sala, G., Scholten, E., 2015. Instrumental characterization of textural properties of fluid food. Modifying Food Texture vol. 2 second ed., Elsevier Ltd. Sala, G., van Aken, G.A., Stuart, M.A.C., van de Velde, F., 2007. Effect of droplet-matrix interactions on large deformation properties of emulsion-filled gels. Journal of Texture Studies 38 (4), 511e535. Sala, G., de Wijk, R.A., van de Velde, F., van Aken, G.A., 2008. Matrix properties affect the sensory perception of emulsion-filled gels. Food Hydrocolloids 22, 353e363, 2008. Sala, G., van Vliet, T., Stuart, M.A.C., van Aken, G.A., van de Velde, F., 2009. Deformation & fracture of emulsion-filled gels: effect of oil content & deformation speed. Food Hydrocolloids 23 (5), 1381e1393. Sanahuja, S., Upadhyay, R., Briesen, H., Chen, J., 2017. Spectral analysis of the stick-slip phenomenon in “oral” tribological texture evaluation. Journal of Texture Studies 48, 318e334. Shama, F., Sherman, P., 1973. Identification of stimuli controlling the sensory evaluation of viscosity II. Oral methods. Journal of Texture Studies 4 (1), 111e118. Stokes, J.R., 2012. Oral tribology. In: Chen, J., Engelen, L. (Eds.), Food Oral Processing: Fundamentals of Eating and Sensory Perception. Wiley Blackwell, Oxford, UK, pp. 265e287. Syed, K.H.G., Saphwan, A., Glyn, O.P., 2011. Hydrogels: methods of preparation, characterization and applications C. In: Angelo (Ed.), Progress in Molecular and Environmental BioengineeringdFrom Analysis and Modeling to Technology Applications. InTech, Rijeka, pp. 117e150. Upadhyay, R., Chen, J., 2019. Smoothness perception as a tactile percept: correlating soft tribology with sensory measurements. Food Hydrocolloids 87, 38e47. Upadhyay, R., Chen, J., Brossard, N., 2016. Mechanisms underlying astringency: introduction to an oral tribology approach. Journal of Physics D: Applied Physics 49, 104003. van Aken, G.A., 2010. Modeling texture perception by soft epithelial surfaces. Soft Matter 6, 826e834. van Vliet, T., Walstra, P., 1995. Large deformation and fracture behaviour of gels. Faraday Discussions 101, 359e370.

CHAPTER

Biopolymer-based scaffolds: development and biomedical applications

29

Ann Mary George, Sai Preetham Reddy Peddireddy, Goutam Thakur, Fiona Concy Rodrigues Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India

1. Introduction A scaffold is a three-dimensional tool that has the potential to regenerate a specific functional tissue on implantation (Baino et al., 2015). A wide array of materials has been exploited for fabrication of scaffolds (Stratton et al., 2016). However, no single materials or the design of scaffold could be appropriate for all applications. However, ideal scaffolds should possess some essential qualities such as biocompatibility, biodegradability, and proper mechanical and physical properties to qualify for successful utilization (Liu and Ma, 2004; Khademhosseini and Langer, 2016). Scaffolds could be porous or gels. Porous structures are used as a template for cellular growth in tissue engineering (TE). Significantly high porosity of the scaffolds allows for the ingress of cells and facilitates space for cellular proliferation and formation of extracellular matrix (ECM). Other than this, they transport nutrients and regulatory factors required for tissue regeneration, drugs for specific healing, and other biomolecular signals. Traditionally, features such as stability, toxicity, and degradation were taken into account for designing synthetic scaffolds. However, as more studies are done on this area, the scaffold designs have become more complicated and now incorporate biological characteristics that give them natural tissuelike properties. Advancements in fabrication methodologies have led to the creation of technology to the production of customized scaffolds according to specific needs. It is now possible to incorporate factors such as multiple degradation mechanisms that affect the degradation rate and strength, controlled drug release, presence of proteins that promote cell adherence, and regeneration (Burdick & Mauck, 2011). The scaffolds have an open structure with interconnected pores that allow the cells to grow inside the structure and provide mechanical stability. If the scaffold is bioresorbable, it degrades periodically as the tissue growth takes over the structure; otherwise the scaffold remains at the specific site for strength and support. Matching the degradation rate to regeneration rate of tissues is essential for maintenance of mechanical stability and for the remains of the degraded scaffold matter to be nontoxic and easily metabolizable. Scaffold must possess adequate surface chemistry and mechanical strength to be utilized in particular applications. For example, scaffolds for orthopedic application must have large load-bearing capacity whereas those for skin graft that should be elastic can be made of collagen fibers to enhance the tissue adherence and proliferation. Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00029-1 Copyright © 2020 Elsevier Inc. All rights reserved.

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Biomaterials play a crucial role in deciding the properties of the scaffold and so does the fabrication method. Biomaterials used can be classified into synthetic and natural. Most of them are derived from materials used for other biomedical applications such as sutures, wound dressings. This includes biodegradable materials of synthetic origin, viz., aliphatic polyesters (PLA, PLGA, PCL, etc.), hydroxyapatite, and materials of natural origin like chitin and collagen. Recent trends in research suggest that biopolymer-based scaffolds have indicated great biocompatibility and are excellent mimics of the ECM systems. Some of the methods applied for the fabrication of scaffolds using these materials include solvent casting and particulate leaching, phase separation, gas forming, and rapid prototyping (Liu et al., 2007). This chapter elucidates the various properties and types of scaffolds, methods of fabrication of such scaffolds, the most suitable biomaterials that have been used to design the scaffolds, and their recent applicability in the fields of drug delivery and TE.

2. Properties of scaffolds The structures should satisfy certain requirements which would make them suitable to be used as scaffolds in TE. Mechanical strength and biological properties are the main constraints to consider while designing a scaffold. These properties will vary according to the tissue characteristics. Selecting the right material and fabrication technique that can make these scaffolds of varying properties is important. The desired properties include biocompatibility, biodegradability, porosity, appropriate mechanical strength, and design.

2.1 Biocompatibility The body creates immunological responses to foreign substances entering the body as a part of defense mechanism and causes the rejection of scaffold or implants. Therefore, any bioengineered structures should cause negligent immune reaction so that the inflammation caused by this does not disrupt the healing process or cause any toxicity in vivo (O’Brien et al., 2008). Apart from that, the structure must provide biomimetic binding sites so that the cells can adhere which can lead to proliferation and differentiation. The biomaterials chosen for this are aliphatic polymers that are biocompatible. The scaffold must not contain any cytotoxic element that can cause cell apoptosis or necrosis. The source for such elements are usually the chemicals used in treating the scaffold such as organic solvent residues from polymer synthesis or initiators used on polymerization or macromolecules from scaffold material (Mondschein et al., 2017).

2.2 Biodegradability Scaffolds are of two classifications, permanent and biodegradable, based on their degradation property. The permanent scaffold must not degrade and should replicate the properties of the soft tissue it is replacing. A biodegradable scaffold is expected to degrade on its own after its purpose is served so that it is replaced by the new growth of cells The by-products should easily exit the body without causing any cytotoxicity. Controlled activity of macrophages is necessary for inflammatory responses so that degradation can occur along with formation of new tissue. The degradation mechanism and degradation rate are vital parameters to be considered while designing a

2. Properties of scaffolds

719

scaffold. Bulk erosion and surface erosion are the two mechanisms that result in breaking down the scaffold and resorption/dissolution of material (O’Brien et al., 2008). The rate of biodegradation of a polymer mainly depends on its chemical properties such as the degree of crystallinity, glass transition temperature, presence of hydrolytically unstable bonds, and the molecular weight (Ye et al., 1997).

2.3 Mechanical properties Generally, any biopolymer scaffold must have mechanical properties that are ideal for the implantation site and should also have desired strength for any surgical procedure required for implantation. The mechanical properties of a scaffold depend on the choice of material and the method of fabrication that affects the structural parameters like pore geometry, size, shape, etc. The properties of the scaffold are selected according to the required application. For example, scaffolds for soft tissue need less strength than those required for orthopedic application. Therefore, the scaffold should have similar mechanical strength to that of the tissue. This strength is bound to decrease over time for biodegradable scaffolds but the combined strength of the newly grown tissue and the degrading scaffold should be comparable because the degraded scaffold material is filled by regenerated tissue. Elastic modulus is an integral property that measures the resistance to elastic deformation when an external force is applied and must be selected accordingly. Ideally, there should not be any plastic deformation in the scaffold as it will lead to loss of its initially designed structure (Chen, 2019). In bone and cartilage reconstruction, they are expected to maintain their structures even after implantation because the stability depends on elasticity, strength, and absorption on material interface and the rate of degradation and regeneration of new tissue (Yang et al., 2001).

2.4 Scaffold architecture and assessment Architecture of scaffold plays a huge role in the strength of the structure and the ability to sustain the regrowth of cells on it. The external structure should be suitable for implantation to the injured site and internally it should be highly porous as the porosity affects the cell viability, and the cell’s ability to adhere, proliferate, and differentiate. Interconnected pore structure is required for the nutrient diffusion, the effective waste removal including metabolic waste and scaffold degradation product and vascularization (O’Brien et al., 2008). Studies show that gyroid-shaped pores show maximum interconnectivity and evenly distributed pore sizes (Mondschein et al., 2017). Bone regeneration and fibrovascular tissue regeneration required ideal pore sizes of 100e350 mm and >500 mm, respectively. A large surface area with suitable biopolymers is favorable for the attachment of cells and their growth. The presence of biomolecules such as adhesive proteins like fibronectin, collagen, RGD peptides, growth factors, insulin, and so forth is also an added advantage for ease of tissue formation. Many natural biopolymers like chitosan already have these molecules which make them suitable whereas for synthetic polymers, they have to be artificially incorporated by covalent attaching, electrostatic adsorption, or self-assembled on the surface of the biomaterial. Surface roughness also plays an important role. Cell differentiation is favored in rough surface as compared to smooth one as in osteoblastic differentiation. As mentioned in this section, the architecture of scaffold plays very important role for its success in tissue generation. Depending on its application, special architectural needs of scaffolds also arise. High

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Chapter 29 Biopolymer-based scaffolds

surface-area-to-volume ratio provides large area for cell anchorage. Similarly, high porosity accounts for higher three-dimensional empty spaces which are required for cell proliferation and tissue growth. Therefore, scaffold architecture could be best characterized by measuring its porosity, pore size, and permeability. The common techniques used for measuring porosity include mercury intrusion porosimetry, gravimetry, micro-CT, etc. Mercury intrusion porosimetry: Here pore volume is measured by forcing mercury into the void space under pressure. However, the process is limited for determining the porosity of scaffolds with closed pores and also not very accurate for scaffolds with large pore sizes (>800 mm) as very little pressure is needed to drive mercury into the pores. Gravimetry: It is also used to determine porosity. First, the external dimensions of the scaffold need to be accurately measured so as to determine its volume (v). Then it can be weighed to estimate the mass (m). Its overall density (d) can be calculated by d ¼ m/v. So, if the density of the material is known (dp), the porosity (p) of the scaffold can be calculated by p ¼ 1  d/dp. Micro-CT: This has been the latest technology used to measure both pore size and porosity with high accuracy and reliability. Image processing: Many current image processing techniques allow researchers to provide more precise quantitative assessment of interpenetrating network, scaffold microstructure, droplet dimensions, etc. The microstructural images were usually processed by background subtraction, normalization, thresholding, binarization, and noise removal as per standard image processing protocol. Although there were software programs (Image J) that help researchers analyze many features such as diameter, length, etc., more mathematical tools such as MATLAB were also used frequently for image processing for determining various microstructural features such as feret diameter, radius, area, eccentricity, etc. and thus eliminating subjectivity for most precise analysis. Thakur et al. (2012) have demonstrated the process of determining SEM-based microstructural analyses using MATLABbased image processing (Thakur et al., 2012) (Fig. 29.1). Permeability on the other hand is also a very crucial parameter for tissue generation as it aids proper mass transfer of nutrients and removal of waste. Porosity is a measure of void space within the scaffold, whereas permeability is defined as the ease of flow of a fluid through the scaffold. Permeability provides more detail insight of scaffold architecture with respect to its appropriate utilization for tissue regeneration. Darcy’s law can be employed to compute the permeability, permeability (k) ¼ QL/(hAt), where Q is the quantity of discharge, L is the length of the sample in the direction of the flow, A is the cross-section of the sample, h is the hydraulic head, and t is the time. High porosity, large pore size, and interconnectivity of the pores account for higher permeability.

2.5 Manufacturing technology The method of fabrication is selected upon the desired properties for the specific TE application. Different biomaterials require their suitable manufacturing method. These techniques can be classified into three categories: conventional, electrospinning, and 3D printing. The traditional techniques employed are gas foaming, melt molding, porogen leaching, phase separation, and freeze-drying. Electrospinning produces fine fibers of nanometer scale that can be used to form the structure. 3D printing and decellularization are the latest advancements in fabrication technique. 3D printing involves the layer-by-layer deposition of biomaterials. Extrusion based, inkjet, and laser assisted are some of the rapid prototyping or 3D-printing methods used in fabrication (Chen, 2019).

3. Types of scaffolds

(A)

(B)

(a)

(a)

(b)

(b)

(C) (a)

(b)

(c)

(d)

(e)

(f)

721

(c)

(c)

FIGURE 29.1 (A) Scanning electron microscopy micrographs of freeze-dried cross-linked gelatin matrices. (B) Segmentation of SEM images. The independently labeled component images are represented for matrices crosslinked at (a) 5 C, (b) 15 C, and (c) 25 C, respectively. (C) Notch box plot showing quantitative estimation of morphological features: (a) radius, (b) ferret diameter, and (c) eccentricity. Each mean value for each figure is expressed and units are represented in mm. Reproduced from Thakur, G., Mitra, A., Basak, A., Sheet, D., 2012. Characterization and scanning electron microscopic investigation of crosslinked freeze dried gelatin matrices for study of drug diffusivity and release kinetics. Micron 43 (2), 311e320 © with permission from Elsevier.

Decellularization is the process in which a scaffold is built by discharging the cellular contents of an organ or tissue by chemicals, and only retaining the extracellular matrix. This is followed by cell seeding or reimplantation to give stem cells, whose cell differentiation can be accelerated by the addition of growth factors (Rana et al., 2017). It is a challenge to create such scaffolds on a large scale as each scaffold needs to be customized for the requirements. Therefore, the manufacturing technology chosen must be cost-effective and able to create multiple structures in a given short time. The storage of such scaffold, delivery to the clinicians, and shelf life are other constraints that are being faced by the manufacturers (O’Brien et al., 2008).

3. Types of scaffolds Scaffolds can be categorized into different types depending on their mechanical strength, stability, porosity, composition, or origin. Typical designs of scaffold include fibers, meshes, foams, sponges, gels, and so forth. Scaffolds are necessary for the regeneration of tissue which is anatomically and functionally similar to the organ or tissue which is to be repaired or replaced. And they are produced using certain biomaterials and their respective fabrication techniques to suite the application. Various types of scaffolds include microsphere scaffold, porous scaffold, fibrous scaffold, hydrogel scaffold, polymerebioceramic scaffold, and acellular scaffold (Dhandayuthpani et al., 2011).

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Chapter 29 Biopolymer-based scaffolds

3.1 Porous scaffold Porous scaffolds have high degree of porosity (>90%) and interconnectivity between the pores. It is required for the cell proliferation, cell seeding, diffusion of nutrients in, and metabolites out. Sponge or foam porous scaffolds provide surface for cells to adhere to, limit the cluster size of the neotissue to the pore size, and allow the cell interaction and nutrient transport (Dhandayuthpani et al., 2011). Ideal pore size may vary for different scaffolds but is usually around 100 mm (Ma, 2004). Pore size for neovascularization is 5 mm, 5e15 mm for ingrowth of fibroblast, 20 mm for hepatocytes ingrowth, 20e125 mm for regeneration of adult mammalian skin, and 200e350 mm for bone regeneration (ElSherbiny and Yacoub, 2013).

3.1.1 Materials and methods A porous scaffold must be made from biocompatible material and degrade linearly with tissue regeneration. The polymers used can be divided into naturally derived polymers such as, fibrin and collagen and synthetic origin polymers like PLA, PEGA, and their copolymer PLGA. Synthetic polymers can have controllable properties and are reproducible in large scale but may have rejection at immunological level. Whereas naturally derived materials are very biocompatible. The porous scaffolds fabrication includes particulate leaching and solvent casting, emulsion freeze-drying, gas foaming, electrospinning, rapid prototyping, and thermally induced phase separation (TIPS). Rapid prototyping which is synonymous with solid freeform fabrication (SFF) is a layer-by-layer manufacturing process using the CAD model of the scaffold. It includes stereolithography, selective laser sintering (SLS), 3D printing, and wax printing. The porous scaffolds formed by these methods are different in architecture (Fig. 29.2). New technology like bioplotter that directly prints cells or a range of processed biomaterial has shown to be better than traditional methods like porogen leaching for producing homogenous interconnected pore network (Hollister, 2005). The advantages and disadvantages of these methods are mentioned in Table 29.1.

3.1.2 Application Porous scaffolds find their use in bone tissue engineering (BTE). The pores should support cell differentiation and cell proliferation by accommodating osteoprogenitor cells and osteoblasts. Porous scaffolds are also used for organ vascularization and peripheral nerve growth. This requires improved structure and increased pore interconnectivity. Sometimes the surface is modified with biomolecules like growth factors to increase the production of ECM (Agrawal and Ray, 2001).

3.2 Microsphere scaffold Laurencin et al., 1996 were the first to introduce microsphere scaffolds in TE by using a polymere ceramic matrix as a bone graft. They would structurally, mechanically, and chemically represent a cancellous bone. Microspheres are spherical, free-flowing, polymer matrices encapsulated with drug so they are generally used for drug delivery application and in TE for gene therapy and treatment of infected bone (Stephens et al., 2000). Their diameter should be ideally in the range of 200e300 mm. The greatest advantage of microsphere scaffolds was that they have good mechanical strength and property of excellent controlled release of bioactive molecules. There are two types of microsphere scaffolds which are (a) microsphere incorporating scaffold and (b) microsphere dissolved scaffold. In

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723

FIGURE 29.2 Typical morphologies of porous polymer foams produced by different techniques and structure of cancellous bone: (A) thermally induced phase separation (TIPS), (B) solvent casting and particle leaching, (C) solid freeform fabrication technique (SFF), (D) microsphere sintering, and (E) cancellous bone. Note the similarity in morphology of these scaffolds to the natural bone. Reprinted with permission from Rezwan, K., Chen, Q.Z., Blaker, J.J., Boccaccini, A.R., 2006. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 27 (18), 3413e3431.

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Table 29.1 Advantages and disadvantages of methods of fabrication of porous scaffold. Fabrication method

Advantages

Disadvantages

Solvent casting and particle leaching

Controlled porosity Controlled pore interconnectivity (if particles are sintered) Porous structure can be customized for targeted tissue Cell encapsulation is viable Good interface with medical imaging High porosities (>95%) High pore interconnectivity Anisotropic and tubular pores possible Control over structure and pore size is possible by varying preparation conditions High porosity (>90%) pore size ranging from 20 to 200 mm possible

Structures are isotropic Use of organic solvents

SFF

TIPS

Emulsion freeze-drying

Electrospinning

Gas foaming

Possible to fabricate fibrous scaffold with fiber dia ranging from several microns down to 100 nanometers Highly porous structure No use of organic solvents Pore size of 100 mm porosity up to 93%

Comparatively lesser resolution Some methods use organic solvents

Long solvent sublimation time (48 h) Scaffolds tend to shrink Small-scale production Use of organic solvents Closed pores may be formed in the final matrix Use of organic solvents No shapes other than cylinder and sheets are possible Closed pores with nonporous surface and with only 10%e30% of interconnected pores may be formed

Modified from Rezwan, K., Chen, Q.Z., Blaker, J.J., Boccaccini, A.R., 2006. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 27 (18), 3413e3431.

the former, microspheres are integrated into premade solid polymer or hydrogel scaffolds composed of organic materials such as chitosan, collagen, or alginate with a top-down approach of fabrication method. Whereas in the latter, microspheres act as the building blocks of the scaffold (Gupta et al., 2017). In microsphere-incorporated scaffold, they control the release of biomolecules on stimuli, create pores, and provide mechanical strength. In microsphere-based scaffolds, there is a 100% pore interconnectivity as the microspheres are packed together randomly or by rapid prototyping. They are held together by cohesive forces such as electrostatic, magnetic, hydrophobic interactions, or chemical cross-linking.

3.2.1 Materials and methods The main methods for fabrication of microspheres are precision particle fabrication (PPF), emulsionesolvent extraction method, and TIPS (Table 29.2). To fuse these microspheres to create a whole macroscopic unit, sintering methods are used. Microspheres with smooth, rough, and porous textures can be created by emulsionesolvent technique. The addition of materials like HAP or chitosan can alter the techniques. The microsphere scaffolds are produced by heat sintering or solvent vapor treatment and are loaded with drugs by emulsion method. They release the drug at the active site over a period of time. Sintering produces porous microspheres that can be used for bone remodeling. Gelatin and similar polymers are used for gel microspheres (Gupta et al., 2017).

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Table 29.2 Advantages and disadvantages of microsphere fabrication methods (Gupta et al., 2017). Fabrication methods

Materials

Solvent evaporation technique

Precision particle fabrication

Collagen, HA

TIPS

Collagen chitosan

Sintering (micro sphere-based scaffold) Heat sintering Solvent-based sintering CO2 sintering Selective laser sintering

· · · ·

Advantages

Disadvantages

Large surface area, high-density cell culture. Used to encapsulate hydrophilic molecules. Low polydispersity. High mechanical stability. Control over microsphere size. Biomaterials can be used. High porosity. Rapid encapsulation. Control over microsphere size. Can be used to make complicated designs. Easy fabrication. Large-scale production. Quick process. Large variety of biomaterials can be used. Bioactive factors can be added. Easy fabrication. Can fabricate complicate designs. Customized grafts can be fabricated. No toxic solvents used. Maintains porosity.

Poor control over size. Solvent toxicity. Low encapsulation efficiency. Fabrication process is complex.

Multiple-step fabrication. Microspheres tend to coalesce.

May require high temperature depending on the material. Denaturation of biopolymers due to high temperature, loss in bioactivity. Solvent toxicity. Exact time requirement. High CO2 pressure might harm the embedded bioactive molecules. Expensive procedure.

3.2.2 Application Composite microspheres are made using chitosan and are used for cartilage and bone applications (Dhandayuthpani et al., 2011). Polymers having low molecular weight are utilized for fabricating porous microspheres which can lead to the fast release of drug, and polymers having high molecular weight, for making microspheres which facilitate slower release of drug (Ravivarapu et al., 2000). Gelatin microspheres and HAP were integrated into alginate-based scaffolds to release calcium cations for BTE scaffold (Jingxuan Yan et al., 2016). These can also be used for nerve regeneration. Valmikinathan et al. developed a spiral-shaped microsphere-based scaffold with increased surface area and demonstrated that Schwann cells have higher rate of cell attachment and proliferation on this compared to tubular-shaped scaffolds (Valmikinathan et al., 2008).

3.3 Hydrogel scaffold A hydrogel is a hydrophilic polymer gel material which finds its applicability in the fields of TE and drug delivery. It has the ability to encapsulate cells and bioactive molecules as well as perform efficient

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mass transfer (Zhu and Marchant, 2011). It contains covalent bonds formed by cross-linking of monomers, physical bonds formed by entanglement of chains, and hydrogen or Van der Waals’ forces between crystallites chains or which lead to two or more chains of macromolecules to come together (Peppas et al., 2000). Natural hydrogels are biocompatible and biodegradable and facilitate cellular activities; however, they may not have the required mechanical strength and may evoke immunological response. Synthetic hydrogel structures can be customized to suit the application. The mechanical strength of the hydrogel depends on the various chemical and physical interactions that make up the structure. Hydrogels can absorb large amount of water or any biological fluid and undergo swelling without dissolving because of the cohesive forces applied by the polymer strands within it (Intini et al., 2018). A hydrogel is an excellent candidate for scaffold as it is biodegradable and has mechanical and structural properties similar to ECM that makes cell adherence and allows cell growth and diffusion of molecules through the network (Drury and Mooney, 2003). A bioactive synthetic hydrogel is designed using short peptide sequences from the bioactive ECM components to produce TE scaffolds with functions mimicking the ECM (Zhu and Marchant, 2011).

3.3.1 Materials and methods Many synthetic and naturally derived materials can be used to form hydrogels. PEO, PLA, PPFderived copolymers, PAA, and PVA are the synthetic polymers, and collagen, fibrin, alginate, chitosan, and HA are of natural origin (Drury and Mooney, 2003). The gel formation takes place by the chemical cross-linking of the covalent bonds in these biomaterials. These materials can be used as scaffold material depending on the application, for example, scaffolds designed to encapsulate cells must be nontoxic and appropriate for the cell to thrive. Matrigel is a gelatin-based protein mixture secreted by EngelbretheHolmeSwarm (EHS) mouse sarcoma cells. It is extremely similar to the extracellular environment as it contains entacin, laminin, collagen type IV, and various growth factors. Therefore, it is used as scaffolds which facilitate tissue vascularization, cell differentiation, and angiogenesis (Zhu and Marchant, 2011). A few techniques used for hydrogel fabrication are emulsification, photolithography, microfluidic synthesis, and micromolding. The advantage with emulsification is that it is easy to produce the microgels but there is little control over shape as only spherical droplets can be synthesized using this method. Photolithography uses UV light to cross-link the polymers and the resolution can go down to submicron scale to millimeters. Microfluidic fabrication can produce hydrogels of varying sizes and shapes and their spatial properties like concentration gradient of two materials used in the hydrogel can be controlled. Micromolding is another technique that mostly uses HA, chitosan, and PEG to create structures of various sizes and shapes (Khademhosseini and Langer, 2007).

3.3.2 Application Hydrogels can be used to deliver protein and cells as they can encapsulate cells without damaging them in the fabrication process. In BTE, bone tissue grows into the porous system of the scaffold as the drug is released and the bone heals. But microspheres cannot be used as a scaffold to support the structure of damaged bone tissue as it is mechanically weak. In addition to collagen, other natural polymers like alginate and its modifications based on hydrogels have been fabricated previously for this. The surface is modified with RGD peptide to facilitate cell migration, proliferation, growth, and organization. The RGD concentration regulates the osteoblast migration (Liu and Ma, 2004).

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Hydrogels can be used to enhance the healing process of a tissue injury where they can act as barriers to avoid restenosis or thrombosis. The high biocompatibility and ability to control and release drugs on stimuli makes them good drug depots. Because of their softness and viscoelastic nature, hydrogels have been used in cardiac TE (El-Sherbiny and Yacoub, 2013).

3.4 Fibrous scaffold Fibers are continuous structures made of biomaterials with a high length-to-width ratio called filaments that can be used to create networks that resemble ECM which makes it an apt compound for making scaffolds used in TE. These can be continuous or short length monofilaments that can be interlaced, intertwined, or interloped to form strands (Li and Cooper, 2011). Conventional textile technologies allow this to be fabricated into complex structures. It is possible to fabricate fibers with diameters that approach the dimensions of collagen fiber bundles, between 50 and 500 nm, and resemble it morphologically and mechanically. Fibrous scaffolds are categorized as microfibrous and nanofibrous scaffolds, depending on the size of individual fibers. A study has shown that chondrocytes seeded in microfibers differentiated to show fibroblast-like morphology whereas those in nanofibrous scaffold maintained the chondrocyte-like morphology (Ko et al., 2008). Nanofiber scaffolds have the advantage of having high surface-area-to-volume ratio that enhances cell adhesion and proliferation (Smith and Ma, 2004). The advantages of using fibers are that they are highly porous and allow for mass transport of nutrients and biomolecules and support tissue growth (Dhandayuthpani et al., 2011).

3.4.1 Materials and methods Microfibrous and nanofibrous scaffolds are fabricated using specific techniques. They are derived from natural origin like silk, cotton, chitosan, wool, and collagen and synthetic polymers like nylon, polyethylene, PLA, PGA, and PLGA. The techniques used to fabricate microfibers include polymer extrusion, fiber spinning, rapid prototyping (FDM, 3D printing, SLS to produce layered laminated scaffold), and textile processing methods like knitting, weaving, braids of polymer fibers (Shalaby and Fabrics, 2004). The techniques used for the fabrication of nanofibers include self-assembly, electrospinning, and phase separation. By far, electrospinning is the best method to fabricate nanofibrous scaffold because a large variety of materials can be used and the production can be scaled up for industry needs (Burdick & Mauck, 2011). Self-assembly can create nanofibers of the smallest range of ECM collagen dimension and electrospinning can make fibers in the upper range, whereas phase separation makes fibers similar to the natural collagen (Smith and Ma, 2004). Surface modifications have to be done on these nanofibers to add specific functional groups to make them functionalized for specific applications. This is usually done by physical blending and coating. Adhesive proteins and ligand molecules are incorporated onto the nanofiber surface by surface grafting polymerization to increase the affinity of ECM. Growth factors, drugs, and genes are mixed directly with the polymers before they are used for electrospinning.

3.4.2 Applications The microfibrous scaffolds are used for wound dressings and soft tissue repair. Microfibrous scaffold can be used to make hernia repair mesh to reinforce the abdomen muscles. Synthetic polymers like

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polypropylene and biopolymers like silk can be used for making nonbiodegradable and biodegradable sutures, respectively (Marois et al., 2000). Vascular grafts have been made using biodegradable polymer meshes and they have shown to improve the pulsatile flow (Nigam and Mahanta, 2014). Cardiovascular grafts are also fibrous; they have indicated the ability to provide a proper environment for the tissue regeneration. Moutus et al. have developed a technique where 3D-woven scaffold structures are blended with chondrocyte hydrogel mixture to create cartilage tissue scaffolds. They have shown to have unique load-bearing properties as compared to other scaffold types (Moutos et al., 2007). Nanofibers have been effectively used in musculoskeletal TE including ligament, cartilage, bone, and skeletal muscle; in skin, vascular, and neural TE; and as well as a vehicle for controlled delivery of drugs, proteins, and DNA (Vasita et al., 2006). Fibrous scaffolds are used for nerve regeneration. Axonal outgrowth of neurites is guided by designing nerve conduits. The fibrous scaffold used for this application should be bioactive to be able to support myelination, outgrowth, and structural support for axons via Schwann cells (Anderson et al., 2015).

3.5 Polymerebioceramic composite scaffold Ceramics like tricalcium phosphate (TCP) and hydroxyapatite (HA) are used in various types of scaffolds for BTE. They exhibit characteristics such as very low elasticity, high mechanical stiffness (Young’s modulus), and hard brittle surface. Although these types of scaffolds have high bone biocompatibility owing to their osteoconductivity and bone-bonding capacity, their low mechanical strength difficulty to process makes them unfit for certain applications. Whereas biopolymer scaffolds are easily fabricated and can be biodegraded, they have poor mechanical characteristics which limit their usage such as for load bearing in orthopedic applications. Therefore, polymer can be introduced into bioceramic scaffolds by various fabrication methods such as coating with polymer solution or sintering bioceramic scaffold with a polymer phase. These composites mimic the natural bone as they are structurally and chemically similar to the mineral phase of bone which is made up of inorganic compounds (mainly partially carbonated HA) and organic compounds (collagen) (O’Brien et al., 2008). Compared to plain polymeric scaffolds, composite scaffolds enhance the mechanical strength of the scaffold and induce the bioactivity (Yunos et al., 2008). It supports cell growth and neotissue formation throughout the scaffold and not just on the surface (Wei and Ma, 2004). The polymer is biodegradable and ensures a rough and continuous structure with high porosity and surface area (Mourin˜o et al., 2013). Therefore, such composites provide with the advantage of both the materials together in a single structure.

3.5.1 Materials and methods Bioceramic material generally includes bioactive and biodegradable glasses and ceramics. Alumina, carbons, silicone nitrides, and zirconia are examples of inert bioceramics. Bioglass 45S5 is a glassceramic developed by Larry Hench in 1968 and HA is semiinert (bioreactive). Aluminum calcium phosphate, HA, plaster of Paris, coralline, and TCP are examples of restorable ceramics. These bioceramics show highly porous structures and are very biocompatible but have low mechanical strength, so a polymer phase is introduced into them. The polymers can be any synthetic or natural polymers that are used for scaffolds such as collagen, alginate, agarose, PLA, PLGA, and PCL.

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Numerous fabrication methods to produce polymerebioceramic composites are available, these include space holder, gas foaming, TIPS, and SFF. TIPS is used to produce composite scaffolds of PLGA and PDLA foams containing bioglass 45S5 (Baino et al., 2015). Suitable biopolymers are also used in this technique. Coating the bioceramic scaffold with a biopolymer and air-drying is another method to introduce polymers in the prepared ceramic scaffold. This improves the mechanical  properties especially the fracture toughness (Reho rek et al., 2013). Electrophoretic deposition is  another method to deposit polymer particles on bioceramic scaffold (Reho rek et al., 2013). Meng et al. (2011) deposited CNTs to glass-ceramic scaffold to culture mesenchymal cells. Instead of introducing polymer components into already prepared scaffolds, the organic and inorganic phases can be mixed on molecular level for solgel-based applications.

3.5.2 Applications The size of the HA particles has shown to cause considerable difference in the properties of the scaffold. Microsized HA (MHA) enhances adsorption of ECM components and proteins. Composites using MHA have shown to suppress apoptosis of osteoblasts by enhanced adsorption of serum proteins such as vitronectin and fibronectin into scaffolds (Woo et al., 2002). HA/polyethylene composite scaffold is currently in the market commercialized under the name “Hapex.” It is used for the repair of orbital floor fractures (Tanner, 2010). Bioglass 45S5 (in wt%: 45% SiO2, 24.5% Na2O, 24.4% CaO, and 6% P2O5) has shown to enhance bone formation due to the presence of Ca, Si, Na, and P ions in critical concentration so as to activate genes in osteoblast cells. Bretcanu et al. (2007) fabricated a fibrous composite of 45S5 coated with poly(3-hydroxybutyrate) that acted as a glue in holding up inorganic particles together and increased its strength from 0.4 to 1.5Mpa (Chen et al., 2006). Collagen is a biopolymer that is usually combined with HAP to fabricate bone tissue scaffold. Collagen was combined with glucosaminoglycan (GAG), a polysaccharide by Prof. Ioannis Yannas of MIT to regenerate skin in patients with severe burn (Yannas et al., 1982). In order to use them in regions subjected to higher load bearing, it was strengthened by introducing a ceramic phase, thereby developing a highly porous collagenehydroxyapatite (CHA) scaffold that is very similar to the structure of bone. The CG scaffold has high porosity that improves the cell infiltration and vascularization, but has low mechanical strength. Whereas the HA scaffold has the required mechanical strength but lacks the cell infiltration and vascularization capacity. The CHA scaffold shows high porosity and has enhanced mechanical properties and permeability that allows improved vascularization and the osteogenic potential of the host cells. The high porosity of the CHA scaffold and the uniform distribution of HA particles (green dots) can be clearly seen in Fig. 29.1 (O’Brien et al., 2008). HA porous scaffolds are sometimes coated with polymers like PCL that is incorporated with antibiotics like vancomycin. The encapsulated drug within the coated scaffold is released much faster and sustainably that directs coated drugs on HA scaffolds (Kim et al., 2005).

4. Scaffold processing and fabrication techniques The characteristics of the scaffold are based on the choice of the correct technique of fabrication. The processing and fabrication technique for the scaffolds can alter its properties significantly. Many of the fabrication techniques that were studied are listed below.

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4.1 Fiber felts or mesh An integral requirement of a scaffold is a high porosity value as a large number of cells have to be accommodated in it. Optimum cell growth rates also require a large value of the total pore area. The interconnecting pore network and the pore diameter are also necessary parameters for tissue growth, nutrient diffusion, and vascularization. Poly(glycolic acid) (PGA) fibers in the forms of tassels and felts were utilized as scaffolds by Mikos et al., 1993. Scaffolds of fiber meshes comprise independent fibers which are woven into the required 3D structure with a variable pore size. In these types of scaffold formation, a large surface area is a defining feature. Studies have shown that fiber felts and meshes may actually lack the necessary mechanical stability to be viable for in vivo use; however, they were successful in showing their feasibility for use in organ regeneration. For the actual use in organ regeneration, they have to be modified into shapes that replicate the actual structure and properties tissues to be used for cell attachment and transplantation. However, actually providing a firm substrate to the transplanted cells was found to be difficult to achieve by this method of fabrication which may result in unwanted degradation. The disadvantages associated with this fabrication technique led to the development of the fiber-bonding technique which had a greater control over other characteristics like structural stability which is lacking in fiber felts or mesh scaffolds.

4.2 Fiber bonding This technique is used to create required designs and shapes for organ implants, wherein nonwoven fibers are bonded together to create structural fiber networks, and these networks are interwoven to create the organ. Furthermore, the fibers are joined to each other physically without any bulk modification and with a constant diameter throughout. Fiber-bonding technique is used in the case where precise control of porosity is required for scaffolds as compared to the previous method. The following is a generalized preparation technique to bond nonwoven fibers together to create scaffolds. The first step is adding a Polymer A which is a nonbonded polymer structure to a solution of another nonbonded polymer structure B. Both the polymers used are required to be immiscible in their melt state and should be incompatible as well as nonreacting with each other. The solvent employed for polymer solution B must also be a nonsolvent for polymer A. In the second step, solvent evaporation takes place leading to formation of a composite in which polymer A fibers are embedded into polymer B matrix. This is followed by further heating the composites beyond the melting point of polymer A results in the fibers getting welded at their crosspoints. Essentially the heat treatment results in the actual fiber bonding, and to get the final bonded fiber structure, the unwanted polymer B is dissolved selectively by using an appropriate solvent. The important features to be considered in this mechanism of fabrication is the choice of solvent, and the properties of the two polymers used, most importantly, their immiscibility and melting temperatures. The drawback of this method is that there might be a case of cell toxicity caused due to solvent residue left behind on the fabricated scaffolds. Also the scaffolds do not have enough mechanical strength to support the growth of load-bearing tissues. Fiber-bonding technique has been used by Mikos et al. in fabricating interconnected fiber networks in the past (Fig. 29.3). In their study poly(glyco1ic acid) (PGA) fibers (polymer A) were embedded in a poly(L-lactic acid) (PLLA) matrix (polymer B). In this method, PLLA was mixed with methylene

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FIGURE 29.3 Schematic of fabrication by fiber-bonding method. Reproduced in modified form with permission from Mikos Ag, Bao Y, Cima LG, Ingber DE, Vacanti JP, Langer R, 1993. Preparation of poly (glycolicacid) bonded fiber structures for cell attachment and transplantation. Journal of Biomedical Materials Research 27(2),183e189.

chloride solution, and this was cast into a Petri dish containing a PGA fiber mesh that was nonwoven. As PLLA and PGA are incompatible with each other, they could be separated upon the application of heat. PLLA was dissolved by the methylene chloride after heating, and the remnants in the Petri dish were that of the required bonded PGA fiber mesh.

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4.2.1 Phase separation According to studies, due to the weak mechanical strength associated with this method, fiber bonding might not be suitable for regeneration of hard tissues. Phase separation technique has been used to solve this problem as this method can be used to produce porous matrices for maximum cell penetration and facilitates the proliferation of cartilage and bone cells relatively efficiently when compared to other techniques. Synthetic or organic biodegradable foams can be used as templates for regeneration of damaged tissues as architecture of the actual tissues can be replicated by the three-dimensional foam structure. This biodegradable foam can be used as a porous polymer scaffold whose various parameters can be adjusted as required. TIPS finds its use in production of microcellular foam structures or microporous membranes that serve as support for cell adhesion. TIPS is a method of preparing a polymer membrane where the polymer is mixed with a substance acting as the solvent at high temperature and then casting this solution into a film, which results in solidification on cooling. This foam has a high porosity and can be seen as a two-phase system which consists of two distinct phases: (1) continuous-polymer phase and (2) continuous-gaseous phase. The process of foam fabrication outlined by Lo et al. (1996) is based on phase separation principle from a homogeneous solution (Lo et al., 1996). Briefly, this technique involves dissolution of polymer in naphthalene molten phenol, or dioxanes at a temperature lesser than the freezing temperature of the used solvent, which induces solideliquid or liquideliquid phase separation. Removal of thermal energy from the single homogenous polymer solution which is at elevated temperature causes the conversion to a two-phase separated constituent that is composed of a polymerlean phase and a polymer-rich phase. The solidified solvent or polymer-rich phase is then removed (by sublimation) leaving behind the required porous scaffold. In this fabrication technique, the foam morphology as well as distribution of pore is dependent on liquideliquid phase separation. However, through this technique, even a minor modification of parameters like polymer type, concentration in solution, ratio of solvent to nonsolvent, quenching temperature, and rate of cooling is shown to affect the resultant porous scaffold morphology significantly. One significant advantage that can be seen in this technique is that bioactive molecules can be incorporated into the matrix with no loss of activity owing to extreme chemical or thermal surrounding. However, the solvent residue left over at the end of the fabrication process might be harmful for in vivo applications and it should be processed appropriately.

4.3 Solvent casting and particulate leaching In this method, biocompatible porous polymer membranes are prepared by dispersing organic particles into biocompatible solution of polymer. This is followed by processing the same (by either freezedrying or casting) and evaporation to yield polymer/salt composite membrane. The desired amount of crystallinity in this method can be controlled by adjusting the heating and cooling rates. Recovery of porous biocompatible membrane involves leaching of salt particles by immersion into water or another suitable solvent (Fig. 29.4). This is then processed and dried and can be used for cell growth and proliferation. Mikos et al., 1994 showed that this technique can be used to prepare scaffold having 93% porosity and median pore diameters of 500 mm. It has been observed that membranes having a higher crystallinity (>20%) will be relatively stronger and will therefore degrade slower than membranes with lower crystallinity.

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FIGURE 29.4 Schematic diagram showing the solvent casting and particulate leaching method. Open access Sampath, U. G. T. M., Ching, Y. C., Chuah, C. H., Sabariah, J. J., Lin, P.C., 2016. Fabrication of porous materials from natural/synthetic biopolymers and their composites. Materials 9 (12), 991.

However, a limitation of this fabrication technique is that only membranes of a relatively lower thickness can be produced (up to 3 mm thickness). However, further studies have been shown by Mikos that even three-dimensional structures can be created using this technique. The 3D structure can be fabricated by polymer membranes of thin cross-sectional layers of the required shape and laminating the membranes being laminated together to produce a 3D matrix possessing the overall required shape. First, the membranes are laminated by wetting one side of each of the membrane. The membranes are placed on top of each other with the wetted surfaces touching, and sufficient force is applied so that the membranes are joined and stay together without slipping. This laminating procedure is repeated until all of the membranes are laminated together to produce the required three-dimensional shape (Mikos et al., 1994).

4.4 Membrane lamination Similar to the three-dimensional structure formation in the previous method, in this technique, first 3D anatomical shape is depicted by a plot. The contour map of the desired three-dimensional shape is drawn, segmented, and then separated into the required layers, as according to the depth and other requirements of porous, biodegradable polymer membranes. Studies show that the preferred thickness of the membrane is between 500 and 2000 mm.

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FIGURE 29.5 Process of lamination of porous membranes. Reprinted with permission from Cai, Z, Cheng G, Geng M, Tang Z, 2004. Layered-lamination Preparing of Tissue Engineering Scaffolds via Emulsion Templates Method.

Generally, membranes were made out of PLLA or PLGA and techniques such as particulate leaching and solvent casting were used to produce the desired shapes, as described above. Coating chloroform or other adhesive chemicals (such as methylene chloride) are used for bonding of membranes adjacent to each other. Two or more membranes to be laminated are placed on the wetted absorbent material (paper, cloth, or sponge wetted with an organic solvent) (Fig. 29.5). The exposed surface of each membrane is subjected to a calculated amount of pressure for a sufficient amount of time so that each bottom surface is wetted sufficiently. For example, even forceps can be used to apply light pressure manually. This method can be used to produce 3D structure of desired shapes.

4.5 Melt molding Melt molding is an easy way to adjust the three-dimensional morphology of polymer scaffolds without the use of harsh chemical solvents. In this method synthetic polymers are preferred (mostly thermoplastic polymers) as they can be easily heated and then subsequently cooled into various solid forms as required. In this technique, the polymers are heated above their glass transition temperature or melting point. This creates a liquid form which is then allowed to cool and solidify in the form of the mold used. Melt molding is used in tandem with particle leaching to introduce porosity into the scaffold. In order to make the scaffold porous, this technique is used in combination with particle leaching. Unlike solvent casting method, however, melt molding does not use polymer solvents; there are no toxic residues left at the end of the process. However, due to the inclusion of heat in this technique, there may be a chance of damage of the bioactive molecules.

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With this method, the pore size and porosity can be directly controlled by the microsphere diameter and by changing the polymer/constituent ratio. Polymer scaffolds of various shapes can be fabricated by modifying the geometry of the mold as required which makes it perfect for industrial and mass production applications (Gorth & Webster, 2011). Among the different processes for the fabrication of porous scaffolds, this method is considered one of the most convenient as rapid production of structures having different sizes and shapes is possible which makes it beneficial for industrial uses and for scaling up (Hou et al., 2003). Further, organic solvents are not required in this method to produce scaffolds (Haugen et al., 2004), thereby making the production of scaffolds easier by melt molding (Wang and Wang, 2014). However, this method also has considerable disadvantages as nonporous layers are formed on the surface of the scaffold and also, porogen compounds might be left behind in the scaffold owing to the difficult process of leaching completely. Requirement of high temperatures in this process may also contribute to the damage of bioactive molecules in this process.

4.6 Polymereceramic fiber composite foam Polymerebioceramic matrix reinforced composites are an essential group of biomaterials for TE applications. Among the technologies employed for its fabrication, solution casting with/without particle leaching and TIPS combined with the process of freeze-drying are employed for the production of polymereceramic composite scaffolds. Apart from these techniques, foam coating and microsphere sintering can also be used which are developed for the union of ceramic and polymeric materials (Yunos et al., 2008). A solvent casting technique is used for specifically forming the polymereceramic fiber composite foam. In this technique, a porogen and short hydroxyapatite (HA) fibers are incorporated into solution of PLGA/methylene chloride. Evaporation of the solvent followed by porogen leaching produces PLGA composite foam having porous structure, reinforced by short fibers of hydroxyapatite. Based on the amount of fiber content, these scaffolds have been shown to have higher compressive strength than nonreinforced materials having same porosity (Yang et al., 2001).

4.7 High-pressure processing This method involves exposure of solid disc to CO2 at high pressure which results in CO2 saturation inside the polymer. Induction of thermodynamic instability takes place by reduction of pressure of CO2 to a certain level which leads to induction of thermodynamic instability, causing nucleation due to the expansion of dissolved CO2, which in turn results in the generation of macropores. One critical advantage of this technique is that there is no involvement of organic solvents. The generation of macropores in the scaffold structure is due to the expansion of gas from within. In this technique, a solid disc of material (PLGA, PLLA, etc.) is subjected to CO2 gas at high pressure which results in CO2 saturation in the polymer and by modifying the pressures of the gas present, a thermodynamic instability is created which results in the expansion of CO2 gas from within, which ultimately results in the formation of macropores. Moreover, macropores are advantageous as they provide minimal diffusional constraints during culture, and allow even spatial cell distribution throughout the scaffold which facilitates homogeneous tissue formation.

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The problem with this method, however, is that it creates a polymer matrix having closed pores that would result in cell-seeding constraints (Yang et al., 2001).

4.8 Hydrocarbon templating Hydrocarbon templating is used mainly in the process of production of polymer foams (Fig. 29.6) that are used to replicate a variety of structures including tissues with complex geometry. A large variety of polymers can be used in this process where formation of pore is preceded by use of particulate phase of a hydrocarbon as a template causing a polymer phase for precipitation of polymer phase and subsequent pore formation. There are two main processes in this method: (1) Fugitive phase leaching (2) Precipitation of polymer

FIGURE 29.6 Steps involved in the preparation of biopolymeric foams. From Shastri, V.P., Martin, I., Langer, R., 2000. Macroporous polymer foams by hydrocarbon templating. Proceedings of the National Academy of Sciences 97(5), 1970. Copyright (2000) National Academy of Sciences, U.S.A.

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A hydrocarbon template is used to achieve an increased control over the properties such as the pore structure, porosity of the scaffold, and other structural and mechanical characteristics of the polymer foam. Polymer foams with very low densities (.05). The sample containing pork fat, soybean oil, and 2.5% rice bran wax treatments had similar lipid oxidation values (P >.05), but the sample with 10% rice bran wax sample provided higher lipid oxidation value than that of pork fat sample. Similarly, da Silva et al. (2019) investigated the usage of oleogels prepared with pork skin, water, and high oleic sunflower oil instead of pork back fat in Bologna-type sausage. The technological, nutritional, oxidative, and sensorial properties were determined. The oleogel addition increased emulsion stability and decreased the cooking loss of final product. Although the replacement boosted the ratio of oleic acid, this treatment did not change oxidative stability. The reformulation of pork back fat by oleogel induced the reducing the fat content of the product. The lipid replacement did not significantly change the ash content of Bologna-type sausages (P