Enzymes Beyond Traditional Applications in Dairy Science and Technology 9780323960106, 0323960103

Table of contents :
Cover
Front-matter
Copyright
List of contributors
Contents
1 Milk enzymes
1.1 Introduction
1.2 Enzymes in milk: significance, nomenclature, reaction catalyzed, and activity levels
1.3 Proteinases
1.3.1 Plasmin
1.3.2 Cathepsin D
1.4 Lipases and esterases
1.4.1 Lipase
1.4.2 Lipoprotein lipase (EC 3.1.1.34)
1.4.3 Bile salt–stimulated lipase
1.4.4 Esterases
1.5 Phosphohydrolases
1.5.1 Alkaline phosphatase
1.5.2 Acid phosphatase
1.5.3 Ribonuclease
1.6 Oxidases
1.6.1 Lactoperoxidase (EC 1.11.1.7)
1.6.2 Catalase (EC 1.11.1.6)
1.6.3 Xanthine oxidase (EC 1.17.3.2)
1.6.4 Superoxide dismutase
1.6.5 Sulfhydryl oxidase (EC 1.8.3--)
1.7 γ-glutamyl transpeptidase (EC 2.3.2.2)
1.8 N-Acetyl-β-d-glucosaminidase (EC 3.2.1.30)
1.9 Lysozyme (EC 3.1.2.17)
1.10 Enzymes from psychrotrophs origin in milk
1.11 Conclusion
References
2 Enzymes in mastitis milk
2.1 Introduction
2.2 Enzymes in mastitis
2.2.1 Proteases
2.2.1.1 Plasminogen and plasmin
2.2.1.2 Elastase, collagenase, and cathepsin
2.2.1.3 Caspases
2.2.2 Esterases
2.2.3 Antioxidant enzymes
2.2.3.1 Glutathione peroxidase (GSH-Px)
2.2.3.2 Catalase
2.2.3.3 Superoxide dismutase (SOD)
2.2.3.4 Xanthine oxidase
2.2.4 Antibacterial enzymes [lactoperoxidase (LPO) and myeloperoxidase (MPO)]
2.2.5 N-acetyl-d-glucosaminidase (NAGase)
2.2.6 Lactate dehydrogenase (LDH)
2.2.7 Phosphatases and aminotransferases
2.3 Efforts in diagnosing mastitis in dairy animals
2.4 Future developments and conclusion
Acknowledgments
References
3 Effect of high-pressure processing on milk enzymes
3.1 Introduction
3.2 Significance of milk enzymes
3.3 Need for alternate processing of milk
3.4 High-pressure processing technology
3.5 Effect of high pressure on the activity and structure of milk enzymes
3.6 Kinetics of high pressure on milk enzyme inactivation
3.7 Effect of high-pressure processing on milk enzyme system
3.7.1 Effect of high-pressure processing on alkaline phosphatase
3.7.2 Effect of high-pressure processing on plasmin
3.7.3 Effect of high–pressure processing on lipoprotein lipase, γ-glutamyltransferase, and lactoperoxidase
3.8 Milk enzymes as high–pressure processing indicator
3.9 Conclusion
References
4 Traditional applications of enzymes in dairy science and technology
4.1 Introduction
4.2 Alkaline phosphatase
4.2.1 Significance of alkaline phosphatase
4.2.2 Methods for estimation of alkaline phosphatase activity
4.2.2.1 Colorimetric methods
4.2.2.2 Fluorimetric methods
4.2.2.3 Immunochemical methods
4.2.2.4 AOAC method for cheese
4.2.3 Effect of mastitis
4.2.4 Alkaline phosphatase activity in nonbovine milk
4.2.5 Reactivation of alkaline phosphatase
4.3 Acid phosphatase
4.4 Milk lipoprotein lipase
4.4.1 Physicochemical characteristics
4.4.2 Concentration in bovine milk
4.4.3 Lipolysis
4.4.4 Significance in dairy industry
4.5 Plasmin
4.5.1 Inactivation of plasmin
4.5.2 Significance of plasmin in milk
4.5.3 Ultrahigh temperature milk
4.5.4 Cheese
4.5.5 Milk protein products
4.5.6 Milk powder products
4.6 Catalase
4.6.1 Physicochemical properties
4.6.2 Catalase activity in milk
4.6.3 Measurement of catalase activity
4.6.4 Significance in dairy industry
4.7 Lactoperoxidase
4.7.1 Physicochemical properties
4.7.2 Concentration in milk and colostrum
4.7.3 Significance of lactoperoxidase enzyme
4.7.4 Lactoperoxidase system in milk
4.8 Xanthine oxidoreductase
4.9 γ-Glutamyl transferase
4.9.1 Physicochemical properties
4.9.2 Significance in dairy industry
4.10 Conclusion
References
5 Methods for identification of bioactive peptides
5.1 Introduction
5.2 Methods of protein digestion
5.2.1 In vitro methods
5.2.1.1 Enzymatic method
5.2.1.2 Microbial method
5.2.2 In vivo methods
5.2.2.1 Aspiration of gut content
5.2.2.2 Measurement in the blood
5.2.2.3 Identification/characterization in the blood
5.3 Methods of isolation and identification/characterization of food-derived peptides
5.4 Methods of function assessment
5.4.1 Function assessment using in vitro methods
5.4.2 Function assessment using in vivo methods
5.5 The importance of quantifying bioactive peptides
5.6 Conclusion
References
6 In-silico methods for milk-derived bioactive peptide prediction
6.1 Introduction
6.2 Methods of milk-derived bioactive peptide prediction
6.2.1 Obtaining the amino acid sequence of milk proteins
6.2.2 In-silico digestion of milk proteins
6.2.3 Molecular docking simulation
6.3 In vitro confirmatory experiments after in silico prediction
6.4 Estimation of bioactive peptide content in food items using in-silico methods
6.5 Conclusion and future perspective
References
7 Production of bioactive peptides from bovine caseins
7.1 Introduction
7.2 Production of bioactive peptides from bovine casein
7.2.1 Hydrolysis by enzymes from plant or microorganism
7.2.2 Degradation by digestive enzymes
7.2.3 Proteolysis during fermentation
7.3 Bioactivities of bovine casein peptides
7.3.1 Antihypertensive activity
7.3.2 Antidiabetic activity
7.3.3 Antioxidant activity
7.3.4 Other bioactivities
7.4 Conclusion and further perspectives
References
8 Production of bioactive peptides from bovine whey proteins
8.1 Introduction
8.2 Generation of whey protein–derived bioactive peptides
8.2.1 Enzymatic hydrolysis
8.2.2 Hydrolysis during gastrointestinal digestion
8.2.3 Fermentation
8.2.4 In silico aided enzymatic release of bioactive peptides
8.3 Analytical techniques for the identification of bioactive peptides
8.3.1 Enrichment and fractionation of bioactive peptides
8.3.2 Peptide characterization
8.4 Biological effects of whey-derived bioactive peptides
8.4.1 Antidiabetic peptides
8.4.2 Antihypertensive peptides
8.4.3 Antimicrobial peptides
8.4.4 Antioxidant peptides
8.4.5 Anticancer peptides
8.4.6 Immunomodulatory peptides
8.4.7 Antiinflammatory peptides
8.4.8 Opioid-like peptides
8.4.9 Satiety hormone-inducing peptides
8.5 Conclusion and future prospects
Acknowledgments
References
9 Bioactive peptides derived from camel milk proteins
9.1 Introduction
9.2 Bioactive peptides from camel milk proteins
9.2.1 Antioxidant
9.2.1.1 Structural activity relationship of camel milk–derived antioxidant peptides
9.2.2 Antihypertensive
9.2.2.1 Structural activity relationship (SAR) of camel milk–derived antihypertensive peptides
9.2.3 Antimicrobial peptides from camel milk
9.2.3.1 Structural activity relationship of camel milk–derived antimicrobial peptides
9.2.4 Antidiabetic peptides derived from camel milk proteins
9.2.4.1 Dipeptidyl peptidase-IV inhibitory peptides from camel milk proteins
9.2.4.2 α-Amylase (AM) inhibitory peptides from camel milk proteins
9.2.4.3 α-Glucosidase (AG) inhibitory peptides from camel milk proteins
9.2.4.4 Structural activity relationship of camel milk–derived antidiabetic peptides
9.2.5 Antiobesity peptides from camel milk proteins and their structural activity relationship
9.2.6 Other biological properties of camel milk–derived hydrolysates
9.3 Future perceptions
References
10 Bioactive peptides from fermented milk products
10.1 Introduction
10.2 Bioactive peptides from fermented bovine milk products
10.2.1 Soful
10.2.2 Yogurt
10.2.3 Koumiss
10.2.4 Kefir
10.2.5 Others
10.3 Bioactive peptides from fermented goat milk products
10.3.1 Yogurt
10.3.2 Kefir
10.3.3 Dahi
10.4 Bioactive peptides from fermented camel milk products
10.4.1 Camel milk
10.4.2 Bioactive peptides from fermented camel’s milk
10.4.3 Bioactive peptides from fermented camel milk products
10.5 Bioactive peptides from fermented mare milk products
10.5.1 Koumiss
10.6 Bioactive peptides from fermented sheep milk products
10.6.1 Koopeh
10.6.2 Yogurt
10.7 Conclusion
References
11 Downstream processing of therapeutic bioactive peptide
11.1 Introduction
11.2 Production mechanisms of bioactive peptides
11.2.1 Enzymatic hydrolysis
11.2.2 Fermentation
11.2.3 Enzymes derived from proteolytic microorganisms
11.3 Downstream processing of bioactive peptides (isolation, purification, and characterization)
11.3.1 Fractionation methods
11.3.2 Membrane separation techniques
11.3.3 Chromatographic methods
11.3.3.1 Size-exclusion chromatography
11.3.3.2 Ion-exchange chromatography
11.3.3.3 Reversed-phase liquid chromatography
11.4 Conclusion
References
12 Enzyme actions during cheese ripening and production of bioactive compounds
12.1 Introduction
12.2 Bioactive compounds
12.2.1 Peptides
12.2.2 Conjugated linoleic acid
12.2.3 Gama aminobutyric acid and L-ornithine
12.2.4 Carotenoids
12.3 Conclusion
References
13 Immobilization of β-galactosidases
13.1 Introduction
13.2 Sources of β-galactosidase
13.3 Structure of β-galactosidase
13.4 Classification of β-galactosidases
13.5 Reactions of β-galactosidase
13.6 Immobilization of β-galactosidase
13.6.1 Functional enzyme aggregates
13.6.2 Gel beads and lattices
13.6.3 Chitosan
13.6.4 Nanoparticles
13.6.5 Metal affinity columns
13.6.6 Methacrylate and its variants
13.7 Conclusion
Acknowledgments
References
14 Low-lactose milk production using β-galactosidases
14.1 Introduction
14.2 Characteristics of β-galactosidases
14.2.1 Sources of β-galactosidases
14.2.2 Reactions catalyzed by β-galactosidases
14.2.3 Optimal reaction conditions for β-galactosidases
14.2.4 Production and purification of β-galactosidases
14.2.5 Sources of industrial β-galactosidase
14.2.6 Technologies for producing low-lactose milk
14.2.7 Future scope
14.3 Immobilized β-galactosidases
14.4 Column reactors with immobilized β-galactosidases
14.5 Conclusions and perspectives
References
15 Production of oligosaccharides, a prebiotic from lactose, using β-galactosidase
15.1 Introduction
15.2 Characteristics of β-galactosidases for the production of galactooligosaccharides
15.2.1 Sources of β-galactosidases
15.2.2 Reactions catalyzed by β-galactosidases for the production of galactooligosaccharides
15.2.3 Optimal reaction conditions for the production of galactooligosaccharides
15.2.4 Production and purification of galactooligosaccharides
15.2.5 Sources of industrial β-galactosidase and galactooligosaccharides
15.2.6 Future scope
15.3 Immobilized β-galactosidase for the production of galactooligosaccharides
15.4 Conclusions and perspectives
References
16 Production of lactulose from cheese whey
16.1 Introduction
16.2 Lactulose production
16.2.1 Isomerization-based lactulose synthesis
16.2.1.1 Chemical method
16.2.1.2 Electro-activation-based isomerization
16.2.1.3 Enzyme-based isomerization
16.2.2 Transgalactosylation-based lactulose synthesis
16.3 Separation of lactulose
16.4 Health benefits of lactulose
16.5 Conclusion
References
17 Determination of lactose in milk and milk-derived ingredients using biosensor-based techniques
17.1 Introduction
17.2 Importance of lactose in milk and dairy ingredients
17.3 Lactose quantification methods
17.4 Biosensors
17.4.1 Biosensors used in dairy foods
17.4.2 Biosensors used for lactose quantification
17.5 Blood glucose meter biosensors as an option for determination of lactose
17.5.1 Blood glucose meter operating principles
17.5.2 Potential issues with a blood glucose meter–based lactose assay
17.5.3 Practical applications reported in literature for use of blood glucose meter in measurement of lactose
17.5.3.1 Measurement of lactose in milk using a blood glucose meter
17.5.3.2 Measurement of lactose in dairy ingredients using a blood glucose meter
17.6 Conclusion
References
18 Enzyme-based analytical methods pertinent to dairy industry
18.1 Introduction
18.2 Urea estimation in milk
18.2.1 Monitoring pH change
18.2.2 Monitoring change in pressure
18.2.3 Potentiometric approach
18.2.4 Spectrophotometric measurement of ammonium ion concentration
18.2.5 Urea biosensor
18.3 Lactose estimation
18.3.1 Spectrophotometric method
18.3.2 By measuring the change in pH
18.4 Estimation of lactate or lactic acid in dairy products
18.5 Estimation of cholesterol in dairy products
18.6 Ascorbic acid estimation in dairy products
18.7 Detection of common adulterants
18.7.1 Detection and estimation of hydrogen peroxide in milk
18.7.2 Detection of glucose in milk
18.7.3 Detection and estimation of sucrose in milk and milk products
18.7.4 Detection of maltodextrin in milk
18.7.5 Paper strip for urea detection
18.7.6 Detection and estimation of starch in milk and milk products
18.8 Conclusion
References
19 Lactate biosensor for assessing milk microbiological load
19.1 Introduction
19.2 Lactic acid for assessing milk microbial load
19.3 Methods of detection
19.3.1 Analytical conventional techniques
19.3.1.1 High-performance liquid chromatography
19.3.1.2 Liquid chromatography–mass spectrometry
19.3.2 Lactate biosensors
19.3.2.1 Electrochemical methods
19.3.2.2 Optical spectroscopic methods
19.3.2.2.1 UV–vis spectroscopy
19.3.2.2.2 Colorimetric
19.3.2.2.3 Fourier transform infrared spectroscopy
19.3.3 Nanotechnology applications in sensors
19.4 Conclusion and future prospective
Acknowledgment
Conflicts of interest
Ethical approval
References
20 Enzymes for cleaning-in-place in the dairy industry
20.1 Introduction: fouling and cleaning-in-place in the dairy industry
20.2 Industrial enzymes and their use for cleaning-in-place in the dairy industry
20.3 Reported studies on the effectiveness of enzymes for cleaning-in-place in the dairy industry
20.3.1 Removal of Type A fouling deposits
20.3.2 Removal of biofilms
20.4 Considerations for the development of optimal enzyme-based cleaning solutions
20.5 Conclusions and future outlook
References
21 Regulatory policies on use of food enzymes
21.1 Introduction
21.2 Regulatory framework regarding food enzymes
21.3 Specific aspects of intellectual property right protection on enzymes
21.4 Government policies toward food in biotechnology
21.4.1 Enzyme regulation in Canada
21.4.2 Enzyme regulation in Australia and New Zealand
21.4.2.1 Proposed amendments
21.4.3 European Union regulation
21.4.4 Scope of enzyme regulation
21.4.5 Limitations
21.4.6 US regulations
21.4.6.1 Petitions for enzyme preparations
21.4.6.2 Generally Recognized as Safe notices for enzyme preparations
21.4.7 FAO/WHO
21.5 Policy and regulatory framework: lower middle income countries
21.6 Future prospect
References
Index

Citation preview

Enzymes Beyond Traditional Applications in Dairy Science and Technology

FOUNDATIONS AND FRONTIERS IN ENZYMOLOGY Series Series Editor: Munishwar Nath Gupta

Series Website: https://www.elsevier.com/books-and-journals/book-series/books-serieslanding-page-request-foundations-and-frontiers-in-enzymology

Enzymes Beyond Traditional Applications in Dairy Science and Technology Edited by Y.S. Rajput Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Rajan Sharma Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-96010-6 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Jonathan Simpson Acquisitions Editor: Megan R. Ball Editorial Project Manager: Lena Sparks Production Project Manager: Kumar Anbazhagan Cover Designer: Mark Rogers Typeset by MPS Limited, Chennai, India

List of contributors Shubhangi Agrawal Novartis, Hyderabad, Telangana, India

Jayendra K. Amamcharla Department of Animal Sciences and Industry, Food Science Institute, Kansas State University, Manhattan, KS, United States

Meisam Barati Department of Clinical Nutrition & Dietetics, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Angela Boyce Department of Chemical Sciences and Bernal Institute, University of Limerick, Limerick, Ireland

Stephany Nefertari Chávez-García Bioprocesses and Bioproducts Research Group. Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila. Saltillo, Coahuila, México

Dora Elisa Cruz-Casas Bioprocesses and Bioproducts Research Group. Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila. Saltillo, Coahuila, México

A.K. Dang Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Sayed Hossein Davoodi Department of Clinical Nutrition & Dietetics, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Priscilla Romina De Gregorio Food Biotechnology Laboratory, University of Vale do Taquari - Univates, Lajeado, RS, Brazil; Biotechnology Graduate Program, University of Vale do Taquari - Univates, Lajeado, RS, Brazil; Reference Center for Lactobacilli - National Council for Scientific and Technological Research (CERELA-CONICET), San Miguel de Tucumán, Tucumán, Argentina

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List of contributors

Hosseini Elahesadat Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Richard J. FitzGerald Department of Biological Sciences, University of Limerick, Limerick, Ireland

Adriana Carolina Flores-Gallegos Bioprocesses and Bioproducts Research Group. Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila. Saltillo, Coahuila, México

Leticia Anael García-Flores Bioprocesses and Bioproducts Research Group. Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila. Saltillo, Coahuila, México

Adriano Gennari Food Biotechnology Laboratory, University of Vale do Taquari - Univates, Lajeado, RS, Brazil; Biotechnology Graduate Program, University of Vale do Taquari - Univates, Lajeado, RS, Brazil

C.G. Harshitha Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

R. Hemamalini Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, India

Masoumeh Jabbari Student Research Committee, Department of Community Nutrition, National Nutrition and Food Technology Research Institute, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Amit Kumar Jain Dairy Chemistry Department, SMC College of Dairy Science, Kamdhenu University, Anand, Gujarat, India

Sunil Kumar Khare Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, India

Thanyaporn Kleekayai Department of Biological Sciences, University of Limerick, Limerick, Ireland

Sumit Kumar Amity Institute of Biotechnology. Amity University, Noida, Uttar Pradesh, India

Kiran Lata Department of Food Processing and Technology, School of Vocational Studies and Applied Sciences (SoVSAS), Gautam Buddha University, Greater Noida, Uttar Pradesh, India

Sajid Maqsood Department of Food Science, College of Agriculture and Veterinary Medicine, United Arab Emirates University, Al Ain, United Arab Emirates

List of contributors

Sandra T. Martín-del-Campo Tecnologico De Monterrey, School of Engineering and Sciences, Querétaro, México

Gloria A. Martínez-Medina Bioprocesses and Bioproducts Research Group. Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila. Saltillo, Coahuila, México

Priti Mudgil Department of Food Science, College of Agriculture and Veterinary Medicine, United Arab Emirates University, Al Ain, United Arab Emirates

P. Murali Krishna Basic and Applied Science, National Institute of Food Technology Entrepreneurship and Management (NIFTEM) Kundli, Haryana, India

Shital D. Nagargoje Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Laxmana Naik Southern Regional Station, ICAR-National Dairy Research Institute, Bengaluru, Karnataka, India

Cathy Verônica Nied Food Biotechnology Laboratory, University of Vale do Taquari - Univates, Lajeado, RS, Brazil

Pranali Nikam Dairy Chemistry Department, College of Dairy Science and Food Technology, Raipur, Chhattisgarh, India

Bibhudatta S.K. Panda Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Satishkumar Parmar Dairy Chemistry Department, SMC College of Dairy Science, Kamdhenu University, Anand, Gujarat, India

Lilia Arely Prado-Barragán Biotechnology Department, Biological and Health Sciences Division, Metropolitan Autonomous University, Ciudad de México, México

R. Vázquez-García Tecnologico De Monterrey, School of Engineering and Sciences, Querétaro, México

Y.S. Rajput Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Rodolfo Ramos-González CONACYT—Universidad Autónoma de Coahuila. Saltillo, Coahuila, México

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Suvartan Ranvir Department of Dairy Chemistry, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, Uttar Pradesh, India

Gurdeep Rattu Department of Biotechnology, School of Applied Sciences, Reva University, Bengaluru, Karnataka, India

Pourahmad Rezvan Department of Food Science and Technology, Varamin- Pishva Branch, Islamic Azad University, Varamin, Iran

Ashwani Sahu Bharat Heavy Electricals Limited (BHEL), New Delhi, India

Smita Sahu Amity IPR Cell, Amity University, Noida, Uttar Pradesh, India

Marta Santos-Hernández Department of Biological Sciences, University of Limerick, Limerick, Ireland

Ahesanvarish Shaikh Dairy Chemistry Department, SMC College of Dairy Science, Kamdhenu University, Anand, Gujarat, India

Rajan Sharma Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Richa Singh Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Azis Boing Sitanggang IPB University, Department of Food Science and Technology, Bogor, Indonesia

Yallappa M. Somagond Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, India

Claucia Fernanda Volken de Souza Food Biotechnology Laboratory, University of Vale do Taquari - Univates, Lajeado, RS, Brazil; Biotechnology Graduate Program, University of Vale do Taquari - Univates, Lajeado, RS, Brazil

Giandra Volpato Federal Institute of Education, Science and Technology of Rio Grande do Sul, Campus Porto Alegre, Porto Alegre, RS, Brazil

Caleb Wagner Department of Animal Sciences and Industry, Food Science Institute, Kansas State University, Manhattan, KS, United States; School of Food Science, Washington State University, Pullman, WA, United States

List of contributors

Gary Walsh Department of Chemical Sciences and Bernal Institute, University of Limerick, Limerick, Ireland

Chenyang Wang School of Food Science and Technology, Guangzhou, P.R. China

Engineering,

South

China

University

of

Engineering,

South

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University

of

Engineering,

South

China

University

of

Mouming Zhao School of Food Science and Technology, Guangzhou, P.R. China

Lin Zheng School of Food Science and Technology, Guangzhou, P.R. China

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Contents

LIST OF CONTRIBUTORS ......................................................................... xvii

Section I Indigenous milk enzymes CHAPTER 1

Milk enzymes ............................................................................ 3 Pranali Nikam, Y.S. Rajput, Rajan Sharma and Suvartan Ranvir 1.1 Introduction ................................................................................ 3 1.2 Enzymes in milk: significance, nomenclature, reaction catalyzed, and activity levels ..................................................... 4 1.3 Proteinases............................................................................... 10 1.3.1 Plasmin ......................................................................... 10 1.3.2 Cathepsin D .................................................................. 10 1.4 Lipases and esterases............................................................. 14 1.4.1 Lipase............................................................................ 14 1.4.2 Lipoprotein lipase (EC 3.1.1.34) .................................. 15 1.4.3 Bile salt stimulated lipase......................................... 15 1.4.4 Esterases ...................................................................... 15 1.5 Phosphohydrolases.................................................................. 16 1.5.1 Alkaline phosphatase................................................... 16 1.5.2 Acid phosphatase ......................................................... 17 1.5.3 Ribonuclease ................................................................ 18 1.6 Oxidases.................................................................................... 19 1.6.1 Lactoperoxidase (EC 1.11.1.7) ..................................... 19 1.6.2 Catalase (EC 1.11.1.6) .................................................. 20 1.6.3 Xanthine oxidase (EC 1.17.3.2) .................................... 21 1.6.4 Superoxide dismutase ................................................. 22 1.6.5 Sulfhydryl oxidase (EC 1.8.3--).................................... 23 1.7 γ-glutamyl transpeptidase (EC 2.3.2.2) .................................. 24 1.8 N-Acetyl-β-D-glucosaminidase (EC 3.2.1.30) ......................... 25

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1.9 Lysozyme (EC 3.1.2.17) ............................................................ 25 1.10 Enzymes from psychrotrophs origin in milk ......................... 26 1.11 Conclusion ................................................................................ 27 References .......................................................................................... 27

CHAPTER 2

Enzymes in mastitis milk ....................................................... 37 Shital D. Nagargoje, Yallappa M. Somagond, Bibhudatta S.K. Panda and A.K. Dang 2.1 Introduction ................................................................................ 37 2.2 Enzymes in mastitis ................................................................... 39 2.2.1 Proteases ........................................................................ 39 2.2.2 Esterases ........................................................................ 41 2.2.3 Antioxidant enzymes ...................................................... 41 2.2.4 Antibacterial enzymes [lactoperoxidase (LPO) and myeloperoxidase (MPO)]................................................ 43 2.2.5 N-acetyl-D-glucosaminidase (NAGase) ........................ 44 2.2.6 Lactate dehydrogenase (LDH)....................................... 45 2.2.7 Phosphatases and aminotransferases ......................... 45 2.3 Efforts in diagnosing mastitis in dairy animals....................... 46 2.4 Future developments and conclusion ...................................... 50 Acknowledgments............................................................................... 50 References .......................................................................................... 50

CHAPTER 3

Effect of high-pressure processing on milk enzymes......... 57 Laxmana Naik, Kiran Lata and Rajan Sharma 3.1 3.2 3.3 3.4 3.5

Introduction ................................................................................ 57 Significance of milk enzymes.................................................... 57 Need for alternate processing of milk ..................................... 58 High-pressure processing technology ..................................... 59 Effect of high pressure on the activity and structure of milk enzymes ............................................................................. 61 3.6 Kinetics of high pressure on milk enzyme inactivation .......... 63 3.7 Effect of high-pressure processing on milk enzyme system......................................................................................... 65 3.7.1 Effect of high-pressure processing on alkaline phosphatase ................................................................... 67 3.7.2 Effect of high-pressure processing on plasmin .......... 68 3.7.3 Effect of high–pressure processing on lipoprotein lipase, γ-glutamyltransferase, and lactoperoxidase .............................................................. 68

Contents

3.8 Milk enzymes as high–pressure processing indicator ...........69 3.9 Conclusion .................................................................................. 70 References .......................................................................................... 70

CHAPTER 4

Traditional applications of enzymes in dairy science and technology ............................................................................... 77 Ahesanvarish Shaikh, Amit Kumar Jain and Satishkumar Parmar 4.1 Introduction .............................................................................. 77 4.2 Alkaline phosphatase............................................................... 78 4.2.1 Significance of alkaline phosphatase ......................... 79 4.2.2 Methods for estimation of alkaline phosphatase activity..................................................... 80 4.2.3 Effect of mastitis .......................................................... 85 4.2.4 Alkaline phosphatase activity in nonbovine milk ............................................................. 85 4.2.5 Reactivation of alkaline phosphatase ......................... 85 4.3 Acid phosphatase ..................................................................... 87 4.4 Milk lipoprotein lipase ............................................................. 88 4.4.1 Physicochemical characteristics ................................ 88 4.4.2 Concentration in bovine milk ...................................... 89 4.4.3 Lipolysis ........................................................................ 89 4.4.4 Significance in dairy industry ...................................... 89 4.5 Plasmin ..................................................................................... 90 4.5.1 Inactivation of plasmin................................................. 91 4.5.2 Significance of plasmin in milk................................... 92 4.5.3 Ultrahigh temperature milk ........................................ 92 4.5.4 Cheese .......................................................................... 93 4.5.5 Milk protein products .................................................. 93 4.5.6 Milk powder products .................................................. 93 4.6 Catalase .................................................................................... 94 4.6.1 Physicochemical properties ........................................ 94 4.6.2 Catalase activity in milk............................................... 95 4.6.3 Measurement of catalase activity ............................... 95 4.6.4 Significance in dairy industry ...................................... 96 4.7 Lactoperoxidase ....................................................................... 96 4.7.1 Physicochemical properties ........................................ 97 4.7.2 Concentration in milk and colostrum......................... 97 4.7.3 Significance of lactoperoxidase enzyme .................... 97 4.7.4 Lactoperoxidase system in milk ................................. 98 4.8 Xanthine oxidoreductase ......................................................... 98

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4.9 γ-Glutamyl transferase ......................................................... 100 4.9.1 Physicochemical properties ...................................... 100 4.9.2 Significance in dairy industry .................................... 100 4.10 Conclusion .............................................................................. 101 References ........................................................................................ 102

Section II Action of enzymes on milk proteins CHAPTER 5

Methods for identification of bioactive peptides................. 119 Meisam Barati, Masoumeh Jabbari and Sayed Hossein Davoodi 5.1 Introduction .............................................................................. 119 5.2 Methods of protein digestion .................................................. 120 5.2.1 In vitro methods ........................................................... 120 5.2.2 In vivo methods ............................................................ 122 5.3 Methods of isolation and identification/characterization of food-derived peptides.......................................................... 126 5.4 Methods of function assessment ............................................ 127 5.4.1 Function assessment using in vitro methods............ 127 5.4.2 Function assessment using in vivo methods ............. 129 5.5 The importance of quantifying bioactive peptides................. 130 5.6 Conclusion ................................................................................ 131 References ........................................................................................ 131

CHAPTER 6

In-silico methods for milk-derived bioactive peptide prediction ................................................................... 137 Meisam Barati, Masoumeh Jabbari and Sayed Hossein Davoodi 6.1 Introduction .............................................................................. 137 6.2 Methods of milk-derived bioactive peptide prediction..........138 6.2.1 Obtaining the amino acid sequence of milk proteins ................................................................ 138 6.2.2 In-silico digestion of milk proteins............................. 143 6.2.3 Molecular docking simulation..................................... 155 6.3 In vitro confirmatory experiments after in silico prediction ........................................................................ 156 6.4 Estimation of bioactive peptide content in food items using in-silico methods ........................................................... 157 6.5 Conclusion and future perspective ......................................... 160 References ........................................................................................ 160

Contents

CHAPTER 7

Production of bioactive peptides from bovine caseins ...... 163 Lin Zheng, Chenyang Wang and Mouming Zhao 7.1 Introduction .............................................................................. 163 7.2 Production of bioactive peptides from bovine casein ........... 164 7.2.1 Hydrolysis by enzymes from plant or microorganism ............................................................. 164 7.2.2 Degradation by digestive enzymes ............................. 166 7.2.3 Proteolysis during fermentation ................................. 166 7.3 Bioactivities of bovine casein peptides................................... 167 7.3.1 Antihypertensive activity .............................................. 167 7.3.2 Antidiabetic activity ...................................................... 170 7.3.3 Antioxidant activity ....................................................... 174 7.3.4 Other bioactivities ........................................................ 177 7.4 Conclusion and further perspectives ..................................... 178 References ........................................................................................ 179

CHAPTER 8

Production of bioactive peptides from bovine whey proteins.................................................................................. 189 Marta Santos-Hernández, Thanyaporn Kleekayai and Richard J. FitzGerald 8.1 Introduction .............................................................................. 189 8.2 Generation of whey protein derived bioactive peptides ...... 190 8.2.1 Enzymatic hydrolysis ................................................... 190 8.2.2 Hydrolysis during gastrointestinal digestion ............. 192 8.2.3 Fermentation ................................................................ 194 8.2.4 In silico aided enzymatic release of bioactive peptides......................................................................... 199 8.3 Analytical techniques for the identification of bioactive peptides..................................................................................... 201 8.3.1 Enrichment and fractionation of bioactive peptides......................................................................... 201 8.3.2 Peptide characterization.............................................. 203 8.4 Biological effects of whey-derived bioactive peptides ..........206 8.4.1 Antidiabetic peptides.................................................... 206 8.4.2 Antihypertensive peptides ........................................... 207 8.4.3 Antimicrobial peptides................................................. 208 8.4.4 Antioxidant peptides..................................................... 210 8.4.5 Anticancer peptides ..................................................... 212 8.4.6 Immunomodulatory peptides ...................................... 213 8.4.7 Antiinflammatory peptides .......................................... 214 8.4.8 Opioid-like peptides ..................................................... 216

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8.4.9 Satiety hormone-inducing peptides............................ 217 8.5 Conclusion and future prospects............................................ 218 Acknowledgments............................................................................. 219 References ........................................................................................ 219

CHAPTER 9

Bioactive peptides derived from camel milk proteins....... 233 Priti Mudgil and Sajid Maqsood 9.1 Introduction .............................................................................. 233 9.2 Bioactive peptides from camel milk proteins........................ 235 9.2.1 Antioxidant .................................................................... 236 9.2.2 Antihypertensive ........................................................... 245 9.2.3 Antimicrobial peptides from camel milk ................... 250 9.2.4 Antidiabetic peptides derived from camel milk proteins ......................................................................... 263 9.2.5 Antiobesity peptides from camel milk proteins and their structural activity relationship.................... 274 9.2.6 Other biological properties of camel milk derived hydrolysates.................................................................. 276 9.3 Future perceptions................................................................... 278 References ........................................................................................ 279

CHAPTER 10

Bioactive peptides from fermented milk products .......... 289 Dora Elisa Cruz-Casas, Stephany Nefertari Chávez-García, Adriana Carolina Flores-Gallegos, Leticia Anael García-Flores, Gloria A. Martínez-Medina, Lilia Arely Prado-Barragán and Rodolfo Ramos-González 10.1 Introduction ............................................................................ 289 10.2 Bioactive peptides from fermented bovine milk products ......................................................................... 291 10.2.1 Soful .......................................................................... 292 10.2.2 Yogurt........................................................................ 293 10.2.3 Koumiss .................................................................... 293 10.2.4 Kefir........................................................................... 294 10.2.5 Others ....................................................................... 295 10.3 Bioactive peptides from fermented goat milk products ......................................................................... 296 10.3.1 Yogurt........................................................................ 296 10.3.2 Kefir........................................................................... 296 10.3.3 Dahi ........................................................................... 297 10.4 Bioactive peptides from fermented camel milk products ...298 10.4.1 Camel milk ............................................................... 298

Contents

10.4.2 Bioactive peptides from fermented camel’s milk ............................................................. 298 10.4.3 Bioactive peptides from fermented camel milk products .................................................................... 299 10.5 Bioactive peptides from fermented mare milk products ...300 10.5.1 Koumiss .................................................................... 301 10.6 Bioactive peptides from fermented sheep milk products ..302 10.6.1 Koopeh ...................................................................... 303 10.6.2 Yogurt........................................................................ 304 10.7 Conclusion .............................................................................. 304 References ........................................................................................ 304

CHAPTER 11

Downstream processing of therapeutic bioactive peptide ................................................................................. 313 PourahmadRezvan and HosseiniElahesadat 11.1 Introduction ............................................................................ 313 11.2 Production mechanisms of bioactive peptides .................... 315 11.2.1 Enzymatic hydrolysis ............................................... 316 11.2.2 Fermentation ............................................................ 316 11.2.3 Enzymes derived from proteolytic microorganisms ....................................................... 317 11.3 Downstream processing of bioactive peptides (isolation, purification, and characterization)........................................ 319 11.3.1 Fractionation methods............................................. 320 11.3.2 Membrane separation techniques .......................... 320 11.3.3 Chromatographic methods...................................... 322 11.4 Conclusion .............................................................................. 325 References ........................................................................................ 325

CHAPTER 12

Enzyme actions during cheese ripening and production of bioactive compounds .................................. 331 R.Vázquez-García and Sandra T. Martín-del-Campo 12.1 Introduction ............................................................................ 331 12.2 Bioactive compounds............................................................. 332 12.2.1 Peptides .................................................................... 332 12.2.2 Conjugated linoleic acid .......................................... 337 12.2.3 Gama aminobutyric acid and L-ornithine ............... 338 12.2.4 Carotenoids .............................................................. 340 12.3 Conclusion .............................................................................. 340 References ........................................................................................ 340

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Section III Action of exogenous enzymes on lactose for value addition CHAPTER 13

Immobilization of β-galactosidases................................... 351 R. Hemamalini, Sumit Kumar and Sunil Kumar Khare 13.1 13.2 13.3 13.4 13.5 13.6

Introduction ............................................................................ 351 Sources of β-galactosidase ................................................... 352 Structure of β-galactosidase................................................. 353 Classification of β-galactosidases ........................................ 353 Reactions of β-galactosidase ................................................ 354 Immobilization of β-galactosidase........................................ 355 13.6.1 Functional enzyme aggregates ............................... 355 13.6.2 Gel beads and lattices ............................................. 356 13.6.3 Chitosan .................................................................... 356 13.6.4 Nanoparticles ........................................................... 356 13.6.5 Metal affinity columns ............................................. 357 13.6.6 Methacrylate and its variants.................................. 357 13.7 Conclusion .............................................................................. 357 Acknowledgments............................................................................. 358 References ........................................................................................ 358

CHAPTER 14

Low-lactose milk production using β-galactosidases ..... 361 Priscilla Romina De Gregorio, Adriano Gennari, Cathy Verônica Nied, Giandra Volpato and Claucia Fernanda Volken de Souza 14.1 Introduction ............................................................................ 361 14.2 Characteristics of β-galactosidases ..................................... 363 14.2.1 Sources of β-galactosidases ................................... 363 14.2.2 Reactions catalyzed by β-galactosidases............... 364 14.2.3 Optimal reaction conditions for β-galactosidases ...................................................... 365 14.2.4 Production and purification of β-galactosidases ...................................................... 367 14.2.5 Sources of industrial β-galactosidase.................... 368 14.2.6 Technologies for producing low-lactose milk ....... 369 14.2.7 Future scope............................................................. 371 14.3 Immobilized β-galactosidases .............................................. 372 14.4 Column reactors with immobilized β-galactosidases......... 376 14.5 Conclusions and perspectives .............................................. 378 References ........................................................................................ 378

Contents

CHAPTER 15

Production of oligosaccharides, a prebiotic from lactose, using β-galactosidase ................................. 383 Priscilla Romina De Gregorio, Adriano Gennari, Cathy Verônica Nied, Giandra Volpato and Claucia Fernanda Volken de Souza 15.1 Introduction ............................................................................ 383 15.2 Characteristics of β-galactosidases for the production of galactooligosaccharides.................................................... 385 15.2.1 Sources of β-galactosidases ................................... 385 15.2.2 Reactions catalyzed by β-galactosidases for the production of galactooligosaccharides ............ 386 15.2.3 Optimal reaction conditions for the production of galactooligosaccharides...................................... 387 15.2.4 Production and purification of galactooligosaccharides .......................................... 388 15.2.5 Sources of industrial β-galactosidase and galactooligosaccharides .......................................... 391 15.2.6 Future scope............................................................. 392 15.3 Immobilized β-galactosidase for the production of galactooligosaccharides ........................................................ 393 15.4 Conclusions and perspectives .............................................. 395 References ........................................................................................ 395

CHAPTER 16

Production of lactulose from cheese whey ...................... 403 Azis Boing Sitanggang 16.1 Introduction ............................................................................ 403 16.2 Lactulose production ............................................................. 405 16.2.1 Isomerization-based lactulose synthesis............... 406 16.2.2 Transgalactosylation-based lactulose synthesis... 414 16.3 Separation of lactulose.......................................................... 416 16.4 Health benefits of lactulose .................................................. 417 16.5 Conclusion .............................................................................. 418 References ........................................................................................ 419

Section IV Action of enzymes for measuring analyte or assessing milk quality or cleaning milk plant CHAPTER 17

Determination of lactose in milk and milk-derived ingredients using biosensor-based techniques ............... 427 Caleb Wagner, Richa Singh and Jayendra K. Amamcharla

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17.1 17.2 17.3 17.4

Introduction ............................................................................ 427 Importance of lactose in milk and dairy ingredients ..........428 Lactose quantification methods............................................ 428 Biosensors .............................................................................. 431 17.4.1 Biosensors used in dairy foods............................... 431 17.4.2 Biosensors used for lactose quantification ........... 431 17.5 Blood glucose meter biosensors as an option for determination of lactose ....................................................... 433 17.5.1 Blood glucose meter operating principles ............ 435 17.5.2 Potential issues with a blood glucose meter based lactose assay.................................... 436 17.5.3 Practical applications reported in literature for use of blood glucose meter in measurement of lactose.......................................... 438 17.6 Conclusion .............................................................................. 439 References ........................................................................................ 440

CHAPTER 18

Enzyme-based analytical methods pertinent to dairy industry .................................................................................. 445 C.G. Harshitha, Rajan Sharma and Y.S. Rajput 18.1 Introduction ............................................................................ 445 18.2 Urea estimation in milk......................................................... 446 18.2.1 Monitoring pH change ............................................. 447 18.2.2 Monitoring change in pressure............................... 447 18.2.3 Potentiometric approach ......................................... 448 18.2.4 Spectrophotometric measurement of ammonium ion concentration ................................. 449 18.2.5 Urea biosensor ......................................................... 449 18.3 Lactose estimation................................................................. 450 18.3.1 Spectrophotometric method ................................... 450 18.3.2 By measuring the change in pH ............................. 452 18.4 Estimation of lactate or lactic acid in dairy products ......... 452 18.5 Estimation of cholesterol in dairy products......................... 455 18.6 Ascorbic acid estimation in dairy products ......................... 457 18.7 Detection of common adulterants ........................................ 459 18.7.1 Detection and estimation of hydrogen peroxide in milk ....................................................... 460 18.7.2 Detection of glucose in milk ................................... 461 18.7.3 Detection and estimation of sucrose in milk and milk products ........................................... 462 18.7.4 Detection of maltodextrin in milk ........................... 463

Contents

18.7.5 Paper strip for urea detection ................................ 463 18.7.6 Detection and estimation of starch in milk and milk products .................................................... 464 18.8 Conclusion .............................................................................. 464 References ........................................................................................ 465

CHAPTER 19

Lactate biosensor for assessing milk microbiological load ...................................................................................... 471 Gurdeep Rattu and P. Murali Krishna 19.1 Introduction ............................................................................ 471 19.2 Lactic acid for assessing milk microbial load..................... 473 19.3 Methods of detection ............................................................. 474 19.3.1 Analytical conventional techniques ........................ 474 19.3.2 Lactate biosensors................................................... 476 19.3.3 Nanotechnology applications in sensors ............... 483 19.4 Conclusion and future prospective....................................... 486 Acknowledgment .............................................................................. 486 Conflicts of interest .......................................................................... 486 Ethical approval ................................................................................ 486 References ........................................................................................ 487

CHAPTER 20

Enzymes for cleaning-in-place in the dairy industry....... 491 Angela Boyce and Gary Walsh 20.1 Introduction: fouling and cleaning-in-place in the dairy industry.......................................................................... 491 20.2 Industrial enzymes and their use for cleaning-in-place in the dairy industry ................................ 493 20.3 Reported studies on the effectiveness of enzymes for cleaning-in-place in the dairy industry ................................ 496 20.3.1 Removal of Type A fouling deposits ....................... 496 20.3.2 Removal of biofilms ................................................. 504 20.4 Considerations for the development of optimal enzyme-based cleaning solutions ........................................ 509 20.5 Conclusions and future outlook............................................ 513 References ........................................................................................ 513

CHAPTER 21

Regulatory policies on use of food enzymes .................... 519 Smita Sahu, Shubhangi Agrawal and Ashwani Sahu 21.1 Introduction ............................................................................ 519 21.2 Regulatory framework regarding food enzymes................. 522

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21.3 Specific aspects of intellectual property right protection on enzymes ................................................. 523 21.4 Government policies toward food in biotechnology ............ 524 21.4.1 Enzyme regulation in Canada ................................. 525 21.4.2 Enzyme regulation in Australia and New Zealand............................................................. 525 21.4.3 European Union regulation ..................................... 526 21.4.4 Scope of enzyme regulation.................................... 528 21.4.5 Limitations ................................................................ 528 21.4.6 US regulations.......................................................... 528 21.4.7 FAO/WHO .................................................................. 531 21.5 Policy and regulatory framework: lower middle income countries.................................................................... 532 21.6 Future prospect...................................................................... 533 References ........................................................................................ 535

INDEX ...................................................................................................... 537

CHAPTER 1

Milk enzymes Pranali Nikam1, Y.S. Rajput2, Rajan Sharma3 and Suvartan Ranvir4 1

Dairy Chemistry Department, College of Dairy Science and Food Technology, Raipur, 2 Chhattisgarh, India, Animal Biochemistry Division, ICAR-National Dairy Research 3 Institute, Karnal, Haryana, India, Dairy Chemistry Division, ICAR-National Dairy Research 4 Institute, Karnal, Haryana, India, Department of Dairy Chemistry, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, Uttar Pradesh, India

1.1

Introduction

Enzymes are delicate organic catalysts mainly secreted by living cells (animals, microorganisms, and plants) and work independently. These are transported from the site of production to the site of action through the circulatory systems. Enzymes can be differentiated from other catalysts on the basis of their chemical nature, specificity, and sensitivity. A distinguished characteristic of any enzyme is its specificity of the reaction that it can catalyze. Most enzymes are proteins, and they function within a narrow pH and temperature range. In general, due to its protein nature, they are fairly sensitive to heat. Milk is good medium for the growth of bacteria. Both pathogenic and nonpathogenic bacteria are reported in milk and contribute to enzyme activity in milk. Because of diverse nature of bacteria in milk, it becomes difficult to numerate enzymes in milk. Nevertheless, about 70 indigenous enzymes in normal bovine milk have been reported (Fox, 2003). Several enzymes in milk are present at very low concentrations. In this chapter, cataloging of milk enzymes, enzyme level in milk of different species, enzyme characteristics, and technological significance of some enzymes in dairy industry are presented. In milk, most of casein, the major protein in milk, is present in micellar (colloidal) form and fat is a part of milk fat globule membrane (MFGM). Milk under centrifugal force separates into fat layer (top layer), micelles at bottom, and serum as middle layer. Because of the use of different milk fractions for different milk products, enzyme activities in these fractions have also been studied. In this chapter, a detailed account of the enzymes which can impact or improve milk quality has been provided. For this purpose, enzymes are grouped as Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00001-1 © 2023 Elsevier Inc. All rights reserved.

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Milk enzymes

proteinases (plasmin and cathepsin D); lipases and esterases [lipase, lipoprotein lipase (LPL), bile salt stimulated lipase (BSSL), and esterase]; phosphohydrolases (alkaline phosphatase, acid phosphatase, and ribonuclease); and oxidases (lactoperoxidase, catalase, xanthine oxidase, superoxide dismutase, and sulfhydryl oxidase). Also, few individual enzymes of significance in milk such as γ-glutamyl transpeptidase (GGTP), N-acetyl-β D-glucosaminidase (NAGase), and lysozyme are also discussed. Psychrotrophs survive in milk stored at refrigerated temperature and produce protease and lipase enzymes which are also touched in this chapter.

1.2 Enzymes in milk: significance, nomenclature, reaction catalyzed, and activity levels The indigenous milk enzymes are minor constituents of milk and are secreted in milk from following sources (Fox & Kelly, 2006a): 1. From blood plasma through defective mammary cells. 2. From secretory cell cytoplasm, some of which may possibly be entrapped within some fat globules by the surrounding MFGM at the time of secretion from the cell. 3. From the MFGM itself, the outer layer of which is obtained from the apical membrane of the mammary cell and which, in turn, originates from the Golgi membranes; this may be the most possible source of secretion of indigenous enzymes in milk. 4. Somatic cells (leukocytes), which may enter the mammary gland from the blood to fight bacterial infection (mastitis), and therefrom enter milk. 5. From contaminating microorganisms which may secrete extracellular enzymes or release intracellular enzymes after the cells have died and lysed. Thus most enzymes enter milk due to their specific and distinct mechanism by which milk constituents, especially the fat globules, are excreted from the secretory cells. For many of these indigenous enzymes, milk does not contain specific substrates while others are sedentary in milk due to inappropriate environmental conditions, for example, optimum temperature, pH, or Eh (oxidation reduction) potential. Nevertheless, many indigenous milk enzymes are of significance (Fig. 1.1). Enzymes (1) assist in milk preservation, (2) exhibit antimicrobial activity, (3) act as markers for heat treatment and mastitis infection, and (4) are responsible for milk deterioration. Milk can be the biological source for isolation of xanthine oxidase, lactoperoxidase and ribonuclease.

1.2 Enzymes in milk: significance, nomenclature, reaction catalyzed, and activity levels

FIGURE 1.1 Significance of indigenous milk enzymes.

Enzymes have been classified by International Union of Biochemistry and Molecular Biology (IUBMB). Six different classes are oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Each class is further divided into subclasses, each subclass is again divided into sub-subclasses, and, finally, each sub-subclass contains several enzymes. Every enzyme is assigned Enzyme Commission (EC) number. For example, the serial classification number for plasmin is EC 3.4.21.7. In this number the first digit “3” from the left represents the class “hydrolases” the second digit “4” represents subclass “enzymes” acting on “proteins”; the third digit “21” represents subsubclass of proteins bond hydrolysis" and the fourth digit “7” represents enzyme catalyzing specific reaction. Milk contains at least 63 enzymes which fall under all six classes of enzymes but majority of them belong to oxidoreductases, transferases, and hydrolases. There are only few reported enzymes from lyases, isomerases, and ligases class. Main constituents of milk are fat, caseins, whey proteins, lactose, and minerals. Enzymes in milk constitue as minor component and can be present in milk fat (fat globule membrane), skim milk or plasma (milk minus fat), and whey or serum (skim milk minus casein) fractions. EC number of indigenous milk enzymes, reaction catalyzed, and their major location in milk fractions are summarized in Table 1.1

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Milk enzymes

Table 1.1 Indigenous milk enzymes, reaction catalyzed, and their major location in milk. EC no.

name

Reaction catalyzed

Locationa

2GSH 1 H2O2 " GS SG 1 2H2O 1 L-Iditol 1 NAD " L-sorbose 1 NADH 1 1 L-lactate 1 NAD " pyruvate 1 NADH 1 H 1 1 L-malate 1 NAD " oxaloacetate 1 NADH 1 H Malate 1 NADP1 " pyruvate 1 CO2 1 NADH Isocitrate 1 NADP1" 2-oxogluterate 1 CO2 1 NADH 6-Phospho-D-gluconate 1 NADP1 " D-ribose-5phosphate 1 CO2 1 NADPH D-Glucose-D-gluconate 1 NADP 1 " D-glucono1,5-lactone-6-phosphate 1 NADPH Xanthine 1 H2O 1 O2 " urate 1 superoxide RCH2NH2 1 H2O 1 O2 " RCHO 1 NH3 1 H2O2 Catalyzes the transfer of fucose form GDP L-fucose to specific oligosaccharides and glycoproteins NADH 1 H1 1 lipoamide " NAD1 1 dihydrolipoamide NADH 1 H1 1 acceptor " NAD1 1 reduced acceptor 2 RSH 1 O2 " RS-SR 1 H2O2 Dihydrolipoamide 1 NAD1 " lipoamide 1 NADH

P P P P P P

H2O2 1 H2O2 " O2 1 2 H2O Donor 1 H2O2 " oxidized donor 1 2 H2O O2 1 O2 1 2 H1 " O2 1 H2O2

L/C S P

(4-L-glutamyl)-peptide 1 an amino acid " peptide 1 4-L-glutamyl-amino acid UDP-galactose 1 D-glucose " UDP 1 lactose

F

Oxidoreductases 1.1.1.9 1.1.1.14 1.1.1.27 1.1.1.37 1.1.1.40 1.1.1.42

Glutathione peroxidase L-Iditol dehydrogenase Lactate dehydrogenase Malate dehydrogenase Malic enzyme Isocitrate dehydrogenase

1.1.1.44

1.2.3.2 1.4.3.6

Phosphoglucuronate dehydrogenase (decarboxylating) Glucose-6-phosphate dehydrogenase Xanthine oxidase Amine oxidase (Cu- containing)

-

Fucosyltransferase

1.6.4.3

Dihydrolipoamide reductase (NAD1)

1.6.99.3

NADH dehydrogenase (cytochrome C reductase) Sulfhydryl oxidase Dihydrolipoamide dehydrogenase (diaphorase) Catalase Lactoperoxidase Superoxide dismutase

1.1.1.49

1.8.3 1.8.1.4 1.11.1.6 1.11.1.7 1.15.1.1

P P F P P F F S P/F

Transferases 2.3.2.2

γ-glutamyl transpeptidase

2.4.1.22

Lactose synthase A protein: UDP galactosyltransferase B protein: α-lactalbumin Glycoprotein 4-β-galactosyltransferase

2.4.1.38

2.4.99.6

CMP-N-acetyl-N-acetyllactosaminide α-2,3-sialyltransferase

2.5.1.3

Thiamine-phosphate pyrophosphorylase

UDP-galactose 1 N-acetyl-D-glucosaminylglycopeptide " UDP 1 4,β-D-galactosyl-N-acetylD-glucosaminyl glycopeptide CMP-N-acetylneuraminate 1 β-D-galactosyl 1,4-N-acetyl-D-glucosaminyl-glycoprotein " CMP 1 α-N-acetylneuraminyl 1-2,3-β-D-galactosyl-1,4-Nacetyl-D-glucosaminyl-glycoprotein 2-Methyl-4-amino-5-hydroxymethyl/pyrimidine diphosphate 1 4-methyl-5-(2-phosphonooxyethyl)thiazole " pyrophosphate 1 thiamine monophosphate

P/S

F

P

F

Continued

1.2 Enzymes in milk: significance, nomenclature, reaction catalyzed, and activity levels

Table 1.1 Indigenous milk enzymes, reaction catalyzed, and their major location in milk. Continued EC no.

name

Reaction catalyzed

2.6.1.1

Aspartate amino transferase

L-aspartate

1 2-oxoglutarate " oxalacetate 1

Locationa P

L-glutamate

2.6.1.2

Alanine amino transferase

2.7.1.30

Glycerol kinase

2.7.7.49

RNA-directed DNA polymerase

2.8.1.1

Thiosulfate sulfur transferase (rhodanese)

1 2-oxogluterate " pyruvate 1 Lglutamate ATP 1 glycerol " ADP 1 sn-glycerol-3phosphate n Deoxynucleoside triphosphate "n pyrophosphate 1 DNA S2O322 1 CN2" SO322 1 SCN2 L-alanine

P F P P

Hydrolases 3.1.1.1

Carboxylesterase

3.1.1.2 3.1.1.3

Arylesterase Triacylglycerol lipase

3.1.3.5 3.1.1.7 3.1.1.8

5'-Nucleotidase Acetylcholine-esterase Cholinesterase

3.1.1.34

Lipoprotein lipase

3.1.3.1

Alkaline phosphatase

3.1.3.2 3.1.3.9

Acid phosphatase Glucose-6-phosphatase

3.1.3.16

Phosphoprotein phosphatase

3.1.4.1

Phosphodiesterase

3.1.6.1 3.1.27.5

Arylsulfatase Ribonuclease (pancreatic)

3.2.1.1

α-amylase

3.2.1.2

β-amylase

3.2.1.17

Lysozyme

3.2.1.21

β-Glucosidase

3.2.1.23

β-Galactosidase

Carboxylic ester 1 H2O " alcohol 1 carboxylate ion A phenyl acetate 1 H2O " a phenol 1 acetate Triacylglycerol 1 H2O " diacylglycerol 1 fatty acid anion Nucleotide 1 H2O" a nucleoside 1 phosphate Acetylcholine 1 H2O " choline 1 acetate An acylcholine 1 H2O " choline 1 carboxylate anion Triacylglycerol 1 H2O " diacylglycerol 1 fatty acid anion An orthophosphoric monoester 1 H2O " alcohol 1 orthophosphate Same as above D-glucose-6-phosphate 1 H2O " D-glucose 1 orthophosphate Hydrolyzes phosphate ester bonds in phosphoproteins A phosphoric diester 1 H2O " a phosphoric monoester 1 alcohol Phenol sulfate 1 H2O " phenol 1 sulfate Endonucleolytic cleavage to 3'- phosphomono- and oligonucleotides ending in Cp or Up Hydrolyzes α-1,4-glucan links in polysaccharides containing three or more glucosyl residues. Hydrolyzes α-1,4-glucan links in polysaccharides by removing successive maltose units from nonreducing end Hydrolyzes β-1,4-links between N-acetylmuramic acid and 2- acetamido-2-deoxy-D-glucose Hydrolysis of terminal nonreducing β-D-glucose residues Hydrolysis of terminal nonreducing β-D-galactose residues in β-D-galactosides

S S S/F F F S/F C F F/C/S F P F M S S P

S F F

Continued

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Table 1.1 Indigenous milk enzymes, reaction catalyzed, and their major location in milk. Continued EC no.

name

Reaction catalyzed

Locationa

3.2.1.24

α-D-Mannosidase

C

3.2.1.30

N-acetyl-β-D-glucosaminidase

3.2.1.31

β-Glucuronidase

3.2.1.51 3.1.6.1 3.4.11.1

α-Fucosidase Arylsulfatase Cytosol aminopeptidase (leucine aminopeptidase) Cystyl-aminopeptidase (oxytocinase) Trypsin Plasmin

Hydrolyzes terminal, nonreducing α-D-mannose units in α-D-mannosides Hydrolyzes terminal, nonreducing 2-acetamido-2deoxy-β-D-glucose residues in chitobiose and higher analogs and in proteins A β-D-glucuronide 1 H2O " alcohol 1 D-glucuronate An α-L-fucoside 1 H2O " an alcohol 1 L-fucose An aryl sulfate 1 H2O " phenol 1 sulfate Aminoacyl-peptide 1 H2O " amino acid 1 peptide Cystyl-peptides 1 H2O " amino acid 1 peptide

3.4.11.3 3.4.21.4 3.4.21.7 3.4. 3.6.1.1 3.6.1.3 3.6.1.9

P

P P P P P C

Cathepsin D Inorganic pyrophosphatase Adenosine triphosphatase (Mg11 activated) Nucleotide pyrophosphatase

Hydrolyzes peptide bonds, preferentially Lys-X, Arg-X Hydrolyzes peptide bonds, preferential cleavage Lys . Arg, higher selectivity than trypsin Hydrolyzes peptide bonds Pyrophosphate 1 H2O " 2 orthophosphate ATP 1 H2O " ADP 1 orthophosphate A dinucleotide 1 H2O " 2 mononucleotides

P/F

Carbonic dehydratase

H2CO3 " CO2 1 H2O

P

P/S P/F F

Lyases 4.2.1.1

Isomerases 5.3.1.9

" D- fructose-6-phosphate

Glucose-6-phosphate isomerase

D-glucose-6-phosphate

Acetyl-CoA carboxylase

ATP 1 acetyl-CoA 1 HCO3 " ADP 1 orthophosphate 1 malonyl-CoA

P

Ligases 6.4.1.2

P

a P, Plasma; S, serum; F, fat globule membrane; C, casein micelles; L, leukocytes, M, milk. Source: Compiled from Farkye (2003); Fox et al. (2015); Kitchen (1985); Shahani et al. (1973); Walstra and Jenness (1984) and updated.

Enzymes in milk can be derived from blood, somatic cells (leukocytes), mammary gland, and microorganisms. Kinetic parameters of principal indigenous milk enzymes, their sources, and reaction catalyzed are summarized in Table 1.2. Several enzymes possess optimum pH close to 7.0 or alkaline pH although milk pH is about 6.8. Optimum temperature for majority of enzymes is about 37 C.

1.2 Enzymes in milk: significance, nomenclature, reaction catalyzed, and activity levels

Table 1.2 Principal indigenous milk enzymes, their source, substrates, kinetic parameters, and reaction catalyzed. Optimum pH

Optimum Temp.

Proteins (αs1-CN, αs2-CN, β-CN)

6.5 8.0

37 C

Hydrolyzes peptide bonds, preferential c-terminal side cleavage Lys . Arg, higher selectivity than trypsin, β-CN . αs-CN . κ-CN

Milk fats, tributyrin, glycerides Milk fats, glycerides

9.0

37 C

9.0

37 C

Phenyl acetate

8.0

37 C

Tributyrln

8.0

37 C

Phenyl propionate

8.0

37 C

Triglycerides 1 H2O " fatty acids 1 partial glycerides 1 glycerol Triglycerides 1 H2O " fatty acids 1 partial glycerides 1 glycerol Phenyl acetate 1 H2O " phenol 1 acetate Carboxylic ester 1 H2O " alcohol 1 carboxylate ions Acylcholine 1 H2O " choline 1 carboxylate anion

Mammary gland Mammary gland Blood

Organic phosphates

9 10

37 C

Organic phosphates

4.0

14 50 C

Phosphodiester bonds

7.0 7.5

37 C

Mammary gland Somatic cells (leukocytes) Blood

H2O2,reducible agents H2O2

6 7

20 C

7.0

37 C

Xanthine, aldehydes, oxypurines, pterin

6 9

37 C

Superoxide dismutase Sulfhydryl oxidase

-

O2 cysteine

7.0 7 8

35 C 45 50 C

V. γ-Glutamyl transferase (transpeptidase, GGTP)

Mammary gland

Glutathione

8 9

45 C

(4-L-glutamyl)-peptide 1 an amino acid " peptide 1 4-L-glutamylamino acid

VI. N-Acetyl-β -Dglucosaminidase

Somatic cells

N-acetyl-β-Dglucosamine

4.2

50 C

Hydrolyzes terminal, nonreducing 2-acetamido-2-deoxy-β-D-glucose residues in chitobiose and higher analogs and in proteins

VII. Lysozyme

Lysosomal enzyme

Cell wall of mucopolysaccharides

7.9, 6.35

37 C

Hydrolyzes β-1,4-links between N-acetylmuramic acid and 2 acetamido-2-deoxy-D-glucose

Enzyme

Source

Substrate

I. Plasmin

Blood

II. Lipase and esterase Lipase

Blood

Lipoprotein lipase A-Esterase (Arylesterase) B-Esterase (Carboxylesterase) C-Esterase (Cholinesterase) III. Phosphohydrolases Alkaline phosphatase Acid phosphatase Ribonuclease

IV. Oxidases Lactoperoxidase Catalase

Xanthine oxidase (oxidoreductase, XOR)

Mammary gland Mammary gland Mammary gland Mammary gland

Reaction catalyzed

Orthophosphoric monoester 1 H2O " alcohol 1 orthophosphate Orthophosphoric monoester 1 H2O " alcohol 1 orthophosphate Endonucleolytic cleavage to 3phosphomono and oligonucleotides ending in Cp or Up Donor 1 H2O2 " oxidized donor 1 2 H2O H2O2 1 H2O2 " O2 1 2 H2O

Xanthine 1 H2O 1 O2 " urate 1 superoxide Aldehyde 1 H2O 1 O2 " Acid 1 H2O2 2O2d2 1 2 H1 " O2 1 H2O2 2 RSH 1 O2 " RS-SR 1 H2O2

9

10

CHAPTER 1:

Milk enzymes

Enzyme level in milk depends on the stage of lactation, animal health, physiology of animal, feed and fodder consumed, as well as on species of animal. Milk may also contain inhibitors and activators of enzymes which may influence measured enzyme activity. World milk production is almost entirely derived from cattle, buffaloes, goats, sheep, and camels. Cattle produce 81% of world milk production, followed by buffaloes with 15%, goats with 2%, sheep with 1%, and camels with 0.5%. The remaining share is produced by other dairy species such as yaks, horses, reindeers, and donkeys. The level of enzymes in milk from cow, buffalo, goat, sheep, camel, and human milk is depicted in Table 1.3. Most commonly studied enzymes are plasmin, LPL, alkaline phosphatase, acid phosphatase, ribonuclease, lactoperoxidase, catalase, xanthine oxidase, superoxide dismutase, sulfhydryl oxidase, γ-glutamyl transpeptidase, N-acetyl-β D-glucosaminidase, and lysozyme. These enzymes are either present at sufficient concentration in milk or are of technological significance in dairy science and milk processing.

1.3

Proteinases

Proteinases hydrolysis peptide bonds in protein. Plasmin, an alkaline milk proteinase, and cathepsin D, an acid proteinase, are present in milk.

1.3.1

Plasmin

Plasmin is main indigenous proteinase which can be active at milk pH. This enzyme has been described in Chapter 4 and readers are advised to refer the chapter.

1.3.2

Cathepsin D

Cathepsin D is the second highest proteinase (Kaminogawa & Yamauchi, 1972), presumably having lysosomal origin found in acid whey (Larsen et al., 1996). In milk, the principal form of cathepsin D proteinase is inactive zymogen and procathepsin D; however, low concentration of mature form of cathepsin D is also observed in milk. Enzyme is also a part of complex plasmin system together with inactive precursor. The presence of cathepsin D in milk is accompanying with somatic cell count (O’Driscoll et al., 1999), but it is still unknown whether its presence is associated with increased production of cathepsin D and/or increased activation of precursors. The αs1-CN (f24 199) is the principal peptide obtained from αs1-casein by the action of cathepsin D, which is also considered as the primary peptide obtained from rennin (chymosin). The proteolytic characteristics of cathepsin D on bovine milk β-casein are quite same as rennin enzyme (Larsen et al., 1996; McSweeney et al., 1995). Cathepsin D is able to cleave κ-casein but has very

Table 1.3 Levels of enzymes in cow, buffalo, goat, sheep, camel, and human milk. Enzyme

Cow milk

Buffalo milk

Goat milk

Sheep milk

Camel milk

Human milk

References

Plasmin

0.07 0.15 μga 5.4c

-

-

5.15 6.90c -

2 150e -

4.8c

0.7 2.4 μga 45.3c

-

20.04c -

-

22 207e -

12.4c

56.8c

-

3.21c -

12.7c

-

-

Rollema et al. (1983) Castillo et al. (2008) Baer et al. (1994) Korycha-Dahl et al. (1983) Fantuz et al. (2001) Rollema et al. (1983) Baer et al. (1994) Korycha-Dahl et al. (1983) Fantuz et al. (2001) Moatsou (2010)

1320b 600b

-

390b -

90b -

-

130b -

1.3c 500b

2.2c -

1.2c -

0.160 0.243c -

57 71d -

-

0.18 0.27c 282.56d 774d 61.39c 30.59c 575c

79.20c 14.46c 253.75c

67d 26.62c 15.79c -

1.819-2.653c 1414d -

15.9 24.3d 21.31c

147c 200.4d -

-

295d

-

-

-

-

Plasminogen

Plasmin 1 Plasminogen Lipoprotein lipase

Alkaline phosphatase

Chandan et al. (1968) Walstra and Jenness (1984) Chávarri et al. (1998) Lorenzen et al. (2011) Prajapati et al. (2017) Walstra and Jenness (1984) Stewart et al. (1958) Kitchen et al. (1970) Chávarri et al. (1998) Dhar et al. (1996) Lorenzen et al. (2010) Sharma et al. (2009) Lorenzen et al. (2011) Prajapati et al. (2017) Yoganandi et al. (2014) Lombardi et al. (2000)

Continued

Table 1.3 Levels of enzymes in cow, buffalo, goat, sheep, camel, and human milk. Continued Enzyme

Cow milk

Buffalo milk

Goat milk

Sheep milk

Camel milk

Human milk

References

Acid phosphatase

13b

-

-

-

-

-

-

-

4.25 mga 0.0277 0.0342c

21.61 29.29c 3.00 mga -

-

3.0 mga 0.059c

10,000 20,000 μga 11 25 mga

-

4250 μga

-

-

-

-

100 200 μga -

19.2 35.4c 22,000b

-

-

-

-

-

-

-

77.25c -

-

0.26c

136c 8.653 10.565c 1.38 6.10c

-

0.07c -

2015d 6.86c 0.97c

-

12.02c -

5190d 0.77c 0.81c

2859d 1.72c

869 1172d -

-

300b

-

-

-

-

-

7.5 36.0c 175c (mastitic milk) 0.86c

0.62c

-

-

6.20c

-

1.18c

0.85c

1.03c

-

-

-

Walstra and Jenness (1984) Kitchen et al. (1970) Chávarri et al. (1998) Chandan et al. (1968) Liu and Williams (1982) Park (2010) Walstra and Jenness (1984) Kitchen et al. (1970) Walstra and Jenness (1984) Morin et al. (1995) Chávarri et al. (1998) Althaus et al. (2001); Seifu et al. (2004) Lorenzen et al. (2010) Sharma et al. (2009) Lorenzen et al. (2011) Dumitrascu ¸ et al. (2012) Walstra and Jenness (1984) Kitchen et al. (1970) Kitchen et al. (1970) Yoganandi et al. (2014) Prajapati et al. (2017)

Ribonuclease

Lactoperoxidase

Catalase

0.0026 0.0037c 11.00 mga 0.366c

-

175b

-

-

-

-

-

15.6 21.4c 9.4c (mastitic milk) 1.83c

-

0.27c

0.69c

-

0.06c

82.45c 120c 0.1 2.5a

96.08c

-

-

-

96.08c 19 113c -

-

3 mga

-

-

-

4143d 8.31c 7081d

8.14c -

603d 2.09c 951d

1878d 4025d

282 366d -

28.8c -

N-Acetyl-β Dglucosaminidase

19.86c -

712d 22.49c -

5.21c 1.51e

-

-

-

Lysozyme

4.06e 130 μga

-

-

1.14e -

-

91.75e -

0.0178 0.0382c

0.0373 0.0734c (milk),0.3017c (colostrum) -

-

-

-

-

250 μga 250 μga

10μga -

150 μga -

4,00,000 μga 40 400 μga

Xanthine oxidase

Superoxide dismutase Sulfhydryl oxidase γ-glutamyl transpeptidase

130 μga 70 μga 100 350 μga a

mass per liter of milk. μmol/min/L. units/mL. d units/L. e nmol/min/mL. b c

-

Walstra and Jenness (1984) Kitchen et al. (1970) Kitchen et al. (1970) Benboubetra et al. (2004) Sharma et al. (2009) Park (2010) Walstra and Jenness (1984) Shin and Oh (2018) Landon (1975) Lorenzen et al. (2010) Sharma et al. (2009) Lorenzen et al. (2011) Dumitrascu ¸ et al. (2013) Lombardi et al. (2000) Sharma et al. (2009) Timms and Schultz (1985) Morin et al. (1995) Walstra and Jenness (1984) Priyadarshini and Kansal (2002b) Chandan et al. (1968) Elagamy et al. (1996) Park (2010)

14

CHAPTER 1:

Milk enzymes

poor milk clotting properties. Two cleavage sites of cathepsin D on α-lactalbumin have been identified but native β-lactoglobulin is quite resistant to cathepsin D (Larsen et al., 1996; McSweeney et al., 1995). At least some cathepsin D is incorporated into cheese curd even after hightemperature short-time (HTST) pasteurization (Swiss varieties cheese) and contributing proteolysis in cheese but its activity is normally dominated by chymosin, which is present at a much higher level.

1.4 1.4.1

Lipases and esterases Lipase

Lipase (EC 3.1.1.3) breaks down triglycerides into free fatty acids and glycerol (Fig. 1.2). From technological point of view, lipase is the most significant indigenous milk enzyme to dairy industry as it causes undesirable rancid flavor in milk due to hydrolytic rancidity while in certain varieties of ripened cheeses, it contributes desirable flavor too. Usually, the substrate (soluble esters) required for enzyme activity is not accessible inherently. But, during the rapid cooling of milk, enzyme dissociates from the casein micelles and interacts to fat globules imparting in “spontaneous lipolysis” and mild mechanical treatments, such as agitation, foaming, freezing or homogenization, cooling/ warming, to unpasteurized milk leads to disruption of MFGM resulting in interaction between the casein micelles and its associated lipase (Corbin, 1965). Apart from to contribute off-flavors in milk and dairy products, lipolysis causes reduction in the surface activity of milk due to liberation of free fatty acids, and as a result, decrease in milk foaming capacity, for example, in cappuccino coffee, and also decrease in its whipping characteristics and churning time (Deeth & Fitz-Gerald, 2009). Milk contains multiple forms of lipase which are due to self-association of lipase or its association with other milk proteins (Fox & Kelly, 2006a) and 90% of lipase in milk is interlinked with the micellar casein. It is sensitive to heat and light.

FIGURE 1.2 Reaction catalyzed by lipase.

1.4 Lipases and esterases

1.4.2

Lipoprotein lipase (EC 3.1.1.34)

LPL is a 90-kDa homodimer wherein each monomer contains 450 amino acid and 8% carbohydrates. The enzyme mainly originates from the vascular endothelial surfaces bounded with heparin sulfate chains playing an important role in lipid synthesis in the mammary gland. The optimum pH and temperature for LPL are 9.0 and 37 C. The catalytic activity kcat of LPL is  3000/s under optimum conditions and milk has adequate lipase (1 2 mg/ L; 10 20 nM) to cause hydrolytic rancidity in short period, that is, within 10 s. However, LPL causes hydrolytic rancidity in most of the milk samples only if the MFGM is damaged, for example, by agitation, foaming, cooling/warming, freezing, or homogenization. But, some bovine milk samples undergo spontaneous lipolysis, with no activation step. Such milk sample contains high level of apolipoprotein C-II, which activates LPL. Normal milk has a higher level of proteose peptone-8 (PP-8), which mainly inhibits LPL (Fox et al., 2015; Girardet et al., 1993; He et al., 2012). Caprine milk contains only B4 % as much lipolytic activity compared to bovine milk, but still very prone to spontaneous rancidity and responsible for “goaty” flavor which may be due to minor branched-chain fatty acids, 4-methyl and 4-ethyloctanoic acids (DeFeo et al., 1982). This difference may due to the distribution pattern of LPL; in bovine milk, 80% of LPL is with casein micelles while in caprine milk only ,10% of the LPL is associated with the micelles. Ovine milk has 10% of the LPL activity to that of bovine milk. Guinea pig milk contains high LPL activity but rat milk has low activity (Hamosh & Scow, 1971).

1.4.3

Bile salt stimulated lipase

In the early years of the 20th century, it was reported that human milk has higher lipolytic activity than bovine milk (Palmer, 1922). Human milk has a second lipase system in addition to LPL, that is, BSSL, which is similar to the broad-specificity pancreatic carboxylic ester hydrolase, also known as cholesterol ester hydrolase (CEH) (Chen et al., 1998). Human BSSL has a total molecular mass of B105 kDa, with 722 amino acid residues containing 15% 20% carbohydrate. BSSL synthesized in mammary gland and human milk is rich in BSSL. It shows great homology with lysophospholipase from rat pancreas and acetylcholine esterase as well as to CEH. BSSL is considered as essential lipase for digestion of lipids in human infants who are lacking or low in both pancreatic lipase and bile salts (Hernell & Bläckberg, 1991; Shahani et al., 1980).

1.4.4

Esterases

In addition to the well-documented lipase system, bovine milk contains several other carboxyl ester hydrolases, collectively referred to as esterases.

15

16

CHAPTER 1:

Milk enzymes

Esterases differ from lipases by their substrate preference for soluble rather than emulsified esters form as well as their preferences for hydrolyzing esters of short rather than long-chain acids. According to Kitchen (1985), milk possesses several esterases, mainly significant are arylesterases (EC 3.1.1.7), carboxylesterase (EC 3.1.1.1), and cholinesterase (EC 3.1.1.8). Arylesterase (also known as solalase/A-esterases) has received considerable attention because of its elevated levels in colostrum and mastitic milk. Since its level in mastitic milk correlates well with other indices of mastitis, it has been suggested as a sensitive indicator of the disease. The enzyme originates from blood, where its activity is up to 2000 times that in milk. Carboxylesterase (B-esterases) activity is elevated in mastitic milk and colostrum, for example, retinyl esterase. It is of interest that the BSSL in human milk, which shows activity identical with pancreatic carboxylesterase, has retinyl esterase activity. Cholinesterases (C-esterases) are most active on choline esters but hydrolyze some aromatic and aliphatic esters slowly; they are inhibited by organophosphates. In normal milk, the ratio of A : B : C esterase activity is about 3 : 10: 1. In abnormal milks, esterase activity is markedly elevated (up to 37 times) (Kitchen, 1981).

1.5

Phosphohydrolases

Phosphohydrolase enzyme catalyzes the hydrolysis of phosphoric acid esters. A great variety of phosphatases existing in nature are phosphomonoesterases, phosphodiesterases, phosphorylases, pyrophosphatases, nucleotidases, and phytases. The technologically significant principal phosphohydrolases reported in milk are alkaline phosphatase (ALP), acid phosphatase (ACP), and ribonuclease.

1.5.1

Alkaline phosphatase

Alkaline phosphatase hydrolyzes monoesters of phosphoric acid (Fig. 1.3) under alkaline condition (pH 9 10.5) and its optimum temperature is 37 C. The phosphatase in milk was first documented in 1925 by F. Demuth (Whitney, 1958). It is naturally present in blood and milk of all mammals. The enzyme is a homodimer of two identical subunits, with MW of 85 kDa and contains 4 atoms of zinc.

FIGURE 1.3 Reaction catalyzed by alkaline phosphatase.

1.5 Phosphohydrolases

Zn11 is essential for its activity but it is also activated by Mg11 (Linden et al., 1974; Linden & Alais, 1976, 1978). It is inhibited by inorganic phosphate and metal chelators. The sequence of amino acids in milk ALP has not been reported but the sequence of human placental and germ cell ALPs show 98% homology (Hoylaerts & Millán, 1991). The milk ALP structure is likely similar to those of Escherichia coli ALP (Fox & Kelly, 2006b). ALP is glycosylated. ALP enzyme is associated mainly in membrane structures of both skim milk and cream but a small amount of activity is found free in serum prepared by ultracentrifugation of skim milk (Groves, 1971). Microsomes (portion of outer MFGM), which contains ALP, is present in skim milk. As this enzyme is more associated with lipid phase, release of this enzyme from cream to the skim milk phase has been used to assess agitation history of raw milk (Stannard, 1975). ALP can be released from membrane on its treatment with butanol or action of phospholipase C enzyme. ALP in milk is largely studied as an index of proper pasteurization which results in killing of non-spore-forming pathogens. Thermal stability of ALP is slightly better than time temperature treatment required for inactivation of pathogens. ALP activity is routinely measured after pasteurization of milk and the absence of ALP activity in milk is indicative of proper pasteurization. Milk is normally pasteurized by holding milk at 72 C for 15 s and is referred to as HTST pasteurization. Under certain milk treatment conditions or storage or both, inactivated ALP is reactivated and ALP test is no longer valid for proper pasteurization (McKellar et al., 1994). High fat content, elevated storage temperatures, and HTST heat treatment in excess of 77.8 C are known to elevate the potential for phosphatase reactivation (Mcfarren et al., 1960). At elevated temperature, sulfhydryl (-SH) groups in whey proteins and ALP are generated. Metal ions can bind to -SH groups. Limited quantity of metal ions compete for -SH groups and this makes some of -SH groups on ALP available for refolding which results in reactivation (Fox & Kelly, 2006b). It has been postulated that Mg11 or Zn11 ion imparts a conformational change in the denatured enzyme, which is necessary for renaturation. Maximum reactivation occurs in products heated at 104 C, adjusted to pH 6.5, containing 64 mM Mg11 incubated at 30 C; and homogenization of raw milk prior to heat processing reduces the magnitude of reactivation (Fox & Kelly, 2006b).

1.5.2

Acid phosphatase

Acid phosphatase in milk is optimally active at acidic pH 4.0, and relatively less thermally labile. It is inactivated at temperature of 88 C during 10-min

17

18

CHAPTER 1:

Milk enzymes

treatment. Unlike ALP, ACP is not activated by Mg11. ACP is strongly inhibited by fluoride ions. The ACP activity in milk is only  2% to that of ALP and reaches maximum at 5 6 days postpartum then slows down till the end of lactation (Andrews et al., 1992). The activity of ACP enzyme in milk increases 4 10 times during mastitis infection (Flynn, 1999). Mastitis milk has ACP of bacterial origin as well. Although ACP enzyme is present in milk at low level than ALP enzyme, its greater heat stability and lower pH make it technologically significant. Dephosphorylation of casein diminishes its heat stability and ability to bind Ca11 ions to react with κ-CN and to form micelles (Fox & Kelly, 2006b). Several small partially dephosphorylated peptides have been isolated from certain cheese varieties such as Cheddar, Parmigiano Reggiano, and Grana Padano. However, it is not known whether indigenous or bacterial acid phosphatase is mainly responsible for dephosphorylation in cheese made from pasteurized milk. It is revealed that ALP enzyme is chiefly responsible for dephosphorylation of peptides in cheese made from raw milk (Fox, 2003; Shakeel-ur-Rehman et al., 2003). Dephosphorylation may be rate limiting for proteolysis during ripening of cheese since most of proteinases and peptidases are inactive on phosphopeptides or phosphoproteins (Fox & Kelly, 2006b). The suitability of ACP as an indicator enzyme for superpasteurization/flash pasteurization of milk has been assessed (Andrews et al., 1987; Griffiths, 1986) and suggested that this enzyme is not suitable marker for this purpose as some alternatives, for example, GGTP or LPO enzymes are most suitable marker (Fox & Kelly, 2006b).

1.5.3

Ribonuclease

Ribonuclease (RNase) degrade RNA by hydrolyzing phosphodiester bond between nucleotides. RNase can be exonuclease or endonuclease. Many types of RNases are now known. RNase A is predominant form secreted in pancreas and is most studied. It contains 124 amino acid residues and molecular mass is 12,600 Da. It was first such enzyme, the amino acid sequence of which was determined. Three-dimensional structure of RNase A is known. It has 4 histidine residues out of which 2 residues, namely, His-12 and His-119 directly participate in catalysis. RNase A is endonuclease and hydrolyzes phosphodiester bond at 3' position of ribose of pyrimidine nucleotide. Four disulfide bonds provide stability to enzyme. RNase occurs in various tissues and secretions, including milk. Bovine milk possess a much higher activity of RNase than blood, serum, or urine of human, rat, or guinea pig, and activity is mostly concentrated in the serum phase; hence, bovine milk can be a commercial source of RNase for isolation. Like pancreatic RNase, the RNase in milk is optimally active at pH 7.5 and is more heat stable at acid pH values than at pH 7.0; in acid whey, adjusted to pH 7.0, 50% of RNase activity was lost on heating at 90 C/5 min and 100% after 20 min, but it

1.6 Oxidases

was completely stable in whey at pH 3.5 when heated at 90 C/20 min (Bingham & Zittle, 1962). Bovine milk possess about three times more RNase as human, ovine, and caprine milk, but porcine milk has a very low activity of RNase (Chandan et al., 1968). Dalaly et al. (1970) reported that milk RNase are usually similar to bovine RNase (pancreatic). Caprine milk has one-third as much RNase enzyme activity in comparison to bovine or buffalo milk. Trace or no RNase enzyme activity persists after UHT heat processing (121 C/10 s) but about 60% persists during thermal processing at 72 C/2 min (Meyer et al., 1987) or at 80 C/15 s (Griffiths, 1986). RNase enzyme activity in raw or in thermally processed milk is stable during repeated freezing and thawing and storage under frozen condition for at least 1 year (Meyer et al., 1987).

1.6 1.6.1

Oxidases Lactoperoxidase (EC 1.11.1.7)

Lactoperoxidase (LPO) is a heme-containing glycoprotein found in milk and other exocrine secretions such as saliva, tears, and airways. LPO was named so because it was isolated from milk in crystalline form for the first time. Through two covalent bonds, LPO binds to heme prosthetic group, a derivative of protoporphyrin IX in its catalytic center. It belongs to the family of mammalian heme-containing peroxidase (XPO) enzymes which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO was first demonstrated in milk in 1881 (Arnold, 1881). It catalyzes the transfer of oxygen from hydrogen peroxide (H2O2) to other substrates such as thiocyanate ion (SCN-) to a short-lived oxidation product hypothiocyanite ion (OSCN-) which inhibit certain bacteria (Fig. 1.4). Thiocyanate is a natural constituent of milk and H2O2 can be

FIGURE 1.4 Reaction catalyzed by lactoperoxidase.

19

20

CHAPTER 1:

Milk enzymes

produced by catalase-negative bacteria or formed in situ through the action of exogenous enzyme, for example, glucose oxidase on glucose which may be added to milk. Thus, in milk, such bacteria possess self-inhibition which is also considered as cold pasteurization. Exogenously added H2O2 together with milk LPO inhibits bacterial growth in milk. LPO is almost lost on heating milk at 80 C and methods have been developed for its detection in milk to assess efficiency of heat treatment of milk at such temperature (Sharma & Rajput, 2014). Microbial membranes usually have low permeability for hypothiocyanite ion but are quite permeable to hypothiocyanous acid. Hypothiocyanite ion or hypothiocyanous acid oxidizes sulfhydryl groups. Thiol enzymes are inhibited by this oxidation. The lactoperoxidase system (lactoperoxidase/thiocyanate/H2O2) in milk is of economic significance because of its antimicrobial effects. It gives potential application in the preservation of raw milk under ambient condition. The efficacy of milk keeping quality by the LPO system is affected by temperature and is high at low temperature of milk storage (Jooyandeh et al., 2011). LPO is a glycoprotein (Mw: 80 kDa) containing one heme-iron prosthetic group per molecule which confers an additional nonenzymatic activity upon this enzyme. Thus it has the ability to oxidize unsaturated fatty acids to form volatile products capable of contributing to oxidized flavors in dairy products. LPO enzymes binds a Ca11 ions, which has a major effect on its stability, including its heat stability. At a pH below  5.0, the Ca11 ion is lost, with a subsequent loss of stability. After xanthine oxidase enzyme, LPO enzyme is the most abundant enzyme in milk, constituting  0.5% of the total whey proteins (  0.1% of total protein; 30 mg/L) (Fox & Kelly, 2006a). Human milk either lacks or has low level of LPO but contains myloperoxidase (Hamosh, 1988; Watanabe et al., 2000). In general, mastitis milk has elevated levels of LPO.

1.6.2

Catalase (EC 1.11.1.6)

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen. It catalyzes the decomposition of H2O2 to H2O and O2. It protects the cell from oxidative damage by reactive oxygen species. Catalase activity is assayed by quantifying the liberation of O2 manometrically or by titrametrically measuring the reduction of H2O2 or measuring reduction in absorbance at 240 nm. 2H2 O2 -2H2 O 1 O2

Bovine liver catalase is a homotetramer of 60 65 kDa subunits (total molecular mass 250 kDa) wherein each subunit contains one heme group. It has

1.6 Oxidases

broad pH optimum between 7 and 11. It is likely that the structure of catalase in milk is similar to liver catalase. In milk, 73% catalase activity is associated with skimmed milk but the specific activity in the cream is 12-fold higher than in skimmed milk. Thus the MFGM fraction is often considered as the base material for catalase isolation from milk (Kitchen et al., 1970). The catalase activity in milk is dependent on feed and stage of lactation. It has been known since long that the mastitis milk has elevated the level of catalase (Kitchen, 1976, 1981; Luedecke et al., 1967). Catalase is quite heatlabile enzyme; heat treatment to milk at 70 C/1 h inhibits the enzyme activity. It is strongly inhibited by some ions, for example, Hg11, Fe11, Cu11, Sn11, CN2, and NO23. The enzyme appeared to have lipid prooxidant properties via its heme-iron.

1.6.3

Xanthine oxidase (EC 1.17.3.2)

Xanthine dehydrogenase (EC 1.17.1.4) predominantly exists as the NADdependent dehydrogenase. It catalyzes successive oxidation of hypoxanthine to xanthine and xanthine to uric acid with the concomitant reduction of NAD1 to NADH which are the last two steps of purine catabolism. During purification of xanthine dehydrogenase, it is largely converted to an O2 -dependent form, xanthine oxidase (XO) (EC 1.17.3.2). The conversion can be triggered reversibly by oxidation of cysteine thiols to form disulfide bonds. Limited proteolysis of xanthine dehydrogenase results in irreversible conversion to XO (Enroth et al., 2000; Wang et al., 2016). In bovine milk, xanthine dehydrogenase is associated with milk fat or MFGM or circular fragments from MFGM (microsomes) which on treatment with pancreatin makes not only enzyme soluble but also generates nick in enzyme leading to its conversion to XO. XO can carry out following reactions. Hypoxanthine 1 O2 1 H2 O-Xanthine 1 H2 O2 1 Xanthine 1 2O2 1 H2 O-Uric acid 1 2O:2 2 1 2H Xanthine 1 O2 1 H2 O-Uric acid 1 H2 O2

DNA and RNA comprise purines and pyrimidines bases. Adenine (purine) is metabolized to hypoxanthine, whereas guanine is converted to xanthine. XO acts on these metabolic products and results in formation of uric acid which undergoes no further metabolism in humans and is excreted by the kidneys. Conversion of xanthine to uric acid is irreversible and generates superoxide anions. Superoxide anion radicals spontaneously or under the influence of enzyme superoxide dismutase (SOD) are converted into hydrogen peroxide and oxygen. 1 2O:2 2 1 2H -H2 O2 1 O2

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Milk enzymes

XO produces H2O2 which can serve as a substrate for lactoperoxidase system in its action as a bactericidal agent. XO along with xanthine dehydrogenase are produced as zymogens that are activated by trypsin. Both xanthine dehydrogenase and xanthine oxidase are single gene product. XO is a homodimer with molecule mass of 290 kDa. XO belongs to the molybdenum-protein family. Each unit contains one molybdenum, one of the flavin adenine dinucleotides (FAD), and two ironsulfur (2Fe-2S) centers of the ferredoxin type. The enzyme contains two separated substrate-binding sites (Kosti´c et al., 2015). In milk, XO is mainly associated with MFGM in which it is the second most abundant protein accounting  20% of the protein of the MFGM (  0.2% of total milk protein;  120 mg/L). XO is also present in microsomes derived from MFGM. The level of XO enzyme activity in human milk, goat milk, and sheep milk is low due to lack of Mo metal ion (Atmani et al., 2004; Benboubetra et al., 2004; Godber et al., 1997). 2 XO can reduce nitrate (NO2 3 ) to nitrite (NO2 ), which is the powerful inhibitor (bactericidal agent) for some bacteria, and then to NO. This property of XO is used in manufacturing of certain varieties of cheeses (Dutch, Swiss) by adding a little amount of sodium nitrate (as a preservative), for preventing late gas blowing defects due to the growth of Clostridium tyrobutyricum, which causes off-flavor in cheeses during ripening (Fox & Kelly, 2006a).

1.6.4

Superoxide dismutase

Superoxide dismutase (SOD) enzyme scavenges superoxide radicals (Od2 2 ) and catalyzes the following reaction. 2O22 1 2H1 -H2 O2 1 O2 

The H2O2 formed is then further reduced to H2O in the presence of catalase, peroxidase, or other suitable reducing agent. SOD is present in animal, insect, bacterial, and plant cells and its principal biological function is to protect tissue against oxygen free radicals. Seven different SODs in insect Bombyx mori are reported (Kobayashi et al., 2019). SODs are metalloenzymes. All mammalian cells possess three isoforms of SOD referred to as SOD1, SOD2, and SOD3 (Mondola et al., 2016). SOD1 is dimer and is present in cytosol, nucleolus, and lumen between outer and inner mitochondrial membrane. SOD1 contains copper and zinc metal ions (McCord & Fridovich, 1969). Each identical subunits of SOD1 enzyme contain 153 amino acid residues (Mw: 16 kDa) which are joined with one or more disulfide (-S-S-) bonds. The active site in each subunit contains one Cu ion ligated by three histidines when in the reduced state and one Zn ion ligated by one aspartic acid and three histidines. One of the histidine ligands of the Zn ion ligands also

1.6 Oxidases

ligates the Cu ion when in the oxidized state (Perry et al., 2010). SOD2 is manganese-containing tetramer and is present in mitochondria (Weisiger & Fridovich, 1973). SOD3 is zinc- and copper-containing extracellular enzyme and exists as tetramer (Marklund, 1982). These metal ions exist in different oxidation states and play crucial roles in electron transfer. The SOD1 is expressed at relatively high levels in blood vessels. SOD activity in milk is about 150 times lower than in blood and the enzyme is largely concentrated in skim milk. SOD1 is homodimer. SOD content in human milk is 2.0 to 2.3 times higher than in bovine milk (Kiyosawa et al., 1993). SOD activity in human and goat colostrum is higher than in milk (Li et al., 2018; Savi´c et al., 2005). The significance of SOD enzyme is to inhibit the lipid oxidation in model systems. SOD may offset the effect of the prooxidant activity of xanthine oxidase. In milk, SOD enzyme is quite thermally stable at temperature 71 C/30 min (no loss of activity during HTST pasteurization). Hence, slight variations in pasteurization temperature are critical to the survival of SOD in heattreated milk and milk products which may lead to contribute to variations milk stability to oxidative rancidity. Homogenization shows little effect on the activity of SOD enzyme in milk. SOD catalyzes the removal of superoxide free radicals (O:2 2 ) and safeguards the cells from harmful effects. Cytosolic Cu/Zn-SOD, mitochondrial MnSOD and extracellular EC-SOD are the major forms of SOD (Matéos et al., 1999). SOD can inhibit lipid peroxidation. In cow milk SOD is exclusively present in skim milk fraction, with a concentration of 0.15 mg to 2.4 mg/L (Fang et al., 2002). Human milk has 2.0 2.3 times higher concentration of SOD than cow milk (Khan et al., 2019).

1.6.5

Sulfhydryl oxidase (EC 1.8.3--)

Sulfhydryl oxidase oxidizes free sulfhydryl groups in proteins and thiolcontaining small molecules by using molecular oxygen as an electron acceptor (Swaisgood, 2003). The enzyme is an aerobic oxidase which catalyzes the following reaction resulting in the formation of hydrogen peroxide. 2RSH 1 O2 -RSSR 1 H2 O2

Two different types of sulfhydryl oxidases in milk are reported. One of them is dependent on metal ion while other requires flavin. Both the enzymes catalyze same reaction. The first mammalian sulfhydryl oxidase, an iron-dependent enzyme that was isolated from bovine milk whey was reported to contain 0.5 atoms of iron per 89 kDa subunit and completely inhibited by EDTA which could be subsequently restored by dialysis against 1-mM ferrous sulfate. Its pH and temperature optima are 7.0 C and 35 C, respectively. The enzyme can

23

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CHAPTER 1:

Milk enzymes

reactivate denatured RNAase with reduction of oxygen to hydrogen peroxide within 1 h and is probably involved in the formation of disulfide bonds during the postsynthesis processing of proteins. It contains 11% carbohydrate (Janolino & Swaisgood, 1975). During UHT processing, thiol groups get oxidized and this results in cooked flavor. Immobilization of sulfhydryl oxidase on glass beads reduces the cooked flavor of UHT processed milk. The process has been patented (Swaisgood, 2003). Flavin-dependent sulfhydryl oxidase is also present in milk and catalyzes the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. It is a soluble 62-kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. It is a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralog lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol (Jaje et al., 2007).

1.7

γ-glutamyl transpeptidase (EC 2.3.2.2)

γ-glutamyl transpeptidase (GGTP) enzyme is ubiquitously present in all life forms and is also known as γ-glutamyl transferase. It catalyzes the transfer of the gamma-glutamyl moiety of glutathione to an acceptor that may be an amino acid, a peptide, or water. Higher eukaryotes mainly utilize GGTP for glutathione degradation. In kidney, GGTP cleaves glutathione and prevents its excretion from the body and thereby conserving cysteine (Terzyan et al., 2015). Mammalian and E. coli GGTPs are similar in structure. However, mammalian GGTP is glycosylated whereas E. coli GGTP is nonglycosylated. E. coli GGTP is synthesized as 580 amino acid long protein of which residues 1 24 correspond to signal peptide. However, mammalian GGTP has anchor domains in their N-terminal regions leading to membrane association. Autocatalysis results in L subunit (residues 25 390) and S subunit (residues 391 580). Functional enzyme has L and S subunit and is heterodimer (Okada et al., 2006). Enzyme is inhibited by cupric ion, ferric ion, diisopropyl fluorophosphate, and iodoacetamide (Fox & Kelly, 2006b). GGTP activity in human and bovine milk varies during lactation, being highest in colostrum. In milk, enzyme is associated with membrane and largely present in skim milk. Colostrum has elevated levels of GGTP. It has been observed that GGTP level in goat milk is about four times lower than either bovine or buffalo milk (Sharma et al., 2009). Inactivation of GGTP is corelated with heat

1.9 Lysozyme (EC 3.1.2.17)

treatment of milk in the range of 70 C 90 C for 16 s (Andrews et al., 1987; Balkishan et al., 2010; Carter et al., 1990) and enzyme is completely inactivated during heat treatment at 78 C for 15 s (Patel & Wilbey, 1989) or 77 C for 16 s (McKellar et al., 1991). Reactivation is not noted as in the case of ALP. Thus, GGTP is a useful marker for heat treatment subjected to milk.

1.8

N-Acetyl-β-D-glucosaminidase (EC 3.2.1.30)

N-Acetyl-β-D-glucosaminidase (NAGase) catalyzes the hydrolysis of nonreducing N-acetyl-β-D-glucosamine terminal residues from N-acetyl-β-D-glucosaminides. Enzyme is of clinical significance in humans. NAGase is lysosomal enzyme that is present in renal proximal tubular cells and increasingly excreted as an indicator of renal dysfunction. Enzyme can act on chromogenic substrate N-acetyl-β-D-glucosamine-p-nitrophenol which results in yellow-colored p-nitrophenol end product under alkaline conditions. NAGase is an intracellular lysosomal enzyme that is released into milk from lysis of mammary tissue and to some degree by damaged epithelial cells of mammary tissue (Fox et al., 1988; Kitchen et al., 1978, 1980) reflecting destruction of udder tissue and also during lysis of inflammatory neutrophils (Kaartinen et al., 1988; Kitchen et al., 1978; Rani et al., 2020). Milk somatic cells contribute less than 15% of the total milk NAGase activity (Rani et al., 2020). NAGase is considered as an effective indicator of clinical and subclinical mastitis infection of udder (Hovinen et al., 2016). The authors suggested that this test performed very well in separating quarters infected with major pathogens from those with minor pathogens. Overall, milk from quarters with clinical mastitis had much higher NAGase activity than in milk from quarters with subclinical mastitis. NAGase has elevated level in colostrum and falls to normal levels within 7 10 days of milking after parturition. NAGase in milk is concentrated in skimmed milk. While milk pH is about 6.8, optimum pH of enzyme is 4.2. NAGase is mostly inactivated at HTST pasteurization (70 C 71 C/15 18 s). Thermal inactivation of NAGase falls between ALP and GGTP and is therefore better suited for assessing efficacy of thermal processing of milk in the range 65 C 75 C/15 s (Andrews et al., 1987; Ardö et al., 1999).

1.9

Lysozyme (EC 3.1.2.17)

Lysozyme hydrolyzes glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in peptidoglycan polymer where NAM is cross-linked to peptides. Peptidoglycan is a part of bacterial cell wall and

25

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CHAPTER 1:

Milk enzymes

Gram-positive bacteria have thick peptidoglycan layer. Lysozyme is lysosomal enzyme and it preferably lysis Gram-positive bacteria. Lysis of Micrococcus lysodeikticus by lysozyme is basis of its assay. It is present in tears, gastric secretions, nasal mucus, and egg white. Egg white is the rich source for lysozyme and it accounts for 3.5% of the total egg white proteins (Cegielska-Radziejewska et al., 2008). The enzyme is commercially available in purified form. Egg white lysozyme (Mw: 14.4 kDa) contains 129 amino acid residues and four disulfide bonds which provide stability to enzyme. Its isoelectric point is 10.7 (Abeyrathne et al., 2014). The enzyme is also present in the milk of several species, including human, horse, buffalo, and donkey (Chandan et al., 1968; Civardi et al., 2002; Jauregui-Adell, 1975; Priyadarshini & Kansal, 2002a) in a significant amount. However, its presence in bovine and camel milk is either negligible or low. Colostrum has five times more lysozyme in comparison to mature milk (Priyadarshini & Kansal, 2003). Lysozyme from buffalo as well as bovine milk is optimally active at pH 7.4. However, the enzyme from these milks differ in thermal stability. Buffalo milk lysozyme is fully stable while the cow milk lysozyme is partly inactivated by pasteurization (LTLT as well as HTST). Lysozyme is relatively stable to heat at acidic pH 3-4 but heat-labile at pH . 7. The level of lysozyme is 10- to 50-fold higher in mastitic cows in comparison to normal milk and the enzyme lacks its relationship with mastitis in buffalo (Priyadarshini & Kansal, 2002b). Sequence homology of lysozyme across the species is of lower order. Human milk lysozyme consists of 130 amino acid residues, as compared to 129 in equine milk enzyme, the extra residue is being Val in former milk. The amino acid sequence of equine milk lysozyme (Mw: 14,647) shows only 51% homology with human milk lysozyme and 50% homology with egg white lysozyme (McKenzie & Shaw, 1985). Lysozyme has 40% sequence homology with whey protein “α-lactalbumin” which acts as an enzyme modifier in the biosynthesis of lactose (Qasba & Kumar, 1997). Due to bactericidal effect, indigenous milk lysozyme enzyme can be helpful in extending the shelf life of milk.

1.10

Enzymes from psychrotrophs origin in milk

Milk is stored at refrigerated temperature to control the growth of mesophilic microorganisms. Some of these microorganisms adapt to low temperatures by synthesizing phospholipids and neutral lipids containing increased proportions of unsaturated fatty acids, resulting in a reduction in the melting point of the lipids and are called psychrotrophs. These psychrotrophs secrete extracellular protease and lipase enzymes. These enzymes are thermostable and survives heat treatments applied to the milk for elimination of microorganisms. The psychrotrophic count represents 78.3% of the total bacterial count in raw

References

refrigerated milk collected in southern Brazil (Ribeiro Júnior et al., 2018). Prolonged action of proteases and lipases make organoleptic changes in fluid milk, bitter or rancid taste in cheeses, or gelation and sedimentation in UHTtreated milk (de Oliveira et al., 2015; Fairbairn & Law, 1986; Matéos et al., 2015; Zhang et al., 2015).

1.11

Conclusion

In contrast to general acceptance of the view that enzymes are produced or transported to other tissues for defined catalytic reaction, it does not hold good for milk enzymes. However, milk has enough substrates for few indigenous enzymes such as lipase and plasmin and their activities are of concern during milk storage or processing. Lipase action on milk fat can result in rancidity. Psychrotrophs present in milk stored at refrigerated temperature produce lipase and protease and their action on substrates in milk makes organoleptic changes in fluid milk. Milk is a good substrate for microorganisms and if good hygiene practice is not followed during milk production and processing, the microorganisms can produce enzymes which can deteriorate milk. These reactions can result in drop in milk pH and ultimately milk is curdled. Milk has several thermally stable enzymes. The knowledge of thermal stability of milk enzymes is exploited to understand heat treatment in terms of duration and temperature, subjected to milk and milk products. In this respect, inactivation of alkaline phosphatase activity is widely accepted as an index of proper pasteurization, albeit reactivation of the enzyme during storage of cream is still of concern. Milk has several antioxidant and antimicrobial enzymes. Lactoperoxidase, which is abundantly present in milk, can be exploited for preservation of milk. Most of the research is limited to bovine milk enzymes and therefore enzymes from milks from goat, sheep, camel, mare, buffalo, donkey, and human need to be studied with equal emphasis. Enumeration of enzymes in milk is difficult and the same can not be free from errors in view of the fact that milk used for measuring enzyme activity cannot be guaranteed for the absence of bacteria. Also, variable number of somatic cells in milk and sensitivity of assay can influence the decision for the presence or absence of enzyme.

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Fox, P. F. (2003). Significance of indigenous enzymes in milk and dairy products. In: Handbook of food enzymology (pp. 255 277). Marcel Dekker. Fox, P. F., & Kelly, A. L. (2006a). Indigenous enzymes in milk: Overview and historical aspects Part 1. International Dairy Journal, 16(6), 500 516. Available from https://doi.org/10.1016/j. idairyj.2005.09.013. Fox, P. F., & Kelly, A. L. (2006b). Indigenous enzymes in milk: Overview and historical aspects Part 2. International Dairy Journal, 16(6), 517 532. Available from https://doi.org/10.1016/j. idairyj.2005.09.017. Fox, P. F., Uniacke-Lowe, T., McSweeney, P. L. H., & O’Mahony, J. A. (2015). Enzymology of milk and milk products (pp. 377 414). Springer Science and Business Media LLC. Available from https://doi.org/10.1007/978-3-319-14892-2_10. Girardet, J. M., Linden, G., Loye, S., Courthaudon, J. L., & Lorient, D. (1993). Study of mechanism of lipolysis inhibition by bovine milk proteose-peptone component 3. Journal of Dairy Science, 76(8), 2156 2163. Available from https://doi.org/10.3168/jds.S0022-0302(93) 77551-7. Godber, B. L. J., Sanders, S., Harrison, R., Eisenthal, R., & Bray, R. C. (1997). 98% of xanthine oxidase in human milk is present in the demolybdo form, lacking molybdopeterin. Biochemical Society Transactions. Griffiths, M. W. (1986). Use of milk enzymes as indices of heat treatment. Journal of Food Protection, 49(9), 696 705. Available from https://doi.org/10.4315/0362-028x-49.9.696. Groves, M. L. (1971). Minor milk proteins and enzymes (pp. 367 418). Elsevier BV. Available from https://doi.org/10.1016/b978-0-12-485202-0.50015-4. Hamosh, M. (1988). Enzymes in milk: Their function in the mammary gland, in milk and in the infant. Biology of human milk, 15, 45 61, Raven Press Nestle. Hamosh, M., & Scow, R. O. (1971). Lipoprotein lipase activity in guinea pig and rat milk. Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism, 231(2), 283 289. Available from https://doi.org/10.1016/0005-2760(71)90140-8. He, S. H., Ma, Y., Wang, J. Q., Li, Q. M., Tang, S., & Li, H. M. (2012). Effects of proteose-peptone fractions from yak milk on lipoprotein lipase lipolysis. International Journal of Dairy Technology, 65(1), 32 37. Available from https://doi.org/10.1111/j.1471-0307.2011.00742.x. Hernell, O. L. L. E., & Bläckberg, L. A. R. S. (1991). Digestion and absorption of human milk lipids. Encyclopedia of human biology (3, pp. 47 56). Academic Press, New York Hovinen, M., Simojoki, H., Pösö, R., Suolaniemi, J., Kalmus, P., Suojala, L., & Pyörälä, S. (2016). N-acetyl-β-D-glucosaminidase activity in cow milk as an indicator of mastitis. Journal of Dairy Research, 83(2), 219 227. Available from https://doi.org/10.1017/S0022029916000224. Hoylaerts, M. F., & Millán, J. L. (1991). Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase. European Journal of Biochemistry, 202(2), 605 616. Available from https://doi.org/10.1111/j.1432-1033.1991.tb16414.x. Jaje, J., Wolcott, H., Fadugba, O., Cripps, D., Yang, A. J., Mather, I. H., & Thorpe, C. (2007). A flavin-dependent sulfhydryl oxidase in bovine milk. Biochemistry, 46(45), 13031 13040. Available from https://doi.org/10.1021/bi7016975. Janolino, V. G., & Swaisgood, H. E. (1975). Isolation and characterization of sulfhydryl oxidase from bovine milk. J Biol Chem, 250(7), 2532 2538. Jauregui-Adell, J. (1975). Heat stability and reactivation of mare milk lysozyme. Journal of Dairy Science, 58(6), 835 838. Available from https://doi.org/10.3168/jds.S0022-0302(75)84646-7. Jooyandeh, H., Aberoumand, A., & Nasehi, B. (2011). Application of lactoperoxidase system in fish and food products: a review. Am. Eurasian J. Agric. Environ. Sci, 10, 89 96.

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Enzymes in mastitis milk Shital D. Nagargoje, Yallappa M. Somagond, Bibhudatta S.K. Panda and A.K. Dang Lactation and Immuno-Physiology Laboratory, ICAR-National Dairy Research Institute, Karnal, Haryana, India

2.1

Introduction

Bovine mastitis is a common inflammatory condition of the udder that affects the dairy sector worldwide. It results from an inflammatory reaction caused by various pathogenic agents invading the mammary gland. Based on the severity of the disease, mastitis is classified into clinical mastitis and subclinical mastitis. The California Mastitis Test (CMT) and bacterial culture in the laboratory are the traditional methods for diagnosing clinical mastitis, but these do not give precise real-time mastitis detection (Hillerton, 1999). Somatic cell count (SCC) differs in milk from noninfected udder, milk from an early stage of infection in the udder (subclinical mastitis), and milk from advanced stage udder infection (clinical mastitis). SCC numbers in milk are an index for evaluating individual lactating animals or herds for mastitis. Due to the apparent slow speed of bacteriological culturing as well as laboratory determination of SCC coupled with high cost per sample, these approaches are not helpful for rapid and early real-time detection. Furthermore, unless under regulated conditions, CMT has little potential to detect subclinical mastitis (Redetzky et al., 2005). Also, mastitis results in an increase in the electrical conductivity of milk. Although the electrical conductivity based method is inexpensive, the method has low sensitivity for detecting specific types and intensities of mastitis. Because of these drawbacks, many researchers have recommended indigenous milk enzymes as cow-side mastitis markers to address the enduring threat of mammary gland infection (Batavani et al., 2007; Moussaoui et al., 2004). Milk somatic cells include milk-producing cells and immune cells. These cells are released in milk during the regular course of milking and are used as an index for measuring dairy cow mammary health and milk quality. Macrophages Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00002-3 © 2023 Elsevier Inc. All rights reserved.

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are present in the healthy mammary gland. During the mammary infection, the blood mammary barrier becomes porous, allowing the transport of ions, various proteins, and enzymes of blood origin into the milk, along with the influx of phagocytic cells. As the pathogen breaches the teat canal and enters the gland, milk macrophages release various chemoattractants. These chemoattractants activate the first line of defense, that is, polymorphonuclear (PMN) cells, which enter the mammary gland from the blood (Alhussien et al., 2016). The accumulated PMNs release various antibacterial peptides, proteases (elastase, cathepsins), other enzymes (lysozyme, collagenase, phosphatases, etc.), and reactive oxygen species (ROS) (Figure 2.1). Milk PMNs show reduced enzymatic activities compared to peripheral blood PMNs, indicating that milk PMNs recruited in mammary glands during mastitis have an immature phenotype and significantly lower enzymatic activities than peripheral blood PMNs (Martinon, 2010). In mastitis, there is a reduction in the mammary gland’s milk synthetic activity. The enzymes associated with milk production decrease, whereas enzymes

FIGURE 2.1 Gross structure of mammary gland showing different enzymes coming in milk at the onset of infection.

2.2 Enzymes in mastitis

involving inflammation increase (Pyörälä, 2003). N-acetyl-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), acid phosphatase (ACP), and alkaline phosphatase (ALP) are found throughout the animal body and produced both intracellularly and extracellularly (Larsen et al., 2010). Mastitis increases the activity of more than 20 enzymes, including NAGase, beta-glucuronidase, LDH, phosphatases, aminotransferase, plasmin, esterase, and different antioxidant enzymes (Zhao & Lacasse, 2008). Based on the activities of different milk enzymes, the enzymes can be used for mastitis diagnosis. However, the enzymes are also native constituents of the healthy milk, but the activity in milk increases due to the host immunological responses and, in some instances, damage to the mammary epithelium. Amid the several diagnostic tests available for mastitis, enzyme-based biomarkers linked to mammary infection could also aid in the invention of a mastitis screening test.

2.2 2.2.1

Enzymes in mastitis Proteases

Proteases in milk can arise from several sources. These can be released into milk by the mammary epithelium, transported from blood to milk (especially in mastitis), or by the invading bacteria. Mastitis milk exhibits a significant amount of proteolytic activity. SCC and proteolysis levels in milk increase in bovine mastitis (Moussaoui et al., 2004). Additionally, many bacteria also release protease, zymogen activators, which, in synergy with cow proenzymes, can boost the proteolytic capability of both fresh and stored milk (Larsen et al., 2010). The apparent positive correlations between NAGase, plasminogen, and plasmin in most milk fractions revealed a significant association between udder health status and the presence of the proteases in subclinical mastitis (Urech et al., 1999).

2.2.1.1 Plasminogen and plasmin In both subclinical and clinical bovine mastitis, there is an elevation in the activity of blood-derived enzymes such as plasminogen. Plasminogen leaks into milk and is converted to plasmin, a proteolytic enzyme that breaks down fibrin and casein proteins (Korhonen & Kaartinen, 1995). The enhanced activation of plasminogen in mastitis milk is most likely due to plasminogen activators generated by activated macrophages and neutrophils (Mullins & Rohlich, 1983). Plasmin is perhaps the essential protease in the milk collected from healthy and infected udders, but as the PMN cell severity of udder inflammation increases, nonplasmin proteases become more significant (Verdi & Barbano, 1988). Milk sodium, a significant predictor of epithelial barrier integrity, has

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Enzymes in mastitis milk

also been strongly correlated to plasmin activity. Plasmin contributes to sodium leakage into milk by degrading epithelial membrane proteins. Plasmin in milk can be used as a biomarker for the diagnosis of mastitis (Bikker et al., 2014). Plasmin is the primary caseinolytic enzyme in milk and releases membrane proteins such as galactosyltransferase and β2microglobulin. Cows with experimentally induced Escherichia coli mastitis can stimulate the plasmin function (Moussaoui et al., 2002). Quarters with subclinical mastitis have a higher capacity for increased plasmin activity and plasminogen concentration. As a result, there is a higher proteolytic index. A 20% rise in plasmin activity and a 30% increase in plasminogen levels are noticed in the case of subclinical mastitis quarters compared to healthy quarters. Milk plasmin activity and SCC are positively correlated. A 2.3-fold escalation in the plasmin activity was associated with an increase in the milk SCC from 100,000 to 1,300,000 cells/mL (Urech et al., 1999).

2.2.1.2 Elastase, collagenase, and cathepsin The neutrophils contain a variety of granules. Enzymes such as elastase and cathepsin are found in the azurophilic granules, whereas collagenases are found in the specialized granules. Massive recruitment of neutrophils during mastitis explains the increase of milk SCC, leading to a part of the increase in proteolytic activity, especially elastase activity. The elastase is a neutral serinetype proteinase mainly associated with neutrophils. It is used as an indicator for determining neutrophils and somatic cells in milk as it is involved in leukocyte migration and phagocytosis, tissue destruction by digestion of extracellular matrix macromolecules. Collagenase aids blood leukocyte movement by destroying extracellular matrix collagen and proteolytic degradation within the lysosome and outside of it. Activities of elastase and collagenase are significantly more in subclinical mastitis milk (Panda et al., 2019). Cathepsin D (aspartic proteinase) is involved in the apoptosis of mammary epithelial cells (Seol et al., 2006) and activity is considerably higher during clinical mastitis and subclinical mastitis (Guerrero et al., 2015). Cathepsin D is found in milk and acid whey, which is involved in reducing milk’s renneting abilities. Pathogens are destroyed by the enzymes elastase and cathepsin G (serine proteinases). There is an altered expression of cathepsin B (cysteine proteinase) during subclinical mastitis and clinical mastitis (Bathla et al., 2020). Elastase and cathepsins can impede milk coagulation containing high SCC (Albenzio et al., 2009).

2.2.1.3 Caspases The apoptosis of bovine mammary epithelial cells can be induced via triggering the caspase-dependent apoptotic pathway (Chen et al., 2017). Extendedspectrum β-lactamase-producing E. coli can potentially damage the epithelial cells in mammary gland tissue when it causes bovine mastitis via increased

2.2 Enzymes in mastitis

mammary epithelial cells’ intracellular ROS generation, mitochondrial superoxide overproduction, and Bcl-2-associated X protein expression, decreased matrix metalloproteinases, and activated caspase-3 (Shi et al., 2020). Formation of neutrophil extracellular traps regulates inflammation and mammary epithelial cell damage in mastitis via activating caspase-1 and caspase-3 (Wei et al., 2019). Caspase-3 is an antiinflammatory factor that inhibits the release of prosurvival and proinflammatory mediators by cleaving the transcription factor nuclear factor kappa B (NF-κB). Different mastitis pathogens promote apoptosis in mammary epithelial cells by a mechanism involving NF-κB (Wesson et al., 2000). Caspase-3 and caspase-7 activities are significantly lowered in clinical mastitis-affected cows (Swain et al., 2014).

2.2.2

Esterases

Esterases are generally classified as carboxylesterases, cholinesterases, and arylesterases. Esterases can hydrolyze a variety of substances as these rapidly cleave ester bonds. Esterase is present in the azurophilic granules of leukocytes, especially granulocytes and monocytes. Mastitic milk has higher esterase activity due to an increase in blood milk permeability, allowing plasma esterase to transfer into milk and increased milk leukocyte count. There is a significant link between esterase activity and SCC. However, the actual source of esterase in cow’s milk has yet to be determined. Since esterase activity in plasma is 1000 2000 times that of milk, a few of the milk esterase activities could be a result of increased blood milk barrier permeability and mobilization of plasma constituents into the mammary gland secretions or the intrinsic properties of nonenzymatic proteins for hydrolysis (Marquardt et al., 1966). There is a rapid rise in milk esterase activity associated with an increase in milk SCC, particularly granulocytes (neutrophils, eosinophils, basophils) and monocytes (Zhao & Lacasse, 2008). Many studies revealed that esterase activity might be employed as a practical cow-side test and a reliable tool for detecting subclinical mastitis (Mirzaei et al., 2019).

2.2.3

Antioxidant enzymes

Enhanced oxidative stress can be caused by an imbalance production of ROS and reduced antioxidant defense system activity after intramammary infection by bacterial pathogens, leading to oxidative damage to DNA, proteins, lipids, and various macromolecules (Brenneisen et al., 2005). The changes in antioxidant enzymes may be essential to counter oxidative stress. Any increase or decrease in blood antioxidant enzyme activity is also associated with a change in enzyme activity in milk (Andrei et al., 2011). The amounts of cellular oxidation biomarkers are greater in the infected udder (Alba et al., 2019).

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2.2.3.1 Glutathione peroxidase (GSH-Px) GSH-Px is the general name of an enzyme family with peroxidase activity that converts reduced glutathione to oxidized glutathione as well as lipid hydroperoxides to their corresponding alcohols or free hydrogen peroxide to water, to protect organisms from oxidative damage (Abd Ellah, 2013). The GSH-Px activity in bovine milk ranges from 12 to 32 IU/mL, and it is substantially linked with selenium content (Przybylska et al., 2007). Rat with mastitis shows a significant decrease in GSH-Px, superoxide dismutase (SOD), and catalase level in serum, tissue, and milk (Eslami et al., 2015). Malondialdehyde and SCC levels are higher in subclinical mastitis milk, and GSH-Px activity is lower than in normal milk (Yang et al., 2011). Lower GSH-Px activity is associated with a higher incidence of mastitis (Ndiweni et al., 1991). The GSH-Px, SOD, and catalase levels in goats’ milk and mammary tissue with gangrenous mastitis were significantly lower (El-Deeb, 2013). In sheep with gangrenous mastitis, erythrocyte GSH-Px activity is much higher than in healthy ewes (Cetin et al., 2005). Subclinical mastitis milk indicated more GSH-Px activity than normal milk (Andrei et al., 2011).

2.2.3.2 Catalase Catalase is an enzyme that initiates the decomposition of hydrogen peroxide into water and oxygen. It is considered an important marker of mastitis development (Ashley & Li, 2013). The enzyme in milk can come from both the mammary gland and bacteria. It is heat labile and can be inactivated in minutes at 65 C. Catalase activity can also be utilized as a mastitis indicator, having substantially greater activity in mastitis milk (Fox & Kelly, 2006). It is certainly linked to somatic cell membranes. There is a positive correlation between catalase activity and milk SCC (Hamed et al., 2008). In order to compensate for the oxidative stress caused by the antioxidant enzyme system, sheep with subclinical mastitis have enhanced catalase activity in their blood (Zengin et al., 2017).

2.2.3.3 Superoxide dismutase (SOD) SOD is an enzyme that converts superoxide radicals to hydrogen peroxide and molecular oxygen, which are subsequently converted to water. The presence of SOD is necessary for milk’s antioxidant stability. Exogenous injection of SOD reduces lipid peroxidation processes, resulting in increased milk stability. Milk SOD activity differs between cow breeds (Lindmark-Månsson & Åkesson, 2000). It is unaffected by the stage of lactation or the cow’s age or with a high SCC. The activity of SOD is independent of the somatic cells in milk (Przybylska et al., 2007). There is no significant change in the distribution of SOD in the milk of normal and subclinical mastitic udders (Yang et al., 2011). In goat milk samples with subclinical mastitis, there is a considerable rise in GPx and a drop in SOD levels which are correlated with milk SCC (Darbaz et al., 2019).

2.2 Enzymes in mastitis

2.2.3.4 Xanthine oxidase Xanthine oxidase and xanthine dehydrogenase are two interconvertible forms. Xanthine oxidase is a metalloflavoprotein that is found in milk fat globules. In milk, this enzyme has several functions. Xanthine oxidase catalyzes the conversion of hypoxanthine to xanthine and produces ROS such as superoxide radicals and hydrogen peroxide, and it can also catalyze the oxidation of xanthine to uric acid. As a result, it is more appropriately known as xanthine oxidoreductase. When combined with the lactoperoxidase (LPO) system in milk, the enzyme complex functions as an antimicrobial, thus protecting the mammary gland against infection. The bactericidal effect in milk is also due to nitric oxide formation. Thus, xanthine oxidase is considered a part of the mammary gland’s immune system, along with LPO and nitric oxide. Significantly lower membrane-bound xanthine oxidase activity is found in mastitis milk fat globule than fresh milk (Verma et al., 2019).

2.2.4 Antibacterial enzymes [lactoperoxidase (LPO) and myeloperoxidase (MPO)] LPO is the enzyme that gives bovine milk its antibacterial characteristic, which is dependent on adequate levels of hydrogen peroxide and thiocyanate ion. It is the second most prevalent enzyme in bovine milk. LPO is produced by alveolar epithelial cells and leukocytes (Alhussien & Dang, 2020; Isobe et al., 2011). Principal functions of this enzyme may be the prevention of the accumulation of toxic peroxide in the bovine udder and the protection of the nursing calf through its antibacterial properties. LPO activity is similar across different parities. In the mammary glands of dairy cows, LPO along with lingual antimicrobial peptide, is known to have a synergistic antibacterial action (Isobe et al., 2011). LPO activity depends on inflammation severity. LPO is elevated in bovine mastitis milk, and this rise is linked with a high milk SCC (Andrei et al., 2011). MPO is an oxidoreductase that oxidizes various chemical compounds. In the presence of hydrogen peroxide, MPO catalyzes the oxidation of halide ions (mainly chlorides) to corresponding acids, acting as a strong oxidizing agent. It is a component of the antibacterial system in neutrophils and monocytes, which participates in the inflammatory responses throughout the body, including the mammary glands. MPO is considered one of the essential lysosomal enzymes stored in the azurophilic granules of neutrophils involved in the oxygen-dependent antimicrobial system, which plays a significant role in innate immunity against invading microorganisms. MPO is a marker for neutrophil granulocyte infiltration and is directly correlated to the early stages of inflammation. In bovines, MPO is identified as a marker of mastitis in dairy cows (Cooray, 1994). However, MPO detection in milk somatic cells

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permits early subclinical mastitis diagnosis and treatment (Alhussien & Dang, 2020).

2.2.5

N-acetyl-D-glucosaminidase (NAGase)

NAGase is an intracellular, lysosomal enzyme released into milk by neutrophils during phagocytosis and cell lysis, as well as from injured mammary epithelial cells (Hovinen et al., 2016). The cytoplasm of mammary epithelial cells is the predominant contributor to the mammary gland NAGase, with only around 5% 15% derived from the blood. Varying types of leukocyte have different NAGase activities (Kitchen et al., 1978). The skim milk fractions contain the most NAGase activity. NAGase activity in milk macrophages is about three times higher than PMN cells in milk and blood (Nagahata et al., 1987). NAGase is released in large quantities into the milk during phagocytosis and cell lysis. Damaged epithelium cells can also release this enzyme. This enzyme’s activity is linked to SCC. The milk NAGase is higher in early lactation and increases to a smaller extent toward the end of lactation (Mattila et al., 1986). Plasmin, NAGase, and Na1 all increased significantly with the number of lactations. These findings suggest that parity causes an increase in epithelial cell breakdown, which is mediated by lysosomal enzymes and plasminogen activation (Piccinini et al., 2007). Milk SCC, LDH, and NAGase activity peak shortly after calving in healthy cows and then gradually decline to low levels 30 40 days later (Chagunda et al., 2006). In healthy udder quarters, variation in the milk NAGase activity between different milking is low (Åkerstedt et al., 2011). N-acetyl-D-glucosaminidase activity is a better indication of subclinical mastitis (Hovinen et al., 2016). An increase in milk SCC and high NAGase activity indicates possible damage to epithelial cells in the udder, which aids in determining the prognosis for mastitis recovery (Nielsen et al., 2005). NAGase has a strong positive correlation with milk SCC, which can also be assessed in frozen milk samples (Kitchen et al., 1984). NAGase is an important enzyme that may be used to distinguish between minor and major pathogen infections since it correctly represents the degree of inflammation (Pyörälä, 2003). NAGase activity varied more in healthy cows than in clinically mastitic animals (Chagunda et al., 2006). The amount of bacterial DNA present in the milk is significantly linked to the quantities of acute-phase proteins and NAGase activity (Kalmus et al., 2013). E. coli bacteria are usually quickly cleared from the udder; however, they cause a significant inflammatory response mainly due to endotoxin (the major virulence factor of Gram-negative infections) (Burvenich et al., 2003; Hogan & Smith, 2003). The Staphylococcus aureus, which causes bovine mastitis, has no effect on the degree of udder parenchymal damage and NAGase activity (Middleton et al., 2002).

2.2 Enzymes in mastitis

Considerably greater SCC and N-acetyl-D-glucosaminidase activity is noticed in sheep and goats infected with mastitis (Leitner, Chaffer, et al., 2004; Leitner, Merin, et al., 2004). The levels of NAGase and plasmin were shown to have a substantial relationship with decreased milk output, suggesting that these markers may be useful in determining production loss due to subclinical mastitis (Mattila et al., 1986).

2.2.6

Lactate dehydrogenase (LDH)

LDH is an enzyme present in the cytoplasm of cells or tissues and is an element of the glycolytic pathway. Because it is liberated from the cells during cell injury and death, it is considered a biomarker of inflammation. Mastitis causes an increase in LDH activity, which comes from parenchyma cells and disintegrating leukocytes rather than just blood plasma (Katsoulos et al., 2010; Mohammadian, 2011). LDH may be of blood origin, indicating increased blood mammary barrier leakage (Lehmann et al., 2013). LDH is an accurate indicator of udder health conditions because it shows significant changes between healthy and mastitic cows at an early stage (Friggens et al., 2007). LDH provides a practical approach for the early detection of bovine mastitis (Chagunda et al., 2006; Hiss et al., 2007). There is a greater correlation between LDH activity and milk SCC in clinically mastitic cows than healthy cows (Chagunda et al., 2006). Compositional variations were discovered when higher LDH levels were observed, suggesting that LDH could be used as a milk quality indicator (Forsbäck et al., 2010). In quarters with subclinical mastitis, LDH activity is reported to be high in the foremilk (Hiss et al., 2007). LDH activity in milk is substantially higher in mastitis samples with Gram-negative bacterial infection than in Gram-positive bacterial infection (Wenz et al., 2010). Cis urocanic acid was reported to reduce mastitis-associated tissue damage in cows. Infused Cis urocanic acid in E. coli infected udder reduces milk NAGase and LDH activity (Bannerman et al., 2009).

2.2.7

Phosphatases and aminotransferases

There is a link between milk alkaline phosphatase (ALP) and mastitis (Babaei et al., 2007). It is most likely that the (basic) milk enzyme comes from the mammary gland itself (Andrews, 1991). Neutrophils have been found to respond to lipopolysaccharides by increasing ALP activity on the cell membrane surface in vitro (Aida & Pabst, 1991). The increased expression and activity of the normal leukocyte ALP are most likely due to the direct or indirect actions of inflammatory cytokines during mastitis (Larsen et al., 2010). LDH and ALP activities in milk are substantially greater in dairy animals that are suffering from subclinical mastitis and mastitis than normal milk (Maiti et al., 2020;

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Enzymes in mastitis milk

Yang et al., 2011). Increased milk SCC and ALP activities are noticed in clinical and subclinical mastitis milk than normal milk of buffaloes (Patil et al., 2015). ALP levels are higher in both Gram-positive and Gram-negative bacteria-infected milk. It is suggested that ALP should be tested in milk to diagnose bovine subclinical mastitis, irrespective of the cause of mastitis. A significant increase in ALP in subclinical mastitis milk could be attributable to mammary epithelium injury as well as a breach in the blood milk barrier that has been specifically affected by bacterial toxins (Katsoulos et al., 2010). Increased aspartate aminotransferase (AST) activity in mastitic milk is linked to the liberation of udder parenchyma cells, disintegrating leukocytes, or both, as well as additional sources such as serum. AST activity is increased in the case of mastitis milk. The increased activity of AST and alanine aminotransferase (ALT) in milk is proportional to the severity of the udder infection and indicates enhanced oxidative stress (Guha et al., 2012). Several studies revealed that elevated LDH, ALP, and AST activities in milk correlate to udder infections in dairy cows (Andrews, 1991; Golshan et al., 2021; Tabatabaee et al., 2021). In subclinical mastitis conditions, milk LDH and ALP were strongly linked with SCC, suggesting that intramammary inflammation in buffaloes and ewes can be diagnosed based on LDH and ALP activities (Nandi, 2021; Sani et al., 2018). A higher acid phosphatase ACP level is associated with a higher leukocyte count (Mukherjee et al., 2005). ACP activity affects casein micellar structure, but the increased ACP activity during mastitis is linked to the emergence of isoenzymes having leukocyte origin (Andrews & Alichanidis, 1975). The respiratory burst and release of granular content leads to disturbance in mammary gland function with the increased LDH and ALP enzyme activities and significantly decreased ACP enzyme activities of the mice mammary gland tissue (Chinchali & Kaliwal, 2014). Table 2.1 shows the level of different enzymes present in healthy, subclinical, and clinical mastitis milk of dairy animals.

2.3

Efforts in diagnosing mastitis in dairy animals

Over the years, various diagnostic approaches for mastitis detection have been developed to identify physical, biochemical, and cellular changes in milk properties. These diagnostic methods are being used to assess the impact of treatments on udder health, but these are not often used to their full potential. A prominent indirect approach for diagnosing mastitis is CMT. CMT has several advantages, including being rapid, inexpensive, and easy, as well as the ability to be employed as a “cow-side” test. In the case of a fresh sample, the CMT has a sensitivity of 66.7% and a specificity of 54.8% for detecting intramammary infection (Edwards & Gibbs, 1994). The test is carried out by adding a detergent

2.3 Efforts in diagnosing mastitis in dairy animals

Table 2.1 Level of different enzymes present in healthy, subclinical, and clinical mastitis milk of dairy animals (Leitner, Chaffer, et al., 2004; Leitner, Merin, et al., 2004; Nandi, 2021). Enzyme level in milk Subclinical mastitis

Enzymes

Animal

Healthy

Plasminogen

Cattle Sheep

1.73 IU/mL 92.2 IU/mL

2.21 IU/mL

Cattle Sheep

0.48 IU/mL 33.90 IU/mL

0.57 IU/mL

Goat

20.32 IU/mL

Cattle Cattle Cattle Cattle Cattle

B15 ng/mL B 6.5 ng/mL B 4 ng/mL 35.75% 0.00%

B18ng/mL B10 ng/mL B 4.5 ng/mL 57.75% 1.32% 2.34 %

Cattle Cattle Goat

32.81 IU/mL 9.85 IU/mg 271.76 IU/L

26.41 IU/mL 14.2 IU/mg

Cattle Sheep Goat

1.82 IU/mL 0.62 IU/mg 2.57 IU/mL

1.6 IU/mL 1.12 IU/mg

Cattle Cattle

,2 IU/mL 19. 18 IU/mL

3 3.9 IU/mL 25.90 IU/mL

$ 5 IU/mL 30. 69 IU/mL

Cattle

0.45 ng/mL

1.56 3.52 ng/mL

5.92 ng/mL

Cattle

Cattle

0.60 IU/mL log NAGase 2.02 to 2.48 μmol/min/L 39. 05 nmol/mL/ min 32.55 IU

0.77 IU/mL log NAGase 2.48 to 7.92 μmol/min/L 69.42 nmol/mL/ min 33. 88 IU

Sheep

22.30 IU

Goat

13.6 IU

Plasmin

Elastase Collagenase Cathepsin Caspases (3 and 7) Leukocyte esterase activity Glutathione peroxidase

Superoxide dismutase

Lactoperoxidase Myeloperoxidase

N-acetyl-Dglucosaminidase

Cattle Cattle

Clinical mastitis 62.5 IU/mL

58.9 IU/mL 39.81 IU/mL B10 ng/mL B 4.2 ng/mL B 4.25 ng/mL 44.16%

300.47 IU/L

2.23 IU/mL

39. 81 IU 95.50 IU

37.9 IU

References Urech et al. (1999) Leitner, Chaffer, et al. (2004) Urech et al. (1999) Leitner, Chaffer, et al. (2004) Leitner, Merin, et al. (2004) Panda et al. (2019) Panda et al. (2019) Panda et al. (2019) Swain et al. (2014) Mirzaei et al. (2019) Yang et al. (2011) Alba et al. (2019) Darbaz et al. (2019) Yang et al. (2011) Alba et al. (2019) Darbaz et al. (2019) Isobe et al. (2011) Piccinini et al. (2007) Alhussien & Dang, 2020 Urech et al. (1999) Nielsen et al. (2005) Chagunda et al. (2006) Piccinini et al. (2007) Leitner, Chaffer, et al. (2004) Leitner, Merin, et al. (2004)

Continued

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Enzymes in mastitis milk

Table 2.1 Level of different enzymes present in healthy, subclinical, and clinical mastitis milk of dairy animals (Leitner, Chaffer, et al., 2004; Leitner, Merin, et al., 2004; Nandi, 2021). Continued Enzyme level in milk Enzymes

Animal

Healthy

Subclinical mastitis

Lactate dehydrogenase

Cattle

2. 52 mmol/min

4. 07 mmol /min

Cattle

485.94 IU/L

1524.04 IU/L

Cattle Cattle Cattle

177.94 IU/L 143.26 IU/L 35.25 IU/L

724.49 IU/L 276.46 IU/L 63.47 IU/L

Buffalo

411.74 IU/mL

Sheep Cattle Cattle Cattle

168.50 U/L 71.85 IU/L 34.51 IU/L 283.38 IU/L

642.33 IU/mL 329.70 116.58 163.91 488.15

Buffalo

16.48 IU/L

Buffalo

16.39 IU/dL

Sheep Cattle Cattle

297.80 IU/L 151.99 IU/L 71.84 IU/L

Alkaline phosphatase

Aspartate aminotransferase

to 963.88

Clinical mastitis

References

1250.07 IU/mL

Chagunda et al. (2006) Batavani et al. (2007) Yang et al. (2011) Maiti et al. (2020) Golshan et al. (2021) Nandi (2021)

42.37 IU/L

Sani et al. (2018) Yang et al. (2011) Maiti et al. (2020) Golshan et al. (2021) Patil et al. (2015)

42.14 IU/dL

Nandi (2021)

U/L IU/L IU/L IU/L

28.11 to 37.73 IU/L 25.96 to 37.07 IU/dL 362.60 IU/L 140.98 IU/L 174.87 IU/L

Sani et al. (2018) Yang et al. (2011) Maiti et al. (2020)

to the milk sample, causing cell lysis, nucleic acid release, and the development of a “gel-like” matrix. When the cell count exceeds a particular level, the sample viscosity interpretation becomes subjective, and there can also be false positives or negative results (Viguier et al., 2009). The most widely used criterion for mastitis monitoring is by measuring SCC content in milk, especially in subclinical form. Inflammation and subclinical mastitis are often indicated by SCC levels exceeding 200,000 cells/mL of milk. Most European nations have set a maximum of 400,000 cells/mL for farm milk commercialization, but a limit of 750,000 cells/mL exists in the United States. Both microscopy and cell staining procedures can be effectively used to assess the SCC at the laboratory level. Cell counters are also available based on imaging methods, Coulter counting, or flow cytometry, for example, in the case of DeLaval and Fossomatic cell counters. The cells are stained with a DNA fluorescent dye, and stained nuclei are counted (www.delaval.com). The Coulter concept is based on the

2.3 Efforts in diagnosing mastitis in dairy animals

electrical conductance of cells suspended in an electrolyte passing through an aperture between electrodes. The quantity and size of the flowing cells are taken into account by the system (Norberg et al., 2004). Most modern farms use sensing tools built into automated milking systems that can provide online measurements to detect physical, biochemical, and cellular changes in milk properties. National authorities and local dairy associations, particularly in developed countries, are using diagnostic tools that include routine analysis, data extraction, and reporting to ensure that milk delivered is of good quality. However, most of these technologies fail to provide information on the pathogens’ biology and timely diagnosis of infection. In biomedical research, transcriptome and proteome analysis are recently introduced. These tests can identify biomarkers, gene expression patterns, and complex molecular processes in normal cell physiology and disease condition (Klopfleisch & Gruber, 2012). Advances in practical proteomic approaches and newly available data on toxins, enzymes, and metabolites identified in the udder aid in developing diagnostic tools with high sensitivity and specificity for diagnosing bovine mastitis (Boehmer, 2011). A biomarker is a measurable and evaluable property that identifies normal biological and pathological processes. To be called a “good” biomarker/indicator, the indicator must be disease specific and unaffected by other stressful conditions. Quantification of biomarkers must be reliable and repeatable. Other indicators, such as enzymes secreted during cell proliferation and tissue degradation, can also be used to diagnose udder infections. Enzymes are released into milk due to an animal’s immunological response to infection and changes in vascular permeability. The enzymes involved in milk production tend to decrease, whereas these under inflammation tend to increase. These enzymes are used as biomarkers in diagnosing mastitis in dairy animals. The number of enzymes produced by phagocytes rises rapidly. NAGase, β-glucuronidase, proteases, and phosphatases are examples of such enzymes that become more active during inflammation (Pyörälä, 2003). Several enzymes, including NAGase, serum amyloid A, haptoglobin, LDH, phosphatases, aminotransferase, plasmin, esterase, and different antioxidant enzymes are used as indicators for mastitis diagnosis (Zhao & Lacasse, 2008). Colourimetric and fluorimetric tests for detecting milk enzyme concentrations, such as NAGase, phosphatases, aminotransferase, and LDH, which rise during the early stages of mastitis, have been developed. At the farm level, a portable spectrophotometer and fluorometric tests are developed to know the catalytic activity of enzymes in raw milk (Hiss et al., 2007; Hovinen et al., 2016). Technological breakthroughs, such as systems that incorporate numerous milk-derived parameters and other enzymatic assays for LDH, NAGase, and phosphatases, may help in early diagnosing and treating subclinical and clinical mastitis. The UdderCheck evaluates LDH activity utilizing paper-based test strips

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and color changes in the presence of an LDH-specific substrate. The results are subjectively contrasted with a color chart. Several research studies have widely analyzed diagnostic sensitivity and specificity. False-positive results often hamper the diagnostic specificity of SCC, CMT, and other methods (HernándezCastellano et al., 2017; Ruegg & Pantoja, 2013).

2.4

Future developments and conclusion

As previously stated, conventional pathogen detection and identification methods are either time-consuming (culture and colony counting) or costly (molecular approaches). It has been extensively documented that a number of enzymes are naturally present in the milk of dairy animals. Enzymes may originate from plasma, blood/milk leukocytes, and milk-producing cells. Properties of enzymes in milk may vary with the health status of animals/mammary glands. In food technology, enzymes are utilized to improve the quality of milk products. Many enzymes come in milk at the onset of inflammation and can be used as an alternative diagnostic tool to detect mastitis and hence could be used as an alternative and effective marker for the screening of milk. However, recent advancements in micro and nanotechnologies have resulted in a new class of analytical systems known as “biosensors.” Improved microfabrication techniques and innovative nanomaterials with better sensing capabilities provide the foundation for more integrated biosensors (Martins et al., 2019). Nevertheless, novel technology addressing the issues of bacterial infection, animal welfare, and antibiotic stewardship is in great demand in the veterinary sector, where mastitis is a key burden. With the discovery of new biomolecules and the development of sensitive instruments, detection of mastitis will be more accurate. This will help in reducing mastitis losses and improving the welfare of dairy animals.

Acknowledgments The authors are thankful to the Department of Biotechnology, Ministry of Science and Technology (BT/PR40092/AAQ/1/789/2020 Dated 28/09/2021) for giving grants to study the activity of immune cells and cytokines in the blood and milk of cows and buffaloes.

References Abd Ellah, M. R. (2013). Role of free radicals and antioxidants in mastitis. Journal of Advanced Veterinary Research, 3(1), 1 7. Aida, Y., & Pabst, M. J. (1991). Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst. Journal of Immunology, 146(4), 1271 1276.

References

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CHAPTER 3

Effect of high-pressure processing on milk enzymes Laxmana Naik1, Kiran Lata2 and Rajan Sharma3 1

Southern Regional Station, ICAR-National Dairy Research Institute, Bengaluru, Karnataka, India, 2Department of Food Processing and Technology, School of Vocational Studies and Applied Sciences (SoVSAS), Gautam Buddha University, Greater Noida, 3 Uttar Pradesh, India, Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

3.1

Introduction

Milk and dairy products naturally contain numerous indigenous enzymes. Selective exogenous enzymes are also added to milk during the preparation of dairy products to induce the specific changes in the final product. In addition to this, milk and milk products contain viable microorganisms which secrete extracellular enzymes or they may release intracellular enzymes upon cell lysis. In this condition, under the controlled environment, the enzymes have been linked to bring up some desirable changes in the body, texture, and flavor of milk and milk products and also believed to improve the digestibility. Many enzymes may also cause undesirable changes and may deteriorate the quality of milk and dairy products. Therefore, like all other foods, milk and milk products are being processed to ensure the safety and also to extend the shelf life. There are many alternate nonthermal food processing techniques available and amongst them high-pressure processing (HPP) seems a very promising technique. As a nonthermal process, it offers new opportunities by altering the physico-bio-chemical properties of food system and other processed products. This chapter briefly covers the application part of HPP with an emphasis on its effect on the milk enzyme system.

3.2

Significance of milk enzymes

Milk, besides having many essential nutrients, namely the proteins, lipids, carbohydrates, minerals, and vitamins, contains numerous biological substances such as immunoglobulins, enzymes, functional peptides, oligosaccharides, Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00003-5 © 2023 Elsevier Inc. All rights reserved.

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hormones, and growth factors. Mammalian milk contains approximately 63 indigenous enzymes (see Chapter 1). Some of the enzymes have antioxidant and antimicrobial characteristics (lysozyme, catalase, superoxide dismutase, lactoperoxidase, xanthine oxidase, ribonuclease, etc.) and are important in terms of milk stability and protection of humans against pathogenic agents (Fox & Kelly, 2006; Korhonen & Pihlanto, 2006). Milk is a perishable commodity and deteriorates rapidly during storage, primarily due to the activity of microorganisms. Certain enzymes have also been linked with various biochemical changes in raw and processed milk, and thus it leads to a loss of stability and reduce the shelf life. Milk being a liquid food is more amenable to enzyme action than the other processed dairy products. Exogenous enzymes are added to induce specific changes in the product. The principal constituents of milk such as lactose, lipids, and proteins can be modified by both indigenous and exogenous enzymes. Many indigenous milk enzymes are of technological significance. For example, sulfhydryl oxidase and superoxide dismutase are involved in the preservation of milk quality. Lipase, proteinase, acid phosphatase, and xanthine oxidoreductase are of great concern in the deterioration of milk quality. Alkaline phosphatase, γ-glutamyltransferase, and lactoperoxidase are used as indices of the thermal history of milk. Concentration of several enzymes especially catalase, N-acetyl-β-D-glucosaminidase, and acid phosphatase increases upon mastitis infection. Lysozyme and lactoperoxidase exhibit the antimicrobial activity. Some of these enzymes may cause desirable flavors, for example, in ripened cheese. The undesirable changes induced by enzymes include hydrolytic rancidity in milk and dairy products by lipase enzymes, bitterness, and/or age gelation of ultra high temperature (UHT) processed milk by plasmin, bittiness in cream by lecithinase, etc. With a few exceptions (e.g., lysozyme and lactoperoxidase), most of the indigenous milk enzymes do not have a beneficial effect on the nutritional or organoleptic attributes of milk; hence, their inactivation is one of the objectives of many dairy processes (Fox et al., 2015).

3.3

Need for alternate processing of milk

Traditionally, humans have developed various methods and techniques to preserve the food. In general, milk is preserved by heat treatment. During the manufacture of dairy products, milk is subjected to a variety of different heating processes ranging from mild heat treatment to UHT processing for varying duration of time. The International Dairy Federation factsheet (IDF Factsheet 001/2018-02) (IDF, 2018) summarizes the different heating types applied to milk. The pasteurization of milk is most widely practiced; generally it is carried out at a temperature below the boiling point of water (Deak, 2014). The

3.4 High-pressure processing technology

general objective of this process is to extend product shelf life by inactivating all non-spore-forming pathogenic bacteria and the majority of vegetative spoilage microorganisms, as well as inhibiting the microbial and enzyme activity (Lindsay et al., 2021). In this modern era of health consciousness, consumers demand for natural, nutritionally healthy, and minimally processed food. Other consumer expectations are enhanced shelf life, fresh-like characteristics, convenience, variety, better taste, appearance, and adequate nutritional value; all these have created great challenges for researchers. High temperatures used for processing the milk can adversely affect the flavor and nutritional values, and, at the same time, keeping the milk fresh for a long time is a difficult task for the dairy sector. Thus a number of nonthermal technologies have been studied in recent years. Over the past decades, HPP has increasingly being acknowledged as unit operation in food processing and preservation; hence, the HPP has gained momentum as a processing technique for various foods including milk.

3.4

High-pressure processing technology

According to the Vetter, (Vetter, 2001) the “high pressure” represents the physical pressure defined as the force load per unit of area (Newton/m2: N/m2) exhibiting the “normal” atmospheric pressure of our natural environment (ambient or barometric pressure). HPP is popularly known as high hydrostatic processing (HHP) or ultra-HHP (UHHP) or isostatic processing and also known as pascalization. HPP is a form of cold processing technology because most of the processing takes place at room temperature (Farr, 1990). However, moderate temperature (up to 65°C) may be used in the HPP process to increase the microbial and enzymatic inactivation levels (SermentMoreno et al., 2014). The concept of food processing using HPP was proposed by Royer in 1895 to kill bacteria and later by Hite in 1899 exploring HPP effects on milk, meat, fruits, and vegetables processing (Naik et al., 2013). Fundamental operational principles underlying HPP may be described using the Le Chatelier’s principle, isostatic rule, electrostriction, and compression of energy and heat. The basic components of a high-pressure system consist of a pressure vessel plus closure, a pressure-generating system, including pumps/intensifiers, pressure-transmitting medium, temperature control device, and material handling systems (San Martín-González et al., 2006; Voigt et al., 2015). All the materials of construction and accessories should withstand the extreme pressures applied and, for this reason, special technologies are required to be applied in the manufacture. HPP is a nonthermal food processing method, wherein the food is subjected to a pressure range from 100 to 800 MPa [1 MPa 5 145.03 PSI (Pounds per square inch) or 10 Bar] in an industrial

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processing conditions. Usually, HPP is accompanied by a moderate increase in temperature, called the adiabatic heating, which depends on the composition of the food product being processed (Balasubramaniam et al., 2004; Hogan et al., 2005). To attain the sterilization effect in order to inactivate bacterial endospores and enzymes of importance in food preservation, much higher pressure levels ( . 1000 MPa) would be required. Next is the generation of commercial pressure by processing units, generally called as pressure-assisted thermal sterilization or pressure-assisted thermal processing, the unit will operate at about 600–800 MPa and 90°C–120°C (Serment-Moreno et al., 2014). In a typical HHP process, the product is packaged in a flexible and waterproof package container and is then conveyed within the baskets into a highpressure chamber filled with a pressure-transmitting (hydraulic) fluid. Air is removed from the vessel with an automatic deaeration valve by means of a low-pressure fast fill-and-drain pump. The hydraulic fluid (normally water) in the chamber is pressurized with a pump, and this pressure is transmitted through the package into the food itself. As represented in Fig. 3.1, in general, the applied pressure acts instantaneously and uniformly through a mass of a food or independently of the size, geometry, and composition, isostatic pressure leaves no evident crushing effect on the products. Pressure is applied for a specific time, usually 3–5 min; in this condition, the applied pressure

FIGURE 3.1 Schematic representation of high-pressure processing of milk.

3.5 Effect of high pressure on the activity and structure of milk enzymes

inactivates foodborne microorganisms and some of enzymes in a few minutes, while minimizing the loss of food quality. The processed product is then removed from the vessel upon depressurizing and the products in baskets are stored and distributed in the conventional manner. HPP allows gentle preservation of food by high pressure without additives. Food labeling information, such as the nutritional value, quality, origin, method of processing, and other standard components of a food label (Naik et al., 2018), helps the consumers to make food choices according to their needs and desires. As consumer recognition of HPP benefits has increased, in this aspect from January 2018, Cold Pressure Council (2022) has introduced the “High Pressure Certified” seal that can be placed on the packaging of products that have undergone the HPP process. The council emphasis that declaration of “High Pressure Certified” seal on the label is going to increase the consumer awareness and also it is going to fuel the market returns. HPP technology can lead to adequate food preservation while causing minimal changes regarding taste, texture, appearance, and nutritional value. This technology is among the most prominent recent innovations in food processing. HPP is a proven tool for a number of industrial processes and promising ones in the future as this technology aims to provide an acceptable shelf life as well as assurance of safety and nutritional value (Balci & Wilbey, 1999; Datta & Deeth, 1999; Huppertz, 2010; Kouassi et al., 2007; Mussa & Ramaswamy, 1997; Naik et al., 2013; Sousa et al., 2016; Stratakos et al., 2019; Tan et al., 2020; Viazis et al., 2007; Voigt et al., 2015).

3.5 Effect of high pressure on the activity and structure of milk enzymes Most enzymes are globular proteins, which catalyze various biochemical reactions and have complex and diverse structure. The HPP method is characterized by three parameters: temperature (T), pressure (p), and exposure time (t). These three parametric HPP approach offers better process efficiency in terms of the lethality of the treatment and its effect on the product quality (Naik et al., 2013). Therefore the enzyme structure and external factors such as applied pressure, time, temperature, pH, and solvent composition regulate the enzyme kinetics. Changes in enzymes can occur as a result of HPP (Cheftel, 1992); for example (1) Conformational changes in enzyme at room temperature can bring reversible or irreversible, partial or complete enzyme inactivation. Pressure, temperature, time, enzyme type, and microenvironmental circumstances (such as pH and the presence of solutes or other biological components) all these influence the severity of the effects; (2) pressurization may denature the macromolecules such as protein, making

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them more reactive to enzymatic reactions; and (3) improved enzyme–substrate interactions can be achieved by altering the cell membrane of intracellular organelles or removing intracellular enzymes from the cell cytoplasm. In terms of molecular structure, the effect of HPP on the milk enzymes can be explained by the alterations in the quaternary, tertiary, and secondary structures of enzymes, which directly affects its active site configuration. Mechanism of enzyme inactivation by HPP is similar to the protein denaturation (Ludikhuyze & Hendrickx, 2001). The application of pressure on the enzyme structure may cause partial or complete and reversible or irreversible unfolding, which can lead to changes in the enzyme activity due to the structural changes of its active site. Induced pressure results in the exposure of hydrophobic amino acids and sulfhydryl group (-SH) groups due to the partial denaturation, a reduction in the total SH content may also be noticed due to new disulfide bond formation and changes in the α-helix, β-sheet, and β-turn structure. On the other hand, no changes on primary structure were evidenced, which was expected since HPP is not considered to break covalent bounds (Eisenmenger & Reyes-De-Corcuera, 2009; Júnior et al., 2017). In most of the commercial HPP, the milk quality is affected by both highpressure-induced activation and inactivation mechanism of indigenous and exogenous milk enzymes. Generally, two types of high-pressure effects on enzymes are reported. In the first case, it was shown that some enzymes can be activated at very modest pressures (approx 100 MPa) (Asaka et al., 1994; Jolibert et al., 1994). In contrast, much higher pressures usually result in enzyme deactivation. Various authors have differentiated pressure-driven inactivation of various enzymes based on the inactivation and regain of enzyme activity and these include (1) complete irreversibly denaturation, (2) complete reversibly denaturation, (3) incomplete and irreversible denaturation, and (4) incomplete and reversible denaturation (Curl & Jansen, 1950a; Miyagawa et al., 1964). In addition to the conformational changes (Asaka et al., 1994), pressure-induced decompartmentalization can cause enzyme activation (Butz et al., 1994; Gomes & Ledward, 1996). This is due to the fact that often enzymes and substrates are separated in intact tissues by compartmentalization and such arrangement would be disturbed on the application of pressure (Butz et al., 1994; Jolibert et al., 1994). Enzymes are a special class of proteins in which biological activity arises from an active site, brought together by the 3D conformation of the molecule. Even a small change in the active site can lead to a loss of enzyme activity (Tsou, 1986). Since protein denaturation is associated with conformational changes, it can change the functionality of the enzyme either by increase or loss of biological activity or may lead to change in substrates

3.6 Kinetics of high pressure on milk enzyme inactivation

specificity (Hendrickx et al., 1998). Enzyme–substrate interaction occurs as a result of pressure-induced membrane breakdown and the consequent leaking of enzyme and substrate. Pressure can speed up or slow down the enzymatic reactions, depending on the reaction volume of the enzyme-catalyzed process (Morild, 1981). There appears to be a minimum pressure for pressure inactivation; therefore, in such case, there is no or very little enzyme inactivation below this point. When pressure crosses this threshold limit, enzyme inactivation accelerates until it reaches a specific point (Balny & Masson, 2009; Cheftel, 1992). In the milk system, the kind of enzyme, pH, milk constituents (lipid, protein, and lactose), storage temperature, contaminants, and other factors can influence the pressure inactivation of milk enzymes. It is observed that the efficiency of HPP on milk enzyme inactivation is improved by applying pressure cycles. Multicyclic process which involves successive applications of high-pressure cycle resulted in higher inactivation of trypsin, chymotrypsin, and pepsin enzymes (Curl & Jansen, 1950a, 1950b). However, repeated steps of pressure buildup and release appeared to have no effect on pectin methyl esterase (Irwe & Olson, 1994). Irreversible modification occurs including the dissociation of oligomeric units into enzyme subunits, conformational changes either at the active site or in the substrate, and possibly due to the aggregation or gelation or the association of hydrophobic molecules (Enrique et al., 2007; Heremans, 1982; Yaldagard et al., 2008). Reversible changes are typically observed in the range of 100–300 MPa (del Pilar Buera et al., 2006) but enzyme activity may also be enhanced within this range (Eisenmenger & Reyes-De-Corcuera, 2009; Enrique et al., 2007; Yaldagard et al., 2008). It has been reported that the majority of the enzymes exhibit higher enzyme inactivation as the temperature and time applied during pressure application are increased; the effect of temperature being more pronounced than the time (Chakraborty et al., 2014). Some enzymes can display high baroresistance, and pressures more than 500 MPa combined with moderate temperatures are required to induce significant inactivation (Serment-Moreno et al., 2014).

3.6 Kinetics of high pressure on milk enzyme inactivation Earlier research on the processing of food by high pressure was mainly a qualitative trial-and-error process (Hendrickx et al., 1998). By looking at the research tendencies in “HPP” and “dairy products” from 1995 until the current date and the number of industrial HPP equipment operating worldwide (Ravash et al., 2020), HPP can be considered as one of the most promising food-processing technologies due to the exponential growth in the

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number of equipment in operation worldwide (Pinto et al., 2020). In a nutshell, at an industrial level, HPP R&D work is characterized by systematic and fundamental approach in which more attention is given to the kinetic aspects and to the specific products. The kinetic parameters related to the pressure, temperature, and time-dependent parameters play important role in the optimization, design, and development of the HPP. It is well known that the effect of pressure alone on the microbial and enzymatic inactivation is not much effective in order to achieve the safe product. Therefore the net combination of high pressure and moderate temperature elevation will lead to microbiologically safe products (Gould, 1973) and economically feasible processes (Mertens, 1992). The time course of isobaric and/or isothermal enzyme inactivation due to pressure and/or temperature can often be described by nth order, 1st (first)-order or fractional conversion models. The latter model, which is a special case of a first-order model, is used when a resistant fraction persists after the inactivation process (Hendrickx et al., 1998). In order to predict the impact of high pressure and thermal processes on the activity of some enzymes in food, first-order kinetic model needs to be implemented (Infante et al., 2011). Basically, it describes the evolution of the activity which depends on the pressure and the temperature that are needed as an input for the model. Published reports say that the kinetic parameters describing the effect of pressure and temperature on microbial and enzymatic inactivation are based on the several simplified versions of primary and secondary models. Furthermore, to predict the level of microbial and enzyme inactivation, application of theoretical, empirical, and semiempirical kinetic models have been proposed (Serment-Moreno et al., 2014). Notably, several of these kinetic models take into account the heat and mass transfer phenomena and the enzymatic inactivation occurring during the process. Researchers have investigated the kinetic parameters such as rate constant (k) and decimal reduction time (D values) based on the first-order rate model by subjecting the fresh raw milk to a selected pressures (200–400 MPa) for various holding times (5–120 min) (Mussa & Ramaswamy, 1997). The pressure sensitivities of the kinetic parameters were evaluated based on the Arrhenius-type volume model and the conventional thermal death time–type model. This finding suggest that higher pressures resulted in higher rates of microbial destruction, enzyme inactivation, as well as color and flavor changes as indicated by the associated lower D values (and higher k values). Further, the rate of microbial destruction was much more rapid than enzyme inactivation or color and viscosity changes. A detailed kinetic study on pressure-temperature inactivation of alkaline phosphatase by HPP in the pressure range of 0.1–725 MPa at temperatures between 25°C and 63°C has been conducted (Ludikhuyze et al., 2000). In this study,

3.7 Effect of high-pressure processing on milk enzyme system

inactivation of alkaline phosphatase was accurately described by a first-order reaction kinetic model formulating a global model describing the D value as a function of pressure and temperature. This study indicates that the D values firstly increased with increasing pressure up to 300 MPa and then decreased with further increase in pressure, showing thermal inactivation to be counteracted by low pressure. Influence of pressures and temperature on the inactivation rate constant of purified plasmin enzyme extract from the milk has been described as first-order kinetic model (Borda et al., 2004). This study indicates that plasmin system was very pressure stable at room temperature, but inactivation occurred with combined high-pressure/temperature treatments. Further, inactivation rate constant was quantified using the Arrhenius equation. At all temperatures studied, a synergistic effect of temperature and high pressure was observed in the range of 300–600 MPa. However, an antagonistic effect of temperature and pressure appeared at pressures above 600 MPa. The indigenous milk enzyme inactivation of γ-glutamyltransferase followed first-order reaction kinetics in the range of 400–800 MPa, whereas a reaction order of 1.5 was found for alkaline phosphatase (Rademacher & Hinrichs, 2006).

3.7 Effect of high-pressure processing on milk enzyme system Milk of all species probably contains the same range of enzymes as that of the bovine milk (Fox et al., 2015). Generally, it is evident that the milk shelf life is influenced by raw milk quality. The native milk enzymes and the heatresistant exogenous enzymes (Barbano et al., 2006) from different microbial sources also affect the milk quality and stability. Therefore the effect of HPP on milk enzyme (Barraquio, 2014) attracted many researchers. According to Rademacher and Kessler (1997), milk treated to a pressure treatment of 400 MPa for 15 min or 600 MPa for 3 min had a shelf life of 10 days when stored at 10°C and 18 days when stored at 4°C. Similar findings reported by Mussa and Ramaswamy (1997) indicated that upon processing milk at a pressure of 350 MPa, it offered an 18-day shelf life at the same storage temperature. Findings by Pereda et al. (2007) indicated that milk processed at 200 MPa and stored at 4°C had shown a shelf life of 14–18 days and at a same storage temperature it had shown 28 days of shelf life upon processing at higher pressure (600 MPa) (Stratakos et al., 2019). Since the enzyme molecules are the protein subsets, the sensitivity of enzymes to HPP depends on their structure and properties. Therefore, generally speaking, dairy enzymes show better resistance against HPP treatment than to thermal processing. Inactivation of milk enzymes occurs at pressures higher than 400 MPa, and the rate of inactivation can increase as the pressure rises. However, not only

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the pressure level, but also the time, type of enzyme, milk composition, and pH levels determine the amount of inactivation (Liepa et al., 2016; Munir et al., 2019; Naik et al., 2013). The pressures less than 350 MPa may boost enzymatic activity (Munir et al., 2019), since the partial unfolding of enzymes promotes their interaction with the substrate. It has also been noticed (Heinisch et al., 1995) that the pressure required varies greatly depending on the enzyme: certain enzymes can be deactivated by a few hundred MPa at ambient temperature, while others can resist 1000 MPa. Indigenous milk enzymes show different behavior against HPP. Among the two phosphatases present in milk, alkaline phosphatase has been reported to be stable as this enzyme can withstand pressure of 800 MPa for 8 min, while the acid phosphatase has been reported to be less stable and gets inactivated at pressures around 200 MPa (Datta & Deeth, 2011). Lactoproxidase has also been shown to quite stable as 50% of its initial activity is retained after HPP at 800 MPa for 4 h at 25 C–60 C (Júnior et al., 2019). Similarly, lysozyme has been shown to withstand pressure of 400 MPa for 30 min (Viazis et al., 2007). At 400 MPa, the activity of plasmin in milk and its products decreased by 75% at 20°C for 30 min (Huppertz et al., 2004) and by 87% at 60°C for 15 min (García-Risco et al., 2000). It has been pointed out that since some of the antimicrobial enzyme activity is retained in HPP-treated milk, this fact may be taken into account while using such milk for preparation of dairy products (Ravash et al., 2020). Generally, it is observed that most of the milk enzymes vary in their sensitivity to high pressure, often a synergistic effect of temperature and high pressure is reported. At 300–600 MPa and temperature of 30°C–65°C, HPP had a synergistic effect on plasmin inactivation. Although it has an antagonistic effect at pressures above 600 MPa, structural modifications stabilize the structure of plasmin and plasminogen (Borda et al., 2004). HPPinduced inactivation of indigenous milk enzymes—alkaline phosphatase, phosphohexoseisomerase, and γ-glutamyltransferase—was studied in the pressure range of 400–800 MPa at temperatures between 5°C and 40°C, and the pressure stability ranking was observed in the order of alkaline phosphatase . γ-glutamyltransferase . phosphohexoseisomerase (Rademacher & Hinrichs, 2006). Further, they reported that reactivation of pressure-treated alkaline phosphatase was observed at low enzyme activity resulting from severe pressure treatment and 2-h storage at 35°C. The influence of process temperature on the pressure-induced inactivation of γ-glutamyltransferase and alkaline phosphatase was limited in the range of 5°C–40°C. Lipase, xanthine oxidase, and lactoperoxidase are resistant to pressures up to 400 MPa. Phosphohexoseisomerase, γ-glutamyltransferase, and alkaline phosphatase in milk are partially inactivated at pressures .350, 400, and 600 MPa, respectively, and almost completely inactivated at around 550, 630, and 800 MPa, respectively (Datta & Deeth, 2011).

3.7 Effect of high-pressure processing on milk enzyme system

In ripened cheeses, the activity of lipase is favorable; pressures of 350–400 MPa for 100 min can increase the activity of this enzyme by up to 140% (Pandey & Ramaswamy, 2004). Malone et al. (2003) have studied the effects of high pressure on enzymes (chymosin, plasmin, and Lactococcus lactis enzymes) involved in cheese preparation. They found that pressure treatment of immature cheeses is likely to change the active enzyme profiles, which could affect the flavor profile of the ripened cheese. Exposing of bovine milk to 400 MPa (Munir et al., 2019) and processing of ewe milk at 200–300 MPa (Alonso et al., 2011) augmented the proteolytic activity during cheese ripening. HPP enhanced the coagulating activity of recombinant chymosin, rennet, and porcine pepsin without changing their nonspecific action (Júnior et al., 2019). However, there is a limit to how much pressure can be given to each enzyme before it loses its activity owing to the denaturation.

3.7.1 Effect of high-pressure processing on alkaline phosphatase The effect of HPP on alkaline phosphatase is of particular interest in milk processing because of its universal use as an indicator of effective pasteurization (Datta & Deeth, 2011; Rankin et al., 2010). It has been indicated that HPP of raw bovine milk at 400, 600, or 800 MPa for 20 min resulted in suppression of alkaline phosphatase activity by 30%, 60%, or 99.7%, respectively (Johnston, 1995). It has been reported that alkaline phosphatase is resistant to pressure treatments up to 400 MPa; however, 50% inactivation can be accomplished at 500 MPa for 90 min or 600 MPa for 10 min, and total inactivation may be obtained at 800 MPa for 8 min (Lopez-Fandiño et al., 1996). To distinguish the thermal and pressure treatments of goat’s milk, it has been demonstrated that HPP for 500 MPa, at 25°C or 50°C for 10 min, did not change the activity of alkaline phosphatase in goats’ milk (Felipe et al., 1997). In a subsequent work by Ludikhuyze et al. (2000), it was concluded that alkaline phosphatase is quite pressure-resistant enzyme, even more than the nonsporogenic pathogenic organism (Mycobacterium tuberculosis) present in milk. Therefore alkaline phosphatase inactivation has not been recommended as an indicator of inactivation of Mycobacterium tuberculosis as this may lead to overprocessing of milk. Alkaline phosphatase enzyme is known to reactivate during cold storage in thermally treated milk; independent of the initial enzyme inactivation, the same trend was also observed in milk samples after pressure treatment (Rademacher & Hinrichs, 2006). The Z-value of alkaline phosphatase enzyme in terms of pressure is quite high (368 MPa) as compared to the microorganisms (168 MPa), therefore its complete inactivation would occur only at very high pressures; hence, alkaline phosphatase is not an appropriate indicator of effective “pasteurization” by HPP treatment (Datta & Deeth, 2011).

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3.7.2

Effect of high-pressure processing on plasmin

Plasmin is a native milk enzyme; it is a natural milk alkaline protease which causes protein degradation during storage leading to bitter flavors and gelation in UHT milks (Datta & Deeth, 2011). It has been reported that the amount of plasmin activity in milk was reduced by 15% at 400 MPa for 8 min, while the reduction was up to 90% when the pressure was increased to 600 MPa (Rademacher & Kessler, 1997). However, a study demonstrated that at lower pressure (400 MPa) upon processing at a higher temperature (60°C) for 15 min resulted in reduction of 86.5% plasmin activity in raw milk (GarcíaRisco et al., 2000). Other workers (Huppertz et al., 2004) have also reported the decrease in plasmin activity by 30% and 75% at pressures of 400 and 600 MPa, respectively, upon processing of milk for 30 min at 20°C. The presence of denatured β-lactoglobulin has been shown to increase plasmin inactivation by forming thiol–disulfide linkages with unfolded β-lactoglobulin (Serra et al., 2009). Plasmin is not inactivated by treatment at 400 MPa for 15 min at room temperature. Plasminogen, the plasmin precursor in milk, is partially inactivated under these conditions. However, treatment at the same pressure at higher temperatures results in inactivation of plasmin, reaching 86.5% at 60°C; this reduces proteolysis and improves the organoleptic quality of long–shelf life milk compared with the same treatments at 25°C (Datta & Deeth, 2011). The influence of HPP on the proteolysis in the raw and stored treated milk was also investigated during the storage of such treated milk. It was observed that pressure treatment at 50 MPa had little effect on proteolysis, but at 300–400 MPa proteolysis was increased, possibly due to changes in micelle structure facilitating increased availability of substrate bonds to plasmin, whereas after 500 MPa, the proteolysis during storage of milk was less than that observed in raw milk (Rastogi, 2013). From the above it may be inferred that HPP treatment of milk has an effect on plasmin activity. However, as the plasmin is inactivated during HPP of milk, changes in the casein micelles structure also happens which may lead to the attack by residual plasmin and thus enhanced proteolysis in HPP–treated milk.

3.7.3 Effect of high–pressure processing on lipoprotein lipase, γ-glutamyltransferase, and lactoperoxidase Lipoprotein lipase is an enzyme present in milk which has been implicated in the hydrolytic rancidity of milk fat. Lipoprotein lipase is a heat–labile enzyme and almost entire enzyme is inactivated during pasteurization of milk. It has been reported that the lipoprotein lipase is quite stable to HPP treatment given to milk in the range of 300–400 MPa for varied time (0– 180 min) (Pandey & Ramaswamy, 2004). The authors have also indicated that activity is enhanced under various pressure/time combination and this could be advantageous for cheese ripening. Same author have also studied

3.8 Milk enzymes as high–pressure processing indicator

the inactivation profile of γ-glutamyltransferase under similar conditions and indicated that although the activity of γ-glutamyltransferase is enhanced initially at low-pressure treatment, at high pressure, there was perceptible inactivation of enzyme following a first-order kinetic. Lactoperoxidase is well known as a process indicator of high–temperature pasteurized milk, but it presents a high stability to pressure. Lactoperoxidase is resistant up to 400 MPa pressure (Rademacher & Kessler, 1997). HPP treatment of bovine milk at 600 MPa for 30 min resulted in 20% inactivation at room temperature, while it is reduced to 16% of the initial activity at 45°C under similar conditions (Seyderhelm et al., 1996). Complete inactivation of lactoperoxidase has been reported at 200 MPa with exposure time of 20 s, at 80°C (Datta et al., 2005). A small increase in the lactoperoxidase activity due to antagonistic effect of high pressure and temperature was noted, which indicated that pressure treatment might lead to preservation of the lactoperoxidase activity (Ludikhuyze et al., 2001). Further, the pressure stability of lactoperoxidase may be due to its monomeric structure, which is stabilized by eight disulfide bonds (Mazri et al., 2012).

3.8 Milk enzymes as high–pressure processing indicator Scientific opinion published by the European Food Safety Authority (EFSA) on “The efficacy and safety of high–pressure processing of food” reports that HPP of food will not present any additional microbial or chemical food safety concerns when compared to other routinely applied treatments (Koutsoumanis et al., 2022). Most stringent HPP conditions industrially used for processing is 600 MPa for 6 min and these would achieve the reductions of relevant hazards based on performance criteria proposed by international standard agencies. Further, based on the literature search regarding the effect of HPP on the inherent milk/colostrum components that could serve as an indicator for pasteurization of milk, it was concluded that alkaline phosphatase is relatively pressure resistant as its complete inactivation occurred only around 800 MPa. The absence of alkaline phosphatase activity in HPP–treated milk has been suggested as overprocessing of milk. Alternative enzymes have been proposed, such as γ-glutamyltransferase, but the HPP effect on them differs greatly depending on the survey and more data is required to verify their suitability. Other enzymes such as xanthine oxidase and acid phosphatase were also studied as potential indicator for HPP of bovine milk. Xanthine oxidase had been resistant to pressure of 400 MPa at 25°C and at higher pressure (600 MPa), 83% of the activity was destroyed within 12 min (Cold Pressure Council, 2022; Olsen et al., 2004; Rastogi, 2013). With reference to the acid phosphatase, it is much less resistant to pressure than alkaline phosphatase and the

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majority of its activity is lost within 10 min of exposure to 500 and 550 MPa (Balci et al., 2002). Further, the findings showed that, after 600 MPa for 10 min, acid phosphatase still showed 25% of activity. A marginally higher inactivation was observed with skimmed milk compared to whole milk for the same time and pressure combination, although activity in skimmed milk was 80% that of whole milk, suggesting acid phosphatase as a potential indicator to discriminate between thermal and high–pressure treatments. Therefore, based on the available information, EFSA report indicates that current data are not robust enough to support the proposal of an appropriate indicator to verify the efficacy of HPP under the current HPP conditions applied by the industry (Koutsoumanis et al., 2022). However, for considering a potential indicator of HPP and for effective implementation of regulations, robust study is required to strengthen the claim regarding the HPP food quality assurance and safety.

3.9

Conclusion

HPP has emerged as one of the potential alternate processing technique for milk and other food products. The effect of HPP on milk enzymes shows activation, inactivation, or no change in the enzyme activity. Generally speaking, mild conditions are able to improve the activity and the stability of several enzymes, whereas extreme process conditions induce the enzyme denaturation with consequent reduction of biological activity. HPP techniques have the ability to inactivate the milk enzymes without affecting the milk quality. However, the resistance to HPP of different enzymes differs from one another, and complete inactivation can sometimes be unachievable even with the most intense treatments. Hence, combination of HPP with mild thermal processing has proven to be more effective to provide safety to milk when compared to HPP alone. Present knowledge cannot pinpoint any enzyme in milk, the levels of which before and after HPP treatment is well correlated with treatment parameters in terms of pressure and temperature.

References Alonso, R., Picon, A., Rodríguez, B., Gaya, P., Fernández-García, E., & Nuñez, M. (2011). Microbiological, chemical, and sensory characteristics of Hispánico cheese manufactured using frozen high pressure treated curds made from raw ovine milk.. International Dairy Journal, 21(7), 484 492. Available from https://doi.org/10.1016/j.idairyj.2011.02.008. Asaka, M., Aoyama, Y., Nakanishi, R., & Hayashi, R. (1994). Purification of a latent form of polyphenoloxidase from La France pear fruit and Its pressure-activation. Bioscience, Biotechnology, and Biochemistry, 58(8), 1486 1489. Available from https://doi.org/10.1271/bbb.58.1486. Balasubramaniam, V. M., Ting, E. Y., Stewart, C. M., & Robbins, J. A. (2004). Recommended laboratory practices for conducting high-pressure microbial inactivation experiments.. Innovative

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Malone, A. S., Wick, C., Shellhammer, T. H., & Courtney, P. D. (2003). High pressure effects on proteolytic and glycolytic enzymes involved in cheese manufacturing. Journal of Dairy Science, 86(4), 1139 1146. Available from https://doi.org/10.3168/jds.S0022-0302(03)73696-0. Mazri, C., Sánchez, L., Ramos, S. J., Calvo, M., & Pérez, M. D. (2012). Effect of high-pressure treatment on denaturation of bovine β-lactoglobulin and α-lactalbumin. European Food Research and Technology, 234(5), 813 819. Available from https://doi.org/10.1007/s00217012-1695-x. Mertens, B. (1992). Under pressure. Food Manufacture, 67, 23 24. Miyagawa, K., Sannoe, K., & Suzuki, K. (1964). Studies on taka-amylase a under high pressure treatment: II. recovery of enzymic activity of pressure inactivated taka-amylase a and its enhancement by retreatment at moderate pressure. Archives of Biochemistry and Biophysics, 106 (C), 467 474. Available from https://doi.org/10.1016/0003-9861(64)90217-6. Morild, E. (1981). The theory of pressure effects on enzymes. Advances in Protein Chemistry, 34 (C), 93 166. Available from https://doi.org/10.1016/S0065-3233(08)60519-7. Munir, M., Nadeem, M., Qureshi, T. M., Leong, TSH., Gamlath, C. J., Martin, GJO., & Ashokkumar, M. (2019). Effects of high pressure, microwave and ultrasound processing on proteins and enzyme activity in dairy systems—A review. Innovative Food Science and Emerging Technologies, 57. Available from https://doi.org/10.1016/j.ifset.2019.102192. Mussa, D. M., & Ramaswamy, H. S. (1997). Ultra high pressure pasteurization of milk: kinetics of microbial destruction and changes in physico-chemical characteristics. LWT - Food Science and Technology, 30(6), 551 557. Available from https://doi.org/10.1006/fstl.1996.0223. Naik, L., Nath, S., Prasad, R., Murthy, B. S., & Nandini, H. S. (2018). Food labelling: An overview. Indian Journal of Dairy and Biosciences, 29(1), 1 8. Naik, L., Sharma, R., Rajput, Y. S., & Manju, G. (2013). Application of high pressure processing technology for dairy food preservation - Future perspective: A review. Journal of Animal Production Advances, 3(8), 232. Available from https://doi.org/10.5455/japa.20120512104313. Olsen, K., Kristensen, D., Rasmussen, J. T., & Skibsted, L. H. (2004). Comparison of the effect of high pressure and heat on the activity of bovine xanthine oxidase. Milchwissenschaft, 59(77–8), 411 413. Pandey, P. K., & Ramaswamy, H. S. (2004). Effect of high-pressure treatment of milk on lipase and γ-glutamyl transferase activity. Journal of Food Biochemistry, 28(6), 449 462. Available from https://doi.org/10.1111/j.1745-4514.2004.02603.x. Pereda, J., Ferragut, V., Quevedo, J. M., Guamis, B., & Trujillo, A. J. (2007). Effects of ultra-high pressure homogenization on microbial and physicochemical shelf life of milk. Journal of Dairy Science, 90(3), 1081 1093. Available from https://doi.org/10.3168/jds.S0022-0302 (07)71595-3. Pinto, C. A., Moreira, S. A., Fidalgo, L. G., Inácio, R. S., Barba, F. J., & Saraiva, J. A. (2020). Effects of high‐pressure processing on fungi spores: Factors affecting spore germination and inactivation and impact on ultrastructure. Comprehensive Reviews in Food Science and Food Safety, 19 (2), 553 573. Available from https://doi.org/10.1111/1541-4337.12534. Rademacher, B., & Hinrichs, J. (2006). Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics, inactivation and potential application. International Dairy Journal, 16(6), 655 661. Available from https://doi.org/10.1016/j.idairyj.2005.10.021. Rademacher, B, & Kessler, H. G. (1997). High pressure inactivation of microorganisms and enzymes in milk and milk products. In K Heremans (Ed.), High pressure bioscience and biotechnology (pp. 291 293). Leuven: Leuven University Press. Rankin, S. A., Christiansen, A., Lee, W., Banavara, D. S., & Lopez-Hernandez, A. (2010). Invited review: The application of alkaline phosphatase assays for the validation of milk product

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pasteurization. Journal of Dairy Science, 93(12), 5538 5551. Available from https://doi.org/ 10.3168/jds.2010-3400. Rastogi, N. K. (2013). High-Pressure Processing of Dairy Products. Recent developments in high pressure processing of foods (pp. 51 65). Springer Nature America, Inc. Available from https://doi.org/10.1007/978-1-4614-7055-7_3. Ravash, N., Peighambardoust, S. H., Soltanzadeh, M., Pateiro, M., & Lorenzo, J. M. (2020). Impact of high-pressure treatment on casein micelles, whey proteins, fat globules and enzymes activity in dairy products: A review. Critical Reviews in Food Science and Nutrition, 1 21. San Martín-González, M. F., Welti-Chanes, J., & Barbosa-Cánovas, G. V. (2006). Cheese manufacture assisted by high pressure. Food Reviews International, 22(3), 275 289. Available from https://doi.org/10.1080/87559120600695157. Serment-Moreno, V., Barbosa-Cánovas, G., Torres, J. A., & Welti-Chanes, J. (2014). High-pressure processing: Kinetic models for microbial and enzyme inactivation. Food Engineering Reviews, 6 (3), 56 88. Available from https://doi.org/10.1007/s12393-014-9075-x. Serra, M., Trujillo, A. J., Guamis, B., & Ferragut, V. (2009). Flavour profiles and survival of starter cultures of yoghurt produced from high-pressure homogenized milk. International Dairy Journal, 19(2), 100 106. Available from https://doi.org/10.1016/j.idairyj.2008.08.002. Seyderhelm, I., Boguslawski, S., Michaelis, G., & Knorr, D. (1996). Pressure induced inactivation of selected food enzymes. Journal of Food Science, 61(2), 308 310. Available from https:// doi.org/10.1111/j.1365-2621.1996.tb14182.x. Sousa, S. G., Delgadillo, I., & Saraiva, J. A. (2016). Human milk composition and preservation: evaluation of high-pressure processing as a nonthermal pasteurization technology. Critical Reviews in Food Science and Nutrition, 56(6), 1043 1060. Available from https://doi.org/ 10.1080/10408398.2012.753402. Stratakos, A. C., Inguglia, E. S., Linton, M., Tollerton, J., Murphy, L., Corcionivoschi, N., Koidis, A., & Tiwari, B. K. (2019). Effect of high pressure processing on the safety, shelf life and quality of raw milk. Innovative Food Science and Emerging Technologies, 52, 325 333. Available from https://doi.org/10.1016/j.ifset.2019.01.009. Tan, S. F., Chin, N. L., Tee, T. P., & Chooi, S. K. (2020). Physico-chemical changes, microbiological properties, and storage shelf life of cow and goat milk from industrial high-pressure processing. Processes, 8(6). Available from https://doi.org/10.3390/PR8060697. Tsou, C. L. (1986). Location of the active sites of some enzymes in limited and flexible molecular regions. Trends in Biochemical Science, 11(10), 427 429. Available from https://doi.org/ 10.1016/0968-0004(86)90178-7. Vetter, G. (2001). Introduction. High pressure process technology: fundamentals and applications (pp. 1 16). Elsevier. Viazis, S., Farkas, B. E., & Allen, J. C. (2007). Effects of high-pressure processing on immunoglobulin A and lysozyme activity in human milk. Journal of Human Lactation, 23(3), 253 261. Available from https://doi.org/10.1177/0890334407303945. Voigt, D. D., Kelly, A. L., & Huppertz, T. (2015). High-pressure processing of milk and dairy products. Emerging dairy processing technologies: Opportunities for the dairy industry (pp. 71 92). Wiley Blackwell. Available from https://doi.org/10.1002/9781118560471.ch3. Yaldagard, M., Mortazavi, S. A., & Tabatabaie, F. (2008). The principles of ultra high pressure technology and its application in food processing/preservation: A review of microbiological and quality aspects. African Journal of Biotechnology, 7(16), 2739 2767. Available from http:// www.academicjournals.org/AJB/PDF/pdf2008/18Aug/Yaldagard%20et%20al.pdf.

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Traditional applications of enzymes in dairy science and technology Ahesanvarish Shaikh, Amit Kumar Jain and Satishkumar Parmar Dairy Chemistry Department, SMC College of Dairy Science, Kamdhenu University, Anand, Gujarat, India

4.1

Introduction

Milk contains nearly 63 different indigenous enzymes with different implications on milk and milk product. Enzymes in milk affect product manufacturing (e.g., plasmin in cheese and fermented milk) and storage (e.g., lipase in milk) of milk. Few enzymes are useful indicators for efficient processing of milk (e.g., alkaline phosphatase, lactoperoxidase, and γ-glutamyl transferase). Level of some enzymes can be used as an indicator of abnormality of milk (acid phosphatase, catalase, N-acetyl-β-D-glucosaminidase, etc.). Flavor profile of milk and milk products is significantly affected by certain enzymes such as lipase and plasmin. Milk from various milch animals shows substantial variation in levels and activity of indigenous enzymes owing to various factors such as physiological stage of the animal, stage of lactation, and feed. It shall be noted that the milk from nonbovine species such as goat, sheep, and camel is gaining popularity. There is definite knowledge gap in regards to the data on enzymology of such milk. The information is required for ensuring safety (e.g., ensuring effective pasteurization), storage stability (e.g., impact of plasmin), and quality of milk from such nonbovine sources. Indigenous enzymes in milk find entry through different routes, major routes are (1) spillover from blood plasma in mammary cells during milk synthesis; (2) through part association of secretory cell cytoplasm in milk fat globules during their excretion from the mammalian secretory cell; (3) the milk fat globule membrane (MFGM) itself, being derived from the apical membrane of the mammary cell. This is probably the source of most of the enzymes in milk; and (4) damaged mammary gland due to mastitis allowing entry of somatic cells of blood in milk (Fox & Kelly, 2006a). Significance and 77 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00004-7 © 2023 Elsevier Inc. All rights reserved.

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traditional applications of selected indigenous enzymes in milk and milk products are discussed in the following sections.

4.2

Alkaline phosphatase

Alkaline phosphatase (ALP, EC 3.1.3.1) belongs to the class of metallo enzymes, which are widely present in nature. It was first identified by Suzuki et al. (1907). Its presence in milk was first recognized and characterized by F. Demuth in 1925 (Rola & Sosnowski, 2010). ALP is a sialic acid containing glycoprotein associated with MFGM (Sharma, 1970). It is a phosphomonoesterase hydrolyzing phosphoric acid monoesters yielding phosphate and the corresponding alcohol. It is substrate specific and tends to hydrolyze true orthophosphate monoesters, the enolic phosphate, phosphoenolpyruvate, the orthophosphoamide, and the phosphocreatine (Morton, 1955). ALP from cow and buffalo milk could dephosphorylate various sugar phosphates at varying rates. ALP hydrolyzes glucose-6-phosphate faster than the glucoseL-phosphate. The ALP also exhibits very low pyrophosphatase activity. The action of ALP on p-nitrophenyl phosphate is faster than glucose-6-phosphate and glucose-1-phosphate (Sharma, 1970). ALP possesses maximum stability in alkaline pH, between 7.5 and 9.5, while the optimal pH lies between 8 and 10 (Fosset et al., 1974; Latner et al., 1971; Murtaza et al., 2017; Sharma, 1970). The Km value for p-nitrophenyl phosphate substrate is at variance which is 6.8 x 10-4 M for skim milk enzyme and 2.8 x 10-4 M for the cream alkaline phosphatase. The isoelectric point of ALP is in range of 5.4 6.0 (Chuang & Yang, 1990; Vega-Warner et al., 1999). Each catalytic site of enzyme contains three metal ions, that is, two Zn12 and one Mg12, necessary for enzymatic activity (Millan, 2006); that two activesite Zn12 ions coordinate the nucleophile and the leaving group, respectively, and a nonbridging oxygen atom of the transferred phosphoryl group is coordinated between the two Zn12 ions. A third metal ion site near the bi-metallo site contains an Mg21 ion and it has been suggested that a Mg21-bound hydroxide ion acts as a general base to deprotonate the serine nucleophile (Zalatan et al., 2008). Milk ALP is a homodimer of two similar subunits, each having molecular mass of about 85 kDa (Fox & Kelly, 2006b), while Bilen et al. (2015) observed the molecular mass of subunits between 72 and 95 kDa, estimated on the basis of mobility on SDS-PAGE. On the other hand, Vega-Warner et al. (1999) reported molecular mass of milk ALP isozyme as 187 kDa with unqualified two identical subunits. Human, cow, and sheep milk are quite similar with respect to molecular mass. Milk ALP and ALP occurring in kidney, brain, and liver have a molecular mass higher than 150 KDa (Linden et al., 1977).

4.2 Alkaline phosphatase

Three forms of ALP, identified as α, β, and γ are present in bovine milk. The α form of ALP prevails in skim milk; however, β form of ALP is found in the MFGM. The β form of ALP has a higher molecular mass due to a complex quaternary conformation. The ALP from cream, when separated using thin-layer chromatography on Sephadex G-200, showed four different fragments of MW-18000, 55000, 140000 (α-ALP), and 570000 (β-ALP) (Claeys et al., 2002b; Copius Peereboom, 1970; Girotti et al., 1994; Vega-Warner et al., 1999). ALP is distributed between cream and skim milk fraction. Specific activity of ALP is substantially higher in cream than in skim milk. In skim milk, enzyme is associated with microsomes (Makhzoum et al., 1996; Martini et al., 2007; Sharma, 1970; Zittle et al., 1950). Microfiltration results in reduction of 50% 59% ALP activity in permeates (Panopoulos et al., 2020; Shakeel-urRehman et al., 2003).

4.2.1

Significance of alkaline phosphatase

The abundance of this enzyme in natural cell systems and biological fluids has made ALP activity measurements one of the most commonly performed procedures (Rankin et al., 2010). It is most commonly used indicator for monitoring effective pasteurization conditions in milk and milk-based drinks. The heat inactivation temperature of ALP is 70 C with the D value of 33 s and Q10 of 60 (Walstra, 1999). Nearly complete inactivation of milk ALP is noted in milk subjected to heat treatment at 70 C for 15 60 s (Lombardi et al., 2000; Sharma, 1970). Fat content does not seem to affect the outcomes of ALP test result applied to pasteurized milk (Claeys et al., 2002b). ALP is commonly used to assess the effectiveness of pasteurization and other heat treatments of milk and milk products owing to (1) relatively higher concentration of enzymes in milk contributing to sensitivity of the assay; (2) enzyme inactivation rate positively correlated with destruction of microorganisms and (3) the availability, cost, and simplicity and rapidity of assay (Lombardi et al., 2000). For the pasteurized milk United States (US) and European Union regulatory authorities prescribe International Standard (ISO 11816-1 [IDF 155-1:2013], 2013) as the reference method for official evaluation of ALP in pasteurized milk. The standard uses fluorimetric method for determining ALP activity in milk with the compliance limit of 350 mU/L for assessing proper pasteurization (IDF, 2016). The ALP test has been adopted by many countries as the standard assay for rapid validation of the milk pasteurization process (Rankin et al., 2010). ALP is slightly more heat resistant than the targeted pathogens, namely, Coxiella burnetii and Mycobacterium tuberculosis, thus inactivation of the enzyme is inferred as destruction of these pathogens and

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resultant assurance of safety of milk and milk products. The thermal inactivation of ALP has been found to follow first-order kinetics, and the denaturation midpoint for milk ALP has been obtained at 56 C for a 30-min heating (Levieux et al., 2007). Indigenous milk ALP can also be inactivated by high-pressure processing in the range 400 to 800 MPa and at temperature ranging from 5 C to 40 C (Rademacher & Hinrichs, 2006). ALP is involved in several essential biological functions, including nutrition, phosphate metabolism, intracellular signaling, as well as modification and transport of metabolites across biological membranes (Zhu et al., 2010).

4.2.2

Methods for estimation of alkaline phosphatase activity

The methods for estimation of ALP activity can be classified into four groups, namely, colorimetric, fluorometric, chemiluminescent, and immunochemical. Of these methods, the first three have been recognized as validated methods for pasteurization verification in the dairy industry. Table 4.1 shows overview of different analytical methods for ALP activity determination.

4.2.2.1 Colorimetric methods Several workers have recommended protocols for ALP assay but differed in variation in sample preparation procedures and substrate used for enzyme (Rankin et al., 2010). Colorimetric methods were the first to be used for detection of alkaline phosphatase in milk (Aschaffenburg & Mullen, 1949; Kay & Graham, 1935; Sanders & Sager, 1946). Mueller and Scharer colorimetric methods are still used by many regulatory authorities to check effectiveness of pasteurization (Aschaffenburg & Mullen, 1949; Scharer, 1938). These colorimetric methods can detect as little as 0.1% raw milk in pasteurized products. During the period of 1950s 80s, colorimetric assays and the phenol measurement methods were used to indicate pasteurization effectiveness by public health authorities. The limit of detection of colorimetric methods is approximately 0.05% 0.2% residual or contaminated raw cow’s milk. Using colorimetric assays the ALP concentration is expressed in terms of micrograms of phenol per milliliter of milk, reflecting the amount of phenol reactant organically extracted from the ortho-phenyl phosphate substrate (Albillos et al., 2011). Aschaffenburg and Mullen test uses p-nitrophenyl phosphate as the substrate which is converted to yellow-colored nitrophenol (Aschaffenburg & Mullen, 1949). This method is slightly more sensitive than the Kay and Graham (1933) method and is considered advantageous as it does not require either extraction or long incubation periods. Subsequently, Tramera and Wight (1950) developed a method for measuring the intensity of color produced in the Aschaffenburg and Mullen phosphatase test, employing

Table 4.1 Overview of different analytical methods for ALP activity determination in dairy products. Method principle

Wavelength (nm)

Substrate (S)/product (P)

Units definition

Detection limit

Fluorimetric

Ex 440/Em . 505

S: monophosphoric ester Fluorophos P: Fluoro-yellow

U 5 μmol/min

Ex 365/Em 450

S: 4-methylumbelliferone- phosphate P: 4-methylumbelliferone

mU 5 nmol/ min

Colorimetric

Scope of use

Short description

References

Remarks

0.006% raw milk

Part 1: liquid dairy products from cow, sheep, and goat Part 2: cheese

Measuring fluorescence in the sample with aromatic monophosphoric ester

ISO 11816-1 [IDF 1551:2013] (2013); ISO 11816-2 [IDF 1552:2016] (n.d.); Rocco (1990); Shakeel-urRehman et al. (2003)

Fluoro-Test FML 200, Fluorophos method

0.006% raw milk

Fluid dairy products

Assay in a 96-microwell plate, 4-MUP as substrate, against a standard curve with 4-MU of known concentration

Ziobro and McElroy (2013)

U 5 μmol/min

Dairy products (fluid and solid)

n.d.

NR

0.006% raw milk

Purified ALP

Reaction of purified ALP from calf with substrate for 30 min at pH 7.9 or 9.6 at 37 C

Fernley and Walker (1965)

Ex 405/Em 519

S: trifluoromethyl-bumbelliferone phosphate P: trifluoromethylbumbelliferone

Relative fluorescent units

0.04% raw milk

Liquid and solid samples especially high fat products

Reverse micellar media (microemulsions) used for determination of enzyme activity with the nonfluorescent substrate

Fenoll et al. (2002)

Dependent on substrate

S: phenyl phosphate, sodium phenyl phosphate P: phenol, phosphate

1 μg phenol/ min per L

0.1% 0.2% raw milk

Whole milk, skim milk, chocolate milk

ALP activity equimolar with release of phosphate from substrate, reaction of phenol with colorimetric compound, Folin Ciocalteu or 2,6dibromoquininechloroimide (BQC)

Shakeel-ur-Rehman et al. (2003)

Previous methods: Scharer (1938), ISO 3356:2009, AOAC 972.17

620 nm

S: phenyl phosphate P: phenol

μg of phenol released/h

NR

Liquid samples

MFO-3 method: kinetic measurement during 1 h with phenyl phosphate, color reaction with phenol: CQC (2,6-dichloroquinone-4

MFO-3 (1981)

Official method in Canada

Continued

Table 4.1 Overview of different analytical methods for ALP activity determination in dairy products. Continued Method principle

Wavelength (nm)

Substrate (S)/product (P)

Units definition

Detection limit

1 μg phenol/mL 5 500 U/L

S: p-nitrophenol phosphate P: phenol

NA

540 nm

Scope of use

Short description

References

6.7x10214 mol/L Phenol 5 6.31x1026 μg phenol/ L

Liquid milk

Electrochemical determination, Graphite Teflon composite tyrosinase biosensor monitors by ALP produced phenol through reduction of oquinone

Serra et al. (2005)

pos/neg

NR

Liquid milk from buffalo, cow and goat

Visual inspection rapid test with using this principle: on strip immobilized pnitrophenyl phosphate reacts with ALP and produces p-nitrophenol that reacts with a chromogen producing a color change

Sharma et al. (2003)

S: 5-bromo-4-hloro 3indolyl phosphate (BCIP) P: phenol

pos/neg

% of raw milk: n.d. (0.87 U/mL based on SD)

Milk

Biosensor, miniaturized: digital image colorimetry with smartphone. ALP antibody immobilized on paper, substrate BCIP

Mahato and Chandra (2019)

S: 3-(2’spiroadamantanane)-4methoxy-4(3v-phosphate phenyl-1,2 dioxetane disodium) salt (Charm reagent) AP P: adamantly1,2-dioxetan

U 5 μmol/min

0.05% 0.2% raw milk

Liquid milk from cow, goat and sheep, flavored drink and cream

Photo-activation of hydrolyzed product (chemiluminescent), kinetic stop reaction, Charm ALPPaslite, Chemi-F-AP

Albillos et al. (2011), ISO 22160:2007 [IDF 209:2007] (2007); Salter et al. (2006)

NA

Blue

Chemiluminescent

green

Remarks

IDF 82A, 1987 and available as rapid test with Dryreagent strips

Charm Test, ISO22160:200, Rapid test: Chemi-F-AP

Adapted from Clawin-Rädecker, I., De Block, J., Egger, L., Willis, C. Da Silva Felicio, M. T., Messens, W. (2021). The use of alkaline phosphatase and possible alternative testing to verify pasteurization of raw milk, colostrum, dairy and colostrum-based products. EFSA Journal, 19 (4). https://doi.org/10.2903/j.efsa.2021.6576

4.2 Alkaline phosphatase

permanent color standards for comparison. Using this method, the relationship was established between phosphatase technique and the official KayGraham test. They proposed a limiting standards corresponding to 2.3 Lovibond Blue Units (LBU) in the Kay Graham test for the 30 min and 2 h procedures of Aschaffenburg and Mullen. Scharer’s rapid phosphatase test was based on the original Kay and Graham method, with significant reduction in analysis time (B75 min). The ALP cleaves a phosphate group from disodium phenyl phosphate substrate. Subsequently, the released phenol group is extracted with butanol, which is reacted with 2,6-dichloroquinone-chlorimide forming indophenol, a bluecolored compound. However, the reagents used as well as the formed indophenol chromate are unstable, resulting in an increased rate of false negative test (Rankin et al., 2010; Scharer, 1938). The butanol used for extraction of phenol in Scharer’s test results in formation of emulsion leads to reduced extraction efficiency. To overcome this, Babson and Greeley (1967) used phenolphthalein monophosphate as the substrate. As ALP releases phenolphthalein, the concentration can be determined by titration with sodium hydroxide. Unlike the Scharer reagents, the phenolphthalein monophosphate substrate and its hydrolysis product are very stable over time and storage conditions typical for this assay applicable to skim milk, milk and light cream.

4.2.2.2 Fluorimetric methods Methods based on fluorescence have replaced phenol-based methods owing to better sensitivity of former methods as it can detect as low as 0.006% of raw milk in pasteurized milk. ISO 11816-1 [IDF 155-1:2013] (2013) and ISO 11816-2 [IDF 155-2:2016] (n.d.) are the currently used reference methods as described by Rocco (1990). These methods use specific substrate fluoro-yellow and measure ALP activity in mU/L of milk. Since 1990 commercial dairy plants utilize two instrument-based methods which use enzyme substrates, namely, enzyme photo-activated systems (EPAS) and produce fluorescence (FluorophosTM Test, Advanced Instruments, Norwood, MA) and luminescence (Charm ALP-PasLiteTM Charm Sciences Inc., Lawrence, MA) signals. In the Fluorophos ALP reaction, ALP activity measurement in fluid dairy products is based on a specially designed enzyme substrate called Fluorophos. Fluorophos is an aromatic ortho-phosphoric monoester compound which is nonfluorescent in solution. However, when ALP is acted on substrate, Fluorophos loses its phosphate radical and becomes a highly fluorescent molecule called Fluoro-yellow. The reaction can be measured with a fluorometer. On the other hand, PasLite test employs an EPAS reagent, which in the presence of alkaline phosphatase emits chemiluminesce. This light is directly proportional to the amount of phosphatase enzyme and it is detected by Charm readers, that is, novaLUM and Charm II analyzers. These

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methods possess certain advantages such as improved speed and precision over conventionally used methods (Bruce, 2001; Rocco, 1990; Wehr & Frank, 2004). These methods measure ALP activity in milliunits per liter (mU/L) and have been shown to detect ,0.3% raw milk in various dairy drinks. Both the fluorescence and chemiluminescence EPAS methods have similar repeatability and reproducibility values in analyses of whole milk of multiple species with lower ALP threshold levels for public safety (Salter et al., 2006). The chemiluminescent assay (Paslite, Charm Sciences Inc., Lawrence, MA) is approved by FDA/NCIMS/ISO/IDF. According to the Regulation (EU) No 2019/6272 and EU Regulation (EC) No. 2074/2005, an ALP test is considered to give a negative result if the measured activity in cows’ milk is not higher than 350 mU/L. This limit is equivalent to the contamination of pasteurized milk with raw milk in a percentage of approximately 0.02 0.05 according to Greenwood and Rampling (1997), 0.05% according to Punoo (2018) and below 0.1% according to Scintu et al. (2000). Fluorophos test system method for measuring ALP activity in milk from cows, sheep, and goats is suitable for 500 mU/L and lower levels of enzyme (Harding & Garry, 2005).

4.2.2.3 Immunochemical methods Since fermented milk products contain ALP of microbial origin as well and immunochemical methods have ability to differentiate microbial and bovine milk ALP, owing to structural differences (Vega-Warner et al., 1999), immunochemical method can be used to detect bovine milk ALP for ascertaining proper pasteurization of fermented milk products. Geneix et al. (2007) developed a simple immunoassay for detection of alkaline phosphatase with milk alkaline phosphatase specificity. It does not react with ALPs of intestinal or bacterial origin. It is said to be first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution.

4.2.2.4 AOAC method for cheese The AOAC phosphatase method for cheese was developed in 1946 (Sanders & Sager, 1946). Based on some laboratory studies, American Public Health Association (APHA) developed a method (Scharer modified field test) in 1953 (Murthy & Cox, 1988). The colorimetric method for the determination of ALP in cheese was developed in the 1930s (IDF, 2016). A fluorimetric method for determination of ALP in cheese (ISO 11816-2 [IDF 155-2:2016], n.d.) was internationally recognized. Egger et al. (2016) studied alkaline phosphatase activity using IDF fluorimetric method in Emmental (hard variety), Raschera (semihard variety), and Chaource (soft cheese variety) and advocated the residual limit for alkaline phosphatase activity as 10 mU/g for clear-cut distinction of cheeses made from pasteurized milk. The Scharer modified method for spectrophotometric quantification of residual enzymatic activity of ALP was found to be efficient in detecting levels of ALP in

4.2 Alkaline phosphatase

cheese, equivalent to levels more than 0.50% of raw milk contamination in pasteurized milk (Soares et al., 2013).

4.2.3

Effect of mastitis

Bogin and Ziv (1973) reported a sixfold rise in bovine milk ALP in experimental mastitis. Many other researchers have reported increased concentration of ALP in milk during mastitic infection in animals (Gain et al., 2015; Guha et al., 2012; Kitchen, 1981; Nandi, 2021). This increase in the concentration is mainly due to increased permeability of the infected udder tissues (Pyörälä, 2003).

4.2.4

Alkaline phosphatase activity in nonbovine milk

Camel milk contains low basal levels of ALP with higher heat stability. Average ALP activities in raw camel milk varies between 15.9 and 24.93 U/L, while 5.8 and 10.2 U/L in pasteurized camel milk (Yadav et al., 2015; Yoganandi et al., 2014). ALP test is not suitable for verifying effectiveness of pasteurization of camel milk (Clawin-Rädecker et al., 2021; Merin et al., 2005; Wernery et al., 2008, 2006). The concentration of ALP in goat milk is about five to ten times lower than bovine milk (Banks & Muir, 2004; Clawin-Rädecker et al., 2021; Mathur, 1975) and is affected by various factors such as breeds, season, stage of lactation, fat content, and udder health. The significant relationship is observed between the concentration of the ALP in the goat milk and the somatic cell count (Banks & Muir, 2004). Heat stability of goat milk ALP when studied in the temperature range of 54 C 69 C is lower in comparison to bovine ´ milk (Wilinska et al., 2007). There is significant reduction in the ALP activity in goat milk upon heat treatment at both 63 C and 95 C (Banks & Muir, 2004). Based on the available evidence from milk samples after pasteurization, there is 95% 99% probability (extremely likely) that pasteurized goat milk and pasteurized sheep milk would have an ALP activity below a limit of 300 and 500 mU/L, respectively (Clawin-Rädecker et al., 2021). Human milk has 40 times lower ALP activity as compared to bovine milk (Heyndrickx, 1962). ALP activity, like the activity of most other enzymes, is greater in human colostrum than in ordinary milk (Heyndrickx, 1962; Sharma & Ganguli, 1971).

4.2.5

Reactivation of alkaline phosphatase

Development of restored phosphatase activity has been termed “reactivation” (Babel et al., 1978). Wright and Tramer (1953) first described reactivation of ALP when they observed that ultrahigh temperature (UHT)-treated milk

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“phosphatase negative immediately after processing” became positive on standing and further indicated microbial phosphatase was not responsible for the phenomenon. They observed that milk phosphatase is irreversibly inactivated by the HTST or Holder process of pasteurization; however, rapid heating of raw milk to temperatures in excess of 100 C results in a reversible inactivation of milk phosphatase. Sulfhydryl (-SH) groups appear to be essential for the reactivation of ALP, perhaps that is why phosphatase becomes reactivated in UHT milk but not in HTST milk (O’Mahony et al., 2013). Sharma (1970) indicated no reactivation of ALP upon pasteurization in case of both cow and buffalo milk. However, heating at higher temperature (90 C 100 C), it exhibited reactivation. Wright and Tramer (1956) and Sharma (1970) observed marked reactivation of ALP in both separated milk and fat-rich milk, though separated milk gave less reactivation than the corresponding whole milk. Wright and Tramer (1956) also indicated that pasteurization at 76.66 C and 82.22 C usually resulted in increased reactivation as compared with that obtained after pasteurization at 71.66 C (161 F). It is suggested that many of the reported cases of pasteurized cream developing a positive phosphatase reaction on storage can be attributed to reactivation of the enzyme. Storage temperature (above 30 C) and higher concentration of magnesium are mainly responsible for reactivation of unfolded ALP. In addition, the presence of Zn21, Mg21, and NaCl increases reactivation of ALP. It has been proposed that Zn21 or Mg21 causes a conformational change in the denatured enzyme that is necessary for renaturation. The level of reactivation is highest at pH 6.5. The reactivation of ALP is spontaneous and is inversely proportional to homogenization pressure but it is independent of fat content (Shakeel-Ur-Rehman & Farkye, 2011). Research done in the area of ALP reactivation has not been able to conclusively establish correlation between reactivation, time temperature combination of pasteurization, or storage time after processing. However, it can be said that milk pasteurized at temperatures higher than 71.7 C has higher proneness to ALP reactivation. Metallic ions (e.g., magnesium acetate) seem to be definitely involved in the reactivation of ALP (Kuzuya et al., 1982; Richardson et al., 1964). Mg21 and Zn21 has stimulating effect on the reactivation of ALP, whereas Co21, Cu21, EDTA, and Sn21 may have inhibitory effect (Fox & Kelly, 2006b; Linden, 1979; Linden et al., 1977; Sharma & Ganguli, 1974). Rankin et al. (2010) described a test to verify whether a pasteurized product would give a false-positive ALP assay due to reactivation. The test was based on increase in ALP activity upon addition of Mg21 to the reaction mixture, which can be used to determine whether an ALP level that exceeds the legal limit is likely to represent a genuine pasteurization failure, or whether it is more likely

4.3 Acid phosphatase

to be due to reactivation. However, difficulties in the interpretation of this test may arise when applied to cream or butter. Generally, susceptibility of ALP to reactivation in the products processed at temperatures above 100 C is more. The reactivation of ALP in case of goat milk was also informed by one of the European marketer of goat milk to EFSA. They observed that in goat milk, ALP becomes reactivated after a certain duration and therefore analyses should be done within 24 h after pasteurization to obtain reliable results (Clawin-Rädecker et al., 2021).

4.3

Acid phosphatase

The reaction catalyzed by phosphatases involves hydrolysis of phosphoric acid esters and thus the enzyme is also called phosphomonoesterase. Based on the optimum pH of activity, phosphatases are categorized as “alkaline” and “acid” phosphatase. Acid phosphatase present in milk has pH optimum at 4.0 and is very heat stable in comparison to ALP. Acid phosphatase in milk is capable of hydrolyzing phenylphosphate and other phosphoric esters. Acid phosphatase activity in cream is about twice to that present in skim milk. Acid phosphatase is present in skim milk, in MFGM, and in membrane material in skim milk (Kitchen, 1985). Acid phosphatase required heat treatment at 88 C for 30 min for complete inactivation (Fox, 2003). The enzyme is relatively heat stable; 70% of the activity remained after heating at 65 C for 15 min (Bingham et al., 1961). About 10% 20% of the activity of acid phosphatase is lost during LTLT pasteurization and the activity is not affected by normal HTST pasteurization (Shakeel-Ur-Rehman & Farkye, 2011). The micellar integrity of caseins is lost due to cleavage of phosphate groups from the serine residue of casein by acid phosphatase (Shakeel-Ur-Rehman & Farkye, 2011). The enzyme is strongly inhibited by fluoride, but the stimulating effect of magnesium, a notable activator of phosphatases, is small. These properties differentiate acid phosphatase from alkaline phosphatase of cows' milk (Mullen, 1950). Acid phosphatase hydrolyzes phosphoproteins, including caseins causing their dephosphorylation. Singh et al. (1997) observed extensive dephosphorylation of some peptides originating from αs1, αs2, and β-caseins in Cheddar cheese. Acid phosphatase is present in milk at a much lower level than alkaline phosphatase and it is active at pH values typical of cheese ripening. Its activity could be of major importance in the cheese ripening process as it is very active against phosphoprotein substrates such as the casein of milk (Andrews, 1992). Phosphopeptides are not bitter and are resistant to proteolytic attack (Dulley & Kitchen, 1972) and thus high acid phosphatase activity during cheese ripening could result in excessive proteolysis and flavor defects.

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According to Andrews (1992), acid phosphatase activity in milk is only 2% that of ALK activity in milk. Andrews and Alichanidis (1975) reported that the activity of acid phosphatase in milk increases 4- to 10-fold during mastitic infection. They also observed that milk from healthy cows contained one acid phophatase while that from mastitic cows contained two additional acid phosphatases which were of leukocyte origin. Chávarri et al. (1998) reported acid phosphatase activity in Ewe’s milk throughout the lactation period and also in cheese during ripening. They observed acid phosphatase activity was increased fourfold in January (early lactation) and then remained constant (17 mU/mL) until the end of lactation. In cheeses which were made in summer, the activity of acid phosphatase increased twofold during the 180 days of ripening, whereas in winter and spring much smaller increases were observed. Akuzawa and Fox (2004) suggested that acid phosphatase from both skim milk and lactic acid bacteria contributes to dephosphorylation of phosphopeptides in cheese, which are produced from casein by coagulant, indigenous milk proteinase, and microbial proteinases during cheese ripening. Bovine milk contains more than one acid phosphatase, which are distributed between the cream and skim milk. The properties of two enzymes isolated from butter milk and skim milk are different. Acid phosphatase enzyme plays a crucial role in animals’ metabolism which affects the technological properties of milk and is indicator of homeostasis in animal tissues, including mastitis (Jó´zwik et al., 2008).

4.4

Milk lipoprotein lipase

Lipases (EC 3.1.1.3) are hydrolases and are primarily responsible for the hydrolysis of triglycerides. This process is commonly known as lipolysis. The enzyme present in milk is known as lipoprotein lipase (LPL). The breakdown products of lipid hydrolysis are glycerol, free fatty acids, and partial glycerides such as monoacylglycerols and diacylglycerols. Lipases act on long chain and insoluble triglycerides while other esterases hydrolyze esters of short-chain fatty acids and soluble esters (Jaeger et al., 1994; Okuda & Fujii, 1968).

4.4.1

Physicochemical characteristics

LPL is well characterized enzyme. Majority of lipolytic activity in bovine milk is attributed to this enzyme (Olivecrona, 1980; Olivecrona et al., 2003). The presence of LPL in milk is due to its spillover from mammary gland. The enzyme does not have specific biological function in milk. LPL is a glycoprotein (Olivecrona et al., 2003). It has a molecular mass of B100 kDa and present as a homodimer (Kinnunen et al., 1976). In milk,

4.4 Milk lipoprotein lipase

more than 80% of this enzyme is found with casein micelles. Heat treatment of milk at 72 C for 15 s inactivates most of the enzyme (Farkye & Imafidon, 1995). It is also unstable to acid. Usually in freshly drawn milk, the fat globules are protected with a membrane and hence, lipolysis does not proceed rapidly.

4.4.2

Concentration in bovine milk

Fresh bovine milk contains about 0.5 2.0 mg/L LPL (Reddy et al., 1986). The content of enzyme in bovine milk depends on the breed, stage of lactation, plane of nutrition, season, and milk production (Deeth & Fitz-Gerald, 1976). The typical LPL activity in milk is enough to make milk rancid in less than 10 min (Olivecrona et al., 2003).

4.4.3

Lipolysis

LPL exhibits positional specificity and not fatty acid specificity (Morley & Kuksis, 1977). It hydrolyzes the fatty acids at the sn-1 and sn-3 positions of the triglyceride molecule (Somerharju et al., 1978). If 2-monoglyceride is rearranged to 1- or 3-monoglyceride, fatty acids can be released (NilssonEhle et al., 1973). As the short-chain fatty acids are esterified mostly in the sn-3 position of bovine milk triglycerides, the profile of released free fatty acids contains mostly short-chain fatty acids (Ouattara et al., 2004). Hydrolysis of phospholipids is less dependent on the activator than triglyceride hydrolysis (Lambert et al., 2000). Agitation and foaming of cold-stored milk causes “spontaneous lipolysis” due to the action of LPL. In milk and milk products, lipases (originated from milk, starter or nonstarter bacteria) catalyze the hydrolysis of ester bond in lipids.

4.4.4

Significance in dairy industry

The significant effect of milk fat lipolysis is in flavor production. Short- and medium-chain fatty acids are considered undesirable owing to their strong flavors. Free fatty acid content at an elevated level ( . 1.5 mmol/L) in whole milk produces flavor which is unacceptable to most people (IDF, 1987). This flavor is variously described as rancid, butyric, or astringent. Lipases also play a role in flavor development in products such as cheese, butter, and margarine (Vakhlu & Kour, 2006). It enhances the flavor of cheeses, accelerates cheese ripening, and is used to manufacture flavorings for cheese analogues (Woo & Lindsay, 1984). In hard Italian and Blue varieties of cheeses, flavors due to lipolysis of fat can be desirable (Deeth & Fitz-Gerald, 1995). Lipases are also used in acceleration of cheese ripening and the lipolysis of butter, fat, and cream (Ghosh et al., 1996; Sharma et al., 2001). The introduction of conjugated linoleic acid in dairy foods has been made possible

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through the immobilization of lipases (Baianu et al., 2003). Commercially, lipases are used in the manufacture of Italian cheeses such as Provolone and Caciocavallo or for the production of enzyme-modified cheese (Hernández et al., 2005). Addition of fungal phospholipase A1 to milk for increasing cheese yield in manufacture of part-skim Mozzarella cheese is another use of lipases (Lilbak et al., 2006). Enhancing flavor and structure in butter and dairy cream is a recent application of lipase (Kurtovic et al., 2011). Nowadays, use of milk whey and other dairy wastes for producing biogas is evolving. Formation of viscous materials and suspensions in anaerobic bioreactors is an important issue causing clogging of the anaerobic reactors. Enzymatic treatments of this material can solve the problem, since it promotes hydrolysis of fats and sludge solids. Lipases, proteases, amylases, and cellulases can contribute to increased efficiency of the process (Leal et al., 2002; Roman et al., 2006).

4.5

Plasmin

Plasmin (EC 3.4.21.7) is serine alkaline protease (Grufferty & Fox, 1988) which is major proteinase in human, ovine, caprine milks, and other mammals (Alichanidis et al., 1986). It is associated with the casein micelle and decomposes β-casein much faster than αs1-casein (Walstra, 1999). The primary role of plasmin is to cleave fibrin in blood and consequently prevents thrombosis (CesarmanMaus & Hajjar, 2005). However, it also infiltrates in milk, wherein it can cleave caseins, but mostly β-casein (Fox & McSweeney, 2003; Grufferty & Fox, 1988). Plasmin is secreted as plasminogen and is activated in blood and milk. The cleavage of plasminogen by urokinase activator results in plasmin (Bastian & Brown, 1996). Plasmin is one of the indigenous proteases, which has a substantial effect on the quality of milk and milk products. Human and bovine plasminogen contain 790 and 786 amino acid residues, respectively. The molecular weight of bovine plasminogen based on amino acid sequence and carbohydrate content is about 88,092 Da (Bastian & Brown, 1996). Molecular weight of blood plasmin based on amino acid sequence is calculated to be 83,200 or 81,000 (Castellino & Powell, 1981). While plasmin and trypsin both hydrolyze Lys-X and Arg-X bonds, plasmin preferentially attacks Lys-X bonds (Bastian & Brown, 1996). Bovine milk plasmin shows optimum activity in the pH range between 7.4 and 7.5 (Humbert & Alais, 1979). Its concentration in milk is 0.3 mg/L (Halpaap et al., 1977; Richardson & Pearce, 1981), while in blood it is 200 mg/L (Halpaap et al., 1977). Concentrations of plasminogen in milk is 2.5 μg/mL (Richardson, 1983). Generally, fresh unheated milk has higher concentration of plasminogen than plasmin (Leite et al., 2021). The plasminogen-to-plasmin ratio in milk is

4.5 Plasmin

between 50:1 and 2:1 (Fox, 2019). Korycha-Dahl et al. (1983) have determined levels of plasmin and plasminogen during lactation and reported 4 5 units of plasmin in 1 mL of bovine milk at both early lactation stage (2 3 months) and late lactation stage (7 8 months), while 26.3 6 2.7 U/mL plasminogenderived activity at the early stage and 45.3 6 13.0 U/mL at the late state of lactation. Plasminogen activators are serine protease and are of two types, namely, tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Plasminogen is largely synthesized in liver. Circulatory blood plasminogen, when comes in contact with clot or cell surface, changes its conformation and becomes susceptible for attack by activators. uPA is the extensively studied and characterized plasminogen activator (Christman et al., 1977). Plasminogen is activated by cleavage of Arg-Val bond by plasmin. uPA also exists in zymogen form and is activated by plasmin. Blood and milk also contain inhibitors of plasminogen activators. Plasminogen, plasmin, plasminogen activator and inhibitor of the activator are present in milk and, therefore measurement of true activity of plasmin in milk is difficult. Plasmin is largely associated with casein micelles and is released on acid coagulation of casein. The plasminogen concentration in fresh milk is 0.18 2.8 mg/L, which is 2 30 times higher compared to plasmin (0.1 0.7 mg/L) (Benfeldt et al., 1995; Ozen et al., 2003; Richardson & Pearce, 1981). Both β- and αs2 caseins are preferred casein substrates for plasmin. However, αs1-casein is also hydrolyzed, although κ-casein and whey proteins are quite resistant to hydrolysis (Aimutis & Eigel, 1982; Fox, 2019). After a 4-h incubation, only 20% of αS2-casein remained intact (Snoeren & Riel, 1979). Plasmin shows high specificity for peptide bonds where carboxyl group is contributed by lysine, and to a lesser extent for arginine. The primary cleavage sites in β-casein are: Lys28-Lys29, Lys105-His106 and Lys107- Glu108, hydrolysis of which yields the γ-caseins [β-CN f29-209 (γ1-CN), f106-209 (γ2-CN) and f108-209 (γ3-CN)], and proteose peptones 5 (β-CN f1-105 and f1-107), 8 slow (β-CN f29-105 and f29-107) and 8 fast (β-CN f1-28) (Bastian & Brown, 1996; Eigel et al., 1979); Lys113-Tyr114 and Arg183-Asp184 are also hydrolyzed fairly rapidly (Fox et al., 2015).

4.5.1

Inactivation of plasmin

Ruminants’ milk subjected to heat treatment (85 C/5 min) results in 93% 100% reduction in original plasmin activity in both the casein and whey fractions (Leite et al., 2021). Plasmin system components have variable heat sensitivities (Lu & Nielsen, 1993; Prado et al., 2006). During mild heat treatment (75 C for 15 s), 81% of original activity of the plasminogen inhibitor

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is lost, while plasminogen activators are more heat stable (D value of 32 s at 140 C), increasing the conversion of plasminogen to plasmin. The different heat sensitivity of the plasmin system components ultimately affects the level of plasminogen being activated to plasmin. For example, bovine milk has more plasminogen (zymogen) than plasmin activity in the casein fraction. When milk was subjected to heat treatment at 63 C/30 min, it led to decrease (25%) in plasminogen activity; however, plasmin activity increased by 100%, thus implying conversion of plasminogen to plasmin (Leite et al., 2021). Plasmin is a highly stable to pressure treatment, except in systems incorporating β-lactoglobulin. However, it can readily hydrolyze β-casein under pressures up to 700 MPa, with similar specificity as observed for atmospheric pressure (Scollard et al., 2000).

4.5.2

Significance of plasmin in milk

Casein being a major protein in bovine milk plays an important role in several dairy product manufacture. Plasmin possesses remarkable casein hydrolyzing ability. In pasteurized milk, UHT-treated milk, and non-fat dry milk (NFDM), proteolysis by plasmin may cause undesirable changes such as precipitation or gelation. Further, uncontrolled proteolysis may also result in poor curd formation in degradation of stored milk proteins intended to be used as functional ingredients in food. Proteolysis induced by plasmin is sometimes essential both from flavor development and textural point of view during ripening of cheese. The loss of plasmin from the casein micelle may slow down the cheese ripening process, consequently increasing processing expenses (Farkye & Fox, 1992a).

4.5.3

Ultrahigh temperature milk

Plasmin is the principal enzyme which can survive in UHT milk. Proteolysis due to plasmin in UHT milk can lead several quality defects, including bitterness, gelation, and fat separation (Fox, 2019). UHT sterilization is mostly performed at temperatures above 140 C which gives adequate sterilizing effect in milk. Also, a sufficiently long shelf life at ambient temperature is only obtained if the residual activity of the milk proteinase (plasmin) is at the most 1% (Walstra, 1999). The UHT milk treated at 145 C for short time such as 0.6 s may have flavor resembling low pasteurized milk. However, such milk can keep good only for 2 3 weeks due to development of off-flavors such as “gluey” and “bitter” primarily due to action of plasmin (Walstra, 1999).

4.5 Plasmin

In general, preheating of milk at 90 C 95 C for 30 60 s (Newstead et al., 2006), 80 C for 300 s (van Asselt et al., 2008), 95 C for 180 s (Rauh et al., 2014), and 90 C for 60 s (Anema, 2017) prior to UHT is recommended for prevention of plasmin-induced proteolysis.

4.5.4

Cheese

Research on high plasmin containing milk indicates that the plasmin albeit does not affect the clotting time during cheese making, it reduces the strength of the cheese gel (Ismail & Nielsen, 2010). Some of the significant changes occurring during ripening of cheese includes proteolysis, increased pH due to ammonia production, and production of flavor compounds. These changes lead to textural modifications in the cheese. The plasmin has differing effect during ripening depending upon the cheese variety, mainly owing to different cooking temperature and pH of the cheese during ripening. Higher plasmin activity either due to plasminogen or added plasmin has been shown to improve the flavor and overall quality of certain cheeses (Banks & Williams, 2004; Farkye & Fox, 1992b; Ismail & Nielsen, 2010; Johnson, 1988). Plasmin is considered to be a valuable enzyme in the area of accelerated ripening of cheese for consequent flavor development and maturation.

4.5.5

Milk protein products

Milk protein products derived both from caseins and whey are becoming popular owing to their nutritional and functional properties. Gazi et al. (2014) studied occurrence of plasmin and plasminogen in certain milk protein products, including sodium caseinate, calcium caseinate, micellar casein isolate, and milk protein concentrate as well as their reconstituted products and observed retention of high levels of plasmin and plasminogen-derived activity in all products. Plasmin activity in the reconstituted commercial whey protein concentrates (both sweet and acid) varies considerably (16.3 330 μg/g of protein), but is significantly lower (2.1 4.4 μg/g of protein, P , .05) in whey isolates (Hayes & Nielsen, 2000). The variability in plasmin activity associated with commercial whey protein products is consistent with the variability known to exist in composition and functional properties of whey protein products. Improvement in functional properties of β-lactoglobulin upon 4% degree of hydrolysis caused by plasmin has been observed (Caessens et al., 1999).

4.5.6

Milk powder products

NFDM is also one of the important products for dairy and food industry. It is also a means by which milk solids are preserved for long-term storage. Therefore maintaining the quality and stability of NFDM is critical for the

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dairy industry. Past research has shown that reconstituted milk powders can undergo proteolysis-like reaction ascribed to plasmin (Newstead et al., 2006). Durkee and Hayes (2008) observed plasmin activity in low-heat NFDM; however, medium-heat NFDM did not, due to induced disulfide bonding between plasmin, plasminogen, and milk proteins. It was also confirmed that proteolysis in the particularly NFDM occurs due to plasmin activity. Thus proteolysis occurring in the low-heat NFDM can alter the functional and sensorial properties.

4.6

Catalase

Catalase is a very common enzyme that is present in almost all organisms that are exposed to oxygen. The enzyme catalase (EC 1.11.1.6) catalyzes the disintegration of hydrogen peroxide to water and oxygen. In bovine milk, catalase is also involved in oxidation of nitrite to nitrate (Silanikove et al., 2009). Catalase protects the living cells from oxidative damage, when cells or other molecules in the body come into contact with oxidizing compounds. Several bacterial species also produce catalase and it is used for their identification purposes. Catalase is different from peroxidase. While peroxidase requires a hydrogen donor or oxidizable compound for splitting H2O2, catalase does not require any such compound (Shahani, 1966).

4.6.1

Physicochemical properties

The catalase molecule usually consists of four identical 60-kDa subunits, with a total molecular weight in the region of 240 kDa and contains four moles of protohaem [A heme complex containing a protoporphyrin bonded ionically to a ferrous iron (Fe21)] (Ito & Akuzawa, 1983; Robinson, 1991; Schonbaum & Chance, 1976). The isoelectric pH of catalase is 5.5; however, the enzyme is stable over wide range of pH between 5 and 10. The enzyme loses the activity outside this pH range. Milk catalase is a heme protein with a molecular weight of 200 kDa. Heating at 70 C for 1 h at pH 7.0 causes complete inactivation. Like other catalases, it is strongly inhibited by Hg21, Fe21, Cu21, Sn21, CN2, and NO32. Various factors such as feed, stage of lactation, and especially mastitic infection affects catalase activity in milk. Although many researchers indicated increased concentration of catalase during mastitis, it is not used as index since somatic cell count. N-acetyl-glucosaminidase activity is superior index of mastitis. Catalase may act as a lipid prooxidant due to the presence of heme iron (i.e., nonenzymatically), although it is not considered significant prooxidant.

4.6 Catalase

The actual reaction requires two hydrogen peroxide molecules to be acted upon by catalase for their conversion to oxygen and water. The reaction requires the presence of a small amount of hydrogen peroxide to bind at the active site in order to generate catalase compound I, which reacts with a second molecule of hydrogen peroxide. The Km value of catalase for H2O2 is in the mmol per liter range; however, its Vmax is quite high. High Km value of catalase facilitates effective degradation of high concentration of hydrogen peroxide found in various cellular and subcellular organelles. H2O2 1 H2O2 - O2 1 2H2O Catalase reaction is considered as dismutation type reaction meaning in which one H2O2 molecule oxidizes another, so that one is converted to O2 and the other to two molecules of H2O.

4.6.2

Catalase activity in milk

Considerable degree of variation in the catalase activity of milk is seen, when compared with other enzymes occurring in milk. The average catalase activity determined using polarographic methods was 1.95 U/mL (Hirvi & Griffiths, 1998). The catalase activity in mastitic milk is generally higher than normal milk. The possible correlation between somatic cell count and catalase activity in milk is often reported. Rifaat et al. (1971) reported catalase activity in buffalo milk ranging from 8.3 to 16.2 units/mL with an average of 10.9 units/mL, while in cow’s milk it ranged from 8.3 to 16.4 units/mL with an average of 12.1 units/mL. Of the total catalase activity, the skim milk is reported to contain 73% and cream 24% (Kitchen et al., 1970). The concentration of catalase is lower in goat milk in comparison to cow milk (Mathur, 1975). The levels of catalase in human milk are 10 times higher than bovine milk (Silanikove et al., 2006). Catalase activity was observed to be 26.21 6 2.78 nmol/min/mL in the human colostrum, while it was 9.39 6 0.53 and 1.84 6 0.39 nmol/min/mL, in transition and mature human milk, respectively Yuksel et al., 2015).

4.6.3

Measurement of catalase activity

The principally common method for measuring catalase activity is the UV spectrophotometric method measuring H2O2 concentration at 240 nm absorbance at high levels of hydrogen peroxide solution ($30 mM). Hadwan (2018) has listed several methods for measuring catalase activity in various matrices, including those involving iodometry (Anderson & Mcwalter, 1937), chemiluminescence (Mueller et al., 1997), and polarimetry (Rørth & Jensen, 1967) and monitoring the production of oxygen via an oxygen electrode (Kroll et al., 1989) or a low-flow gas meter (Guwy et al., 1999). Most of these

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methods are time-consuming in nature and thus these are not feasible for clinical application. Alternatively, catalase activity can be measured quantitatively by titration of the unreacted excess of hydrogen peroxide (Euler & Josephson, 1927). However, this method may be associated with difficulty in end-point determination (Goldblith & Proctor, 1955). Willits and Babel (1965) suggested use of disk flotation test for the measurement of catalase activity in milk, where a disk was dropped into a 16 3 125 mm test tube containing 5 mL of 3% hydrogen peroxide in tris-HC1 buffer (pH 7.2). Ito and Akuzawa (1983) used spectrophotometric method using titanium tetrachloride reagent for measuring catalase activity in milk. Many oxygen electrode-based methods have been reported by various workers for the measurement of catalase activity in milk (Jha & Souza, 2011; Kroll et al., 1989).

4.6.4

Significance in dairy industry

Catalase enzyme in milk has several applications in dairy industry. The catalase test has been used for estimating somatic cell count in milk. This test is based on the decomposition of hydrogen peroxide by the enzyme catalase which is present in milk. Milk normally contains some catalase; however, udder infections result in increased catalase activity. This increased activity is chiefly due to somatic cell count, body cells, blood, and bacteria, especially staphylococci and aerobic spore formers (Janzen & Cook, 1967; Shahani, 1966). Some workers have associated catalase activity in raw milk with population of Gram-negative bacteria. This bacterial population of raw milk can in turn be related to the shelf life of processed milk. Catalase also has specific role in protecting from oxidative damage by converting nitrite to nitrate (Silanikove et al., 2009).

4.7

Lactoperoxidase

Peroxides are chemical compounds in which two oxygen atoms are linked together by a single covalent bond. Peroxide-containing compounds can be used as bleaching agents, initiators in polymerization reactions, H2O2 production, etc. Peroxidase (POD: H2O2-Oxydoreductase) groups of enzymes catalyze oxidation of a substrate by hydrogen peroxide. Reactive oxygen species generated during metabolism are converted into harmless molecules by oxidoreductase enzymes (Davies, 1995). These are heme-containing proteins and being present in all living things. Hydrogen peroxide acts as an electron acceptor in a reaction wherein organic and inorganic substrates are being oxidized by these enzymes (Hussain et al., 1995).

4.7 Lactoperoxidase

Lactoperoxidase (LP, EC 1.11.1.7) is one of the member of these peroxidases. It is present in milk indigenously and possesses oxidoreductase activity. On isolation from milk, it is named “lactoperoxidase.” Characteristics of peroxidase enzyme from milk are identical to that of peroxidases from animals and human (Reiter & Härnulv, 1984). It contains an iron molecule and the conformation is stabilized by a chelated calcium ion. The Ca21 ion disappears under pH 5.0 reducing the stability of the enzyme (Kussendrager & Van Hooijdonk, 2000).

4.7.1

Physicochemical properties

The lactoperoxidase (LP) enzyme possesses 612 amino acids and 8% 10% carbohydrate. It is a glycoprotein. The enzyme is a single polypeptide chain with a molecular weight of B78 kDa (Ozdemir et al., 2001). The isoelectric pH of the enzyme is 9.2 and it contains heme as its prosthetic group (Atamer et al., 1999; Kussendrager & Van Hooijdonk, 2000; Pourtois et al., 1991). The heme group is covalently bound to the polypeptide chain through a disulfide bridge (Thanabal & La Mar, 1989). One iron atom is present per lactoperoxidase molecule amounting it to 0.07%. It is very active in acidic pH (Wever et al., 1982). A calcium ion is important for the structural integrity of LP (Booth et al., 1989).

4.7.2

Concentration in milk and colostrum

In bovine milk, the concentration of LP is 30 mg/L. Concentration wise, it is next to xanthine oxidase (de Wit & van Hooydonk, 1996). The concentration of LP is low in bovine colostrum but increases rapidly to reach a maximum after 3 6 5 days postpartum (Korhonen, 1977).

4.7.3

Significance of lactoperoxidase enzyme

The LP enzyme helps generating products with a wide antimicrobial activity through catalysis of oxidation of molecules in the presence of hydrogen peroxide and demonstrates a significant protective effect in bovine milk. The concentration of thiocyanate and hydrogen peroxide is important for activation of the LP system in milk. Transformation of thiocyanate into hypothiocyanate (an antibacterial compound) is carried out by the LP system in the presence of hydrogen peroxide (Gülçin et al., 2009, 2006; Haddadin et al., 1996). However, the end products of these compounds are safe for human health as they are oxidized. Several bacterial and fungal strains are destoyed by this system (Gülçin, 2007, 2009; Gülçin et al., 2009, 2006). Lactoperoxidase shows a broad antifungal activity (Jacob et al., 1998; Sisecioglu et al., 2009). Mastitis, a disease of concern for a dairy industry, is a bacterial inflammation in mammals. Different

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concentrations of thiocyanate-H2O2 are capable of reducing bacterial growth by damaging the cell membranes and inhibiting activities of several cytoplasmic enzymes (Sisecioglu et al., 2010; Uguz & Ozdemir, 2005). The biocidal activity of the LP is attributed to hypothiocyanate which interacts with the thiol groups of various protein critical for the survival of pathogens. The oxidation of sulfhydryl (-SH) groups makes the bacterial cytoplasmic membrane lose its ability to transport glucose, potassium ions, amino acids, and peptides (Sisecioglu et al., 2010). The enzyme is of biological significance owing to its natural protection system against the invading organisms. It catalyzes oxidation of the thiocyanate ion into the antibacterial hypothiocyanate and functions as a nonimmune biological defense system of mammals (Kumar et al., 1995).

4.7.4

Lactoperoxidase system in milk

Lactoperoxidase is an enzyme naturally present in biological secretions such as raw milk, colostrum, and saliva. Lactoperoxidase content in bovine milk is around 10 60 mg/L (Ekstrand, 1994). It forms antimicrobial compound(s) from thiocyanate (SCN2) in the presence of hydrogen peroxide. The whole system comprising thiocyanate, H2O2, and lactoperoxidase enzyme forms the lactoperoxidase system (LPS). Thiocyanate content in fresh milk is 1 10 mg/L and is insufficient to activate the LPS. Due to the action of natural catalase, peroxidase, or superoxide dismutase, fresh milk does not have hydrogen peroxide. Around 8 10 mg hydrogen peroxide per liter is required for LPS. In the LPS system, the SCN2 is oxidized to the antimicrobial OSCN2 (Gaya et al., 1991; Reiter & Härnulv, 1984). Gram-negative bacteria, including Pseudomonads are more vulnerable to the lactoperoxidase system than Gram-positive bacteria (Björck, 1978). However, the system inhibits both types of bacteria (Beumer et al., 1985; Kamau et al., 1990; Siragusa & Johnson, 1989). Only 5%-10% lactoperoxidase activity is retained in milk subjected to heating at 80 C for 15 s (Sharma et al., 2009). Barrett et al. (1999) hypothesized that lactoperoxidase may have a role in keeping up the quality of pasteurized milk treated at 72 C for 15 s. It can increase the shelf life of raw milk. In infant formula, ice cream, cream and cheeses, lactoperoxidase system can be used as a preservation process (Ekstrand, 1994).

4.8

Xanthine oxidoreductase

Xanthine oxidase (XO; EC 1.1.3.22) and xanthine dehydrogenase (XDH; EC 1.1.1.204) are the two forms of xanthine oxidoreductase (XOR). Xanthine oxidase in milk is concentrated in the MFGM. Milk xanthine oxidase has a

4.8 Xanthine oxidoreductase

molecular weight of 300 kDa and it consists of two identical subunits. The optimum pH of xanthine oxidase is 8.5. It is capable of oxidizing aldehydes and purines with the concomitant reduction of O2 to H2O2. This enzyme requires flavin adenine dinucleotide, iron and molybdenum cations, and an acid-labile sulfur compound as cofactors. Xanthine oxidase is one of the principal proteins of the MFGM (Fox, 2003). Xanthine oxidase is fairly heat stable, being more stable than lipase and alkaline phosphatase. Average xanthine oxidase activity in milk is 110 mU/mL (Cerbulis & Farrell, 1977). According to El-Gazzar et al. (1999), xanthine oxidase activity of buffalo milk from an Egyptian breed (0.045 6 0.016 U/mL) is lower than that of cow milk (0.070 6 0.007 U/mL). Kitchen et al. (1970) studied xanthine oxidase activity for normal milk and mastitic milk. They reported that xanthine oxidase activity was 15.6 21.4 unit/mL in normal milk and 9.4 units/mL in mastitic milk. Xanthine oxidase activity is observed in the milk of the sheep and goat, but the milks of sow, mare, and human are devoid of this enzyme (Modi et al., 1959). Xanthine oxidase activity in goat milk is significantly lower than in bovine or buffalo milks (Sharma et al., 2009). Bovine milk is rich in xanthine oxidase and the enzyme has been implicated in the development of oxidized flavors in milk (Aurand et al., 1967). Xanthine oxidase acts as a prooxidant. Milk which undergoes spontaneous change in rancidity contains about 10 times higher xanthine oxidase level than normal and spontaneous oxidation can be induced in normal milk by the addition of xanthine oxidase to about 4 times normal levels (Fox et al., 2015). The xanthine oxidase activity of milk is affected by various processing treatments which damage or alter the MFGM. Xanthine oxidase activity is increased by 100% on storage at 4 C for 24 h, by 50% 100% on heating at 70 C for 5 min and by 60% 90% on homogenization. These treatments cause the release of xanthine oxidase from the MFGM into the aqueous phase (Fox, 2003). Its activity in milk increases with several heat treatments, homogenization, by protease and lipase action (Shahani, 1966). Xanthine oxidase accomplish the reduction of oxygen for producing the reactive oxygen species (ROS), superoxide and hydrogen peroxide. It can also reduce nitrite, yielding reactive nitrogen species (RNS), such as nitric oxide and peroxynitrite. Due to the generation of ROS and RNS, milk xanthine oxidoreductase may play an antimicrobial defensive role in the neonatal gut, complementing endogenous enzyme of the intestinal epithelium. Xanthine oxidoreductase is also involved in secretion of milk fat globules in a process dependent on the enzyme protein rather than on its enzymic activity, which is known to vary greatly with time after parturition and also between species (Harrison, 2006).

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4.9

γ-Glutamyl transferase

γ-glutamyl transferase (GGT, EC 2.3.2.2) or γ-glutamyl transpeptidase (GGTP) is an enzyme found in many organ and tissues of mammals. It is a glycoprotein having 12 isozymes differing in content of sialic acid (Cao et al., 2021; Fox & Kelly, 2006b). GGTP catalyzes the reaction of a broad range of γ-glutamyl amides (γ-Glu-Xaa) with water (hydrolysis) and amino acids or dipeptides (Yaa) (transpeptidation) as shown below (Hiratake et al., 2013). γ-Glu-Xaa 1 H2O - Glu 1 Xaa (hydrolysis) γ-Glu-Xaa 1 Yaa - γ-Glu-Y 1 Xaa (transpeptidation)

4.9.1

Physicochemical properties

About 70% of GGT activity is found in milk fat membrane material in skim milk, much higher than the activity associated with MFGMs. The enzyme, has a molecular mass of B80 kDa and consists of two subunits of 57 and 25 kDa (Baumrucker, 1979; Cao et al., 2021). The enzyme, which associates strongly (Kenny, 1977; Tate & Meister, 1976), is optimally active at pH 8.5 9 at B45 C and has an isoelectric point of 3.85. It is strongly inhibited by diisopropylfluorophosphate, iodoacetamide, and metals, for example Cu21 and Fe31 (Farkye, 2003). Milk membrane GGT has characteristics that are similar to the enzyme from other mammalian tissues (Meister et al., 1976). The GGT activity in the whole milk is observed in the following order: cow . sheep . goat milk. The enzyme activity is comparatively higher in whole milk than skimmed milk. The average enzymatic values in whole sheep, goat, and cow milk observed were 4025 6 294, 951 6 193, and 7081 6 505 U/L, respectively (Dumitra¸scu et al., 2013). Calamari et al. (2015) has reported mean value of milk GGT activity during lactation to range from 3863 to 3024 U/L.

4.9.2

Significance in dairy industry

The inactivation of GGT occurs in the temperature range of 65 C and 76 C (Zehetner et al., 1995). Since the commercial pasteurization temperature falls between this range, this enzyme has potential to be used for monitoring effectiveness of pasteurization (Zehetner et al., 1995; Ziobro & McElroy, 2013). Vetsika et al. (2014) considered GGT as a useful marker for bovine milk pasteurization. GGT is comparatively more heat stable than ALP. Its activity in bovine milk is reduced by about 80% with pasteurization (72 C 75 C for 15 s), but it may be especially useful for testing higher heat treated milk (heated between 75 C and 80 C) (Farkye, 2003). Dos Anjos

4.10 Conclusion

et al. (1998) observed complete inactivation at 70 C after 10 min and at 80 C after 1 min. Thus GGT has the potential to act as an indicator within the temperature region between that covered by ALP and LP as well as to define the limit between pasteurized and high-pasteurized milk (Claeys et al., 2002a; Zehetner et al., 1995). Lorenzen et al. (2010) compared the effects of isochrone heating (35 C 85 C for 90 s) on the residual activities of ALP, GGT and LPO in bovine, ovine and caprine milk. They demonstrated that the heat stability of these enzymes is in the following order: ALP , GGT , LPO. They observed residual enzyme activities to be higher in goat and sheep milk in comparison to cow milk. Lombardi et al. (2000) demonstrated that in addition to ALP, GGT would be suitable as a potential marker for heat denaturation in buffalo milk, with GGT having the advantage that its concentration is higher. St˘anciuc et al. (2011) studied kinetics of inactivation of GGT in milk and cream in the temperature range of 60 C and 85 C. They observed enzyme activity in cream to be significantly higher (3 to 4 times) than skim and whole milk. It was also noted that residual enzyme activities in heated samples was considerably higher in skim milk in lower temperature range and lower at higher temperature when compared with whole milk and cream. Dumitra¸scu et al. (2014) reported z-values in skimmed and whole milk as 8.02 C 6 0.23 C and 7.09 C 6 0.09 C in goat milk, 5.97 C 6 0.08 C and 5.88 C 6 0.027 C in sheep milk, and 5.80 C 6 0.05 C and 5.83 C 6 0.01 C in cow milk, respectively. However, the incidences of reactivation as observed in the case of ALP has not been seen in the case of GGT; hence, it can be advantageous for the checking effectiveness of pasteurization, particularly for cow milk. As higher inactivation temperatures are required for GGT in comparison to ALP, the chances of residual GGT activity remaining in the milk are higher if conventional pasteurization temperature is used (72 C for 15 s).

4.10

Conclusion

Considerable progress is made in the field of milk enzymology. It is still important to understand the origin, mechanism of secretion, and compartmentalization of indigenous milk enzymes in different milk fractions. Studies need to be designed to understand the in vivo function of milk enzyme, rather than the extrapolation from in vitro studies that simulate in vivo conditions. Observed variation in the activity of indigenous enzymes of milk is due to several factors like, sources origin of enzymes , presence of activator and inhibitors in milk, effect of physiology and health of lactating animal, lactation period, etc.

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Alkaline phosphatase hold significance in dairy industry because of its use as index enzyme for milk pasteurization; however, its reactivation in certain condition is of concern. The levels and inactivation pattern of alkaline phosphatase from nonbovine milk warrant further research in order to ensure its use as index of pasteurization. Plasmin, an indigenous proteolytic enzyme, can result in age gelation in UHT milk and is of consequence in cheese ripening; especially its impact on flavor development as well as nutritional and functional properties of cheese. Milk lipase can lead to rancid flavor besides it helps in flavor development in cheeses made from raw milk. Xanthine oxidase and catalase are implicated in oxidative flavor formation by virtue of the production of superoxide by the former and the heme iron content of the latter. Although LPS is promised to improve storage life of milk, its potential is not fully exploited.

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Sisecioglu, M., Cankaya, M., & Ozdemir, H. (2009). Effects of some vitamins on lactoperoxidase enzyme activity. International Journal for Vitamin and Nutrition Research, 79(3), 188 194. Available from https://doi.org/10.1024/0300-9831.79.3.188. Sisecioglu, M., Gulcin, I., Cankaya, M., Atasever, A., & Ozdemir, H. (2010). The effects of norepinephrine on lactoperoxidase enzyme (LPO). Scientific Research and Essays, 5(11), 1351 1356. http://www.academicjournals.org/sre/PDF/pdf2010/4Jun/Sisecioglu%20et%20al.pdf. Snoeren, T. H. M., & Riel, J. A. M. V. (1979). Milk proteinase, its isolation and action on αs2and β-casein. Milchwissenschaft, 34. Soares, C. F., Fonseca, L. M., Leite, M. O., & Oliveira, M. C. P. P. (2013). Application of Scharer’s quantitative method for the determination of residual alkaline phosphatase activity in standard Minas cheese. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia, 65(4), 1223 1230. Available from https://doi.org/10.1590/S0102-09352013000400039. Somerharju, P., Kuusi, T., Paltauf, F., & Kinnunen, P. K. J. (1978). Stereospecificity of lipoprotein lipase is an intrinsic property of the active site of the enzyme protein. FEBS Letters, 96(1), 170 172. Available from https://doi.org/10.1016/0014-5793(78)81086-2. St˘anciuc, N., Dumitrascu, L., Râpeanu, G., & Stanciu, S. (2011). γ-Glutamyl transferase inactivation in milk and cream: A comparative kinetic study. Innovative Food Science & Emerging Technologies, 12(1), 56 61. Available from https://doi.org/10.1016/j.ifset.2010.11.003. Suzuki, U., Yoshimura, K., & Takaishi, M. (1907). Uber ein enzyme phytase: das anhydro-oxymethylendiphosphorsaure spaltet. Bulleting Collection of Agricultural Tokyo Imp. University, 7, 503 512. Tate, S. S., & Meister, A. (1976). Subunit structure and isozymic forms of gamma-glutamyl transpeptidase. Proceedings of the National Academy of Sciences, 73(8), 2599 2603. Available from https://doi.org/10.1073/pnas.73.8.2599. Thanabal, V., & La Mar, G. N. (1989). A nuclear overhauser effect investigation of the molecular and electronic structure of the heme crevice in lactoperoxidase. Biochemistry, 28(17), 7038 7044. Available from https://doi.org/10.1021/bi00443a039. Tramer, J., & Wight, J. (1950). The phosphatase test of Aschaffenburg and Mullen: Use of permanent colour standards and comparison with the Kay-Graham test. Journal of Dairy Research, 17(2), 194 199. Available from https://doi.org/10.1017/S0022029900005756. Uguz, M. T., & Ozdemir, H. (2005). Purification of bovine milk lactoperoxidase and investigation of antibacterial properties at different thiocyanate mediated. Applied Biochemistry and Microbiology, 41(4), 349 353. Available from https://doi.org/10.1007/s10438-005-0059-8. Vakhlu, J., & Kour, A. (2006). Yeast lipases: Enzyme purification, biochemical properties and gene cloning. Electronic Journal of Biotechnology, 9(1), 69 85. Available from https://doi.org/ 10.2225/vol9-issue1-fulltext-9. van Asselt, A. J., Sweere, A. P. J., Rollema, H. S., & de Jong, P. (2008). Extreme high-temperature treatment of milk with respect to plasmin inactivation. International Dairy Journal, 18(5), 531 538. Available from https://doi.org/10.1016/j.idairyj.2007.11.019. Vega-Warner, A. V., Wang, C. H., Smith, D. M., & Ustunol, Z. (1999). Milk alkaline phosphatase purification and production of polyclonal antibodies. Journal of Food Science, 64(4), 601 605. Available from https://doi.org/10.1111/j.1365-2621.1999.tb15093.x. Vetsika, F., Boukidi, K., & Roussis, I. G. (2014). γ-glutamyl-transferase, Xanthine oxidase and total free sulfhydryls as potential markers for pasteurization treatments in dairy technology. Journal of Food and Nutrition Research, 53(4), 324 332. Available from http://www.vup.sk/en/. Walstra, P. (1999). Dairy technology: Principles of milk properties and processes. CRC Press. Wehr, H. M., & Frank, J. F. (2004). Standard methods for the examination of dairy products (pp. 327 404). American Public Health Association.

References

Wernery, U., Fischbach, S., Johnson, B., & Jose, S. (2008). Evaluation of alkaline phosphatase (ALP), γ-glutamyl transferase (GGT) and lactoperoxidase (LPO) activities for their suitability as markers of camel milk heat inactivation. Milchwissenschaft, 63(3), 265 267. Wernery, U., Maier, U., Johnson, B., George, R. M., & Braun, F. (2006). Comparative study on different enzymes evaluating heat treatment of dromedary milk. Milchwissenschaft, 61(3), 281 285. Wever, R., Kast, W. M., Kasinoedin, J. H., & Boelens, R. (1982). The peroxidation of thiocyanate catalysed by myeloperoxidase and lactoperoxidase. Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, 709(2), 212 219. Available from https://doi.org/10.1016/0167-4838 (82)90463-0. ´ Wilinska, A., Bryjak, J., Illeová, V., & Polakovic, M. (2007). Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer. International Dairy Journal, 17(6), 579 586. Available from https://doi.org/10.1016/j.idairyj.2006.08.008. Willits, R. E., & Babel, F. J. (1965). Disc flotation test for measurement of catalase activity in milk. Journal of Dairy Science, 48(10), 1287 1289. Available from https://doi.org/10.3168/ jds.S0022-0302(65)88449-1. Woo, A. H., & Lindsay, R. C. (1984). Concentrations of major free fatty acids and flavor development in Italian cheese varieties. Journal of Dairy Science, 67(5), 960 968. Available from https://doi.org/10.3168/jds.S0022-0302(8/4)81394-6. Wright, R. C., & Tramer, J. (1953). 517. Reactivation of milk phosphatase following heat treatment. II. Journal of Dairy Research, 20(3), 258 273. Available from https://doi.org/10.1017/ S0022029900006919. Wright, R. C., & Tramer, J. (1956). Reactivation of milk phosphatase following heat treatment. 4. The influence of certain metallic ions. Journal of Dairy Research, 23(2), 248 257. Yadav, A. K., Kumar, R., Priyadarshini, L., & Singh, J. (2015). Composition and medicinal properties of camel milk: A Review. Asian Journal of Dairy and Food Research, 34(2), 83. Available from https://doi.org/10.5958/0976-0563.2015.00018.4. Yoganandi, J., Jain, A. K., Mehta, B. M., Wadhwani, K. N., & Aparnathi, K. D. (2014). Comparative studies on selected enzymes activities of camel, cow and buffalo milk. Journal of Camel Practice and Research, 21(2), 249 252. Available from https://doi.org/10.5958/22778934.2014.00044.7. Yuksel, S., Yigit, A. A., Cinar, M., Atmaca, N., & Onaran, Y. (2015). Oxidant and antioxidant status of human breast milk during lactation period. Dairy Science and Technology, 95(3), 295 302. Available from https://doi.org/10.1007/s13594-015-0211-z. Zalatan, J. G., Fenn, T. D., & Herschlag, D. (2008). Comparative enzymology in the alkaline phosphatase superfamily to determine the catalytic role of an active-site metal ion. Journal of Molecular Biology, 384(5), 1174 1189. Available from https://doi.org/10.1016/j.jmb.2008.09.059. Zehetner, G., Bareuther, C., Henle, T., & Klostermeyer, H. (1995). Inactivation kinetics of γ-glutamyltransferase during the heating of milk. Zeitschrift Für Lebensmittel-Untersuchung Und -Forschung, 201(4), 336 338. Available from https://doi.org/10.1007/BF01192728. Zhu, J. Y., Ye, G. Y., Fang, Q., & Hu, C. (2010). Alkaline phosphatase from venom of the endoparasitoid wasp, Pteromalus puparum. Journal of Insect Science, 10(1). Available from https:// doi.org/10.1673/031.010.1401. Ziobro, G. C., & McElroy, K. M. (2013). Fluorometric detection of active alkaline phosphatase and gamma-glutamyl transferase in fluid dairy products from multiple species. Journal of Food Protection, 76(5), 892 898. Available from https://doi.org/10.4315/0362-028X.JFP-12-302. Zittle, C., Monica, D., & S, E. (1950). Effects of borate and other ions on the alkaline phosphatase of bovine milk and intestinal mucosa. Arch Biochem, 26, 112 122.

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CHAPTER 5

Methods for identification of bioactive peptides Meisam Barati1, Masoumeh Jabbari2 and Sayed Hossein Davoodi1,3 1

Department of Clinical Nutrition & Dietetics, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 2Student Research Committee, Department of Community Nutrition, National Nutrition and Food Technology Research Institute, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 3Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5.1

Introduction

Food-derived peptides are digestion-resistant and released from dietary proteins during the digestion process. Some of food-derived peptides are called bioactive peptides (BPs). BPs are digestion-resistant and absorbable peptides that can induce beneficial biological effects. The beneficial effects of BPs such as antidiabetic, anticancer, antihypertensive, and cholesterol-lowering properties have been widely reported (Barati et al., 2020b). Some BPs have been identified freely in food matrix; while, most of them are released from the proteins through microbial or enzymatic digestion. Currently, more than 4000 of BPs have been registered in related databases. BPs have some common structural features including (1) their peptide length between 2 and 20 amino acids (AAs), (2) the presence of hydrophobic AAs, and (3) the presence of arginine, lysine, or proline residues (Barati et al., 2020c). The first step for the identification of BPs is digestion of food proteins. In vitro methods have been mostly used to digest food proteins. The enzymatic digestion and microbial fermentation of proteins are the commonly used in vitro methods for proteolysis (Wang et al., 2017). On the other hand, some in vivo methods have been also introduced to better simulation of digestion in gastrointestinal tract (GIT) (Barati et al., 2020c). Characterization of released food-derived peptides is the second step for BPs' identification. The high-resolution mass spectrometry (MS) is used for the characterization of food-derived peptides. Selecting an appropriate technique among the various types of MS techniques is an 119 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00005-9 © 2023 Elsevier Inc. All rights reserved.

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important issue (Nongonierma & FitzGerald, 2017). The final step for BPs' identification is function assessment of food-derived peptides to find BPs. There are several different methods for function assessment of food-derived peptides. The function assessment methods are also divided into in vitro and in vivo methods. An enzyme exposure to food-derived peptides and oral administration of a peptide to animals or humans are the two commonly used methods for function assessment (Aguilar-Toala et al., 2017; Jauhiainen et al., 2012). In this regard, as illustrated in Fig. 5.1, the current chapter aims to describe the available methods for the identification of food-derived peptides and BPs.

5.2 5.2.1

Methods of protein digestion In vitro methods

Digestion of food proteins to find BPs is mostly performed by in vitro methods. The enzymatic digestion and microbial fermentation of proteins are commonly used in vitro methods for proteolysis. In the following sections, the in vitro methods of proteolysis are described.

5.2.1.1 Enzymatic method In this process, dietary proteins are subjected to enzymatic digestion at the optimum pH and temperature. For food-derived peptide characterization, the enzymatic digestion has priority over the microbial fermentation due to the short reaction time and ease of predictability in the enzymatic method. In this regard, to obtain the food-derived peptides, various enzymes are used for proteolysis. However, the temperature, pH, and enzyme activity should be optimized for proteolysis when the digestive enzyme and protein materials are incorporated together (Cruz-Casas et al., 2021; Monari et al., 2020). Although, there are no special recognized enzymes for the generation of specific BPs, it has been suggested that the hydrolysis by subtilisin (isolated from Bacillus subtilis) can be the best method for more generation of low-molecular-weight (LMW) peptides (Khiari et al., 2014). It should be noted that sonication of protein isolates and/or their exposure to reducing agents before proteolysis can increase the degree of hydrolysis. More production of LMW peptides creates more chance for the generation of BPs (Khiari et al., 2014; Majumder & Wu, 2010). The proportion of enzyme to substrate has been proposed as an important factor for reaching a satisfactory level of hydrolysis. In this context, the type of food-derived peptides can be related to the temperature, enzyme activity, and kind of enzyme(s) used (Cruz-Casas et al., 2021). On the other hand, the efficiency of the digestion process may be affected by the protons. These protons are released during the digestion process and could change the pH of the medium. By adding alkali or acid solutions, the pH fluctuations can be corrected. Titration with alkali solutions usually has some consequences such as accumulation of the added salts in the hydrolysates. The

5.2 Methods of protein digestion

1

Digestion/ Proteolysis

In-vitro

Enzymatic Digestion

In-vivo

Microbial Fermentation

Supernatant

AGC Method

Identification in blood

Measurement in blood

2

3

PQR VY PL VK DM 4 EL PGPIPN PK TL VL PF

ACE-inhibitory peptides PQR, VY, PL, VK, DM Antioxidant peptides EL, VY Anticancer peptides PGPIPN DPP-IV inhibitor peptides PK, TL, VY, VL, PF

FIGURE 5.1 The process flow diagram for the identification of food-derived BPs. (1) Proteolysis by in vitro or in vivo methods; (2) isolating LMWcontaining supernatant from precipitate after digestion; (3) identification/characterization of the LMW peptides using MS techniques; (4) finding the relevant function for the identified peptides. AGC, Aspiration of gut content; BPs, Bioactive peptides; DPP-IV, Dipeptidyl peptidase IV; LMW, low molecular weight; MS, mass spectrometry.

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accumulated salts themselves can disrupt proteolysis. Therefore, to avoid the mentioned problem, proteolysis should be performed in a buffer (Daliri & Oh, 2017). The MS-grade enzymes are highly recommended for enzymatic digestion. These kinds of enzymes have reduced autocatalytic activity. Also, they are highly purified and chemically modified for maximum activity and stability (Boo et al., 2018). Sometimes, protein isolation and purification are done before enzymatic digestion. In this case, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional gel electrophoresis are mostly used for isolation and purification. Using these gel electrophoresis methods, proteins are separated based on the size and/or charge. So, the digestion can be performed more selectively (Gomez et al., 2019; Smolikova et al., 2020). After the enzymatic hydrolysis, the mixture should be centrifuged to separate the LMW peptides containing solution from the precipitates. Then, the solution should be prepared for characterization/identification of food-derived peptides.

5.2.1.2 Microbial method In this method, to produce the proteolytic enzymes, yeast or bacteria are cultured on protein substances. This process leads to protein hydrolysis and production of food-derived peptides. At the beginning of this process, microorganisms are grown in a broth medium at the optimized condition to enter the exponential phase. Then, the microorganisms are collected and suspended in the sterile distilled water containing glucose. The prepared solution is used as an initiator for fermentation of a protein substrate (Cruz-Casas et al., 2021). The hydrolysis rate is more dependent on the following factors: the protein type, bacterial species, and fermentation duration. For example, Lactobacillus helveticus can generate more LMW peptides in milk because of its high proteinase activity (Griffiths & Tellez, 2013). Furthermore, a combination of various microorganisms can increase the degree of hydrolysis (Rizzello et al., 2017). On the other hand, transfer of protease genes into the fermenting microbes using the expression-cloning technique is a novel approach to enable the microbes to produce new food-derived peptides (Yao et al., 2010). After the fermentation process, the mixture is centrifuged, and the supernatant is recovered. Further hydrolysis of the supernatant by proteolytic enzymes can produce the smaller peptides.

5.2.2

In vivo methods

BPs identified after in vitro digestion do not exert their functions in vivo necessarily. This can be related to digestion of these peptides through GIT or their unabsorbability. Proteins and peptides are susceptible to digestion in the GIT by the various proteases and peptidases. So, many peptides characterized after in vitro proteolysis cannot be absorbed in the small intestine. Therefore the enzymatic and microbial methods are not suitable for simulation of protein digestion in the GIT (Barati et al., 2020b). It should be noted that, regardless of the type of

5.2 Methods of protein digestion

digestion method, most of identified BPs are di- or tripeptide. Due to the high capacity of intestine to absorb di- and tripeptides, the BPs are absorbed into the general circulation with limited degradation by the gut proteases/ peptidases (Shen & Matsui, 2019). In Table 5.1, the characteristics of the proteolysis methods are summarized. In the following sections, the common in vivo methods for proteolysis are presented.

5.2.2.1 Aspiration of gut content Aspiration of gut content (AGC) after food ingestion is used to isolate foodderived peptides. In this regard, the appearance of food-derived peptides in the GIT has been investigated by the gastrointestinal intubation technique (Sayd et al., 2018; Tu et al., 2019). In this method, a tube is passed through the nasal cavity into the stomach and/or small intestine for sampling of gut contents after an overnight fasting. The gut content is aspirated following meal ingestion. The duodenum is an important part of intestine for digestion. So, the duodenum content is mainly sampled in AGC method. The duodenal content is taken at 10 cm lower than pylorus (Barbé et al., 2014). Then, the aspirated content is centrifuged to separate supernatant. When the identification step is not supposed to perform at the same time, the aspirated content is mixed with a protease inhibitor and frozen in -70
C. The AGC method has some limitations. For instance, the AGC is an invasive in vivo method. The identified food-derived peptides in this technique may not necessarily be absorbable through GIT. In the AGC method, the brushborder and serum peptidases cannot be involved in protein digestion. On the other hand, this method has its own advantages. The AGC is a suitable approach for assessment of the interactions between food matrix and food-derived peptides (Barati et al., 2020b; Wada et al., 2017). There are limited studies which have been conducted to evaluate the effects of food matrix on the nature of generated peptides. Food-derived peptides are susceptible to reaction with the food matrix. The bioavailability of food-derived peptides can be reduced by these interactions. Moreover, generation of new compounds with bioactive or harmful characteristics is another consequence of these interactions (Udenigwe & Fogliano, 2017).

5.2.2.2 Measurement in the blood In this method, previously defined BPs (such as VPP or IPP) or protein sources of BPs (such as collagen or whey protein) are administered orally. After oral administration of protein/peptide material, serum levels of predefined BPs are measured (Taga et al., 2019). Before and after the food ingestion, serum samples are collected. Different food-derived and endogenous peptides with diverse molecular weights can be detected in the blood after the food ingestion. However, this method cannot identify all of them. Only

123

Table 5.1 The characteristics of the proteolysis methods. The enzymes involved in the digestion

Proteolysis methods In vitro methods

In vivo methods

Enzymatic digestion Microbial fermentation AGC method Measurement in blood Identification in blood

The method type*

Stomach enzymes

Pancreatic enzymes

Brushborder enzymes

Serum peptidase

Invasiveness

Applicability

Microbial enzymes

Characterization

No

High

Optional

Optional

Optional

Optional

Optional

Characterization

No

High

Yes

No

No

No

No

Characterization Measurement

Yes No

Low High

No No

Yes Yes

Yes Yes

No Yes

No Yes

Characterization

No

Low

No

Yes

Yes

Yes

Yes

AGC, Aspiration of gut content. *The methods have been used for the identification of food-derived peptides after proteolysis.

5.2 Methods of protein digestion

the predefined structures can be measured by this method (Asai et al., 2019). On the other word, this method can be considered as a measurement method, not an identifying method. Shigemura et al. in a pilot study on human subjects measured the serum levels of cyclic Pro-Hyp, an in vitro confirmed BP, before and after the ingestion of collagen hydrolysate. The study results showed that the serum levels of the BP significantly increased in the plasma after ingestion of collagen hydrolysate. After 2 h, the BP reached a maximum level and then decreased (Shigemura et al., 2018).

5.2.2.3 Identification/characterization in the blood There are a pool of proteins and peptides in the blood. A large part of this pool contains globulins and hormones. Also, degradation of endogenous proteins produces some peptide fragments which are added into this pool. Foodderived peptides are also added to this pool after the food ingestion. So, combination of the endogenous and exogenous peptides is a barrier to identify the food-derived peptides in the blood (Caira et al., 2022). Theoretically, there is a two-step method for the identification of food-derived peptides in the blood as described in the following: step one is discrimination between endogenous and food-derived peptides and step two is characterization/identification of food-derived peptides. Chabance et al. (1995) identified κ-casein-derived peptides in the plasma of infants after milk ingestion. They separated and identified the peptide profile of infants' plasma after milk ingestion. Then, the identified peptide profile was compared to the plasma peptide profile of overnight-fasted control subjects. Their findings showed that after milk ingestion, the κ-casein-derived peptides with IAIPPKKIQDK and MAIPPKKNQDK sequences were entered the blood (Chabance et al., 1995). However, this is the last study which has been conducted for the identification of food-derived peptides in the blood. A comprehensive research on the identification of foodderived peptides in the blood has been done by Chabance and colleagues. However, they used the tools/equipment with limited ability for the characterization of peptides (Chabance et al., 1995). Nowadays, due to the development of the novel MS techniques, this method has been achieved a great potential for the identification of peptides. Over the last two decades, many studies have been conducted to characterize the serum endogenous LMW peptides as diagnostic biomarkers using the MS technique. Tirumalai et al. (2003) used an MS technique to characterize the LMW peptides in serum. In a similar study, several LMW peptides have been reported as diagnostic biomarkers of hepatocellular carcinoma (HCC) by An et al. (2010). They assessed the serum samples of patients with HCC and healthy subjects. Characterization of LMW peptides in the both serum samples has been done by the MS method that has been developed by Tirumalai et al. (2003). Then, the obtained LMW peptides profile in the HCC patients

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was compared to the healthy controls. Their findings proposed HVQPQPQPKPQVQLHVQSQT, NGFKSHALQLNNRQI, VPPNNSNAAEDDLP TVELQGVVPR, and DDPDAPLQPVTPLQLFEGRRN as diagnostic biomarkers of HCC (An et al., 2010). The method that has been used in the study of An et al. (2010) can be considered as a model for the identification of foodderived peptides in the blood. Identification of LMW peptide profile under the fasting and postingestion conditions and the between-group comparison of these profiles can propose as a comprehensive method for the characterization/identification of food-derived peptides.

5.3 Methods of isolation and identification/ characterization of food-derived peptides The methods for digestion and obtaining the hydrolysate of food proteins are comprehensively described in the previous section. In this section, the methods for identification/characterization of food-derived peptides have been described. For separation of the supernatant containing LMW peptide, after collecting the samples (e.g. serum or plasma, gut content, hydrolysate from enzymatic digestion or fermentation), the mixture should be sonicated and centrifuged to separate the precipitates. Then, the supernatant is acidified by trifluoroacetic acid (TFA) and diluted in guanidine hydrochloride (GuHCL). The GuHCL is one of the strongest protein denaturants. GuHCL allows LMW peptides to filtrate easily. Then centrifugal ultrafiltration or cross-flow membrane filtration is performed to eliminate the bulky constituent proteins. This process leads to the enrichment of the LMW peptides (An et al., 2010). Gel filtration also can be used for quick separation and then LMW peptides can be concentrated. Furthermore, this method can isolate these peptides according to their sizes and/or charge (Gomez et al., 2019). The LMW-enriched solution should be desalted on C18 cartridge to eliminate GuHCL and other ions. Using freeze-drying, the desalted solution is evaporated and prepared power is used measuring LMW peptide content and its characterization/identification. For this, the dried sample is redesolved in acetonitrile or formic acid solutions. Some studies used TFA solution for sample preparation (An et al., 2010). Fractionation of the peptides is performed on RP-HPLC column. Each fraction is analyzed by MALDI-TOF/MS, LC-MS/MS, LC-ESI-QTOF-MS, RPHPLC-MALDI-TOF-MS, LC-ESI MS/MS, LC-MALDI-TOF MS/MS, and/or IRMALDESI-MS (Fideler et al., 2019; Singhal et al., 2015). In selecting the kind of MS technique, it should be considered if the technique has the potential to characterize di- and tripeptides. For instance, MALDI-TOF/MS is not an appropriate technique for the characterization/identification of di- and

5.4 Methods of function assessment

tripeptides. However, there are some BPs among the food-derived peptides, and di- and tripeptides which promise for further investigation (Barati et al., 2020b). To identify the sequence of peptides, all MS and/or MS/MS data should be analyzed in the protein database search programs. MASCOT (http://www.matrixscience.com/search_intro.html), SEQUEST (https://proteomicsresource.washington.edu/protocols06/sequest.php), and PepArML (http://peparml.sourceforge.net) are the most famous databases for translating MS output into the amino acid (AA) sequence (Kong et al., 2017). As mentioned in the previous section, to identify/characterize the food-derived peptides in the blood, profile of serum LMW peptides should be examined under the fasting and postingestion conditions using the mentioned MS techniques. Identifying the unknown MS peaks (the peaks that appears after food ingestion) could be considered as the most important step in this approach. Comparing Tandem MS spectra from fasting and postingestion serum samples using statistical tests such as principal component analysis provides the data related to the unknown peaks. After the identification of unknown MS peaks, these should be translated to AA sequence using the before-mentioned search programs (Fideler et al., 2019; Kong et al., 2017).

5.4

Methods of function assessment

In this chapter, the methods for identification of food-derived peptides are comprehensively described. Among food-derived peptides, some of them have beneficial effects such as hypotensive, antioxidant, and antimicrobial effects on the body. The food-derived peptides with beneficial biological function(s) are called BPs. Function assessment of food-derived peptides is performed by in silico, in vitro, and in vivo methods. In chapter 6, the in silico methods for prediction of BPs are comprehensively described. Once a food-derived peptide has been identified, it should be synthesized for function assessment. The most important methods for peptide synthesis are chemical techniques. There are two chemical techniques for the production of peptides, including solution-phase synthesis and solid-phase peptide synthesis. Introducing and describing the methods for peptide synthesis is beyond the aim of the current chapter. In the next sections, function assessment using in vitro and in vivo methods has been described.

5.4.1

Function assessment using in vitro methods

Evaluation of enzyme activity in the presence of food-derived peptides is an important in vitro method for the assessment of food-derived peptides' function. Degradation of internally quenched fluorescent substrate is measured to assess the enzyme function in this in vitro method. The "o-aminobenzoylglycyl-p-nitro-

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Methods for identification of bioactive peptides

L-phenylalanyl-L-proline"

and "Gly-Pro p-nitroanilide" are internally quenched fluorescent substrate for angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), respectively (Ke˛ska & Stadnik, 2022; Samaei et al., 2021). Generation of fluorescence is measured after incubation of a food-derived peptide with a given enzyme in the presence of the internally quenched fluorescent substrate. Enzyme inhibition is reported as percentage. The half maximal inhibitory concentration (IC50) is a parameter to assess the inhibitory function of a compound when exposed to a given enzyme. By this parameter, one can determine the needed amount of a particular inhibitory substance, such as peptides, to inhibit the function of the desired enzyme by 50%. The lower IC50 indicates that the given food-derived peptide is a more potent inhibitor when exposed to a given enzyme (Kumagai et al., 2021). Kumagai et al. in their in vitro study hydrolyzed a protein isolate from red algae by thermolysin and identified 42 peptides from the hydrolysate. Among the obtained fragments, ACE-inhibitory function of LRM, ARY, and YLR was assessed and IC50 was reported as 0.15, 1.3, and 5.8 μM, respectively. This means that LRM (IC50: 0.15 μM) is a more potent ACE-inhibitory peptide compared to the other identified peptides (Kumagai et al., 2021). Also, one of the most potent ACE-inhibitory peptide is LRY with IC50 of 0.044 μM (44 nM) (Kumagai et al., 2021). On the other hand, the IC50 for captopril, the original inhibitor of ACE, is in the range of 1.7915.1 nM (Henda et al., 2013). In another in vitro method, the antibacterial activity of food-derived peptides is examined by disk diffusion assay. In this method, activation of microorganisms is done in the nutrient broth. On the freshly prepared strain, the peptide-containing solutions are spotted. Then, the incubation of plates at the optimum condition is done. After the incubation time, the diameter of inhibition zone (the zone in which growth is inhibited around the spotted droplet) is measured. Then, the inhibitory activity of each peptide/fraction is reported. In this regard, streptomycin (a kind of antibiotic) and sterilized water are positive and negative controls, respectively (Al-Mohammadi et al., 2020). Pellegrini et al. (2001) identified VAGTWY, AASDISLLDAQSAPLR, and VLVLDTDYK as antimicrobial peptides after digestion of bovine β-lactoglobulin by trypsin. The microbial inhibitory activity is calculated by the following equation: Inhibitory activity ð%Þ 5

  Ac-As 3 100 Ac

where "Ac" is inhibition zone of positive control and "As" is inhibition zone of food-derived peptide In another different method, the antioxidant capacity of food-derived peptides is assessed. In the assessment of antioxidant capacity, the 2,20 -azino-bis3-ethylbenzothiazoline-6-sulfonic (ABTS) assay is a commonly used method.

5.4 Methods of function assessment

The method is based on the reduction of ABTS cations by adding foodderived peptides. This process leads to reagent decolorization which is measurable by spectrophotometry. There are some determinant factors in reagent decolorization, including reaction time, type, and concentration of foodderived peptides (Durand et al., 2021; S¸ anlidere Aloˆglu & Öner, 2011). There are some other methods for measurement of antioxidant activity, including oxygen radical absorbance capacity, 2,2 diphenyl-1 picrylhydrazyl (DPPH), enhanced chemiluminesence, electron spin resonance, ferric reducing antioxidant power, and total radical-trapping antioxidant potential (Munteanu & Apetrei, 2021). In recent years, anticancer BPs have attracted the attention of many researchers. For anticancer function assessment of food-derived peptides, the cancer cell lines should be exposed to these peptides (Soon et al., 2020). After this exposure, growth and apoptosis of cancer cells should be respectively evaluated by proliferation assays and flow cytometry (Su et al., 2014). When a peptide can induce apoptosis and decline cancer cell growth, real-time PCR and Western blot are the techniques used for clarification of the anticancer mechanism(s) (Fan et al., 2022). In a similar method, for hypolipidemic function assessment of food-derived peptides, the peptides are mostly exposed to the HepG2 cell line (human liver cancer cell), and then the behavior of the cells is evaluated (Lammi et al., 2015). There are many methods for function assessment of food-derived peptides. Here, some more common methods are described briefly. Any in vitro obtained functions should be confirmed in vivo. So, in the next section, in vivo methods of function assessment for food-derived peptides have been explained.

5.4.2

Function assessment using in vivo methods

Human and animal subjects are involved in in vivo methods for function assessment of food-derived peptides. Many animal and human studies have been evaluated the effects of oral administration of in vitro identified BPs on given functions in the body. Collagen is one of the important sources of BPs. Several in vitro and in vivo studies have been confirmed the bioactivity of these peptides (Barati et al., 2020a). Sato et al. (2020) in an in vitro study showed that Pro-Hyp, a collagen-derived BP, can exert its wound healing effect by induction of fibroblast growth. The same research group examined the effect of oral administration of Pro-Hyp on wound healing in mice. They showed that the peptide can accelerate wound healing accompanied by less scarring after wounding in mice (Jimi et al., 2021). In a similar study, Sato et al. in an in vitro study characterized the Wakame-derived peptides with ACE-inhibitory function after digestion with protease S. The in vitro part of their study represented VY (IC50: 35.2 μM), IY (IC50: 6.1 μM), AW (IC50:

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18.8 μM), FY (IC50: 42.3 μM), VW (IC50: 3.3 μM), IW (IC50: 1.5 μM), and LW (IC50: 23.6 μM) as antihypertensive BPs. In the in vivo part, the researchers evaluated the effect of oral administration of the peptides (1 mg/kg) on blood pressure in spontaneously hypertensive rats (SHR). The in vivo results revealed that VY, IY, FY, and IW significantly decrease systolic blood pressure in SHR (Sato et al., 2002). When the bioactivity of food-derived peptides is confirmed in animal studies, it can be evaluated in humans. It should be noted that the supplementation with BPs in human studies should be initially performed with a very low dose. Also, clinical trials in this field should be done with extreme caution and after a comprehensive review of the literature. This is because of the potential adverse effects of supplementation with food-derived peptides on endocrine and renin-angiotensin systems over the long term (Barati et al., 2021). IPP (IC50: 5 μM) and VPP (IC50: 9 μM) are two important ACE-inhibitory peptides, called lactotripeptides, have been widely studied until now. The in vitro and animal studies have been confirmed the blood pressurelowering effects of the peptides. Jauhiainen et al. in a randomized-controlled trial evaluated the effects of oral administration of IPP and VPP on blood pressure in hypertensive subjects. In this study, 89 mildly hypertensive subjects were divided into the following groups: (1) subjects who ingested a fermented milk drink with 5 mg per day of IPP and VPP during the first 3 months, and a milk drink with 50 mg per day of lactotripeptides for the following three months (n 5 45) and (2) subjects who ingested a placebo milk drink without lactotripeptides (n 5 44). The results showed that the intervention could slightly lower the elevated blood pressure compared to the baseline, but this reduction was not significant compared to the placebo group (Jauhiainen et al., 2012). Although Jauhiainen et al. reported nonsignificant effects of the lactotripeptides on blood pressure in hypertensive subjects, a recent systematic review and metaanalysis by Liao et al. (2021) revealed that supplementation with foodderived peptides such as IPP and VPP can significantly reduce both systolic and diastolic blood pressure.

5.5

The importance of quantifying bioactive peptides

The development of an efficient method is highly required for quantifying BPs in food items. This can provide a better understanding of BPs role in health and disease. Although several studies including in vitro (Gomez et al., 2019), in vivo (Carrizzo et al., 2019), and meta-analysis (Liao et al., 2021) have been done to evaluate the short-term effects of BPs on human health, there are so limited studies on their long-term effects. Observational studies (cohort and case-control) are commonly used for investigation of the long-term effects of food ingredients. Observational studies cannot assess this long-term effect

References

without quantification of BP content of food items (Barati et al., 2021; Jabbari et al., 2022). Quantification of BP content of food items is so time-consuming and expensive. To reduce the cost and time of projects related to BP quantification, identification and prediction methods should be used together. For the first time, our research team predicted the BP content of dairy products by introducing an in silico model (Barati et al., 2020c). This in silico model can be improved using the identification methods.

5.6

Conclusion

In the current chapter, methods of BP identification including proteolysis methods, identification of food-derived peptides, and function assessment of the obtained fragments have been discussed. Regardless of the digestion method, most of identified BPs are di- or tripeptide. These BPs are absorbed into the general circulation because of the high capacity of the intestine to absorb di- and tripeptides. So, BPs can enter into the general circulation without a wide degradation by the gut proteases/peptidases.

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Cruz-Casas, D. E., Aguilar, C. N., Ascacio-Vald‘s, J. A., Rodr guez-Herrera, R., Ch vez-Gonz lez, M. L., & Flores-Gallegos, A. C. (2021). Enzymatic hydrolysis and microbial fermentation: The most favorable biotechnological methods for the release of bioactive peptides. Food Chemistry: Molecular Sciences, 3. Available from https://doi.org/10.1016/j.fochms.2021.100047. Daliri, eB., & Oh, D. (2017). Bioactive peptides. Foods, 6(5). Available from https://doi.org/ 10.3390/foods6050032. Durand, E., Beaubier, S., Ilic, I., fine, F., Kapel, R., & Villeneuve, P. (2021). Production and antioxidant capacity of bioactive peptides from plant biomass to counteract lipid oxidation. Current Research in Food Science. Current Research in Food Science, 4, 365397. Available from https://doi.org/10.1016/j.crfs.2021.05.006. Fan, X., Guo, H., Teng, C., Zhang, B., Blecker, C., & Ren, G. (2022). Anti-colon cancer activity of novel peptides isolated from in vitro digestion of quinoa protein in Caco-2 cells. Foods, 11 (2). Available from https://doi.org/10.3390/foods11020194. Fideler, J., Johanningsmeier, S. D., Ekel”f, M., & Muddiman, D. C. (2019). Discovery and quantification of bioactive peptides in fermented cucumber by direct analysis IR-MALDESI mass spectrometry and LC-QQQ-MS. Food Chemistry, 271, 715723. Available from https://doi. org/10.1016/j.foodchem.2018.07.187. Gomez, H. L. R., Peralta, J. P., Tejano, L. A., & Chang, Y. W. (2019). In silico and in vitro assessment of portuguese oyster (Crassostrea angulata) proteins as precursor of bioactive peptides. International Journal of Molecular Sciences, 20(20). Available from https://doi.org/10.3390/ ijms20205191.

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In-silico methods for milk-derived bioactive peptide prediction Meisam Barati1, Masoumeh Jabbari2 and Sayed Hossein Davoodi1,3 1

Department of Clinical Nutrition & Dietetics, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Science and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 2Student Research Committee, Department of Community Nutrition, National Nutrition and Food Technology Research Institute, Faculty of Nutrition and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 3 Cancer Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6.1

Introduction

The in vitro and in vivo identification of bioactive peptides (BPs) is expensive and time-consuming. Therefore simulation of the procedures for BPs’ identification before any experiments, saves time and cost. Some research works in BP field such as their quantification are almost impossible without in-silico simulation (Barati et al., 2020a, 2020b). The in-silico methods allow digestion of proteins and prediction of BPs among the obtained fragments. The National Center for Biotechnology Information (NCBI) and Universal Protein Resource Knowledgebase (UniProtKB) are the most important sources for obtaining amino acid (AA) sequence of proteins (Barati et al., 2020a). Selection of the appropriate enzyme(s) for digestion is the most important step for BPs’ prediction. For instance, digestion of a protein by trypsin may increase the antioxidant function of the obtained hydrolysate; while, hydrolysis of the same protein by pepsin promotes antihypertensive activity of the hydrolysate. Based on the purpose of research, in-silico digestion repeatedly occurs with different enzymes and proteins to reach the desirable results (Majumder & Wu, 2010). The in silico BPs’ prediction is performed according to the quantitative structure
activity relationship (QSAR) principles. The basic principle behind QSAR modeling is that the activity or a function of a specific chemical can be described by its molecular or physicochemical descriptors (Tripaldi et al., 2018). BP databases play the most important role in BPs’ prediction. But the existing databases are not complete. It takes a long time for these databases Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00006-0 © 2023 Elsevier Inc. All rights reserved.

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to contain the entire existing BPs. For instance, BIOPEP-UWM database, one of the most important databases for BPs, contains only 4426 BPs (Minkiewicz et al., 2019). However, the number of potentially bioactive di-, tri-, and tetrapeptides is about 160,000 (Prasasty & Istyastono, 2019). So, relying only on databases in BPs’ prediction reduces the quality of simulation. Molecular docking, a kind of in silico method, fills this weakness of BP databases. Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. The goal of ligand-protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known three-dimensional (3D) structure (Vidal-Limon et al., 2022). Considering the role of in silico simulations on saving cost and time in in vitro studies, the current chapter is describing the in silico methods for BPs’ prediction.

6.2 6.2.1

Methods of milk-derived bioactive peptide prediction Obtaining the amino acid sequence of milk proteins

For BPs’ prediction in a protein, the AA sequence of the protein should be obtained. AA sequences of proteins can be done in The UniProt Knowledgebase (UniProtKB: https://www.uniprot.org/uniprot/ and NCBI (https://www.ncbi. nlm.nih.gov/) (Apweiler et al., 2004; Xu & Xu, 2004). The significant contributors of milk proteins are αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, and α-lactalbumin (Barati et al., 2020b). The two most important filters that should be considered in searching through UniProtKB are: (1) Reviewed/unreviewed filter: The results obtained after searching a protein in UniProtKB are divided into “Reviewed” and “Unreviewed.” The reviewed sequences are confirmed by repeated studies and UniProtKB/NCBI staff and/ or collaborators, while unreviewed status means that the reported sequence is not confirmed by experimental works (UniProt, 2017). Therefore in BPs prediction only reviewed sequences should be considered. (2) The second filter in the UniProtKB is popular organism in which the desired animal must be selected. For instance, after searching β-casein in the database, the “Reviewed” and “Bovine” filters should be activated if the confirmed AA sequence of β-casein from cow’s milk is in question. For each protein, UniProtKB introduces signal peptide and AA chain. Then, the BPs’ prediction occurs using the AA chain (UniProt, 2017). In Table 6.1, the obtained AA sequence of milk proteins from different species, including cow (Bos taurus), buffalo (Bubalus bubalis), human (Homo sapiens), donkey (Equus asinus), sheep (Ovis aries), goat (Capra hircus), horse (Equus caballus), and camel (Camelus dromedarius), from UniProtKB or NCBI, is summarized. Each protein in UniProtKB database is identified by a unique and stable entry code (UniProt, 2017). As it can be

Table 6.1 The amino acid sequence of milk proteins from different species obtained from UniProtKB or NCBI databases. αs1-casein

αs2-casein

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Cow (Bos taurus)

.sp|P02662|16-214 RPKHPIKHQGLPQE VLNENLLRFFVAPFP EVFGKEKVNELSKD IGSESTEDQAMEDI KQMEAESISSSEEI VPNSVEQKHIQKE DVPSERYLGYLEQ LLRLKKYKVPQLEI VPNSAEERLHSM KEGIHAQQKEPM IGVNQELAYFYPE LFRQFYQLDAYP SGAWYYVPLGTQ YTDAPSFSDIPNP IGSENSEKTTMPLW

.sp|P02663|16-222 KNTMEHVSSSE ESIISQETYKQEK NMAINPSKEN LCSTFCKEVVR NANEEEYSIGS SSEESAEVATE EVKITVDDKHYQ KALNEINQFYQK FPQYLQYLYQGP IVLNPWDQVKRN AVPITPTLNREQL STSEENSKKTVDM ESTEVFTKKTKLTE EEKNRLNFLKKISQ RYQKFALPQYLKT VYQHQKAMKPWI QPKTKVIPYVRYL

.sp|P02668|22-190 QEQNQEQPIRCE KDERFFSDKIAKYIP IQYVLSRYPSYGLNY YQQKPVALINNQFLP YPYYAKPAAVRSPA QILQWQVLSNTVPA KSCQAQPTTMARHP HPHLSFMAIPPKKNQ DKTEIPTINTIASGEPT STPTTEAVESTVATLE DSPEVIESPPEINTV QVTSTAV

.sp|P02666|16-224 RELEELNVPGEIV ESLSSSEESITRIN KKIEKFQSEEQQQ TEDELQDKIHPFAQ TQSLVYPFPGPIPN SLPQNIPPLTQTPV VVPPFLQPEVMGVS KVKEAMAPKHKEMP FPKYPVEPFTESQSL TLTDVENLHLPLPLL QSWMHQPHQPLPP TVMFPPQSVLSLSQ SKVLPVPQKAVPYP QRDMPIQAFLLYQE PVLGPVRGPFPIIV

.sp|P00711|20-142 EQLTKCEVFRELK DLKGYGGVSLPEW VCTTFHTSGYDTQA IVQNNDSTEYGLFQ INNKIWCKDDQNPH SSNICNISCDKFLDD DLTDDIMCVKKILD KVGINYWLAHKA LCSEKLDQWLCEKL

.sp|P02754|17-178 LIVTQTMKGLDIQ KVAGTWYSLAM AASDISLLDAQS APLRVYVEELKP TPEGDLEILLQK WENGECAQKK IIAEKTKIPAVFK IDALNENKVLVL DTDYKKYLLFC MENSAEPEQS LACQCLVRTP EVDDEALEKF DKALKALPMH IRLSFNPTQLE EQCHI

Buffalo (Bubalus bubalis)

.sp|O62823|16-214 RPKQPIKHQGLPQG VLNENLLRFFVAPF PEVFGKEKVNELST DIGSESTEDQAMED IKQMEAESISSSEEI VPISVEQKHIQKED VPSERYLGYLEQLL RLKKYNVPQLEIVP NLAEEQLHSMKE GIHAQQKEPMIG VNQELAYFYPQL FRQFYQLDAYPS GAWYYVPLGTQ YPDAPSFSDIPNPI GSENSGKTTMPLW

.sp|P11840|22-190 QEQNQEQPIRCEK EERFFNDKIAKYIPIQ YVLSRYPSYGLNYY QQKPVALINNQFLP YPYYAKPAAVRSP AQILQWQVLPNTV PAKSCQAQPTTMT RHPHPHLSFMAIPP KKNQDKTEIPTINT IVSVEPTSTPTTE AIENTVATLEAS SEVIESVPETNT AQVTSTVV

.sp|Q9TSI0|16-224 RELEELNVPGEI VESLSSSEESIT HINKKIEKFQSE EQQQMEDELQ DKIHPFAQTQSL VYPFPGPIPKSL PQNIPPLTQTP VVVPPFLQPE IMGVSKVKEA MAPKHKEMPF PKYPVEPFTES QSLTLTDVENL HLPLPLLQSWM HQPPQPLPPTV MFPPQSVLSLS QSKVLPVPQKA VPYPQRDMPIQ AFLLYQEPVLGP VRGPFPIIV

.sp|Q9TSN6|19-142 AEQLTKCEVFRE LKDLKDYGGVSL PEWVCTAFHTS GYDTQAIVQNN DSTEYGLFQIN NKIWCKDDQN PHSSNICNISCD KFLDDDLTDDI MCVKKILDKVGI NYWLAHKALC SEKLDQWLCEKL

.sp|P02755|19-180 IIVTQTMKGLDIQ KVAGTWYSLAM AASDISLLDAQSA PLRVYVEELKPT PEGDLEILLQKW ENGECAQKKIIAE KTKIPAVFKIDALN ENKVLVLDTDYKK YLLFCMENSAEPE QSLACQCLVRTP EVDDEALEKFDK ALKALPMHIRLS FNPTQLEEQCHV

From NCBI . NP_001277794.1 MKFFIFTCLLAV ALAKHTMEHVS SSEESIISQETYK QEKNMAIHPSKE NLCSTFCKEVIRNA NEEEYSIGSSSEE SAEVATEEVKITV DDKHYQKALNEIN QFYQKFPQYLQYL YQGPIVLNPWDQ VKRNAVPITPTLN REQLSTSEENSK KTVDMESTEVFT KKTKLTEEDKNRL NFLKKISQHYQKF AWPQYLKTVYQY QKAMKPWTQPKT NVIPYVRYL

Continued

Table 6.1 The amino acid sequence of milk proteins from different species obtained from UniProtKB or NCBI databases. Continued αs1-casein

αs2-casein

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Human (Homo sapiens)

.sp|P47710|16-185 RPKLPLRYPERLQ NPSESSEPIPLES REEYMNGMNR QRNILREKQTD EIKDTRNESTQN CVVAEPEKMES SISSSSEEMSLS KCAEQFCRLNE YNQLQLQAAHA QEQIRRMNENS HVQVPFQQLNQ LAAYPYAVWYY PQIMQYVPFPPF SDISNPTAHENY EKNNVMLQW

Human milk does not contain αs2casein.

.sp|P07498|21-182 EVQNQKQPACH ENDERPFYQKTA PYVPMYYVPNS YPYYGTNLYQRR PAIAINNPYVPRT YYANPAVVRPH AQIPQRQYLPNS HPPTVVRRPNLH PSFIAIPPKKIQDKI IIPTINTIATVEPT PAPATEPTVDSV VTPEAFSESIITST PETTTVAVTPPTA

.sp|P00709|20-142 KQFTKCELSQLLK DIDGYGGIALPELIC TMFHTSGYDT QAIVENNESTEY GLFQISNKLWCK SSQVPQSRNICDI SCDKFLDDDITDD IMCAKKILDIKGIDY WLAHKALCTEK LEQWLCEKL

Human milk does not contain β-lactoglobulin.

Donkey (Equus asinus)

.sp|P86272|1-202 RPKLPHRHP EIIQNEQDSREKVL KERKFPSFALH TPREEYINELNR QRELLKEKQKD EHKEYLIEDPE QQESSSTSSSE EVVPINTEQKR IPREDMLYQHT LEQLRRLSKY NQLQLQAIYAQ EQLIRMKENSQ RKPMRVVNQE QAYFYLEPFQP SYQLDVYPYAA WFHPAQIMQHV AYSPFHDTAKLI ASENSEKTDII PEW

.sp|B7VGF9|16-236 KHNMEHRSSSE DSVNISQEKFKQ EKYVVIPTSKES ICSTSCEEATRN INEMESAKFPTE VYSSSSSSEESA KFPTEREEKEVE EKHHLKQLNKIN QFYEKLNFLQYL QALRQPRIVLTPW DQTKTGASPFIPIV NTEQLFTSEEIPK KTVDMESTEVVT EKTELTEEEKNYL KLLNKINQYYEKF TLPQYFKIVHQHQ TTMDPQSHSKTN SYQIIPVLRYF

From NCBI . XP_014702750.1 MKSFFLVVNILAL TLPFLGAEVQNQ EQPTCRKNDERF FDLKTVKYIPIYYV LNSSPRNEPIYY QHRLAVLINNQH MPYQYYARPAA VRPHVQIPQWQV LPNIYPSTVVRHP RPHPSFIAIPPKKL QEKTVIPKINTIA TVEPTPIPTPEP TVNNAVIPDASS EFIIASTPETTTVP VTSPVV

.sp|P05814|16-226 RETIESLSSSEESI TEYKQKVEKVKH EDQQQGEDEH QDKIYPSFQPQP LIYPFVEPIPYGFL PQNILPLAQPAV VLPVPQPEIMEV PKAKDTVYTKGR VMPVLKSPTIPFF DPQIPKLTDLENL HLPLPLLQPLMQ QVPQPIPQTLALP PQPLWSVPQPKVL PIPQQVVPYPQRA VPVQALLLNQ ELLLNPTHQIYPVT QPLAPVHNPISV .sp|P86273|1-226 REKEELNVSSET VESLSSNEPDSS SEESITHINKEKS QKFKHEGQQQ REVEHQDKISRF VQPQPVVYPYAE PVPYAVVPQNILV LAQPPIVPFLQPEI MEVSQAKETILP KRKVMPFLKSPIV PFSERQILNPTNG ENLRLPVHLIQP FMHQVPQSLLQ TLMLPSQPVLSP PQSKVAPFPQPV VPYPQRDTPVQA FLLYQDPQLGLTG EFDPATQPIVP VHNPVIV

.sp|P28546|1-123 KQFTKCELSQVL KSMDGYKGVTL PEWICTIFHSSGY DTQTIVKNNGK TEYGLFQINNK MWCRDNQILPS RNICGISCNKFL DDDLTDDVMCA KKILDSEGIDYW LAHKPLCSEKL EQWLCEEL

.sp|P19647|1-163 TDIPQTMQDLDL QEVAGRWHSVA MVASDISLLDSES APLRVYVEELRPT PEGNLEIILREGAN HVCVERNIVAQK TEDPAVFTVNYQ GERKISVLDTDYAH YMFFCVGPCLPSA EHGMVCQYLART QKVDEEVMEKFS RALQPLPGHVQII QDPSGGQERCGF

Sheep (Ovis aries)

Goat (Capra hircus)

.sp|P04653|16-214 RPKHPIKHQGLS SEVLNENLLRFV VAPFPEVFRKENI NELSKDIGSESIE DQAMEDAKQMK AGSSSSSEEIVP NSAEQKYIQKED VPSERYLGYLEQ LLRLKKYNVPQL EIVPKSAEEQLH SMKEGNPAHQ KQPMIAVNQELAY FYPQLFRQFYQLD AYPSGAWYYLPLG TQYTDAPSFSDIP NPIGSENSGKIT MPLW .sp|P18626|16-214 RPKHPINHRGLS PEVPNENLLRFV VAPFPEVFRKENI NELSKDIGSESTE DQAMEDAKQMK AGSSSSSEEIVP NSAEQKYIQKED VPSERYLGYLEQ LLRLKKYNVPQL EIVPKSAEEQLH SMKEGNPAHQK QPMIAVNQELAY FYPQLFRQFYQL DAYPSGAWYYLP LGTQYTDAPSFS DIPNPIGSENSGK TTMPLW

.sp|P04654|16-223 KHKMEHVSSSEE PINISQEIYKQEKN MAIHPRKEKLC TTSCEEVVRNA DEEEYSIRSSSE ESAEVAPEEVKI TVDDKHYQKALN EINQFYQKFPQY LQYLYQGPIVLNPW DQVKRNAGPFTPT VNREQLSTSEENS KKTIDMESTEVFTK KTKLTEEEKNRLN FLKKISQYYQKFA WPQYLKTVDQHQ KAMKPWTQPKT NAIPYVRYL .sp|P33049|16-223 KHKMEHVSSSE EPINIFQEIYKQE KNMAIHPRKEKL CTTSCEEVVRN ANEEEYSIRSSS EESAEVAPEEI KITVDDKHYQK ALNEINQFYQK FPQYLQYPYQG PIVLNPWDQVK RNAGPFTPTVN REQLSTSEENS KKTIDMESTE VFTKKTKLTEE EKNRLNFLKKIS QYYQKFAWPQY LKTVDQHQKAM KPWTQPKTNAIP YVRYL

.sp|P02669|22-192 QEQNQEQRICC EKDERFFDDKIA KYIPIQYVLSRYPS YGLNYYQQRPVA LINNQFLPYPYYA KPVAVRSPAQT LQWQVLPNAVPA KSCQDQPTAMA RHPHPHLSFMAI PPKKDQDKTEIPA INTIASAEPTVHST PTTEAVVNAVDN PEASSESIASAPE TNTAQVTSTEV

.sp|P02670|22-192 QEQNQEQPICCE KDERFFDDKIAKY IPIQYVLSRYPSY GLNYYQQRPVALI NNQFLPYPYYAKP VAVRSPAQTLQW QVLPNTVPAKSCQ DQPTTLARHPHPHL SFMAIPPKKDQDKT EVPAINTIASAEPT VHSTPTTEAIVNT VDNPEASSESIAS ASETNTAQVTSTEV

.sp|P11839|16-222 REQEELNVVGET VESLSSSEESITHI NKKIEKFQSEE QQQTEDELQDK IHPFAQAQSLVY PFTGPIPNSLPQNI LPLTQTPVVVPPF LQPEIMGVPKVKE TMVPKHKEMPFPK YPVEPFTESQSLTL TDVEKLHLPLPLVQ SWMHQPPQPLPPT VMFPPQSVLSLSQP KVLPVPQKAVPQRD MPIQAFLLYQEPVL GPVRGPFPILV

.sp|P33048|16-222 REQEELNVVGETVE SLSSSEESITHINKK IEKFQSEEQQQTE DELQDKIHPFAQA QSLVYPFTGPIPN SLPQNILPLTQTP VVVPPFLQPEIM GVPKVKETMVPK HKEMPFPKYPVE PFTESQSLTLTDV EKLHLPLPLVQS WMHQPPQPLSP TVMFPPQSVLSL SQPKVLPVPQKA VPQRDMPIQAFL LYQEPVLGPVRG PFPILV

.sp|P09462|20-142 EQLTKCEVFQE LKDLKDYGGVSLP EWVCTAFHTSG YDTQAIVQNNDS TEYGLFQINNKIW CKDDQNPHSRN ICNISCDKFLDDD LTDDIMCVKKILD KVGINYWLAHKAL CSEKLDQWLCEKL

.sp|P67976|19-180 IIVTQTMKGLDIQK VAGTWHSLAMA ASDISLLDAQSA PLRVYVEELKPT PEGNLEILLQKW ENGECAQKKIIAE KTKIPAVFKIDAL NENKVLVLDTD YKKYLLFCMEN SAEPEQSLACQ CLVRTPEVDN EALEKFDKALKA LPMHIRLAFNPTQ LEGQCHV

.sp|P00712|20-142 EQLTKCEVFQKLK DLKDYGGVSLPEW VCTAFHTSGYDTQ AIVQNNDSTEYGLF QINNKIWCKDDQNP HSRNICNISCDKFLD DDLTDDIVCAKKILD KVGINYWLAHKALC SEKLDQWLCEKL

.sp|P02756|19-180 IIVTQTMKGLDIQK VAGTWYSLAMAA SDISLLDAQSAPLRV YVEELKPTPEGNLEI LLQKWENGECAQK KIIAEKTKIPAVFKID ALNENKVLVLDTDY KKYLLFCMENSA EPEQSLACQCLV RTPEVDKEALEK FDKALKALPMHIRL AFNPTQLEGQCHV

Continued

Table 6.1 The amino acid sequence of milk proteins from different species obtained from UniProtKB or NCBI databases. Continued Horse (Equus caballus)

Camel (Camelus dromedarius)

αs1-casein

αs2-casein

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

From NCBI . NP_001075352.1 MKLLILTCLVAVA LARPKLPHRQPEII QNEQDSREKVLK ERKFPSFALEYIN ELNRQRELLKE KQKDEHKEYLIED PEQQESSSTSSS EEVVPINTEQK RIPREDMLYQHT LEQLRRLSKYNQ LQLQAIHAQEQLI RMKENSQRKPMR VVNQEQAYFYLEP FQPSYQLDVYPY AAWFHPAQIMQ HVAYSPFHDTAK LIASENSEKTDII PEW .sp|O97943|16-230 RPKYPLRYPEVFQ NEPDSIEEVLNKRK ILELAVVSPIQFRQE NIDELKDTRNEPTE DHIMEDTERKESGS SSSEEVVSSTTEQK DILKEDMPSQRYLE ELHRLNKYKLLQL EAIRDQKLIPRVKLS SHPYLEQLYRINED NHPQLGEPVKVVT QEQAYFHLEPFPQ FFQLGASPYVAWY YPPQVMQYIAHPS SYDTPEGIASEDG GKTDVMPQWW

From NCBI . NP_001164238.1 MKFFIFTCLLAVA LAKHNMEHRSSS EDSVNISQEKFK QEKYVVIPTSKE SICSTSCEEATRN INEMESAKFPTE REEKEVEEKHHL KQLNKINQFYEKL NFLQYLQALRQPR IVLTPWDQTKTGDS PFIPIVNTEQLFTSE EIPKKTVDMESTEV VTEKTELTEEEKN YLKLLYYEKFTLPQ YFKIVRQHQTTMD PRSHRKTNSYQIIP VLRYF

.sp|P82187|21-185 EVQNQEQPTCH KNDERFFDLKT VKYIPIYYVLNSS PRYEPIYYQHRLA LLINNQHMPYQYY ARPAAVRPHVQIP QWQVLPNIYPSTV VRHPCPHPSFIAIPP KKLQEITVIPKINTIAT VEPTPIPTPEPTVN NAVIPDASSEFIIAS TPETTTVPVTSPV VQKL

.sp|Q9GKK3|16-241 REKEELNVSSETV ESLSSNEPDSSSE ESITHINKEKLQK FKHEGQQQREVE RQDKISRFVQPQP VVYPYAEPVPYAV VPQSILPLAQPPILP FLQPEIMEVSQAK ETILPKRKVMPFLK SPIVPFSERQILNPT NGENLRLPVHLIQ PFMHQVPQSLLQ TLMLPSQPVLSPP QSKVAPFPQPVV PYPQRDTPVQAF LLYQDPRLGPTGE LDPATQPIVAV HNPVIV

.sp|P08334|1-123 KQFTKCELSQVLK SMDGYKGVTLPE WICTIFHSSGYDTQ TIVKNNGKTEYGLF QINNKMWCRDNQI LPSRNICGISCDKFL DDDLTDDVMCAKK ILDSEGIDYWLAH KPLCSEKLEQWL CEEL

.sp|P02758|19-180 TNIPQTMQDLDLQE VAGKWHSVAMAAS DISLLDSESAPLRVY IEKLRPTPEDNLEIIL REGENKGCAEKKIF AEKTESPAEFKINYL DEDTVFALDTDYKN YLFLCMKNAATPGQ SLVCQYLARTQMVD EEIMEKFRRALQPL PGRVQIVPDLTRM AERCRI

.sp|O97944|16-193 KHEMDQGSSSEES INVSQQKFKQVKKV AIHPSKEDICSTFCE EAVRNIKEVESAE VPTENKISQFYQKW KFLQYLQALHQGQI VMNPWDQGKTRAY PFIPTVNTEQLSISE ESTEVPTEESTEVF TKKTELTEEEKDH QKFLNKIYQYYQT FLWPEYLKTVYQY QKTMTPWNHIKRYF

.sp|P79139|21-182 EVQNQEQPTCFE KVERLLNEKTVKYF PIQFVQSRYPSYGIN YYQHRLAVPINNQF IPYPNYAKPVAIRLH AQIPQCQALPNIDP PTVERRPRPRPSF IAIPPKKTQDKTVN PAINTVATVEPPVI PTAEPAVNTVVIA EASSEFITTST PETTTVQITSTEI

.sp|Q9TVD0|16-232 REKEEFKTAGEA LESISSSEESITHIN KQKIEKFKIEEQ QQTEDEQQDKIY TFPQPQSLVYSH TEPIPYPILPQNF LPPLQPAVMVPF LQPKVMDVPKTK ETIIPKRKEMPLL QSPVVPFTESQS LTLTDLENLHLPL PLLQSLMYQIPQ PVPQTPMIPPQS LLSLSQFKVLPV PQQMVPYPQRAM PVQAVLPFQEPVP DPVRGLHPVPQP LVPVIA

.sp|P00710|1-123 KQFTKCKLSDELK DMNGHGGITLAE WICIIFHMSGYD TETVVSNNGNRE YGLFQINNKIWCR DNENLQSRNIC DISCDKFLDDDLTD DKMCAKKILDKEGI DYWLAHKPLC SEKLEQWQCEKW

Camel milk does not contain β-lactoglobulin.

A, Alanine; R, Arginine; N, Asparagine; D, Aspartate; C, Cysteine; E, Glutamate; Q, Glutamine; G, Glycine; H, Histidine; I, Isoleucine; L, Leucine; K, Lysine; M, Methionine; F, Phenylalanine; P, Proline; S, Serine; T, Threonine; W, Tryptophan; Y, Tyrosine; V, Valine. The bold sequences were obtained from NCBI. NCBI: The National Center for Biotechnology Information

6.2 Methods of milk-derived bioactive peptide prediction

seen in, the UniProtKB entry code for bovine αs1-casein, αs2-casein, κ-casein, β-casein, α-lactalbumin, and β-lactoglobulin is P02662, P02663, P02668, P02666, P00711, and P02754, respectively. When a protein is not found in UniProtKB (e.g., αs2-casein of buffalo milk, κ-casein of donkey milk, αs2casein, and αs1-casein of horse milk), so, NCBI should be examined. The NCBI contains a large number of databases such as Gene, Genome, Nucleotide, and Protein. To find a protein in NCBI, the Protein database must be selected. Like UniProtKB, the search results should be limited to the reference sequences (RefSeq) database and the desired organism in the NCBI database. For instance, to find bovine αs1-casein in the Protein database, the search results should be limited to “RefSeq” and “Bos taurus.” The RefSeq database contains reviewed sequence of proteins and genes. The reference sequence of αs2-casein and αs1-casein from horse milk as well as αs2-casein from buffalo milk, obtained from NCBI, can be viewed. A unique accession number has been assigned for each protein in NCBI. As it can be seen in Table 6.1, the accession number for αs1-casein of horse milk is NP_001075352.1. The accession number of reviewed/reference sequences is started with NP (Midic & Obradovic, 2012). The accession number for donkey milk κ-casein is XP_014702750.1 and it is not reviewed sequence. When an accession number is started with XP, it means that the sequence is hypothetical and obtained from bioinformatics analysis, not from experimental work. In fact, obtaining XP_ sequences are mostly achieved by means of in silico translation of the related gene(s) (Barati et al., 2020b; Midic & Obradovic, 2012). The αs1-casein, αs2-casein, β-casein, κ-casein, β-lactoglobulin, and α-lactalbumin are the main proteins of milk. But AA sequence of each protein from different species is different from others. For example, human milk αs1casein has fewer residues than those from other mammalian species (Parastouei et al., 2022). The multiple alignments of milk proteins from different species reveal these differences. The “Clustal omega” tool (https:// www.ebi.ac.uk/Tools/msa/clustalo/) from the European Bioinformatics Institute (EMBL-EBI) database is one of the most important available tools for protein alignment (Sievers & Higgins, 2021). In Fig. 6.1, the multiple alignments for AA sequence of β-casein from cow, buffalo, human, donkey, sheep, goat, horse, and camel milks are shown. As it can be seen, there are many differences in their sequences. These differences are important in BPs’ prediction. In this part, obtaining the AA sequence of proteins from NCBI and UniProtKB was described. In the next part, in silico digestion of proteins is examined.

6.2.2

In-silico digestion of milk proteins

In the previous section, the available databases for obtaining AA sequence of proteins were reviewed. Before any in silico digestion, the potential of each

143

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CHAPTER 6:

In-silico methods for milk-derived bioactive peptide prediction

FIGURE 6.1 The multiple alignment for AA sequence of β-casein from cow, buffalo, human, donkey, sheep, goat, horse, and camel milks.

protein to produce BPs with given function must be achieved. So, the occurrence frequency (A) should be calculated by the following equation: A 5 Na where a is the number of fragments with a given function potentially reside in a protein; N is the number of AA resides in the protein.

6.2 Methods of milk-derived bioactive peptide prediction

The BIOPEP-UWM (https://biochemia.uwm.edu.pl/biopep-uwm/) is a database of BPs. By means of the database, “A” parameter can be calculated. Higher occurrence frequency score indicates that the higher number of BPs, with the given function, can be isolated from the examined protein (Tripaldi et al., 2018). BIOPEP-UMW provides the “ENZYME(S) ACTION” tool with 34 proteases for in silico digestion of proteins. Using this tool, in silico digestion can be performed under the simultaneous action of up to three proteases. If your simulation needs more than three enzymes or a protease that is not contained in the tool (Tripaldi et al., 2018), tools in the other databases such as Expasy PeptideCutter (https://web.expasy.org/peptide_cutter/) should be used (Gasteiger et al., 2003). In Table 6.2, the obtained fragments from in silico simultaneous action of trypsin, chymotrypsin, and pepsin of milk proteins from different species are reported. The BPs’ prediction occurs among the obtained fragments from in silico digestion. The obtained fragments are searched against BIOPEP-UMW database to find BPs and their exact function. About 55 groups of BPs with different biological functions such as angiotensin-converting enzyme inhibitory (ACE-I), antioxidant, renin inhibitory, and dipeptidyl peptidase IV (DPP-IV) inhibitory have been defined in BIOPEP-UMW. If there is no defined activity for a fragment, it’s not necessarily mean that the fragment is not bioactive; this means that these peptides/fragments have not been registered in the BIOPEP-UWM database so far. So, in this case, other databases should be tried. Iwaniak et al. (2021) in a chapter summarized the existing databases for predicting BPs. The release frequency (AE) and relative release frequency (W) are the most important parameters to be evaluated after in silico digestion of a protein. The AE and W for each function such as ACE-I, renin inhibitory, DPP-IV inhibitory, and antioxidant activities are calculated according to the following equations (Minkiewicz et al., 2019). AE 5

d N

W5

AE A

d is the number of fragments with a given function released from a protein by in silico digestion; N is the number of AA resides in the protein; A is the occurrence frequency. As mentioned before, “A” parameter is used to indicate the potential of each protein to produce BPs with a given function (Minkiewicz et al., 2019). For instance, “A” parameter score for renin-inhibitory BPs in κ-casein of bovine milk is 0.02 and for ACE-I BPs is 0.46. So, κ-casein of bovine milk is a richer source of BPs with ACE-I function than renin inhibitory activity. Using “A” score, different proteins can be compared to find a good source of a desired function. The AE parameter is another useful score to evaluate the effects of

145

Table 6.2 The fragments obtained from in silico digestion of milk proteins from different species using simultaneous action of trypsin, pepsin, and chymotrypsin. αs1-casein

αs2-casein

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Cow(Bos taurus)

.sp|P02662|16-214 R - PK - H PIK - H - QGL - PQEVL - N - EN - L - L - R - F - F - VAPF - PEVF - GK - EK - VN - EL - SK DIGSESTEDQAM - EDIK - QM EAESISSSEEIVPN - SVEQK - H IQK - EDVPSER - Y - L - GY - L - EQL - L - R - L - K - K - Y - K VPQL - EIVPN - SAEER - L - H SM - K - EGIH - AQQK - EPM IGVN - QEL - AY - F - Y - PEL F - R - QF - Y - QL - DAY PSGAW - Y - Y - VPL - GTQY TDAPSF - SDIPN - PIGSEN SEK - TTM - PL - W

.sp|P02663|16-222 K - N - TM - EH VSSSEESIISQETY - K QEK - N - M - AIN - PSK EN - L - CSTF - CK - EVVR - N - AN - EEEY SIGSSSEESAEVATEEVK ITVDDK - H - Y - QK - AL N - EIN - QF - Y - QK - F PQY - L - QY - L - Y QGPIVL - N - PW - DQVK R - N - AVPITPTL - N - R EQL - STSEEN - SK - K TVDM - ESTEVF - TK - K TK - L - TEEEK - N - R - L - N - F - L - K - K - ISQR Y - QK - F - AL - PQY - L K - TVY - QH - QK - AM K - PW - IQPK - TK - VIPY - VR - Y - L

.sp|P02668|22-190 QEQN QEQPIR - CEK - DER - F - F - SDK - IAK - Y - IPIQY - VL - SR - Y PSY - GL - N - Y - Y - QQK - PVAL - IN - N - QF - L - PY - PY - Y - AK - PAAVR - SPAQIL - QW - QVL SN - TVPAK - SCQAQPTTM - AR H - PH - PH - L - SF - M - AIPPK K - N - QDK - TEIPTIN TIASGEPTSTPTTEAVESTVATL EDSPEVIESPPEIN - TVQVTSTAV

.sp|P02666|16-224 R - EL - EEL - N VPGEIVESL SSSEESITR - IN - K - K - IEK - F QSEEQQQTEDEL QDK - IH - PF AQTQSL - VY - PF PGPIPN - SL - PQN IPPL - TQTPVVVPPF L - QPEVM - GVSK VK - EAM - APK - H K - EM - PF - PK - Y PVEPF - TESQSL - TL - TDVEN - L - H - L PL - PL - L - QSW - M - H - QPH - QPL PPTVM - F - PPQSVL SL - SQSK - VL PVPQK - AVPY - PQR - DM - PIQAF - L - L Y - QEPVL - GPVR GPF - PIIV

.sp|P00711|20-142 EQL - TK - CEVF - R EL - K - DL - K - GY GGVSL - PEW - VCTTF - H - TSGY DTQAIVQN - N DSTEY - GL - F - QIN N - K - IW - CK DDQN - PH - SSN ICN - ISCDK - F - L DDDL - TDDIM - CVK K - IL - DK - VGIN - Y W - L - AH - K - AL CSEK - L - DQW - L CEK - L

.sp|P02754|17-178 L - IVTQTM - K - GL DIQK - VAGTW - Y SL - AM - AASDISL - L - DAQSAPL - R - VY VEEL - K - PTPEGDL EIL - L - QK - W - EN GECAQK - K - IIAEK TK - IPAVF - K - IDAL N - EN - K - VL - VL DTDY - K - K - Y - L L - F - CM - EN SAEPEQSL - ACQCL VR - TPEVDDEAL - EK - F - DK - AL - K - AL PM - H - IR - L - SF N - PTQL - EEQCH - I

Buffalo (Bubalus bubalis)

.sp|O62823|16-214 R - PK QPIK - H - QGL - PQGVL - N EN - L - L - R - F - F - VAPF PEVF - GK - EK - VN - EL STDIGSESTEDQAM - EDIK - QM - EAESISSSEEIVPISVEQK - H IQK - EDVPSER - Y - L - GY - L - EQL - L - R - L - K - K - Y - N VPQL - EIVPN - L - AEEQL - H SM - K - EGIH - AQQK - EPM IGVN - QEL - AY - F - Y - PQL F - R - QF - Y - QL - DAY PSGAW - Y - Y - VPL - GTQY PDAPSF - SDIPN - PIGSEN SGK - TTM - PL - W

From NCBI . NP_001277794.1 M - K - F - F - IF - TCL L - AVAL - AK - H - TM EH - VSSSEESIISQETY K - QEK - N - M - AIH PSK - EN - L - CSTF CK - EVIR - N - AN EEEY SIGSSSEESAEVATEEVK - ITVDDK - H - Y - QK AL - N - EIN - QF - Y QK - F - PQY - L - QY - L - Y - QGPIVL - N - PW DQVK - R - N AVPITPTL - N - R - EQL - STSEEN - SK - K TVDM - ESTEVF - TK - K - TK - L - TEEDK - N - R -L-N-F-L-K-KISQH - Y - QK - F - AW PQY - L - K - TVY - QY QK - AM - K - PW TQPK - TN - VIPY - VR Y-L

.sp|P11840|22-190 QEQN QEQPIR - CEK - EER - F - F - N DK - IAK - Y - IPIQY - VL - SR - Y PSY - GL - N - Y - Y - QQK - PVAL - IN - N - QF - L - PY - PY - Y - AK - PAAVR - SPAQIL - QW - QVL PN - TVPAK - SCQAQPTTM - TR H - PH - PH - L - SF - M - AIPPK K - N - QDK - TEIPTIN TIVSVEPTSTPTTEAIEN - TVATL EASSEVIESVPETN - TAQVTSTVV

.sp|Q9TSI0|16-224 R EL - EEL - N VPGEIVESL SSSEESITH - IN - K - K - IEK - F - QSEEQQQM - EDEL - QDK - IH - PF - AQTQSL - VY - PF PGPIPK - SL - PQN IPPL - TQTPVVVPPF L - QPEIM - GVSK - VK - EAM - APK - H - K EM - PF - PK - Y PVEPF - TESQSL - TL - TDVEN - L - H - L PL - PL - L - QSW - M - H - QPPQPL PPTVM - F - PPQSVL SL - SQSK - VL PVPQK - AVPY - PQR - DM - PIQAF - L - L Y - QEPVL - GPVR GPF - PIIV

.sp|Q9TSN6|19-142 AEQL - TK - CEVF - R - EL - K - DL - K - DY GGVSL - PEW - VCTAF - H - TSGY DTQAIVQN - N DSTEY - GL - F - QIN N - K - IW - CK DDQN - PH - SSN ICN - ISCDK - F - L DDDL - TDDIM - CVK K - IL - DK - VGIN - Y W - L - AH - K - AL CSEK - L - DQW - L CEK - L

.sp|P02755|19-180 IIVTQTM - K - GL DIQK - VAGTW - Y SL - AM - AASDISL - L - DAQSAPL - R - VY VEEL - K - PTPEGDL EIL - L - QK - W - EN GECAQK - K - IIAEK TK - IPAVF - K - IDAL N - EN - K - VL - VL DTDY - K - K - Y - L L - F - CM - EN SAEPEQSL - ACQCL VR - TPEVDDEAL - EK - F - DK - AL - K - AL PM - H - IR - L - SF N - PTQL - EEQCH
V

Human (Homo sapiens)

.sp|P47710|16-185 R - PK - L PL - R - Y - PER - L - QN PSESSEPIPL - ESR - EEY - M N - GM - N - R - QR - N - IL - R - EK - QTDEIK - DTR - N ESTQN - CVVAEPEK - M ESSISSSSEEM - SL - SK CAEQF - CR - L - N - EY - N QL - QL - QAAH - AQEQIR - R M - N - EN - SH - VQVPF - QQL - N - QL - AAY - PY - AVW - Y Y - PQIM - QY - VPF - PPF SDISN - PTAH - EN - Y - EK - N - N - VM - L - QW

Human milk is not contained αs2-casein.

.sp|P07498|21-182 EVQN - QK QPACH - EN - DER - PF - Y - QK TAPY - VPM - Y - Y - VPN - SY PY - Y - GTN - L - Y - QR - R PAIAIN - N - PY - VPR - TY - Y AN - PAVVR - PH - AQIPQR - QY L - PN - SH - PPTVVR - R - PN - L - H - PSF - IAIPPK - K - IQDK IIIPTIN TIATVEPTPAPATEPTVDSVVTPEAF - SESIITSTPETTTVAVTPPTA

.sp|P05814|16-226 R - ETIESL SSSEESITEY - K - QK VEK - VK - H EDQQQGEDEH - QDK - IY - PSF - QPQPL - IY - PF - VEPIPY - GF - L - PQN - IL - PL AQPAVVL - PVPQPEIM - EVPK - AK - DTVY TK - GR - VM - PVL - K - SPTIPF - F - DPQIPK - L - TDL - EN - L - H L - PL - PL - L - QPL M - QQVPQPIPQTL AL - PPQPL - W SVPQPK - VL PIPQQVVPY - PQR AVPVQAL - L - L - N QEL - L - L - N - PTH QIY - PVTQPL - APVH - N - PISV

.sp|P00709|20-142 K - QF - TK - CEL - SQL - L - K - DIDGY GGIAL - PEL - ICTM F - H - TSGY DTQAIVEN - N - ESTEY - GL - F - QISN - K - L - W - CK - SSQVPQSR - N - ICDISCDK - F - L - DDDITDDIM - CAK K - IL - DIK - GIDY - W - L - AH - K - AL CTEK - L - EQW - L CEK - L

Human milk does not contain β-lactoglobulin.

Donkey (Equus asinus)

.sp|P86272|1-202 R - PK - L PH - R - H - PEIIQN - EQDSR EK - VL - K - ER - K - F - PSF AL - H - TPR - EEY - IN - EL - N - R - QR - EL - L - K - EK - QK DEH - K - EY - L IEDPEQQESSSTSSSEEVVPIN TEQK - R - IPR - EDM - L - Y QH - TL - EQL - R - R - L - SK Y - N - QL - QL - QAIY - AQEQL - IR - M - K - EN - SQR - K - PM - R - VVN - QEQAY - F - Y - L EPF - QPSY - QL - DVY - PY AAW - F - H - PAQIM - QH VAY - SPF - H - DTAK - L IASEN - SEK - TDIIPEW

.sp|B7VGF9|16-236 K - H - N - M - EH - R SSSEDSVN - ISQEK - F K - QEK - Y - VVIPTSK ESICSTSCEEATR - N - IN EM - ESAK - F - PTEVY SSSSSSEESAK - F - PTER - EEK - EVEEK - H - H - L - K - QL - N - K - IN - QF Y - EK - L - N - F - L - QY - L - QAL - R - QPR - IVL TPW - DQTK - TGASPF IPIVN - TEQL - F TSEEIPK - K - TVDM ESTEVVTEK - TEL - TEEEK -N-Y-L-K-L-L-NK - IN - QY - Y - EK - F TL - PQY - F - K - IVH - QH - QTTM - DPQSH - SK - TN - SY - QIIPVL - R - Y - F

From NCBI . XP_014702750.1M - K - SF - F - L - VVN - IL - AL TL - PF - L - GAEVQN QEQPTCR - K - N - DER - F - F DL - K - TVK - Y - IPIY - Y - VL N - SSPR - N - EPIY - Y - QH - R - L - AVL - IN - N - QH - M - PY QY - Y - AR - PAAVR - PH VQIPQW - QVL - PN - IY PSTVVR - H - PR - PH - PSF IAIPPK - K - L - QEK - TVIPK IN - TIATVEPTPIPTPEPTVN - N AVIPDASSEF IIASTPETTTVPVTSPVV

.sp|P86273|1-226 R EK - EEL - N VSSETVESL - SSN EPDSSSEESITH - IN K - EK - SQK - F - K H - EGQQQR - EVEH QDK - ISR - F VQPQPVVY - PY AEPVPY - AVVPQN - IL - VL - AQPPIVPF - L QPEIM - EVSQAK ETIL - PK - R - K - VM - PF - L - K - SPIVPF SER - QIL - N - PTN GEN - L - R - L - PVH L - IQPF - M - H QVPQSL - L - QTL - M - L - PSQPVL SPPQSK - VAPF PQPVVPY - PQR DTPVQAF - L - L - Y QDPQL - GL - TGEF DPATQPIVPVH - N PVIV

.sp|P28546|1-123 K QF - TK - CEL - SQVL - K - SM - DGY - K GVTL - PEW - ICTIF H - SSGY - DTQTIVK N - N - GK - TEY - GL - F - QIN - N - K - M W - CR - DN - QIL PSR - N - ICGISCN - K - F - L - DDDL TDDVM - CAK - K - IL DSEGIDY - W - L - AH - K - PL - CSEK - L EQW - L - CEEL

.sp|P19647|1-163 TDIPQTM - QDL - DL QEVAGR - W - H SVAM - VASDISL - L DSESAPL - R - VY VEEL - R - PTPEGN L - EIIL - R - EGAN - H - VCVER - N - IVAQK TEDPAVF - TVN - Y QGER - K - ISVL DTDY - AH - Y - M - F - F - CVGPCL PSAEH - GM - VCQY L - AR - TQK VDEEVM - EK - F - SR - AL - QPL - PGH VQIIQDPSGGQER CGF

Sheep (Ovis aries)

.sp|P04653|16-214 R - PK - H PIK - H - QGL - SSEVL - N - EN - L - L - R - F - VVAPF - PEVF R - K - EN - IN - EL - SK DIGSESIEDQAM - EDAK - QM K - AGSSSSSEEIVPN - SAEQK -

.sp|P04654|16-223 K - H - K - M - EH - VSSSEEPIN - ISQEIY - K - QEK - N - M - AIH - PR - K - EK - L CTTSCEEVVR - N ADEEEY - SIR -

.sp|P02669|22-192 QEQN - QEQR - ICCEK - DER - F - F - DDK - IAK Y - IPIQY - VL - SR - Y - PSY - GL - N - Y - Y - QQR - PVAL - IN - N QF - L - PY - PY - Y - AK - PVAVR - SPAQTL - QW - QVL - PN -

.sp|P11839|16-222 R - EQEEL - N VVGETVESL SSSEESITH - IN - K - K - IEK - F QSEEQQQTEDEL -

.sp|P09462|20-142 EQL - TK - CEVF - QEL - K - DL - K - DY GGVSL - PEW - VCTAF - H - TSGY DTQAIVQN - N -

.sp|P67976|19-180 IIVTQTM - K - GL DIQK - VAGTW - H SL - AM - AASDISL - L - DAQSAPL - R - VY VEEL - K - PTPEGN -

Continued

Table 6.2 The fragments obtained from in silico digestion of milk proteins from different species using simultaneous action of trypsin, pepsin, and chymotrypsin. Continued αs1-casein

αs2-casein

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Y - IQK - EDVPSER - Y - L - GY - L - EQL - L - R - L - K - K - Y N - VPQL - EIVPK - SAEEQL - H - SM - K - EGN - PAH - QK QPM - IAVN - QEL - AY - F - Y PQL - F - R - QF - Y - QL - DAY - PSGAW - Y - Y - L - PL GTQY - TDAPSF - SDIPN PIGSEN - SGK - ITM - PL - W

SSSEESAEVAPEEVK ITVDDK - H - Y - QK - AL N - EIN - QF - Y - QK - F PQY - L - QY - L - Y QGPIVL - N - PW - DQVK R - N - AGPF - TPTVN - R - EQL - STSEEN - SK - K TIDM - ESTEVF - TK - K TK - L - TEEEK - N - R - L - N - F - L - K - K - ISQY Y - QK - F - AW - PQY - L - K - TVDQH - QK - AM K - PW - TQPK - TN AIPY - VR - Y - L

AVPAK - SCQDQPTAM - AR - H PH - PH - L - SF - M - AIPPK - K DQDK - TEIPAIN - TIASAEPTVH STPTTEAVVN - AVDN PEASSESIASAPETN - TAQVTSTEV

QDK - IH - PF AQAQSL - VY - PF TGPIPN - SL - PQN IL - PL - TQTPVVVPPF - L - QPEIM - GVPK VK - ETM - VPK - H - K - EM - PF - PK - Y PVEPF - TESQSL - TL - TDVEK - L - H - L PL - PL - VQSW - M H - QPPQPL - PPTVM - F - PPQSVL - SL SQPK - VL - PVPQK AVPQR - DM - PIQAF L - L - Y - QEPVL GPVR - GPF - PIL - V

DSTEY - GL - F - QIN N - K - IW - CK DDQN - PH - SR - N ICN - ISCDK - F - L DDDL - TDDIM - CVK K - IL - DK - VGIN - Y W - L - AH - K - AL CSEK - L - DQW - L CEK - L

L - EIL - L - QK - W EN - GECAQK - K IIAEK - TK - IPAVF - K - IDAL - N - EN - K VL - VL - DTDY - K - K - Y - L - L - F - CM EN - SAEPEQSL ACQCL - VR TPEVDN - EAL - EK F - DK - AL - K - AL PM - H - IR - L - AF N - PTQL - EGQCH - V

Goat (Capra hircus)

.sp|P18626|16-214 R - PK - H PIN - H - R - GL - SPEVPN - EN - L - L - R - F - VVAPF - PEVF R - K - EN - IN - EL - SK DIGSESTEDQAM - EDAK - QM K - AGSSSSSEEIVPN - SAEQK Y - IQK - EDVPSER - Y - L - GY - L - EQL - L - R - L - K - K - Y N - VPQL - EIVPK - SAEEQL - H - SM - K - EGN - PAH - QK QPM - IAVN - QEL - AY - F - Y PQL - F - R - QF - Y - QL - DAY - PSGAW - Y - Y - L - PL GTQY - TDAPSF - SDIPN PIGSEN - SGK - TTM - PL - W

.sp|P33049|16-223 K - H - K - M - EH - VSSSEEPIN - IF - QEIY - K - QEK - N M - AIH - PR - K - EK - L CTTSCEEVVR - N - AN EEEY - SIR SSSEESAEVAPEEIK ITVDDK - H - Y - QK - AL N - EIN - QF - Y - QK - F PQY - L - QY - PY QGPIVL - N - PW - DQVK R - N - AGPF - TPTVN - R - EQL - STSEEN - SK - K TIDM - ESTEVF - TK - K TK - L - TEEEK - N - R - L - N - F - L - K - K - ISQY Y - QK - F - AW - PQY - L - K - TVDQH - QK - AM K - PW - TQPK - TN AIPY - VR - Y - L

.sp|P02670|22-192 QEQN QEQPICCEK - DER - F - F - DDK IAK - Y - IPIQY - VL - SR - Y - PSY - GL - N - Y - Y - QQR - PVAL - IN - N - QF - L - PY - PY - Y - AK PVAVR - SPAQTL - QW - QVL - PN - TVPAK - SCQDQPTTL - AR - H PH - PH - L - SF - M - AIPPK - K DQDK - TEVPAIN - TIASAEPTVH STPTTEAIVN - TVDN PEASSESIASASETN - TAQVTSTEV

.sp|P33048|16-222 R - EQEEL - N VVGETVESL SSSEESITH - IN - K - K - IEK - F QSEEQQQTEDEL QDK - IH - PF AQAQSL - VY - PF TGPIPN - SL - PQN IL - PL - TQTPVVVPPF - L - QPEIM - GVPK VK - ETM - VPK - H - K - EM - PF - PK - Y PVEPF - TESQSL - TL - TDVEK - L - H - L PL - PL - VQSW - M H - QPPQPL - SPTVM - F - PPQSVL - SL SQPK - VL - PVPQK AVPQR - DM - PIQAF L - L - Y - QEPVL GPVR - GPF - PIL - V

.sp|P00712|20-142 EQL - TK - CEVF - QK - L - K - DL - K - DY GGVSL - PEW - VCTAF - H - TSGY DTQAIVQN - N DSTEY - GL - F - QIN N - K - IW - CK DDQN - PH - SR - N ICN - ISCDK - F - L DDDL - TDDIVCAK - K - IL - DK - VGIN - Y W - L - AH - K - AL CSEK - L - DQW - L CEK - L

.sp|P02756|19-180 IIVTQTM - K - GL DIQK - VAGTW - Y SL - AM - AASDISL - L - DAQSAPL - R - VY VEEL - K - PTPEGN L - EIL - L - QK - W EN - GECAQK - K IIAEK - TK - IPAVF - K - IDAL - N - EN - K VL - VL - DTDY - K - K - Y - L - L - F - CM EN - SAEPEQSL ACQCL - VR TPEVDK - EAL - EK - F - DK - AL - K - AL PM - H - IR - L - AF N - PTQL - EGQCH - V

Horse (Equus caballus)

From NCBI . NP_001075352.1M - K - L - L - IL - TCL - VAVAL - AR - PK - L - PH - R - QPEIIQN EQDSR - EK - VL - K - ER - K - F - PSF - AL - EY - IN - EL N - R - QR - EL - L - K - EK QK - DEH - K - EY - L IEDPEQQESSSTSSSEEVVPIN - TEQK - R - IPR - EDM - L -

From NCBI . NP_001164238.1 M - K - F - F - IF - TCL L - AVAL - AK - H - N M - EH - R - SSSEDSVN - ISQEK - F - K - QEK Y - VVIPTSK ESICSTSCEEATR - N IN - EM - ESAK - F PTER - EEK - EVEEK - H

.sp|P82187|21-185 EVQN QEQPTCH - K - N - DER - F - F DL - K - TVK - Y - IPIY - Y - VL - N - SSPR - Y - EPIY - Y - QH - R - L - AL - L - IN - N - QH - M - PY QY - Y - AR - PAAVR - PH VQIPQW - QVL - PN - IY - PSTVVR - H - PCPH - PSF - IAIPPK - K - L QEITVIPK - IN TIATVEPTPIPTPEPTVN - N -

.sp|Q9GKK3|16-241 R - EK - EEL - N VSSETVESL - SSN EPDSSSEESITH - IN K - EK - L - QK - F - K - H - EGQQQR - EVER - QDK - ISR - F VQPQPVVY - PY AEPVPY - AVVPQSIL PL - AQPPIL - PF - L -

.sp|P08334|1-123 K QF - TK - CEL - SQVL - K - SM - DGY - K GVTL - PEW - ICTIF H - SSGY - DTQTIVK N - N - GK - TEY - GL - F - QIN - N - K - M W - CR - DN - QIL PSR - N - ICGISCDK F - L - DDDL - TDDVM

.sp|P02758|19-180 TN - IPQTM - QDL DL - QEVAGK - W - H - SVAM - AASDISL - L - DSESAPL - R - VY IEK - L - R - PTPEDN L - EIIL - R - EGEN - K - GCAEK - K - IF AEK - TESPAEF - K IN - Y - L - DEDTVF -

Camel (Camelus dromedarius)

Y - QH - TL - EQL - R - R - L SK - Y - N - QL - QL - QAIH AQEQL - IR - M - K - EN SQR - K - PM - R - VVN QEQAY - F - Y - L - EPF QPSY - QL - DVY - PY - AAW - F - H - PAQIM - QH - VAY SPF - H - DTAK - L - IASEN SEK - TDIIPEW

- H - L - K - QL - N - K IN - QF - Y - EK - L - N F - L - QY - L - QAL - R QPR - IVL - TPW - DQTK - TGDSPF - IPIVN TEQL - F - TSEEIPK - K TVDM - ESTEVVTEK TEL - TEEEK - N - Y - L K - L - L - Y - Y - EK - F - TL - PQY - F - K - IVR QH - QTTM - DPR - SH R - K - TN - SY - QIIPVL -R-Y-F

AVIPDASSEF IIASTPETTTVPVTSPVVQK - L

QPEIM - EVSQAK ETIL - PK - R - K - VM - PF - L - K - SPIVPF SER - QIL - N - PTN GEN - L - R - L - PVH L - IQPF - M - H QVPQSL - L - QTL - M - L - PSQPVL SPPQSK - VAPF PQPVVPY - PQR DTPVQAF - L - L - Y QDPR - L - GPTGEL DPATQPIVAVH - N PVIV

- CAK - K - IL DSEGIDY - W - L - AH - K - PL - CSEK - L EQW - L - CEEL

AL - DTDY - K - N - Y - L - F - L - CM - K - N - AATPGQSL - VCQY L - AR - TQM VDEEIM - EK - F - R R - AL - QPL - PGR VQIVPDL - TR - M AER - CR - I

.sp|O97943|16-230 R - PK - Y PL - R - Y - PEVF - QN EPDSIEEVL - N - K - R - K - IL EL - AVVSPIQF - R - QEN - IDEL - K - DTR - N - EPTEDH - IM EDTER - K ESGSSSSEEVVSSTTEQK - DIL K - EDM - PSQR - Y - L - EEL H-R-L-N-K-Y-K-L-LQL - EAIR - DQK - L - IPR - VK L - SSH - PY - L - EQL - Y - R IN - EDN - H - PQL - GEPVK VVTQEQAY - F - H - L - EPF PQF - F - QL - GASPY - VAW Y - Y - PPQVM - QY - IAH PSSY - DTPEGIASEDGGK TDVM - PQW - W

.sp|O97944|16-193 K - H - EM - DQGSSSEESIN VSQQK - F - K - QVK - K VAIH - PSK - EDICSTF CEEAVR - N - IK EVESAEVPTEN - K - ISQF - Y - QK - W - K - F - L QY - L - QAL - H QGQIVM - N - PW - DQGK - TR - AY - PF - IPTVN TEQL SISEESTEVPTEESTEVF TK - K - TEL - TEEEK - DH - QK - F - L - N - K - IY QY - Y - QTF - L - W - PEY - L - K - TVY - QY - QK TM - TPW - N - H - IK - R Y-F

.sp|P79139|21-182 EVQN QEQPTCF - EK - VER - L - L - N EK - TVK - Y - F - PIQF - VQSR - Y - PSY - GIN - Y - Y - QH - R - L AVPIN - N - QF - IPY - PN - Y - AK - PVAIR - L - H - AQIPQCQAL - PN - IDPPTVER - R - PR - PR - PSF IAIPPK - K - TQDK - TVN - PAIN TVATVEPPVIPTAEPAVN TVVIAEASSEF ITTSTPETTTVQITSTEI

.sp|Q9TVD0|16-232 R - EK - EEF - K TAGEAL ESISSSEESITH - IN - K - QK - IEK - F - K IEEQQQTEDEQQDK IY - TF - PQPQSL - VY - SH - TEPIPY - PIL PQN - F - L - PPL QPAVM - VPF - L QPK - VM - DVPK - TK - ETIIPK - R - K - EM PL - L - QSPVVPF TESQSL - TL - TDL EN - L - H - L - PL - PL - L - QSL - M - Y QIPQPVPQTPM IPPQSL - L - SL - SQF - K - VL - PVPQQM VPY - PQR - AM PVQAVL - PF QEPVPDPVR - GL - H - PVPQPL - VPVIA

.sp|P00710|1-123 K QF - TK - CK - L SDEL - K - DM - N GH - GGITL - AEW ICIIF - H - M - SGY DTETVVSN - N - GN R - EY - GL - F - QIN N - K - IW - CR - DN EN - L - QSR - N ICDISCDK - F - L DDDL - TDDK - M CAK - K - IL - DK EGIDY - W - L - AH - K - PL - CSEK - L - EQW - QCEK - W

Camel milk does not contain β-lactoglobulin.

A, Alanine; R, Arginine; N, Asparagine; D, Aspartate; C, Cysteine; E, Glutamate; Q, Glutamine; G, Glycine; H, Histidine; I, Isoleucine; L, Leucine; K, Lysine; M, Methionine; F, Phenylalanine; P, Proline; S, Serine; T, Threonine; W, Tryptophan; Y, Tyrosine; V, Valine. The bold sequences were obtained from NCBI. NCBI: The National Center for Biotechnology Information.

150

CHAPTER 6:

In-silico methods for milk-derived bioactive peptide prediction

different enzymes on releasing BPs with a given function from a protein. For example, The AE score for ACE-I BPs in trypsin-digested κ-casein from bovine milk is 0.02, while the score is 0.04 when the casein is digested with simultaneous action of trypsin (EC 3.4.21.4), pepsin (EC 3.4.23.1), and chymotrypsin (EC 3.4.21.1). This means that if a researcher wants to increase ACE-I function of κ-casein hydrolysate, the simultaneous digestion should be used. The “W” parameter is another score that can be calculated by the following fraction: W5

The obtained BPs with given f unction af ter in 2 silico digestion of a protein potentially existing BPs with the same f unction in the protein ð6:1Þ

For instance, “W” score for trypsin-digested κ-casein of bovine milk is 0.02, while simultaneous digestion of the protein with trypsin (EC 3.4.21.4), pepsin (EC 3.4.23.1), and chymotrypsin (EC 3.4.21.1) increases the score to 0.07. Finally, evaluation of in silico digestion of proteins using A, AE, and W parameters introduces precious information. Researchers can use these information for the selection of protein(s) and enzyme(s) for in vitro experiments. In Table 6.3, the mentioned scores of milk proteins from different species were summarized for ACE-I and renin inhibitory peptides after calpain II (EC 3.4.22.53), chymotrypsin C (EC 3.4.21.2), trypsin (EC 3.4.21.4), trypsin 1 pepsin (EC 3.4.23.1), or trypsin 1 pepsin 1 chymotrypsin (EC 3.4.21.1) hydrolysis. As it shown in Table 6.3, milk proteins are good source of ACE-I (high A score), but not renin inhibitory peptides (low A score). Also, the collected data showed that digestion with calpain II and chymotrypsin C can retrieve ACE-I peptides from milk proteins better than trypsin, trypsin 1 pepsin, and/or trypsin 1 pepsin 1 chymotrypsin. Other than BIOPEPUWM database, there are many databases to find biological function(s) of obtained fragments from in silico digestion. For instance, FermFooDb (https://webs.iiitd.edu.in/raghava/fermfoodb/), YADAMP (http://yadamp.unisa.it/), and AHTPDB (http://crdd.osdd.net/raghava/ahtpdb/) databases contain fermented food
derived BPs, antimicrobial BPs, and antihypertensive BPs, respectively (Iwaniak et al., 2021). The prediction of BPs for the whole proteome of an organism cannot be done by the above mentioned steps because of its time-consuming nature. SpirPep (http://spirpepapp.sbi.kmutt.ac.th/Home.html.) is an in silico digestion-based platform and enables the researchers to discover BPs from the whole proteome of an organism. In this platform, the AA sequence of more than 3000 proteins can be entered simultaneously to discover BPs (Anekthanakul et al., 2018). There are limited numbers of BPs registered in BP databases. So, it is possible that a large number of retrieved peptides from in silico digestion are not found within these databases. For these peptides, another in silico strategy should be used to estimate their potential activity. PeptideRanker (http://

Table 6.3 The A, AE, and W scores of milk proteins from different species for ACE-I and renin inhibitory peptides after calpain II (EC 3.4.22.53), chymotrypsin C (EC 3.4.21.2), trypsin (EC 3.4.21.4), trypsin 1 pepsin (EC 3.4.23.1), or trypsin 1 pepsin 1 chymotrypsin (EC 3.4.21.1) hydrolysis. αs1-casein

Cow (Bos taurus)

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Function

Digested with

A

AE

W

A

AE

W

A

AE

W

A

AE

W

A

AE

W

A

AE

W

ACE-I

Trypsin

0.51

0.02

0.03

0.38

0.02

0.04

0.46

0.02

0.02

0.62

0.01

0.02

0.39

0.00

0.01

0.51

0.03

0.05

Trypsin, Pepsin

0.51

0.02

0.03

0.38

0.02

0.04

0.46

0.01

0.02

0.62

0.03

0.04

0.39

0.00

0.01

0.51

0.04

0.07

Trypsin, Pepsin, Chymotrypsin

0.51

0.03

0.04

0.38

0.02

0.05

0.46

0.04

0.07

0.62

0.02

0.03

0.39

0.04

0.11

0.51

0.05

0.09

Calpain II

0.51

0.06

0.10

0.38

0.05

0.11

0.46

0.07

0.12

0.62

0.05

0.06

0.39

0.05

0.12

0.51

0.04

0.07

Chymotrypsin C

0.51

0.07

0.10

0.38

0.03

0.07

0.46

0.05

0.10

0.62

0.11

0.14

0.39

0.03

0.07

0.51

0.07

0.09

Trypsin

0.02

0.00

0.00

0.02

0.00

0.12

0.02

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

Trypsin, Pepsin

0.02

0.00

0.19

0.02

0.00

0.25

0.02

0.00

0.24

0.01

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.25

Trypsin, Pepsin, Chymotrypsin

0.02

0.00

0.19

0.02

0.00

0.12

0.02

0.01

0.49

0.01

0.00

0.00

0.03

0.00

0.00

0.02

0.01

0.49

Calpain II

0.02

0.00

0.19

0.02

0.00

0.25

0.02

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

Chymotrypsin C

0.02

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

Trypsin

0.45

0.01

0.02

0.32

0.00

0.01

0.40

0.00

0.01

0.58

0.02

0.03

0.37

0.00

0.01

0.50

0.03

0.05

Trypsin, Pepsin

0.45

0.01

0.02

0.32

0.01

0.03

0.40

0.01

0.02

0.58

0.03

0.04

0.37

0.00

0.02

0.50

0.04

0.07

Trypsin, Pepsin, Chymotrypsin

0.45

0.03

0.04

0.32

0.03

0.07

0.40

0.03

0.06

0.58

0.02

0.03

0.37

0.04

0.12

0.50

0.05

0.09

Renin inhibitory

Buffalo (Bubalus bubalis)

αs2-casein

ACE-I

Renin inhibitory

Calpain II

0.45

0.06

0.10

0.32

0.05

0.11

0.40

0.04

0.08

0.58

0.05

0.06

0.37

0.04

0.12

0.50

0.04

0.07

Chymotrypsin C

0.45

0.07

0.11

0.32

0.04

0.08

0.40

0.05

0.11

0.58

0.10

0.13

0.37

0.03

0.08

0.50

0.05

0.09

Trypsin

0.02

0.00

0.00

0.03

0.00

0.09

0.02

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.02

0.00

0.00

Trypsin, Pepsin

0.02

0.00

0.19

0.03

0.00

0.18

0.02

0.00

0.24

0.01

0.00

0.00

0.00

0.00

0.00

0.02

0.00

0.25

Trypsin, Pepsin, Chymotrypsin

0.02

0.00

0.19

0.03

0.00

0.09

0.02

0.01

0.49

0.01

0.00

0.00

0.00

0.00

0.00

0.02

0.01

0.49

Calpain II

0.02

0.00

0.19

0.03

0.00

0.18

0.02

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.02

0.00

0.00

Chymotrypsin C

0.02

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.02

0.00

0.00

Continued

Table 6.3 The A, AE, and W scores of milk proteins from different species for ACE-I and renin inhibitory peptides after calpain II (EC 3.4.22.53), chymotrypsin C (EC 3.4.21.2), trypsin (EC 3.4.21.4), trypsin 1 pepsin (EC 3.4.23.1), or trypsin 1 pepsin 1 chymotrypsin (EC 3.4.21.1) hydrolysis. Continued αs1-casein

Human (Homo sapiens)

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Function

Digested with

A

AE

W

A

AE

W

A

AE

W

A

AE

W

A

AE

W

A

AE

W

ACE-I

Trypsin

0.3

0.00

0.01

-

-

-

0.37

0.00

0.00

0.53

0.01

0.01

0.34

0.00

0.02

-

-

-

Trypsin, Pepsin

0.3

0.01

0.03

-

-

-

0.37

0.00

0.01

0.53

0.03

0.04

0.34

0.00

0.01

-

-

-

Trypsin, Pepsin, Chymotrypsin

0.3

0.04

0.10

-

-

-

0.37

0.02

0.04

0.53

0.05

0.07

0.34

0.02

0.06

-

-

-

Calpain II

0.3

0.03

0.09

-

-

-

0.37

0.05

0.09

0.53

0.06

0.07

0.34

0.05

0.14

-

-

-

Chymotrypsin C

0.3

0.07

0.18

-

-

-

0.37

0.07

0.12

0.53

0.06

0.08

0.34

0.04

0.12

-

-

-

Trypsin

0.04

0.00

0.00

-

-

-

0.01

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.00

-

-

-

Trypsin, Pepsin

0.04

0.00

0.00

-

-

-

0.01

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.24

-

-

-

Trypsin, Pepsin, Chymotrypsin

0.04

0.00

0.00

-

-

-

0.01

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.24

-

-

-

Calpain II

0.04

0.00

0.12

-

-

-

0.01

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.24

-

-

-

Chymotrypsin C

0.04

0.00

0.00

-

-

-

0.01

0.00

0.00

0.01

0.00

0.00

0.03

0.00

0.00

-

-

-

Trypsin

0.28

0.01

0.05

0.20

0.00

0.00

0.31

0.00

0.00

0.26

0.00

0.02

0.34

0.00

0.00

0.37

0.00

0.00

Trypsin, Pepsin

0.28

0.02

0.08

0.20

0.01

0.04

0.31

0.01

0.02

0.26

0.01

0.03

0.34

0.01

0.04

0.37

0.00

0.01

Trypsin, Pepsin, Chymotrypsin

0.28

0.03

0.11

0.20

0.01

0.03

0.31

0.04

0.09

0.26

0.02

0.06

0.34

0.04

0.10

0.37

0.03

0.06

Renin inhibitory

Donkey (Equus asinus)

αs2-casein

ACE-I

Renin inhibitory

Calpain II

0.28

0.04

0.12

0.20

0.05

0.14

0.31

0.04

0.10

0.26

0.03

0.08

0.34

0.05

0.14

0.37

0.07

0.16

Chymotrypsin C

0.28

0.05

0.16

0.20

0.06

0.18

0.31

0.06

0.13

0.26

0.05

0.13

0.34

0.03

0.08

0.37

0.03

0.08

Trypsin

0.03

0.00

0.00

0.01

0.00

0.00

0.01

0.00

0.00

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.00

Trypsin, Pepsin

0.03

0.00

0.22

0.01

0.00

0.00

0.01

0.00

0.25

0.01

0.00

0.00

0.02

0.00

0.33

0.02

0.00

0.00

Trypsin, Pepsin, Chymotrypsin

0.03

0.00

0.11

0.01

0.00

0.11

0.01

0.00

0.25

0.01

0.00

0.00

0.02

0.00

0.33

0.02

0.00

0.00

Calpain II

0.03

0.01

0.33

0.01

0.00

0.22

0.01

0.00

0.00

0.01

0.00

0.00

0.02

0.00

0.33

0.02

0.00

0.00

Chymotrypsin C

0.03

0.00

0.00

0.01

0.00

0.00

0.01

0.00

0.00

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.00

Sheep (Ovis aries)

ACE-I

Renin inhibitory

Goat (Capra hircus)

ACE-I

Renin inhibitory

Trypsin

0.41

0.00

0.00

0.34

0.01

0.04

0.40

0.00

0.01

0.46

0.01

0.02

0.36

0.00

0.01

0.49

0.03

0.05

Trypsin, Pepsin

0.41

0.01

0.01

0.34

0.02

0.05

0.40

0.01

0.02

0.46

0.03

0.05

0.36

0.00

0.01

0.49

0.04

0.07

Trypsin, Pepsin, Chymotrypsin

0.41

0.03

0.05

0.34

0.03

0.08

0.40

0.04

0.07

0.46

0.03

0.05

0.36

0.04

0.12

0.49

0.05

0.09

Calpain II

0.41

0.05

0.09

0.34

0.05

0.12

0.40

0.04

0.08

0.46

0.06

0.09

0.36

0.04

0.12

0.49

0.04

0.08

Chymotrypsin C

0.41

0.06

0.11

0.34

0.05

0.12

0.40

0.05

0.10

0.46

0.09

0.14

0.36

0.03

0.08

0.49

0.06

0.10

Trypsin

0.02

0.00

0.00

0.02

0.00

0.11

0.01

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Trypsin, Pepsin

0.02

0.00

0.16

0.02

0.00

0.11

0.01

0.00

0.33

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Trypsin, Pepsin, Chymotrypsin

0.02

0.00

0.16

0.02

0.00

0.11

0.01

0.01

0.66

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.33

Calpain II

0.02

0.00

0.16

0.02

0.00

0.11

0.01

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Chymotrypsin C

0.02

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Trypsin

0.38

0.00

0.00

0.35

0.01

0.04

0.38

0.00

0.01

0.46

0.01

0.02

0.37

0.00

0.01

0.50

0.03

0.05

Trypsin, Pepsin

0.38

0.01

0.02

0.35

0.02

0.05

0.38

0.01

0.03

0.46

0.03

0.05

0.37

0.01

0.03

0.50

0.04

0.07

Trypsin, Pepsin, Chymotrypsin

0.38

0.03

0.06

0.35

0.04

0.09

0.38

0.04

0.08

0.46

0.03

0.05

0.37

0.05

0.13

0.50

0.05

0.09

Calpain II

0.38

0.05

0.10

0.35

0.05

0.11

0.38

0.04

0.09

0.46

0.06

0.09

0.37

0.04

0.11

0.50

0.04

0.08

Chymotrypsin C

0.38

0.06

0.11

0.35

0.05

0.12

0.38

0.04

0.09

0.46

0.09

0.15

0.37

0.04

0.09

0.50

0.06

0.10

Trypsin

0.02

0.00

0.00

0.02

0.00

0.12

0.01

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Trypsin, Pepsin

0.02

0.00

0.16

0.02

0.00

0.12

0.01

0.00

0.33

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Trypsin, Pepsin, Chymotrypsin

0.02

0.00

0.16

0.02

0.00

0.12

0.01

0.01

0.66

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.33

Calpain II

0.02

0.00

0.16

0.02

0.00

0.12

0.01

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Chymotrypsin C

0.02

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

0.01

0.00

0.00

0.00

0.00

0.00

0.01

0.00

0.00

Continued

Table 6.3 The A, AE, and W scores of milk proteins from different species for ACE-I and renin inhibitory peptides after calpain II (EC 3.4.22.53), chymotrypsin C (EC 3.4.21.2), trypsin (EC 3.4.21.4), trypsin 1 pepsin (EC 3.4.23.1), or trypsin 1 pepsin 1 chymotrypsin (EC 3.4.21.1) hydrolysis. Continued αs1-casein

Horse (Equus caballus)

κ-casein

β-casein

α-lactalbumin

β-lactoglobulin

Function

Digested with

A

AE

W

A

AE

W

A

AE

W

A

AE

W

A

AE

W

A

AE

W

ACE-I

Trypsin

0.28

0.01

0.05

0.23

0.00

0.01

0.33

0.00

0.00

0.28

0.00

0.01

0.34

0.00

0.00

0.33

0.01

0.02

Trypsin, Pepsin

0.28

0.03

0.10

0.23

0.02

0.06

0.33

0.00

0.00

0.28

0.01

0.03

0.34

0.01

0.04

0.33

0.01

0.02

Trypsin, Pepsin, Chymotrypsin

0.28

0.05

0.14

0.23

0.02

0.06

0.33

0.01

0.03

0.28

0.02

0.05

0.34

0.04

0.10

0.33

0.02

0.05

Calpain II

0.28

0.05

0.14

0.23

0.06

0.15

0.33

0.04

0.08

0.28

0.04

0.09

0.34

0.05

0.14

0.33

0.07

0.17

Chymotrypsin C

0.28

0.06

0.17

0.23

0.06

0.15

0.33

0.06

0.13

0.28

0.06

0.14

0.34

0.03

0.08

0.33

0.04

0.11

Trypsin

0.03

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.20

Trypsin, Pepsin

0.03

0.00

0.24

0.02

0.00

0.00

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.33

0.01

0.00

0.00

Trypsin, Pepsin, Chymotrypsin

0.03

0.00

0.12

0.02

0.00

0.09

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.33

0.01

0.00

0.00

Calpain II

0.03

0.00

0.00

0.02

0.00

0.18

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.33

0.01

0.00

0.00

Chymotrypsin C

0.03

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

0.02

0.00

0.00

0.02

0.00

0.00

0.01

0.00

0.00

Trypsin

0.34

0.00

0.02

0.25

0.00

0.00

0.36

0.01

0.02

0.31

0.00

0.01

0.31

0.00

0.00

-

-

-

Trypsin, Pepsin

0.34

0.03

0.07

0.25

0.01

0.02

0.36

0.01

0.03

0.31

0.03

0.05

0.31

0.01

0.04

-

-

-

Trypsin, Pepsin, Chymotrypsin

0.34

0.01

0.03

0.25

0.03

0.08

0.36

0.03

0.07

0.31

0.05

0.10

0.31

0.06

0.18

-

-

-

Renin inhibitory

Camel (Camelus dromedarius)

αs2-casein

ACE-I

Renin inhibitory

Calpain II

0.34

0.05

0.11

0.25

0.03

0.08

0.36

0.05

0.10

0.31

0.05

0.09

0.31

0.04

0.13

-

-

-

Chymotrypsin C

0.34

0.07

0.16

0.25

0.06

0.14

0.36

0.04

0.08

0.31

0.08

0.16

0.31

0.03

0.09

-

-

-

Trypsin

0.02

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.00

-

-

-

Trypsin, Pepsin

0.02

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.24

-

-

-

Trypsin, Pepsin, Chymotrypsin

0.02

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.16

0.02

0.00

0.14

0.03

0.00

0.24

-

-

-

Calpain II

0.02

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

0.03

0.01

0.50

-

-

-

Chymotrypsin C

0.02

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.00

0.02

0.00

0.00

0.03

0.00

0.00

-

-

-

ACE-I, Angiotensin-converting-enzyme inhibitor

6.2 Methods of milk-derived bioactive peptide prediction

distilldeep.ucd.ie/PeptideRanker/) tool evaluates the likelihood of the fragments’ bioactivity. The tool provides a score between 0 and 1 for each peptide where “0” represents the least and “1” represents the most likelihood of bioactivity. This tool does not report the exact function and only indicates tendency of fragments to be bioactive (Mooney et al., 2012). On the other hand, the most important in silico method for the functional assessment of peptides with unknown activity is molecular docking simulation.

6.2.3

Molecular docking simulation

As mentioned above, for functional assessment of peptides retrieved from in silico digestion which have unknown function, molecular docking is the preferred method before in vitro evaluation. All BPs exert their function(s) by interacting with the body’s biomolecules. Antihypertensive BPs inhibit ACE and/or renin, while antidiabetic peptides inhibit DPP-IV. In this kind of simulation, the exact target(s) should be defined. For instance, if hypotensive function of a peptide is in question, the potential targets are ACE and renin. On the other hand, β-hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase is a target for hypocholesterolemic BPs. In addition, α-amylase, trypsin, etc. can be a target for the functional assessment of digestion-resistant peptides (Liang et al., 2021). The first step for this method is providing 3D structure of the targets. There are many ways to find these 3D structures. The most important way is searching the target in the Research Collaboratory for Structural Bioinformatics Protein Database (RCSB-PDB: https://www.rcsb.org/). The RCSB-PDB contains 3D structure of proteins mostly obtained from Nuclear Magnetic Resonance spectroscopy or X-ray in vitro. It is possible that the 3D structure of the selected target is not provided by the RCSB-PDB database (Burley et al., 2019). In this situation, the 3D structure simulation of the desired target(s) should be considered. Website of I-TASER (https://zhanggroup.org/I-TASSER/) is the most common and user-friendly source for 3D structure prediction (Yang et al., 2015; Zheng et al., 2021). On the other hand, some software such as MODELLER are used for 3D structure prediction of targets (Webb & Sali, 2014). In molecular docking, the 3D structure of the retrieved peptides or ligands should be predicted in addition to the 3D structure of the target(s). One of the most important sources of 3D structure of BPs is the data reported by Prasasty and Istyastono (2019) in which 3D structure of 168400 di, tri, and tertapeptides is provided. For 3D structure modeling and energy minimization of peptides, Gaussian (Meyer & Hauser, 2020), GROMACS (Frederix et al., 2011), and Molecular Operating Environment (MOE) software are mostly used (Prasasty & Istyastono, 2019). After providing 3D structure of target(s) and ligand(s), the molecular docking can be performed. Molecular docking can be performed online or by related

155

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In-silico methods for milk-derived bioactive peptide prediction

software. The FeptideDB (http://www4g.biotec.or.th/FeptideDB/index.php) is an online tool to assess ACE-I function of peptides. The ACE 3D structure is available in FeptideDB by default and the users only need to prepare the 3D structure (PDB file) of ligand(s) (Panyayai et al., 2019). The GalaxyPEPDOCK (https://galaxy.seoklab.org/index.html) is another tool for molecular docking. In this tool, it is mandatory to upload the PDB file of the target protein. But, for ligands uploading the related Text file is adequate (Ko et al., 2012). There are software-based tools in addition to the online tools for docking simulation. The most popular software for molecular docking are Auto-dock and MOE. Comparing docking scores (S score) of in silico retrieved peptides with an original inhibitor can provide an indicator for identifying BPs. For instance, Lisinopril is an original inhibitor for ACE and is used for treatment of hypertension. When S score for docking of Lisinopril and ACE is -12.6746, the peptides with S score near -12.6746 can be categorized as ACEI peptides (Minkiewicz et al., 2019). Prasasty and Istyastono (2019) determined the S score less than -11.5 as the cut-point for selection of ACE-I peptides. It should be noted that the reliability assessment must be performed before running the docking procedures. In this regard, the root mean square deviation value should be less than 2 A between the native and redocked ligands’ conformation. After screening the in silico digestion
retrieved peptides and finding peptides with the given function, human gastrointestinal absorption (HIA) and toxicity assessment should be done. The most important tools for HIA and toxicity assessment are SwissADME (http://www.swissadme.ch/) and ToxinPred (https://webs.iiitd.edu.in/raghava/toxinpred/algo. php), respectively (Minkiewicz et al., 2019).

6.3 In vitro confirmatory experiments after in silico prediction In this chapter, sequential steps for in silico prediction of BPs have been described. It has been suggested that in silico method is a cost-saving and simple method for the prediction of BPs. But, the obtained results must be confirmed by in vitro or in vivo experiments (Barati et al., 2020b). Majumder et al. in an in silico approach predicted that digestion of egg proteins with thermolysin and pepsin produces IRY, LKP, and IQY as the potent ACE-I BPs. It was surprising that pretreatment of the protein by reducing agents and in vitro digestion with pepsin and thermolysin leads to release IRYCT, LKPI, and IQYCA (Majumder & Wu, 2010). Dietary proteins lose most of their secondary, tertiary, and quaternary structures through the gastrointestinal tract. However, some features of secondary structures could not be lost easily. Disulfide bonds are an example of these features. Thus disulfide bonds

6.4 Estimation of bioactive peptide content in food items using in-silico methods

should be considered during in silico BP prediction. Ignoring disulfide bonds can be the most important challenge for in silico prediction of BPs. For BP prediction, primary structure (AA sequence) of a protein is used. However, in primary structure, posttranslational modifications such as disulfide bonds and glycosylated residues are not shown. By solving this challenge, BPs with new structures can be yielded (Barati et al., 2020a).

6.4 Estimation of bioactive peptide content in food items using in-silico methods It is highly required to develop an efficient method for quantification of BPs. This can provide a better understanding of BPs’ role in health and disease. Different types of studies including in vitro (Kwon et al., 2011), in vivo (Carrizzo et al., 2019), and meta-analysis (Fekete et al., 2015) have been conducted to evaluate the short-term effects of BPs on human health. However, there are so limited studies on the long-term effects of BPs. For investigation of the long-term effects of food ingredients, observational studies (cohort and case-control) are commonly used (Barati et al., 2020a). The longterm effect assessment by observational studies cannot be done without quantification of BPs’ content. For the first time, our research team predicted the BPs’ content of dairy products by introducing an in silico model (Barati et al., 2020b).The estimation is done by the following formula:  BP content of bovine milk 5  1

     A B 3 103 3 a 1 3 103 3 b A1 B1

           C D E F 3 103 3 c 1 3 103 3 d 1 3 103 3 e 1 3 103 3 f C1 D1 E1 F1

ð6:2Þ

where A1, A, and a are molecular weight (MW), concentration per 100-g milk, and the number of peptides obtained after in silico digestion with given function for αs1-casein. B1, B, and b are MW, concentration per 100-g milk, and the number of peptides obtained after in silico digestion with given function for αs2-casein. C1,C, and c are MW, concentration per 100-g milk, and the number of peptides obtained after in silico digestion with given function for β-casein. D1, D, and d are MW, concentration per 100-g milk, and the number of peptides obtained after in silico digestion with given function for κ-casein. E1, E, and e are MW, concentration per 100-g milk, and the number of peptides obtained after in silico digestion with given function for β-lactoglobulin. F1, F, and f are MW, concentration per 100-g milk, and the number of peptides obtained after in silico digestion with given function for α-lactalbumin.

157

Table 6.4 The predicted concentration of BPs in milk and milk proteins from different species.

Species

Unit

Bovine

mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein

Buffalo

Human

Sheep

Total peptide fragment

LMW peptides

HMW peptides

ACE inhibitory peptides

DPP-III inhibitory peptides

Antioxidant peptides

DPP-IV inhibitory peptides

Immunomodulatory peptides

6.61

3.90

2.70

1.38

0.14

0.33

1.61

0.12

192.19

113.55

78.63

40.13

4.31

9.69

47.01

3.49

188.86

112.25

76.61

38.92

4.35

9.35

42.96

4.07

212.36

121.52

90.84

47.45

4.07

11.74

71.42

0

7.48

4.35

3.13

1.47

0.13

0.32

2.20

0.12

189.49

110.24

79.24

37.26

3.33

8.27

55.74

3.25

188.39

110.37

78.01

36.13

3.18

7.91

54.31

3.73

211.53

118.83

92.70

47.78

4.33

11.59

70.90

0

0.14

0.08

0.05

0.02

0.002

0.006

0.03

0.0002

189.08

111.16

77.92

28.74

3.02

9.17

48.74

0.36

192.50

120.15

72.34

29.00

5.33

5.33

53.43

0.64

184.68

99.44

85.23

28.41

0

14.20

42.61

0

10.73

6.16

4.56

2.15

0.33

0.36

3.14

0.17

195.05

112.15

82.90

39.18

6.11

6.56

57.19

3.13

190.16

109.38

80.77

37.46

6.22

5.73

54.27

3.63

214.94

123.08

91.86

48.30

4.74

11.36

72.94

0

Goat

Camel

Donkey

Horse

mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein mmol per 100 g milk mmol per 100 g milk protein mmol per 100 g casein mmol per 100 g whey protein

5.78

3.29

2.48

1.20

0.13

0.14

1.81

0.16

193.89

110.63

83.26

40.56

4.56

4.93

60.92

5.65

191.98

109.24

82.74

39.67

4.66

4.36

60.45

6.41

215.20

125.27

89.92

49.26

3.48

9.52

66.67

0

4.30

2.57

1.73

0.89

0.16

0.08

1.00

0.01

176.97

105.79

71.18

36.77

6.59

3.35

41.20

0.81

169.42

97.93

71.49

31.29

7.70

2.75

37.63

0.94

221.76

152.46

69.30

69.30

0

6.93

62.73

0

1.92

1.05

0.86

0.38

0.07

0.09

0.47

0.02

183.02

100.45

82.56

36.61

6.97

9.44

45.21

2.03

180.48

109.10

71.37

43.94

8.74

10.41

65.08

4.92

196.51

100.30

96.21

33.53

6.12

9.32

33.24

0

2.75

1.65

1.00

0.37

0.03

0.08

0.79

0.03

180.25

108.32

71.93

24.56

2.36

5.51

51.76

2.50

171.07

103.60

67.46

21.02

0.59

5.19

55.45

3.64

200.49

118.77

81.71

32.32

6.25

6.25

43.75

0

ACE-I, Angiotensin-converting-enzyme inhibitors; DPP: Dipeptidyl peptidase; HMW, high molecular weight, i.e., peptides with more than three residues; LMW, Low molecular weight, i.e., di and tripeptides.

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Using the above mentioned formula, BP content of milk and milk proteins from different species was calculated and reported in Table 6.4. Although, the proposed model has its own limitations but it is the only available model for quantification of BPs’ content in food items, until now. Barati et al. have quantified the concentration of BPs in bovine dairy products by the above mentioned formula (Barati et al., 2020b). Then, they conducted two risk assessment studies on the association of bovine dairy-derived BPs with breast cancer (Jabbari et al., 2022) and hypertension (Barati et al., 2021). Their findings showed that the long-term consumption of the dairyderived BPs had an inverse association with breast cancer risk (Jabbari et al., 2022). On the other hand, there was an increased risk between the long-term consumption of bovine dairy-derived BPs with hypertension risk in a cohort population (Barati et al., 2021).

6.5

Conclusion and future perspective

In the current chapter the in silico methods for BPs prediction is described in detail. In silico simulation enables researchers to continue their research more purposeful. Also, by means of these simulations, researchers can reduce errors and spend less time and cost in their research. Even, some investigations such as BPs’ quantification are almost impossible without in silico simulation. BPs’ quantification is an important subject in nutrition research field and there is only a primary in silico work to predict BPs’ content of food items. Combining in silico and in vitro methods is the only way to provide optimal models for estimating the content of BPs in food items.

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In-silico methods for milk-derived bioactive peptide prediction

Meyer, R., & Hauser, A. W. (2020). Geometry optimization using Gaussian process regression in internal coordinate systems. The Journal of Chemical Physics, 152(8), 084112. Available from https://doi.org/10.1063/1.5144603. Midic, U., & Obradovic, Z. (2012). Intrinsic disorder in putative protein sequences. Proteome Science, 10(Suppl 1), S19. Available from https://doi.org/10.1186/1477-5956-10-S1-S19. Minkiewicz, I., & Darewicz, I. (2019). BIOPEP-UWM database of bioactive peptides: Current opportunities. International Journal of Molecular Sciences, 20(23), 5978. Available from https:// doi.org/10.3390/ijms20235978. Mooney, C., Haslam, N. J., Pollastri, G., Shields, D. C., & Kurgan, L. (2012). Towards the improved discovery and design of functional peptides: Common features of diverse classes permit generalized prediction of bioactivity. PLoS ONE, 7(10), e45012. Available from https://doi.org/10.1371/journal.pone.0045012. Panyayai, T., Ngamphiw, C., Tongsima, S., Mhuantong, W., Limsripraphan, W., Choowongkomon, K., & Sawatdichaikul, O. (2019). FeptideDB: A web application for new bioactive peptides from food protein. Heliyon, 5(7), e02076. Available from https://doi.org/10.1016/j.heliyon.2019. e02076. Parastouei, K., Jabbari, M., Javanmardi, F., Mahmoudi, Y., Barati, M., Khalili-Moghadam, S., et al. (2022). Estimation of bioactive peptide content of milk from different species using an insilico method. Amino Acids. Available from https://doi.org/10.1007/s00726-022-03152-6. Prasasty, V. D., & Istyastono, E. P. (2019). Data of small peptides in SMILES and threedimensional formats for virtual screening campaigns. Data in Brief, 27, 104607. Available from https://doi.org/10.1016/j.dib.2019.104607. Sievers, F., & Higgins, D. G. (2021). The clustal omega multiple alignment package. Methods in Molecular Biology (2231, pp. 3
16). Humana Press Inc. Available from https://doi.org/ 10.1007/978-1-0716-1036-7_1. Tripaldi, P., Pérez-González, A., Rojas, C., Radax, J., Ballabio, D., & Todeschini, R. (2018). Classification-based QSAR models for the prediction of the bioactivity of ACE-inhibitor peptides. Protein and Peptide Letters, 25(11), 1015
1023. Available from https://doi.org/10.2174/ 0929866525666181114145658. Vidal-Limon, A., Aguilar-Toalá, J. E., & Liceaga, A. M. (2022). Integration of molecular docking analysis and molecular dynamics simulations for studying food proteins and bioactive peptides. Journal of Agricultural and Food Chemistry, 70(4), 934
943. Available from https://doi. org/10.1021/acs.jafc.1c06110. Webb, B., & Sali, A. (2014). Protein structure modeling with MODELLER. Methods in Molecular Biology, 1137. Available from https://doi.org/10.1007/978-1-4939-0366-5_1. Xu, D., & Xu, Y. (2004). Protein databases on the internet. Current Protocols in Molecular Biology / Edited by Frederick M. Ausubel ... [et al.], 19. Available from https://doi.org/10.1002/ 0471142727.mb1904s68. Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., & Zhang, Y. (2015). The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12(1), 7
8. Available from https://doi. org/10.1038/nmeth.3213. Zheng, W., Zhang, C., Li, Y., Pearce, R., Bell, E. W., & Zhang, Y. (2021). Folding non-homologous proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods, 1(3), 100014. Available from https://doi.org/10.1016/j.crmeth.2021.100014.

CHAPTER 7

Production of bioactive peptides from bovine caseins Lin Zheng, Chenyang Wang and Mouming Zhao School of Food Science and Engineering, South China University of Technology, Guangzhou, P.R. China

7.1

Introduction

Casein is the major protein in bovine milk, accounting for B80% of the total milk protein. It is mainly composed of four unique proteins, including αs1, αs2, β, and κ-casein. They have open and flexible conformation and consist of hydrophilic and hydrophobic segments (Ranadheera et al., 2016). Due to its unique composition and properties, casein serves as an important ingredient in the food industry. Over the past two decades, casein has also been demonstrated to be a good source of bioactive peptides which are beneficial for human health. These peptide fragments are inactive within the parent protein but can be released by the action of proteolytic hydrolysis. The peptides are mainly obtained in three different ways: (1) hydrolysis by enzymes from plant or microorganism such as Alcalase, Neutrase, and papain; (2) degradation by digestive enzymes such as pepsin, trypsin, and chymotrypsin; and (3) fermentation by proteolytic starter cultures. To date, there have been many studies demonstrating the beneficial effects of casein hydrolysates in vitro and in vivo, such as antihypertensive, antioxidative, antidiabetic, and immunomodulatory activities (Healy et al., 2016; Ishida et al., 2011; Rousseau-Ralliard et al., 2010; Shazly et al., 2019). Additionally, a great number of casein-derived peptides have also been identified with diverse bioactivities depending on their amino acid composition and sequence (García-Tejedor et al., 2015; Nakamura et al., 1995). Generally, the length of these bioactive peptides varies from 2 to 20 amino acid residues. For instance, several angiotensin-converting enzyme (ACE) inhibitory peptides, including IPP, VPP, HLPLP, RYLGY, and AYFYPEL, have been identified and demonstrated to reduce blood pressure in vivo (Anadón et al., 2010; Nakamura et al., 1995; Sánchez-Rivera et al., 2016). In addition, peptides such as IPI, IPIQY, LPYPY, and VPYPQ containing a Pro residue at Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00007-2 © 2023 Elsevier Inc. All rights reserved.

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the P2 position of N-terminus are demonstrated to be dipeptidyl peptidaseIV (DPP-IV) inhibitors with the potential for prevention of type-2 diabetes (Nongonierma & Fitzgerald, 2014a; Zheng et al., 2019). At present, some casein-derived bioactive peptides have been developed as supplements in functional food and numerous products are already on the market. For example, the antihypertensive product Calpis sour milk (Calpis Food Industry Co., Ltd., Tokyo, Japan) contains the tripeptides, IPP and VPP. This work intends to provide an overview of the production of bioactive peptides from casein and the bioactive peptides most reported in the literature regarding antihypertensive, antidiabetic, and antioxidant peptides.

7.2 Production of bioactive peptides from bovine casein 7.2.1

Hydrolysis by enzymes from plant or microorganism

The most common way to produce bioactive peptides from casein is through enzymatic hydrolysis by commercial proteases from plant (e.g., papain, bromelain, and ficin) or microorganism (e.g., Alcalase, Neutrase, Protamex, and Flavourzyme). Generally, they are used singly or in combination to prepare casein hydrolysates. For example, Lacroix and Li-Chan (2012a) reported that sodium caseinate was hydrolyzed by 11 different commercially available proteases to produce DPP-IV inhibitory peptides, including Alcalase, Protease A “Amano” 2, bromelain, Corolase PP, Flavourzyme, Protease N “Amano” K, Protamex, Protin SD-NY10, thermolysin, Umamizyme K, and Validase BNPL. The sodium caseinate hydrolysates obtained by thermolysin and bromelain displayed the highest DPP-IV inhibitory activity (with 50% and 47% inhibition at 0.487 mg/mL, respectively), while the sodium caseinate hydrolysates with the lowest DPP-IV inhibitory activity (33%) were produced with Protease A “Amano” 2 and Protease N “Amano” K. Additionally, Neutrase, Protamex, Flavourzyme, or papain are also widely used to prepare DPP-IV inhibitory peptides or antioxidant peptides from bovine casein (Nongonierma, Lalmahomed, et al., 2017; Nongonierma, Maux, et al., 2017; Nongonierma, Mazzocchi, et al., 2017; Shazly et al., 2019; Zhang et al., 2016; Zheng et al., 2019). Among them, one of the most outstanding proteases is Alcalase, also called subtilisin A. It is a serine alkaline protease with broad specificity. It is widely used to prepare antioxidant peptides from casein (Rao et al., 2020; Shazly et al., 2019; Wang et al., 2016; Xie et al., 2014). For example, buffalo casein hydrolysate obtained by Alcalase and bovine casein hydrolysate obtained by trypsin showed the best antioxidant properties of hydrolysates (Shazly et al., 2019). However, Alcalase seemed to be less

7.2 Production of bioactive peptides from bovine casein

suitable to produce DPP-IV or ACE inhibitory peptides. Zheng et al. (2019) compared the DPP-IV inhibitory IC50 values of casein hydrolysates prepared by Alcalase, Neutrase, Protamex, bromelain, and papain and found that the casein hydrolysates obtained by Alcalase showed the lowest DPP-IV inhibitory activity with a highest IC50 value of 3.41 mg/mL, while the casein hydrolysates obtained by papain showed the highest DPP-IV inhibition with an IC50 value of 0.90 mg/mL. Additionally, few studies were reported to use Alcalase for the preparation of antihypertensive peptides. It might be the reason that the activity of antioxidant peptides depends more on the amino acid composition, such as some hydrogen or electron donors, that is, Tyr, Trp, Cys, and Met (Hernández-Ledesma et al., 2007; Zheng et al., 2016), while the activities of DPP-IV or ACE inhibitory peptides are not only related to amino acid composition but also related to the position of constitutive amino acids of the peptide sequence (Ding et al., 2021; Nongonierma & FitzGerald, 2019). Therefore the release of DPP-IV or ACE inhibitory peptides might require proteases with some more specific cleavage sites. However, Alcalase with a broad specificity could break many peptide bonds and generate protein hydrolysates with a high hydrolysis degree, making it less suitable to prepare DPP-IV or ACE inhibitory peptides. In addition to the type of proteases, the hydrolysis conditions including pH, temperature, enzyme:substrate ratio (E:S), and time are also important. Nongonierma and FitzGerald (2019) studied the in vitro DPP-IV inhibitory potential of bovine milk protein isolate (MPI) hydrolyzed with Neutrase 0.8L and found that temperature and time had an effect (P , .05), while E:S had no effect (P..05) on DPP-IV IC50 values. The hydrolysis conditions could affect the degree of hydrolysis (DH) and the release of bioactive peptides. Therefore the conditions are generally optimized to obtain casein peptides with desired DH and bioactivities. In order to obtain casein hydrolysates with better bioactivities and yield, some pretreatment approaches are also adopted, including thermal, high hydrostatic pressure, microwave, and ultrasound (Hu et al., 2017; Uluko et al., 2015). Uluko et al. (2015) found that the radical scavenging activity of milk protein concentrate hydrolysates was improved when thermal, microwave, and ultrasound pretreatments were used, especially ultrasound pretreatment. In addition to commercial proteases, some crude extracts or protease isolated from plants or microorganisms were also used to prepare ACE inhibitory or antioxidant peptides from casein. For example, Nascimento et al. (2021) purified an extracellular serine fungus protease produced by Acremonium sp. L1-4B isolated from the Antarctic continent to hydrolyze bovine and caprine sodium caseinate to prepare antioxidant and antihypertensive peptides. The use of aspartic proteases from stigmas of mature artichoke (Cynara scolymus L.) flowers to obtain hydrolytic enzymes (cinarases) in the production of antioxidant

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and ACE inhibitory peptides from bovine casein was also reported and the optimal hydrolysis conditions of artichoke flower extracts were also carried out (Bueno-Gavilá et al., 2020).

7.2.2

Degradation by digestive enzymes

Bioactive peptides can also be released from bovine casein by the action of digestive enzymes such as pepsin, trypsin, and chymotrypsin. Among them, the most prominent enzyme is trypsin which has been shown to release a large number of bioactive peptides, including ACE inhibitory (De Oliveira et al., 2020; Holder et al., 2013), DPP-IV inhibitory (Nongonierma, Maux, et al., 2017; Zhang et al., 2016), antioxidative, immunomodulatory, and anxiolytic-like peptides (dela Peña et al., 2016). For example, Nongonierma, Mazzochi, et al. (2017) studied the release of DPP-IV inhibitory peptides from bovine MPI during trypsin hydrolysis using a design of experiments approach and found that the generated MPI hydrolysates displayed relatively low DPP-IV IC50 values. Some known DPP-IV inhibitory peptides such as INNQFLPYPY, LPL, VPL, and YPVEPF from casein were identified within the MPI hydrolysates. Trypsin specifically cleaves at the carboxylic side of Lys and Arg, producing not very long peptides with basic Lys or Arg at C-terminus. For example, an anxiolytic decapeptide that corresponds to residues 91-100 (YLGYLEQLLR) of bovine αs1-casein could be released by trypsin (Laurent Miclo et al., 2001). Numerous preclinical and clinical studies have demonstrated the anxiolytic effects of industrial casein tryptic hydrolysate (Hafeez et al., 2021). However, in some circumstances, it seems unfavorable to utilize these specific proteases singly that produce a low DH in casein. Therefore a successive treatment with pepsin and pancreatin (a mixture of trypsin, chymotrypsin, elastase, etc.) in order to simulate gastrointestinal digestion has been widely used to produce bioactive peptides from bovine casein (Bottani et al., 2020; Contreras et al., 2013; Mudgil et al., 2021; Tagliazucchi et al., 2018; Zheng et al., 2019). For example, a β-caseinderived DPP-IV inhibitory peptide VPYPQ could be released efficiently from casein following hydrolysis by a combination of papain and in vitro digestion, reaching up to 3211.15 μg/g.

7.2.3

Proteolysis during fermentation

Proteolysis during fermentation is also an effective way to produce bioactive peptides from casein. To date, various ACE inhibitory peptides (such as IPP, VPP, LHLPLP, and HLPLP) have been produced by fermentation (GarcíaTejedor et al., 2015; Miclo et al., 2012; Rojas-Ronquillo et al., 2012). Casein fermentation is mainly catalyzed by lactic acid bacteria (LAB) for the production of bioactive peptides. LAB are able to synthesize cell-surface proteases to

7.3 Bioactivities of bovine casein peptides

hydrolyze casein during fermentation and subsequently release various peptides into the medium (Chai et al., 2020). Rojas-Ronquillo et al. (2012) determined inhibition of ACE and antithrombotic properties of peptides released from bovine caseins during fermentation by Lactobacillus casei Shirota and Streptococcus thermophilus and found that both species released ACE inhibitory peptides, whereas only L. casei Shirota produced antithrombotic activity (Rojas-Ronquillo et al., 2012). In addition to LAB, yeast strains are also used for fermentation. García-Tejedor et al. (2013) produced casein-derived antihypertensive hydrolysates by yeasts. In this study, the potential of 20 dairy yeast strains belonging to Debaryomyces hansenii, Kluyveromyces lactis, and Kluyveromyces marxianus species was examined for the production of antihypertensive hydrolysates and permeate fractions with MW , 3 kDa of four casein hydrolysates with high ACE inhibition were demonstrated to exert in vivo antihypertensive effect in spontaneously hypertensive rats.

7.3 7.3.1

Bioactivities of bovine casein peptides Antihypertensive activity

Hypertension is a leading risk factor of cardiovascular diseases, characterized by elevated blood pressure with systolic blood pressure $ 140 mmHg and/or diastolic blood pressure $ 90 mmHg, at rest (Xie et al., 2021). ACE is the key target for developing antihypertensive agents. Casein-derived ACE inhibitory peptides are displayed in Table 7.1. Some ACE inhibitory peptides have been demonstrated to display antihypertensive activity in vivo. Caseinderived ACE inhibitory peptides were first discovered in tryptic hydrolysates from bovine αs1-casein (Yamamoto et al., 1994). Ile-Pro-Pro (IPP) and ValPro-Pro (VPP) are two most well-known food-derived antihypertensive peptides. IPP is located in both β- and κ-caseins (fragments 74-76 and 108-110, respectively), whereas VPP exists only in β-casein (fragments 84-86). IPP and VPP were initially identified as ACE inhibitory peptides (Nakamura et al., 1995). These two peptides were further shown to reduce blood pressure in hypertensive subjects using double-blind, placebo-controlled trials focusing on the intervention (Cicero et al., 2010; Ishida et al., 2011). IPP and VPP were initially identified from sour milk fermented by Lactobacillus helveticus and Saccharomyces cerevisiae (Nakamura et al., 1995). Not until 2007, IPP and VPP were also identified in Swiss cheese (Miguel et al., 2007). The use of microbial enzymes to produce IPP and VPP is limited. Mizuno et al. (2004) further purified an endopeptidase and an aminopeptidase from Sumizyme FP, which is capable of releasing IPP from β-casein. These two enzymes were identified as neutral protease I (NP I) and leucine aminopeptidase (LAP). Among them NP I first cleaved the X-Pro-Pro (X refers to any

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Table 7.1 Antihypertensive peptides from bovine casein. Casein type αS1-casein

αS2-casein

Peptide sequence

IC50 of ACE inhibition (µM)

Activity in vivo

References

FFVAPFPEVFGK TTMPLW AYFYPE

18.0 12.0 836.2

/ / /

Tauzin et al. (2002)

YKVPQL

22.0

/

RYLGY

0.71

AYFYPEL

6.58

RY RYL RYLG YLGY LGY FYPEL TYKEE

54.4 106.6 224.7 41.9 21.5 80.6 83.0

Spontaneously hypertensive rats Spontaneously hypertensive rats / / / / / / /

YQKFPQY

20.08

NMAINPSK ALNEINQFY ALNEINQFYQK FPQYLQY FALPQY FALPQYLK TVY ITP WIQP YQK FPQY TKVIP

60.0 219.0 264.0 14.0 4.3 4.3 15.0 14.9 14.2 312.2 300.7 400.0

AMKPW

580.0

MKPWIQP

300.0

AMKPWIQP

600.0

MKP

0.3

Spontaneously hypertensive rats / / / / / / / / / / / Spontaneously hypertensive rats Spontaneously hypertensive rats Spontaneously hypertensive rats Spontaneously hypertensive rats Spontaneously hypertensive rats

Yamamoto et al. (1994) Maeno et al. (1996) Contreras et al. (2009)

Contreras et al. (2013)

Donkor et al. (2007) Contreras et al. (2009) Tauzin et al. (2002)

Norris et al. (2015) Contreras et al. (2013) Maeno et al. (1996)

Yamada et al. (2013)

Continued

7.3 Bioactivities of bovine casein peptides

Table 7.1 Antihypertensive peptides from bovine casein. Continued Casein type

Peptide sequence

IC50 of ACE inhibition (µM)

β-casein

KVLPVPQ

1000

LQSW

500

YQEPVL RINKK SLPQN LQP MAP KYPVEPFTESQSLTL SKVLPVPQ PPQSVLSLSQSKVLPVPQ RDMPIQAF LLYQQPVLGPVRGPFPIIV DELQDKIHPFAQTQSLVYPFPGPIPNS VLGPVRGPFP LHLPLP VRGPFPIIV LHLPLPL LVYPFPGPIPNSLPQNIPP VPP IPP

β-casein/ κ-casein κ-casein

Activity in vivo

References Maeno et al. (1996)

45.5 79.3 29.5 1617.2 0.8 338.1 39 476.1 209 442.4 121.6 137.0 5.5 599.0 425.0 5.2 9.0 5.0

Spontaneously hypertensive rats Spontaneously hypertensive rats / / / / / / / / / / / / / / / / / /

ARHPH

59.5

/

RYPSYG DERF MAIPPKK

400.6 118.8 /

/ / Spontaneously hypertensive rats

Donkor et al. (2007) Jiang et al. (2010)

amino acids) sequence from C-terminus of β-casein, then LAP processed IPPcontaining peptide into IPP afterward. In addition, the VPP and IPP precursors in β-casein were Val-Pro-Pro-Phe (VPPF) and Ile-Pro-Pro-Leu (IPPL), in which C-terminal residues can be successfully cut off by pancreatic carboxypeptidase A (Rutella et al., 2016). The ACE inhibitory activity of peptides was confirmed in vivo by i.v. administration to rats, but larger amounts were required in comparison with in vitro studies (López-Fandiño et al., 2006). This indicated that these peptides are easily metabolized in vivo, as there has been disagreement between in vitro and in vivo studies of other ACE

Donkor et al. (2007) Tonouchi et al. (2008) Yamamoto et al. (1994)

Tauzin et al. (2002)

Nakamura et al. (1995)

Miguel et al. (2007)

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inhibitory peptides. LHLPLP was another famous ACE inhibitory peptide, corresponding to β-casein f(133-138), that was identified as one of the major peptides responsible for the ACE inhibitory activity of fermented milk products (Quirós et al., 2007). Different from IPP and VPP, LHLPLP was hydrolyzed by cellular peptidases to peptide HLPLP before the transport across the intestinal epithelium using Caco-2 cells. After both intravenous and oral administration, HLPLP could be degraded into smaller fragments LPLP and HLPL. Furthermore, HLPLP and all its possible derived peptides showed potent antihypertensive activity in spontaneously hypertensive rats (Sánchez-Rivera et al., 2016). It suggested that the antihypertensive effect of HLPLP is due to the concomitant action of the parent peptide and several novel derived fragments.

7.3.2

Antidiabetic activity

Casein contains a large number of Pro residues, which increases the vulnerability to cleavage by DPP-IV and potential for the release of peptides with DPP-IV inhibitory activity. Using an in silico approach, caseins from cow’s milk appeared to be the richest potential sources of DPP-IV inhibitors in the 34 proteins by comparative potency of dietary proteins as a source of DPP-IV inhibitory peptides (Lacroix & Li-Chan, 2012b). Among them, bovine κ-casein displayed the highest potency index for DPP-IV inhibitory peptides (Nongonierma & Fitzgerald, 2014b). Response surface methodology was used to optimize the release of DPP-IV inhibitory peptides during hydrolysis of bovine casein. Caseinate hydrolysates were generated with Protamex (a bacillus proteinase), while hydrolysis time had the highest influence on DPP-IV inhibitory properties (Nongonierma, Maux, et al., 2017). The ability of proteases to produce DPP-IV inhibitory peptides was also evaluated, and the highest in vitro DPP-IV inhibitory activity was observed in the hydrolysate of sodium caseinate by bromelain compared to thermolysin, pepsin, trypsin, and pancreatin (Hsieh et al., 2016). The DPP-IV inhibition peptides from casein was displayed in Table 7.2. LPQNIPPL from water-soluble extract of a Gouda-type cheese showed the highest DPP-IV inhibitory activity. The oral administered of LPQNIPPL reduced postprandial area under the blood glucose curve in mice (Uenishi et al., 2012). FLQP, the potent DPP-IV inhibitors, were identified using theoretical digestion of caseins with a prolyl oligopeptides activity (Nongonierma & FitzGerald, 2013). In silico digestion of milk protein-derived peptides with gastrointestinal enzyme activities was used to predict the release of peptides with a Pro residue at position 2 from the N-terminus. Five casein-derived peptides (IPIQY, LPLPL, YPYY, LPYPY, and IPI) generated with gastrointestinal enzymes showed DPP-IV inhibition activity (Nongonierma & Fitzgerald, 2014a). Furthermore, structure
activity relationship modeling of milk protein-derived peptides with DPP-IV

7.3 Bioactivities of bovine casein peptides

Table 7.2 Antidiabetic peptides from bovine casein.

Casein type

Peptide sequence

IC50 of DPP-IV inhibitory (µM)

αS1casein

VPL

15.8

WY

281

LW

993.4

IP

149.6

LP

712.5

YP

658.1

VPYPQ

41.45

VP VPY VPYP YPQ FLQP

262.5 104.37 123.89 271 65.3

DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity

LPVPQ




DPP-IV inhibitory activity

LPLPL LPL

325 241.4

LPP

563.3

LPPL

428.9

VLGP

580.4

LQP

1181.1

WIQP

237.3

VLGP

580.4

VR

826.1

DPP-IV inhibitory activity DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice DPP-IV inhibitory activity; the blood glucose levels in mice

αS1/αS2/ β/κ-casein

β-casein

Action mechanism

References postprandial

Umezawa et al. (1984)

postprandial

Nongonierma and Fitzgerald (2013) Nongonierma and FitzGerald (2013)

postprandial postprandial postprandial postprandial postprandial

Zheng et al. (2019)

postprandial

Nongonierma and FitzGerald (2013) Nongonierma and Fitzgerald (2016) Nongonierma and Fitzgerald (2014a)

postprandial

Nongonierma and Fitzgerald (2016)

postprandial postprandial

Nongonierma and FitzGerald (2013)

postprandial postprandial postprandial postprandial

Continued

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Table 7.2 Antidiabetic peptides from bovine casein. Continued

Casein type

β/κ-casein

Peptide sequence

IC50 of DPP-IV inhibitory (µM)

VPP




IPP




IPPKKNQDKTE




IPIQY YPYY LPYPY IPI YPY

35.2 194.4 108 3.5 243.4

Action mechanism

References

Enhancement of glucose uptake and activation of AMP-activated protein kinase in differentiated C2C12 myotubes Enhancement of glucose uptake and activation of AMP-activated protein kinase in differentiated C2C12 myotubes Improvement of insulin resistance in HepG2 cells DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity DPP-IV inhibitory activity; the postprandial blood glucose levels in mice

Iwasa et al. (2021)

Song et al. (2017) Nongonierma and Fitzgerald (2014a)

inhibitory activity was established, but there was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved (Nongonierma & Fitzgerald, 2016). Since glucose-dependent insulinotropic peptide and glucagon-like peptide 1 (GLP-1) are subject to rapid degradation by DPP-IV, the inhibition of DPPIV was associated with insulin and incretin release. Indeed, acute and longterm supplementation with casein hydrolysate in mice revealed a lowering in glucose and lipid and increase in insulin secretion (Drummond et al., 2018). Moreover, a hydrolyzed casein diet could promote Islet neogenesis to protect diabetic rats against developing diabetes (Wang et al., 2000), and the protection effect derived from alterations in both the target islet tissue and key cells of the gut immune system (Malaisse et al., 2000). In healthy subjects and patients with type-2 diabetes, ingestion of high doses of casein hydrolysate also stimulated insulin secretion (Jonker et al., 2011). It is very well demonstrated that protein is the most satiating component of food, which not only increases energy expenditure but also manifests reduction in energy intake through mechanisms that influence appetite control. Moreover, the increased insulin and GLP-1 not only helps in modifying the glycemic response but is also strongly associated with satiety and decreased food intake by

7.3 Bioactivities of bovine casein peptides

suppressing appetite. Many studies reported that casein and casein hydrolysate had a stronger effect on suppression of food intake and hunger. For example, casein showed strong effects on cholecystokinin (CCK) release and pronounced effects on GLP-1 release in STC-1 cells (Geraedts et al., 2011). Satiety hormones such as ghrelin, CCK, and peptide YY (PYY) are also directly or indirectly involved in the regulation of food intake. Moreover, α-casein and β-casein of casein were more beneficial in stimulating GLP-1 secretion (Gillespie & Green, 2016). Additionally, the encapsulation of starch with casein could decrease incretin hormone secretion and glucose release than starch alone (Bruen et al., 2012). Casein hydrolyzed by a chymosin enzyme, Maxiren 180 (enzyme activity: 180 International Milk Clotting Units (IMCU)/mL), also increased secretion of the satiety hormone via activating calcium signaling in the enteroendocrine cell line. Furthermore, administration of this hydrolysate to mice reduced the cumulative food intake over an 8-h period (O’Halloran et al., 2018), while postprandial blood glucose, plasma active GLP-1, amino acids, insulin, and food consumed were not significantly different in pigs that consumed this hydrolysate compared to sodium caseinate in a dairy beverage (Kondrashina et al., 2018). Casein gastrointestinal digests were also reported to increase CCK and GLP-1 secretion and expression in STC-1 cells. The release of GLP-1 was maximized with casein gastric digests, and casein jejunal digests behaved as more potent CCK inducers than in vitro casein gastrointestinal digests (SantosHernández et al., 2018). In addition, it was reported that gastric emptying of a casein hydrolysate does not differ compared to intact casein, and, thus, mechanisms other than gastric emptying might contribute to the satiety and appetite effects of casein and casein hydrolysates (Horner et al., 2019). Casein hydrolysate could also repair impaired intestinal barrier function which contributes to the prevention of type 1 diabetes in the diabetes-prone Bio-Breeding rat. Beneficial effect of hydrolyzed casein diet on intestinal barrier function was associated especially with gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) (Visser et al., 2012). Other antidiabetic mechanisms of casein hydrolysates have also been reported. Casein glycomacropeptide hydrolysates obtained with papain reduced hepatic insulin resistance in high-fat diet-fed C57BL/6J mice (Song et al., 2018), and the mechanism was involved in modulating gut microbiota (Yuan et al., 2020). Casein hydrolysate would also attenuate NLRP3 inflammasome
mediated IL-1β secretion in adipose tissue and improve obesity induced insulin resistance (Healy et al., 2016). A milk casein hydrolysate
derived peptide was reported to enhance glucose uptake through the AMP-activated protein kinase signaling pathway in skeletal muscle cells (Iwasa et al., 2021).

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7.3.3

Antioxidant activity

Caseins were recognized as an important source of peptides with an antioxidative potential, and a large number of casein-derived antioxidant peptides were prepared by animal and plant enzyme hydrolysis, gastrointestinal digestion, and microbial fermentation. All the subunits of casein were able to inhibit Fe-induced peroxidation in liposomal models, while α-casein had the strongest inhibitory effect (Cervato et al., 1999). Caseinophosphopeptides were also reported to inhibit lipid oxidation in oil-in-water emulsions and in ground beef via binding transition metals (Díaz et al., 2003; Díaz & Decker, 2004). Díaz et al. (2003) reported that casein hydrolysates obtained by trypsin were more effective inhibitors of lipid oxidation than the enriched caseinophosphopeptides. The in vitro gastrointestinal digestion model is often widely used to prepare antioxidant peptide fractions from casein. It was reported that peptides above 3000 Da were more easily digested by gastric digestion than those below 3000 Da, and the peptides below 1000 Da exhibited the best initial and surviving antioxidant activities in 2, 20 -azinobis (3ethylbenzothiszoline-6-sulfonic acid) diammonium salt (ABTS1), hydroxyl radical scavenging (OH) activities, and oxygen radical absorbance capacity (ORAC) values (Chen & Li, 2012). Moreover, amino acid composition of peptides affected the antioxidant activity of peptides. The acidic peptide fractions had higher bioavailability and a higher residual ratio of antioxidant activity (Wang et al., 2016). The high hydrophobic peptide fractions preferred remaining antioxidant activity, but poor digestive stability in simulated gastrointestinal digestion (Xie et al., 2015). Microbial fermentation was always used to produce antioxidant peptides from bovine casein. For example, the antioxidant activity of bovine casein hydrolysate was found after fermentation with Bifidobacterium longum KACC91563, while the ,3-kDa fraction exhibited the highest antioxidant activity compared with the other fractions in the ABTS assay (Chang et al., 2013). Similarly, chhurpi produced using Lactobacillus delbrueckii WS4 had a relatively higher yield, protein hydrolysis, and antioxidant activity as compared to other starter strains (Chourasia et al., 2022). Other microorganisms, such as Lactobacillus kefiri strain (Ayrancı et al., 2019) and a Bacillus metalloendopeptidase (PROTIN SDNY10), were also reported to produce antioxidant peptides after fermentation with bovine casein (Megrous et al., 2020). Antioxidative peptides from caseins are presented in Table 7.3. These peptides showed antioxidative activities via multiple reaction mechanisms, and several different antioxidant assays were always used to acquire comprehensive information about overall antioxidative capacity of the peptides. Common assays for evaluating antioxidant activity were establishing scavenging activity of free radicals include 1,1-diphenyl-2-picrylhydrazyl (DPPH), ABTS1, OH, ORAC, inhibition of lipid peroxidation, iron (Fe21) chelating

7.3 Bioactivities of bovine casein peptides

Table 7.3 Antioxidant peptides from bovine casein. Casein type αS1casein

αS2casein

β-casein

Peptide sequence YFYPEL EL PEL YPEL FYPEL YFYPEL

Action mechanism

References

21

RYLGY AYFYPEL RY RYL RYLG YLG YLGY LGY AYFYPE APSFSDIPNPIGSENSE

H2O2-Fe and DPPH radical scavenge activity H2O2-Fe21 and DPPH radical scavenge activity H2O2-Fe21 and DPPH radical scavenge activity H2O2-Fe21 and DPPH radical scavenge activity ORAC; H2O2-Fe21 and DPPH radical scavenge activity Superoxide anion, hydroxyl radical, and DPPH radical scavenging activity ORAC ORAC ORAC ORAC ORAC ORAC ORAC ORAC ORAC Mitigation of oxidative damage of Caco-2 cell

RYLGYLE YLGYLE YFYPEL YQLD FSDIPNPIGSEN FSDIPNPIGSE YFYP LHSMK YQKFPQY

Mitigation Mitigation Mitigation Mitigation Mitigation Mitigation Mitigation Mitigation ORAC

YQK FPQY QGPIVLNPWDQVKR

ORAC ORAC Mitigation of oxidative damage of Caco-2 cell

VKEAMAPK

Inhibition of enzymatic and non-enzymatic lipid peroxidation TEAC TEAC ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell; ABTS and DPPH radical scavenge activity; Mitigation of oxidative damage of Caco-2 cell ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell

HLPLP WSVPQPK AVPYPQR KVLPVPEK VPYPQR

of of of of of of of of

oxidative oxidative oxidative oxidative oxidative oxidative oxidative oxidative

damage of damage of damage of damage of damage of damage of damage of damage of

Caco-2 cell Caco-2 cell Caco-2 cell HepG2 cell HepG2 cell HepG2 cell HepG2 cell Caco-2 cell

Suetsuna et al. (2000)

Pihlanto (2006) Contreras et al. (2009) Contreras et al. (2013)

Tonolo, Fiorese, et al. (2020) Amigo et al. (2020)

Zhao et al. (2021)

Contreras et al. (2009) Contreras et al. (2013) Tonolo, Fiorese, et al. (2020) Pihlanto (2006) Hernández-Ledesma et al. (2007) Pihlanto (2006); Tonolo et al. (2018) Tonolo et al. (2019)

Continued

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Table 7.3 Antioxidant peptides from bovine casein. Continued Casein type

κ-casein

Peptide sequence

Action mechanism

References

VLPVPEK

ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell

EDELQDKIHPF

Mitigation of oxidative damage of Caco-2 cell

ARHPHPHLSFM

ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell ABTS and DPPH radical scavenging activity; Mitigation of oxidative damage of Caco-2 cell Mitigation of oxidative damage of Caco-2 cell

Pihlanto (2006); Tonolo et al. (2019) Tonolo, Fiorese, et al. (2020) Pihlanto (2006); Tonolo et al. (2018) Tonolo et al. (2019)

RHPHPHLSFM ARHPHPHLSF HPHPHLSFM NTVPAKSCQAQPTTM

Tonolo, Fiorese, et al. (2020)

activity, and reducing power. Pihlanto (2006) studied structure
activity relationship of casein-derived antioxidative peptides with free radical scavenging activity. Most of them were composed of 5
11 amino acids including hydrophobic amino acids, proline, histidine, tyrosine, or tryptophan in the sequence. Suetsuna et al. (2006) also reported four peptides (EL, PEL, YPEL, and FYPEL) separated from casein protein hydrolysate showed strong free radical scavenging activities, and EL sequence is important for the activity (Suetsuna et al., 2000). In addition, it is necessary to evaluate the antioxidant activity of peptide in a cellular environment. The four caseinderived peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM), previously recognized to possess in vitro antioxidant capabilities, were also able to prevent cell viability against oxidative stress induced by H2O2 or TbOOH in Caco-2 cells, causing a decrease of reactive oxygen species (ROS) production and increase of the thiol-related antioxidant enzymes, thioredoxin reductase and glutathione reductase (Tonolo et al., 2018). Similarly, six milk-derived antioxidant peptides obtained in vitro simulating gastrointestinal digestion were able to cross the apical membrane of Caco-2 cell layer and release in the basolateral compartment. These peptides also protected the cells against oxidative damage in Caco-2 cells (Tonolo et al., 2019). The key signal pathway activated by antioxidant peptides was studied. KVLPVPEK was reported to activate the nuclear factor erythroid-2-related factor 2 (Nrf2) nuclear translocation and triggered the overexpression of the antioxidant enzymes Trx1, TrxR1, GR, NQO1, and SOD1. Furthermore, molecular modeling shows that KVLPVPEK was able to hinder the interaction of Nrf2 with

7.3 Bioactivities of bovine casein peptides

Keap1 (Tonolo, Folda, et al., 2020). Similarly, four antioxidant peptides (YQLD, FSDIPNPIGSEN, FSDIPNPIGSE, and YFYP) were found to possess high DPPH scavenging ability. Moreover, the four peptides stimulated mRNA and protein expression of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as Nrf2, but decreased the production of ROS and malondialdehyde (MDA) in HepG2 cells (Zhao et al., 2021).

7.3.4

Other bioactivities

Biologically active peptides derived from casein have also been reported with other activities, including opioid-like function, immunostimulating activity, antimicrobial activities, and the ability to enhance mineral absorption. The common structural characteristics of endogenous opioid peptides are the presence of a Tyr residue at the N-terminus and the presence of other aromatic residue, Phe or Tyr, in the third or fourth position. Bovine caseins contain the analogous structure, which are a potential source of opioids peptide. Particularly, αS1-casein-derived peptides, such as YVPFP (αS1-casein-(158162)) (Kampa et al., 1996), YFYPEL (αS1-casein-(144-149)) (FernándezTomé et al., 2016), and YFYPE (αS1-casein-(144-148)) (Fernández-Tomé et al., 2016), could bind to opioid receptors and decrease the cell proliferation of breast cancer cells. However, other casein-derived peptides, the structures of which do not fulfill the requirements of opioid ligands, have also been demonstrated for regulation of mucin production in cells and in animals, such as the peptide β-CN f (94-123) found in yogurts and the derived fragments (94-108) and (117-123). Also, αS1-casein-derived peptide AYFYPEL, containing a residue different from Tyr at N-terminal position, could interact with opioid receptors (Laura Sánchez-Rivera et al., 2020). Research on various casein hydrolysates has shown that they possess immunoregulation activities. Stuknyte et al. (2011) reported that 3-kDaultrafiltered casein hydrolysates deriving from digestion with proteinases of Lactobacillus acidophilus ATCC 4356 and Lactococcus lactis subsp. lactis GR5 showed antiinflammatory activity by decreasing NF-κB activity in Caco-2 cells. Similarly, 5-kDa ultrafiltration permeate fractions of casein hydrolysates produced using different enzymes (Alcalase 2.4L, Prolyve 1000, Flavourzyme, TPCK-Trypsin, and Promod 144 MG) and gastrointestinal digestive enzymes (Mukhopadhya et al., 2015) displayed antiinflammatory effects. C-terminal sequence of bovine β-casein had a stimulatory effect on primed lymph node cells, and the corresponding peptides were identified, including β-casein (191-209) (Bonomi et al., 2011), β-casein (192209) (Coste et al., 1992), and β-casein (193-209) (Coste et al., 1992). Casein-derived peptides QEPVL and its derivative QEPV stimulated

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lymphocyte proliferation contributing to their antiinflammation activity (Jiehui et al., 2014). Two opioid peptides of β-casomorphin-7 (YPFPGPI) and β-casokinin-10 (YQQPVLGPVR) suppressed lymphocyte proliferation at lower concentrations but stimulated at higher concentrations (Kayser & Meisel, 1996). Similarly, two peptides (YG and YGG) with a Tyr residue at N-terminal position significantly enhanced the peripheral blood lymphocyte proliferation (Kayser & Meisel, 1996). However, whether peptides with opioid structure have immune-enhancing activity remains unknown. Peptides obtained during hydrolysis of casein have been shown to inhibit microorganism growth. Casecidin was the first antimicrobial peptides identified following chymosin digestion of casein at pH 6 or 7, which inhibited in vitro Staphylococci, sarcina, Bacillus subtilis, Diplococcus pneumoniae, and Streptococcus pyogenes (Lahov & Regelson, 1996). Isracidin, consisted of the Nterminal segment (1-23) of αs1-casein B, was demonstrated as antibiotics in the protection of mice against infection by Staphylococcus aureus strain Smith (Lahov & Regelson, 1996). Antimicrobial mechanisms of caseinderived peptides seem to be not only involved in direct antimicrobial effects but also related to host defense by immunomodulatory properties. Biopeptides could stimulate innate and adaptive immunity, producing a variety of inflammatory mediators for killing of microorganisms. Tryptic casein hydrolysate showed antimicrobial activity through nonspecific immunityenhancing effects. YQQPVLGPVRGPFPIIV, derived from bovine β-casein (residues 193-209), enhanced antimicrobial activity of macrophages by upregulating MHC class II antigen expression and increasing their phagocytic activity. A number of in vitro studies have shown the mineral-absorbing property of casein peptides, especially caseinophosphopeptides (CPPs). CPPs play their mineral-binding role due to the presence of the phosphoric group in the serine, which forms a polar acidic region that facilitates the isolation of divalent metals such as calcium, zinc, copper, manganese, and iron. The presence of SerP-SerP-SerP-Glu-Glu embedded in the bioactive peptide is a distinctive feature for all functional CPPs, and therefore CPPs always contain the peptides' fragment derived from β-casein-4P (1-25), αS1-casein-5P (59-79), αS2casein-4P (1-21), and αS2-casein-4P (46-70).

7.4

Conclusion and further perspectives

Bioactive peptides derived from bovine casein have been widely reported to display antioxidant, antidiabetic, antihypertensive, antibacterial, and anxiolytic activities. Their structure
activity relationships have also been studied extensively. However, there is still a high level of uncertainty on the role of

References

casein bioactive peptides in human health. Most of these bioactivities reported were studied in vitro, while evidence from in vivo or clinical studies is limited. More in vivo animal and clinical studies should be focused on to validate the findings from in vitro studies. Additionally, there is a need to identify and quantify bioactive peptides in biological tissues and fluids. It may help to provide sufficient scientific evidence to support healthpromoting effects of bioactive peptides. However, it is more challenging when compared to drug composition due to their low bioavailability. Some bioactive peptides can also be generated from endogenous proteins, which highlights the difficulty. With the development of mass spectrometry, it is easier to characterize peptide in hydrolysates. However, the comprehensive identification of short peptides (,6 amino acid residues) within complex casein hydrolysates is still challenging. Thus there is lack of adequate information on the identity of bioactive peptides within casein hydrolysates. Furthermore, although a large number of bioactive peptides have been identified and their activities have also been demonstrated, few studies focused on their content within the hydrolysates. Whether the activity of casein hydrolysates is attributed to these peptides is still unknown. It should be noted that casein hydrolysates consisted of numerous peptides, it may be infeasible to attribute a specific beneficial effect to a particular peptide. Furthermore, the release of some desired peptides is challenging due to the limited cleavage sites of commercially available proteases. There is a need to develop some more suitable proteases and peptidases by recombined enzyme technology. Some novel technologies such as membrane and chromatographic separation technique could also be applied to enrich active peptide fractions from casein hydrolysates. With the development of bioactive peptides concerning to in vivo effects, bioavailability, and production, more casein-derived bioactive peptides can be developed as supplements in functional food to promote health effects in human in the future.

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CHAPTER 8

Production of bioactive peptides from bovine whey proteins Marta Santos-Hernández, Thanyaporn Kleekayai and Richard J. FitzGerald Department of Biological Sciences, University of Limerick, Limerick, Ireland

8.1

Introduction

Whey protein is a coproduct of cheese making or from processes involving the coagulation of milk that contains a variety of proteins including β-lactoglobulin (β-lg), α-lactalbumin (α-la), bovine serum albumin (BSA), immunoglobulins, and other minor proteins with high nutritional value along with biological and functional properties. Bioactive peptides (BAPs) from milk proteins are of particular interest in food science and nutrition. These peptides may be involved in biological activities such as antidiabetic, antioxidative, antihypertensive, immunomodulatory, antiinflammatory, antimicrobial, along with their promotion of gut hormone release and their opiate properties. BAPs occur within the sequence of intact protein molecules and they can be released during in vivo gastrointestinal digestion or during fermentation. BAPs can also be released during in vitro hydrolysis with different enzymes arising from animal sources, from proteolytic microorganisms, or with plant and microbial proteases. Moreover, because whey is an ingredient in several food formulations, a whey-derived product rich in BAPs may incorporate significant added value to different food products. This chapter aims to provide an update of the methods used to (1) generate bovine whey protein
derived BAPs (enzymatic hydrolysis, hydrolysis during gastrointestinal digestion, fermentation, along with the recent developments in in silico aided enzymatic release of BAPs), (2) the characterization and identification techniques used to identify BAP sequences, and (3) the main bioactivities of whey protein
derived BAPs described to date, including whey protein
derived antidiabetic, antihypertensive, antimicrobial, antioxidant, anticancer, antiinflammatory, immunomodulatory, opioid-like, and satiety hormone-inducing peptides. 189 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00008-4 © 2023 Elsevier Inc. All rights reserved.

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8.2 Generation of whey protein
derived bioactive peptides 8.2.1

Enzymatic hydrolysis

Different proteolytic enzymes can be used to release peptides from whey proteins, such as digestive enzymes along with other animal, plant, and microbial proteases. These peptides may display a number of physiological roles and bioactive properties. Bacterial, digestive, and fungal enzymes such as Alcalase, chymotrypsin, Flavourzyme, Neutrase, and Protamex appear to have been the most used to date in the production of whey protein hydrolysates (WPHs) (Morais et al., 2015; O’Keeffe et al., 2017). Alcalase is a commercially available serine endopeptidase derived from Bacillus licheniformis; chymotrypsin is a digestive enzyme that hydrolyzes peptide bonds on the carboxyl side of aromatic (Phe, Tyr, and Trp) or nonpolar amino acids (Leu and Met) (Gauthier & Pouliot, 2003); Flavourzyme is a protease from Aspergillus oryzae with both exo- and endopeptidase activities (Merz et al., 2016); and Neutrase and Protamex (from Bacillus amyloliquefaciens and Bacillus subtilis, respectively) are also considered endopeptidases but with different specificities (Madsen et al., 1997). Table 8.1 shows some of the whey protein
derived BAPs reported to date and the enzymes used to release these sequences from their parent protein molecules. Recent studies evaluated the conditions under which Alcalase generates BAPs during whey protein hydrolysis. Mansinhbhai et al. (2021) optimized the hydrolysis conditions of Alcalase, that is, an enzyme:substrate (E:S) of 2% (v/w) during 8 h hydrolysis, in order to obtain an hydrolysate with high antioxidant activity. Some of the peptides identified following reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) separation and analysis were partially matched with sequences in the BIOPEP (https://biochemia. uwm.edu.pl/biopep-uwm/) and in the antihypertensive peptide (AHTPDB; http://crdd.osdd.net/raghava/ahtpdb/) databases having antioxidant and angiotensin-converting enzyme (ACE) inhibitory activity (Mansinhbhai et al., 2021). da Cruz et al. (2020) hydrolyzed bovine cheese whey proteins with Alcalase immobilized on a glyoxyl-corncob powder support and different peptide fractions were subsequently evaluated for their antioxidant and antimicrobial activities. An Alcalase hydrolysate of α-la contained the antioxidative fragment 56IWCKDDQNPH68 (Báez et al., 2021). Evaluation of the antioxidant and ACE inhibitory properties of different WPHs from double-cream cheese whey using different enzymes and hydrolysis conditions was performed previously (Bustamante et al., 2021). Different hydrolysates were generated using Alcalase, chymotrypsin, or Flavourzyme while varying the E:S and the incubation time. This study reported that the optimal

8.2 Generation of whey protein
derived bioactive peptides

Table 8.1 Example of whey-derived bioactive peptides identified following hydrolysis of whey protein starting materials with different enzymatic activities/preparations. Substrate

Protein

Enzyme activity

Sequence

Fragment

Bioactivity

References

Cheese whey

β-lg β-lg

Alcalase Chymotrypsin

KGL IRL

Antioxidant Antioxidant

Bustamante et al. (2021)

α-La Cheese whey

α-la β-lg β-lg

Alcalase Chymotrypsin Chymotrypsin

IWCKDDQNPH RVY HIRL

Antioxidant ACE inhibition ACE inhibition

Báez et al. (2021) Bustamante et al. (2021); Li et al. (2011)

β-lg β-lg

Chymotrypsin Chymotrypsin

LIVTQ VLDTDY

α-la β-lg

Flavourzyme Thermoase PC10F Thermoase PC10F

GVSLPEW LDIQKVAGTW

f (24-26) f (173175) f (59-68) f (56-58) f (146149) f (17-21) f (110115) f (39-45) f (9-19)

IQKVAGTW

f (11-19)

Thermoase PC10F Thermoase PC10F Thermoase PC10F

LKPTPEGDLEIL

f (46-57)

LKPTPEGDLE

f (46-55)

VLDTDY

f (94-99)

Thermoase PC10F Thermoase PC10F

LDTDY

f (95-99)

LKALPMH

f (140146)

LSFNPTQ LKGYGGVSLPE

f (149155) f (15-25)

GYGGVSLPEW

f (36-45)

WLAHKAL

f (104110)

Whey protein isolate

β-lg β-lg β-lg β-lg β-lg β-lg β-lg α-la α-la α-la

Thermoase PC10F Thermoase PC10F Thermoase PC10F Thermoase PC10F

ACE inhibition ACE inhibition ACE inhibition ACE inhibition ACE inhibition, DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition ACE inhibition, DPP-IV inhibition ACE inhibition

Lacroix et al. (2016)

Lacroix and Li-Chan (2014) Lacroix et al. (2016)

ACE inhibition, DPP-IV inhibition ACE inhibition DPP-IV inhibition ACE inhibition ACE inhibition, DPP-IV inhibition

Lacroix et al. (2016); Lacroix and Li-Chan (2014)

α-la, α-lactalbumin; β-lg, β-lactoglobulin; ACE, angiotensin-converting enzyme; DPP-IV, dipeptidyl peptidase-IV.

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conditions for the production of antioxidant and ACE inhibitory hydrolysates were at an E:S of 1:20 (w/w) for all the enzymes and an incubation period of 7, 2, and 6 h with Alcalase, chymotrypsin, and Flavourzyme, respectively. Moreover, some peptides identified by UHPLC-ESI-MS/MS analysis had features that may contribute to the observed antioxidant activity (Bustamante et al., 2021). The same authors hydrolyzed whey protein concentrate (WPC) using these three enzymes and evaluated the antioxidant and ACE inhibitory properties of the hydrolysates therefrom before and after simulated gastrointestinal digestion using the standardized INFOGEST protocol (Minekus et al., 2014). Bustamante et al. (2021) identified some peptides with antioxidant activity, which were previously described (Li et al., 2011). For instance, the α-laderived fragment 120INY122 in the Alcalase hydrolysate, the β-lg-derived fragments 173IRL175, 18IVTQTM23, 110VLDTDY116, and 17LIVTQTM23 in the chymotrypsin hydrolysate, and the β-lg-derived fragment 59VEEL62 in the Flavourzyme hydrolysate had features which were consistent with the three main structural features of antioxidant peptides (see Section 8.4.4). In addition, other peptides were previously identified as ACE-inhibiting peptides, that is, the β-lg-derived fragments 56RVY58, 146HIRL149, 17LIVTQ21, and 110VLDTDY115 identified in the chymotrypsin hydrolysate and α-la-derived 39GVSLPEW45 in the Flavourzyme hydrolysate (Lacroix et al., 2016; Mullally et al., 1997; Otte et al., 2007). An integrated enzymatic membrane reactor, which retained the enzyme Protamex or Corolase 2TS, was used to prepare dipeptidyl peptidase-IV (DPP-IV) inhibitory and radical scavenging hydrolysates, demonstrating that hydrolysis with Protamex produced hydrolysates having the highest DPP-IV inhibitory activity (O’Halloran et al., 2019). Combination of enzymes, that is, trypsin, Neutrase, papain, and Protease S during studies on the production of low-allergenic whey-derived products suggested that using trypsin with either Neutrase or papain was the most effective in the removal of β-lg and in producing low molecular mass peptides with reduced antigenic properties (Hyun et al., 2007). Other enzymes used for the production of BAPs from whey protein are the Cardosins (Tavares et al., 2012, 2011). Plant origin enzymes, such as Cardosin A and B, obtained from the dried flowers of cardoon (Cynara cardunculus L.) are interesting enzymes for the production of functional foods because of their natural origin, low cost, and ease of extraction. These studies identified peptides released during hydrolysis with cardosin having ACE inhibitory activity.

8.2.2

Hydrolysis during gastrointestinal digestion

In order to exert physiological effects in vivo, BAPs must be released prior to or during intestinal digestion of food proteins and then reach their target sites at the luminal side of the intestinal tract or after absorption in the peripheral organs. Therefore, in order to understand the metabolic and physiological

8.2 Generation of whey protein
derived bioactive peptides

consequences of the interaction between dietary proteins and the gastrointestinal tract, the effective in vivo release of peptides needs to be established. Milk protein
derived digestion peptides and, particularly, peptides released in the jejunum after ingestion of casein and whey protein in healthy human subjects were characterized (Boutrou et al., 2013). Following the identification of 105 peptides from β-lg and 85 from α-la in the human jejunum, some of these peptides were later investigated for their potential bioactive properties. For instance, 72RVYVEELKPTPEGDLEI56 was previously reported as an specific immunoglobin E (sIgE) binding peptide (Zenker et al., 2020), while 78 IPAVFK83 has ACE inhibitory activity (Power et al., 2014). Earlier studies also identified peptides released throughout gastrointestinal digestion, that is, peptides have been found in human jejunal samples (Sanchón et al., 2018) and in samples from pig duodenum (Egger et al., 2017). These studies developed a comparison of the proteomic and peptidomic profiles between the in vivo identified peptides and the peptides identified in simulated gastrointestinal digests following digestion using the INFOGEST protocol (Brodkorb et al., 2019). Similar proteomic and peptidomic profiles were found within the in vitro and the in vivo digested samples. Since in vivo studies are not always ethically and economically feasible, the use of in vitro digestion models mimicking the gastrointestinal tract has been proposed as an alternative to in vivo experiments. Peptides from β-lg were identified after in vitro gastric digestion with ACE inhibitory activity, whereas different peptide sequences were detected after complete digestion having, DPP-IV inhibitory, antioxidative, and IgE binding properties (Kopf-Bolanz et al., 2014). Peptides were identified following simulated gastrointestinal digestion of bovine whey proteins which were previously reported in human jejunum effluents, some of which possessed DPPIV inhibitory activity (Corrochano et al., 2019). The same authors identified peptides after 4 h of in vitro gastrointestinal digestion which were transported across a Caco-2/HT29 intestinal barrier, and which inhibited free radical formation in situ in muscle and liver cells (Corrochano et al., 2019). The antioxidant benefits to muscle cells of the peptides was investigated in a mouse muscle cell line (C2C12) and in hepatic (HepG2) cells. Peptides from β-lg 52GDLE55, 142ALPM145, α-la 99VGIN102, and BSA 568AVEGPK573 presented an antioxidant effect, and these peptides along with β-lg 43VEELKPT49 also promoted the secretion of interleukin (IL)-1β from stimulated macrophages. The bioactivity of some of these peptides was previously reported (Picariello et al., 2013). For instance, although peptide β-lg 43VEELKPT49 was transported across the intestinal barrier, this peptide was cytotoxic to HepG2 and C2C12 cells undermining its ability to reduce oxidative stress at 2.5 mM in HepG2 cells. β-Lg peptide 76TKIPAV81 was identified in an antioxidant fraction of infant formula postsimulated gastrointestinal digestion

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(Hernández-Ledesma, Quirós, et al., 2007). A simulated gastrointestinal digestion of bovine milk proteins was performed using pepsin and Pancreatin in order to identify peptides resistant to gastrointestinal digestion which were transported through the Caco-2 cell monolayer (Picariello et al., 2013). Trypsin has been used to prehydrolyze an α-la-rich WPC before in vitro simulated gastrointestinal digestion followed by evaluation of the DPP-IV inhibitory activity of the peptides generated (Jia et al., 2020). The generation of WPC hydrolysates was aided using a central composite design to optimize four conditions during hydrolysis with trypsin, that is, incubation temperature, substrate concentration, enzyme concentration and hydrolysis duration. After simulated gastrointestinal digestion of the intact protein substrate and the trypsin hydrolyzed digests, some peptides were identified by RP-HPLC-MS/MS showing a DPP-IV inhibitory effect in vitro, although their effectiveness would need to be confirmed following in vivo analysis.

8.2.3

Fermentation

BAPs can also be released during the manufacture of fermented dairy products as a result of the action of the different proteolytic systems present (including coagulants, starter, and nonstarter culture proteases, endogenous milk proteases, and the proteolytic activities of the secondary cultures), which take places during the ripening process (Ardö et al., 2017; Kleekayai et al., 2021). The majority of the BAPs released during fermentation reported to date are mainly derived from the caseins which represent the major proteins (B80% of total proteins) found in bovine milk. Furthermore, the flexibility within casein protein structures may promote their susceptibility to proteolysis in comparison to whey proteins which have more rigid and compact structures in their native state (Fox et al., 2015). This could explain the greater number of casein-derived peptides, as oppose to whey-derived peptides, identified in fermented dairy products reported to date. The enhancement of the biofunctional properties of fermented dairy products and the BAPs released have been associated with the release of proteolytic activity during the fermentation process (Virtanen et al., 2007). Lactic acid bacteria (LAB) are one of the most extensively used group of starter organisms for the development of a range of fermented dairy products, such as yoghurt, drinking yoghurt, and cheese. Cell envelope proteinases (CEPs) derived from LAB are considered as the main proteolytic activities responsible for the release of BAPs in fermented dairy products (Hafeez et al., 2014). The proteolytic activity of endogenous milk proteases, for example, plasmin, elastase, and cathepsin D, B, and G, has been also linked with the release of β-lg-derived BAPs in an unfermented whey protein isolate (WPI) sample (Ali et al., 2019). These peptides include the ACE inhibitor 87IIAEKTKIPA96, an

8.2 Generation of whey protein
derived bioactive peptides

anticancer peptide 1LIVTQTMK8, the antibacterial peptide 84IDALNENK91, and the antibacterial and DPP-IV inhibitor 92VLVLDTDYK100. In addition, the diversity in the genes encoding the proteolytic system of the starter culture used can affect growth rate, proteolytic activity, and the release of BAPs. The proteolytic systems in two Lactobacillus species, that is, Lactobacillus helveticus LH-2 and Lactobacillus acidophilus La-5, were characterized (Ali et al., 2019). They reported that the CEP and aminopeptidase activities in L. helveticus LH-2 was higher than that of L. helveticus La-5. This may be linked to the presence of different genes encoding the proteolytic systems, thereby resulting in different proteolytic cleavage patterns on the major bovine milk proteins (β-, αs1-, αs2-, κ-casein, and β-lg) between the two cultures. The bioactive properties of fermented dairy products reported to date are mainly associated with ACE inhibitory, antihypertensive, antimicrobial, and antioxidant activities. Table 8.2 summarized a number of whey-derived peptides possessing ACE inhibitor and hypotensive effects which have been identified in fermented dairy products such as WPI fermented with L. helveticus LH-2 and L. acidophilus La-5 (Ali et al., 2019), in commercial cheddar cheeses (Lu et al., 2016), in cheese whey fermented with Kluyveromyces marxianus isolated from Parmigiano Reggiano (Martini et al., 2021) or with L. helveticus PR4 (Pontonio et al., 2021), and in lactoferrin (LF) fermented with K. marxianus Km2 (García-Tejedor et al., 2014). A number of LAB species, for example, L. acidophilus, Levilactobacillus brevis, Ligilactobacillus animalis, Lactococcus lactis, L. helveticus, and Lactiplantibacillus plantarum, have been extensively used as a starter culture for the generation of ACE inhibitory peptides (Pontonio et al., 2021). The use of mixed strain LAB cultures and the encapsulation of bacterial cultures have been reported to improve the bioactive properties of fermented dairy products when compared to the use of a single strain and/or a cell-free approach. For instance, WPC incubated with encapsulated B. subtilis cells exhibited higher 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS1) scavenging (39.79%) and ACE inhibitory activities (40.27%) compared to the sample obtained using cell-free systems (having 32.35% and 34.65% antioxidant and ACE inhibition, respectively) (Alvarado Pérez et al., 2019). This approach provides benefits in relation to recycling of the bacterial culture, while still maintaining the proteolytic activity. Encapsulation of a commercial probiotic culture Lactoferm ABY 6 [containing Streptococcus salivarius ssp. thermophilus (80%), L. acidophilus (13%), Bifidobacteriu bifidum (6%), and Lactobacillus delbrueckii ssp. bulgaricus (1%)] with a tryptic hydrolyzed WPC-alginate showed an enhancement in the in vitro antioxidative properties [measured using 2,2-diphenyl-1-picrylhydrazyl

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Table 8.2 Examples of some whey protein
derived bioactive peptides identified in different fermented dairy products. Bioactive property

Whey protein

Sequencea

Fragment

β-lg

GLDIQKVAGT

f (9-18)

ACE inhibitory, antibacterial

GLDIQKVAGTW

f (9-19)

ACE inhibitory

LAMA

f (22-25)

ACE inhibitory

AASDISLLDAQSAPLR

f (25-40)

Antibacterial

AVF

f (80-82)

Antiinflammatory

VLVLDTDYK

f (92-100)

LLF

f (103-105)

DPP-IV inhibitory, antimicrobial ACE inhibitory

LF

f (104-105)

ACE inhibitory

IR

f (148-149)

ACE inhibitory

LF

f (52-53)

ACE inhibitory

YGL

f (50-52)

ACE inhibitory

DKVGINYW

f (97-104)

ACE inhibitory

LAHKAL

f (105-110)

ACE inhibitory

RHPYFYAPELLYYANK

f (168-183)

ACE inhibitory

FP

f (221-222)

ACE inhibitory

DAFLGSFLYEYSR

f (329-341)

ACE inhibitory

α-la

BSA

Sample

References

WPI fermented with Lactobacillus helveticus LH-2/Lactobacillus acidophilus La-5 WPI fermented with L. helveticus LH-2 Commercial cheddar cheeses WPI fermented with L. helveticus LH-2 Cheese whey fermented with Kluyveromyces marxianus isolated from Parmigiano Reggiano Cheese whey fermented with K. marxianus isolated from Parmigiano Reggiano Commercial cheddar cheeses Commercial cheddar cheeses Commercial cheddar cheeses Commercial cheddar cheeses Commercial cheddar cheeses, cheese whey fermented with K. marxianus isolated from Parmigiano Reggiano Cheese whey fermented with K. marxianus isolated from Parmigiano Reggiano Commercial cheddar cheeses Cheese whey fermented with L. helveticus PR4 Commercial cheddar cheeses Cheese whey fermented with L. helveticus PR4

Ali et al. (2019)

Ali et al. (2019) Lu et al. (2016) Ali et al. (2019) Martini et al. (2021)

Martini et al. (2021) Lu et al. (2016) Lu et al. (2016) Lu et al. (2016) Lu et al. (2016) Lu et al. (2016); Martini et al. (2021)

Martini et al. (2021) Lu et al. (2016) Pontonio et al. (2021) Lu et al. (2016) Pontonio et al. (2021)

Continued

8.2 Generation of whey protein
derived bioactive peptides

Table 8.2 Examples of some whey protein
derived bioactive peptides identified in different fermented dairy products. Continued Whey protein

Sequencea

Fragment

Bioactive property

LF

DPYKLRP

f (70-76)

Hypotensive

PYKLRP

f (71-76)

Hypotensive

YKLRP

f (72-76)

Hypotensive

GILRP

f (130-134)

Hypotensive

Sample

References

LF fermentation with K. marxianus Km2 LF fermentation with K. marxianus Km2 LF fermentation with K. marxianus Km2 LF fermentation with K. marxianus Km2

García-Tejedor et al. (2014) García-Tejedor et al. (2014) García-Tejedor et al. (2014) García-Tejedor et al. (2014)

α-la, α-lactalbumin; β-lg, β-lactoglobulin; ACE, angiotensin-converting enzyme; BSA, bovine serum albumin; DPP-IV, dipeptidyl peptidase-IV; LF, lactoferrin; WPI, whey protein isolate. a Peptide sequence with the one-letter code.

(DPPH) and ABTS1 scavenging and total reducing power assays] in a fermented whey beverage (Kruni´c & Rakin, 2022). This approach was also reported to improve probiotic stability over 28-day storage compared to other carriers, that is, alginate, alginate-chitosan, and alginate-WPC. In addition to the choice of microbial species and strains, other parameters, including type of substrate, fermentation conditions (e.g., fermentation temperature and duration), and the inclusion of growth supplements, have been reported to have a significant impact on the biofunctional properties and the BAPs released during fermentation. The impact of the bacterial culture used (L. helveticus strains T80, T105, B734, and DSMZ 20075, and L. acidophilus La-05) on the antioxidant activity of a fermented dairy product was studied (Skrzypczak et al., 2019). It was reported that the antioxidant potency of fermented products depended on the type of substrate [whole milk powder, α-la, casein glycomacropeptide (CGMP), WPI, and WPC (i.e., WPC30, WPC40, WPC60, WPC80)] and the bacterial strains used. The highest ABTS1 scavenging activity (95.86%), ferric reducing oxidant power (FRAP: 0.581%), and ferrous ion chelating activity (75.31%) were obtained from WPI fermented with L. helveticus T80, CGMP fermented with L. helveticus T105, and α-la fermented with L. acidophilus La-05. The optimized fermentation conditions for the generation of an antimicrobial peptide preparation derived from cheese whey fermented with pure and mixed cultures of L. acidophilus LA-5 and Bifidobacterium lactis BB-12 were studied (Amiri et al., 2022). It was reported that the single strain LA-5 culture incubated at

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38.71 C for 26.15 h with 4.45% yeast extract supplementation showed the most promising antimicrobial properties against foodborne pathogens having minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranging from 8 to 32 and 16 to 64 μg/mL, respectively. A traditional Ukrainian fermented baked milk (using a starter culture containing L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus) supplemented with 9% (w/w) electro-activated whey showed an enhancement of DPPH scavenging activity [giving a half-maximal inhibitory concentration (IC50) of 2.54 mg/mL] compared to an untreated whey supplemented sample (IC50: 4.08 mg/mL) and control sample without whey supplementation (IC50: 3.83 mg/mL) (Aidarbekova & Aider, 2022). The higher antioxidant activity in the electro-activated whey supplement has been linked to protein unfolding which leads to an exposure of sulfhydryl groups as well as the formation of Maillard reaction product intermediates, that is, Schiff bases (Kareb et al., 2018). In addition, it has been reported that an electro-activation process can lead to hydrolysis of intact proteins, resulting in the release of smaller molecular mass components (Aidarbekova & Aider, 2021) which may explain the enhancement in the antioxidative properties observed. A number of studies have reported on the utilization of liquid cheese whey as a fermentation substrate for the generation of functional food products and BAPs. Fermentation of sweet whey with Bacillus clausii at 25 C for 3 days exhibited antimicrobial activity against foodborne pathogens (Salmonella typhimurium, Escherichia coli, Shigella flexneri, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis showing zones of inhibition ranging between 9.8 and 15.7 mm) and antioxidant activity (having ABTS1 and DPPH scavenging activity of 85% and 80%, respectively) (Rochín-Medina et al., 2018). It was noted that the bioactive properties observed showed a positive correlation with the highest proteolytic activity and the soluble peptide content released at day 3 of the fermentation period. Liquid whey obtained from a mixture of ewe, goat, and cow cheese manufacture fermented with a commercial LAB starter culture (containing L. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. lactis biovar diacetylactis, S. thermophilus, and L. delbrueckii subsp. bulgaricus) at 37 C for 6 days exhibited antibacterial activity against L. monocytogenes and E. coli O157 having MIC values of 0.2 and 0.4 μg/mL, respectively (Santos et al., 2021). Four whey-derived BAPs, including the α-la-derived ACE inhibitory peptides 50YGL52 and 97DKVGINYW104, the β-lg-derived antiinflammatory peptide 80AVF82, and the β-lg-derived DPP-IV inhibitor and antimicrobial peptide: 92VLVLDTDYK100, in acid whey fermented with K. marxianus were isolated from Parmigiano Reggiano (Martini et al., 2021). Two BSA-derived peptides, 329DAFLGSFLYEYSR341 and 168RHPYFYAPELLYYANK183, were identified in a potent ACE inhibitory fraction purified from a ricotta cheese whey fermented with L. helveticus PR4 at 37 C for 24 h (Pontonio et al., 2021).

8.2 Generation of whey protein
derived bioactive peptides

Ripening is considered as a key process for the development of unique flavors and textures in cheese products. Enzymes, including proteolytic activities, are the main agents contributed to the cheese ripening process (Ardö, 2021). The BAPs released during the ripening process vary depending on the ripening period. A number of previously reported whey-derived ACE inhibitory peptides were identified in commercial cheddar cheeses including three peptides from α-la (105LAHKAL110, 50YGL52, 52LF53), four peptides from β-lg (103LLF105, 147IR148, 148RL149, 22LAMA25), and one peptide from BSA (221FP222) (Lu et al., 2016). In addition, quantitative analysis revealed that the concentrations of the ACE inhibitory peptides detected in the cheese samples varied depending on the duration of the ripening period and the manufacturing process used. The latter can be linked to the specific processing conditions employed such as the starter culture used. The release of ACE inhibitory peptides during cheese ripening has been associated with the action of endogenous milk enzymes (e.g., plasmin, cathepsins B, D, and G), residual chymosin from cheese making, proteinase and peptidases from the LAB in cheese, and CEPs [e.g., X-prolyl-dipeptidyl aminopeptidase (X-PDAP) and aminopeptidase P (PepP)] (Lu et al., 2016).

8.2.4

In silico aided enzymatic release of bioactive peptides

The use of computational methods to predict the presence of BAPs in specific protein molecules has become a powerful tool since it provides a targeted approach for the selection of substrate and proteolytic activity for peptide release thus reducing the use of chemical reagents thereby enhancing the resultant cost-effectiveness. Various in silico approaches are currently being employed to help in the targeted discovery and release of protein-derived BAPs (Liu et al., 2019; Nongonierma & FitzGerald, 2017). For instance, databases, such as BIOPEP-UWM (University of Warmia and Mazury; http://www.uwm.edu.pl/biochemia/index.php/en/biopep), Pepbank (http://pepbank.mgh.harvard.edu/), PeptideLocator (Mooney et al., 2013), PeptideRanker (Mooney et al., 2012), PeptideDB, and the Milk Bioactive Peptides DataBase (MBPDB; https://mbpdb.nws.oregonstate.edu/), provide very useful resources for the identification of bioactive sequences within protein molecules, the primary structures of which are known. Other databases include specific enzyme cutter tools which are able to predict those proteolytic activities which could release specific BAPs, for example, “enzyme action” from BIOPEP-UWM or “PeptideCutter” from ExPASy (Udenigwe, 2014), along with the online tool PeptidePredictor (www.peptidepredictor.com). There are also specific BAP databases, as reviewed elsewhere (Iwaniak et al., 2019), such as the Collection of Antimicrobial Peptides (CAMP) and the Antimicrobial Peptide Database [APD; (Wang et al., 2016)]. In terms of in silico studies on whey protein
derived peptides, peptides within WPC Alcalase hydrolysates were identified which corresponded with sequences within the BIOPEP and the AHTPDB databases reported

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to display antioxidant and ACE inhibitory activity (Mansinhbhai et al., 2021). The primary sequences of bovine α-la and β-lg were digested in silico using the PeptideCutter program from ExPASY to predict the potential sites cleaved by pepsin at pH 1.3 and trypsin and chymotrypsin (Tulipano et al., 2015). Once the peptide list was generated, the DPP-IV inhibitory activity was evaluated comparing the amino acids at the N-terminal of each peptide with the results of the sequence alignment between food protein
derived DPP-IV inhibitory peptide sequences from the literature (Tulipano et al., 2015). However, the results obtained from in silico predictions should be corroborated with in vitro or in vivo studies. An in silico digestion of bovine α-la with pepsin, trypsin, chymotrypsin, glutamyl endopeptidase, and elastase was reported (Nongonierma, Le Maux, et al., 2016). In silico digestion with elastase was predicted to release the greatest number of previously identified DPP-IV inhibitory sequences. Subsequent confirmatory in vitro digestion with this enzyme using a design of experiments approach along with in vitro assessment confirmed the DPP-IV inhibitory activity of the digests. The authors mentioned the requirement for in vivo investigations to evaluate their potential role as DPP-IV inhibitors in humans. A comparison between the peptides released after simulated digestion with those predicted by in silico methods, SWISS-PROT and BIOPEP, was performed (Chatterjee et al., 2015). It identified β-lg peptides released after in vitro digestion, that is, 9GLDIQK14 and 142ALPMHIR148 with ACE inhibitory activity, whereas 78IPAVFK83 and 92 VLVLDTDYK100 had an antibacterial property, as indicated by the peptide databases. These four peptides were also identified following in silico digestion of bovine (Bos taurus) β-lg with gastrointestinal proteinase activities. However, given that there are many different web portals handling and offering access to different databases with different information, it is important to select a specific database relevant to the final in silico-based objective. Other in silico approaches which are available to aid the identification of BAP sequences include the application of quantitative structure
activity relationship (QSAR) or modeling (QSAM) studies. These approaches attempt to connect the observed biological activity with the sequence/structure of a given peptide (Kubinyi, 1997; Udenigwe, 2014; Zhou et al., 2008). The structure of peptides with different biological activities (ACE inhibitory activity, renin inhibitory activity, antioxidant, antimicrobial, and DPP-IV inhibitory activity) has been studied using QSAR models. This is facilitated via possession of a library of the peptides containing a particular bioactivity along with some molecular descriptors such as molecular size, charge, hydrophobicity, and physicochemical properties. This allows classification of peptides at different levels in terms of their structure. QSAR has been used for modeling the ACE inhibitory activity of peptides derived from milk and casein (Lin et al., 2018; Pripp et al., 2004). These authors used physicochemical properties as descriptors of the amino acids in QSAM of ACE-inhibiting peptides and found a

8.3 Analytical techniques for the identification of bioactive peptides

correlation between ACE inhibition and hydrophobicity, charge properties (positive charge), and molecular volume of the amino acids at the C-terminal region (Pripp et al., 2004). Recent QSAR studies on milk protein
derived ACE inhibitory peptides employed a quantum topological molecular similarity (QTMS) index categorization of the amino acids within different peptide sequences (Bahadori et al., 2019). The utility of applying QTMS amino acid indices in predicting the ACE inhibitory activity of some milk-derived peptides was demonstrated (FitzGerald & Meisel, 2000). This approach was also employed to predict the ACE inhibitory potential of casein and whey protein sequences derived from human, goat, bovine, and sheep milks. The prediction of peptides with biological activities using in silico approaches offers the possibility for high-throughput screening of large numbers of peptides for a specific biofunctional property. However, the results obtained experimentally may not always reflect the predicted results, therefore there is a requirement for confirmatory studies to validate the in silico predictions.

8.3 Analytical techniques for the identification of bioactive peptides 8.3.1

Enrichment and fractionation of bioactive peptides

Following the production of a promising sample having a potent specific bioactive property, the sample can be subjected to fractionation to allow for enrichment in order to aid in the ultimate identification of the specific BAPs responsible for the bioactive outcome under assessment. This facilitates BAP separation from a complex mixture which can contain residual intact protein substrate, inactive peptides, free amino acids, as well as proteolytic enzyme(s) and the microorganisms which may have been used during enzymatic hydrolysis and fermentation. A range of separation techniques such as precipitation, membrane processing, and chromatography can be employed to fractionate complex hydrolysates. Hence, the BAPs therein can be separated on the basis of their physicochemical characteristics such as molecular mass, net charge, isoelectric point, and hydrophilicity/hydrophobicity. The advantages and disadvantages associated with each separation technique have been reviewed previously (Agyei et al., 2016). A sequential series of different separation modes is routinely employed during BAP enrichment and purification. Generally, this involves initial separation based on molecular mass using membrane processing followed by liquid chromatography (LC) in different modes, for example, reverse phase (RP), for further fractionation based on other physicochemical properties, for example, based on hydrophobicity/hydrophilicity. Membrane processing is one of the most extensively used techniques at both lab- and larger scale for the enrichment of BAPs. This technique provides

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versatility due to the wide range of molecular mass cutoff membranes which are available, such as membranes having nominal molecular mass cutoff of 0.5, 1, 5, and 10 kDa. The application of membrane processing for the enrichment of whey-derived BAP fractions has been addressed elsewhere (Dullius et al., 2018; Fernández et al., 2014; Kleekayai et al., 2021). Nonetheless, a challenge associated with conventional batch-type membrane processing is membrane fouling. A continuous process, so-called membrane bioreactor, has been proposed as a means to overcome this limitation (Cen et al., 2019; Ratnaningsih et al., 2021; Sáez et al., 2019). Furthermore, integrated enzyme membrane reactors/bioreactors have been developed to facilitate the simultaneous generation and separation of BAPs as well as the recovery of the enzyme/microorganism in a single operation. This has been shown to increase the yield of whey-derived DPP-IV inhibitory peptides by up to 8.7%, when an enzyme membrane reactor was used as opposed to the conventional enzymatic hydrolysis process (O’Halloran et al., 2019). In addition, integration of membrane processing with an electrical field, known as electro-membrane filtration, has been developed to enable a more selective enrichment of BAPs, that is, between neutral and charged peptides having similar molecular mass. This application has been employed for the separation of anionic (e.g., 84IDALNENK91 and 41VYVEELKPTPEGDLEILLQK59) and cationic (e.g., 142ALPMHIR148, 76TKIPAVFK83, 92VLVLDTDYKK101, and 15 VAGTWY20) whey-derived BAPs obtained from a tryptic digest of β-lg (Suwal et al., 2017). Further details regarding advancements in membrane-based separation techniques for the selective enrichment of BAPs have been extensively discussed elsewhere (Ratnaningsih et al., 2021). Nonetheless, it is advised to optimize the membrane processing conditions on a case-by-case basis in order to achieve maximum generation and recovery of the specific BAP(s) of interest. Compared to membrane processing, chromatographic techniques have somewhat limited application for food ingredient applications due to the batch processing mode of operation and thus the cost efficiency of such processes for commodity-type ingredient production. Hence, while this technique can separate BAPs with higher purity, it is currently mainly applied in the pharmaceutical industry (Punia et al., 2020). However, peptide purification can be accomplished using different types of chromatographic matrices such as ion-exchange, gel permeation, reverse-phase, and affinity chromatography columns. Furthermore, inline multidimensional chromatographic systems have been developed to improve purification efficiency, as previously described (De Luca et al., 2021). These techniques are often used in the discovery of BAPs at lab-scale, which in turn require further characterization, that is, using mass spectrometry for peptide sequence identification. Column-based adsorption chromatography using activated carbon as an

8.3 Analytical techniques for the identification of bioactive peptides

absorbent may be employed for larger scale applications. This technique has been successfully used for the separation of Trp-containing ACE inhibitory dipeptides (i.e., IW and AW) derived from an α-la hydrolysate (Hippauf et al., 2014). In addition, it has been used for the preparation of a low Phecontaining WPC hydrolysate for phenylketonuria sufferers (Bu et al., 2020). It should be noted that fractionation of BAPs may result in the reduction and or loss of overall bioactive properties due the potential of differential partitioning of peptides with synergistic effects occurring during the course of separation. Therefore the purification steps employed need to be carefully chosen and should ideally be bioassay guided in all cases. The utilization of information derived from in silico analyses may provide useful information for the development of strategies to choose appropriate purification techniques (Agyei et al., 2016; Dullius et al., 2018). Further information regarding in silico approaches is provided in Section 8.2.4. In silico aided enzymatic release of BAPs herein.

8.3.2

Peptide characterization

The identification and characterization of bioactive sequences is a requirement in order to help understand their potential mechanism(s) of action as well as provide information for the development of enrichment/fractionation strategies especially at larger scale. MS, coupled with different LC methods, can allow the high-resolution separation and identification of peptides which is essential in order to establish a relationship between a particular activity and the peptide sequence. Given its high specificity, sensitivity, and reproducibility, MS analysis can provide qualitative and quantitative information on food proteins and peptides that are firstly ionized and then separated on the basis of their mass-to-charge ratio. The utility of this approach was illustrated by Guo et al. (2019) who performed whey protein hydrolysis with L. helveticus LB 10 proteinases immobilized in sodium alginate before fractionation of the hydrolysate by gel filtration. The transport of different fractions across a Caco-2 cell monolayer was subsequently evaluated. The potential bioavailability of different peptides with in vitro ACE inhibitory activity which partitioned into the basolateral medium was then determined following analysis using matrix-assisted laser desorption/ionization
quadrupole-time-of-flight and ultrahigh-performance liquid chromatography (UPLC)
MS/MS (Guo et al., 2019). Most of the recent literature (Table 8.3) reports on the identification of BAP sequences using RP-UPLC and -HPLC as a chromatographic separation technique, followed by electrospray ionization (ESI) as an ion source and an orbitrap MS system as the mass analyzer. The biological activities of the peptides identified have been assigned using in silico databases (BIOPEP or

203

Table 8.3 Recent examples of the application of MS in the identification of bioactive peptides derived from bovine whey proteins. Ion source-mass analyzer UHPLC-ESI- hybrid quadrupole
Orbitrap Q Exactive HPLC-MS/MS Velos Orbitrap

Test sample

Sequence

Bioactivity

References

By-product of cheese-making

FGK VRY

ACE inhibitor ACE inhibitor

Montone et al. (2021)

Hydrolyzed WPC80

AASDISLLDAQSAPLR TPEVDDEALEK VLVLDTDYK LIVTQTMK LDAQSAPLR IDALNENK

Antimicrobial DPP-IV inhibitor, antimicrobial DPP-IV inhibitor, antimicrobial Cytotoxic ACE inhibitor Stimulates proliferation, Antimicrobial ACE inhibitor Stimulates proliferation reduced vasoconstrictor endothelin-1 release ACE inhibitor Hypocholesterolemic Hypocholesterolemic ACE inhibitor DPP-IV inhibitory ACE inhibitor DPP-IV inhibitor ACE inhibitor ACE inhibitor ACE inhibitor ACE inhibitor ACE inhibitor ACE inhibitor ACE inhibitor

Hinnenkamp and Ismail (2021)

VLDTDYK ALPMHIR

UPLC-ESI-QqQ

Whey hydrolysates

VGINYWLAHK VYVEELKPTPEG DLEILLQK DAQSAPLRVY LKPTPEGDL KGYGGVSLPEW LKGYGGVSLPE DKVGINY RVY HIRL LIVTQ VLDTDY GVSLPEW GGVSLPE

Bustamante et al. (2021)

Nano-HPLC-QTOF

Fermented whey

UPLC-triple quadrupoletime-of-flight mass spectrometer UPLC-ESI-Orbitrap Elite

α-La-rich whey protein concentrate hydrolysate with trypsin Hydrolysis of whey proteins by Enterococcus faecalis

MALDI-QTOF and UPLCESI-MS/MS

Whey hydrolysates with proteinase from Lactobacillus helveticus LB 10

RPKHPIKHQGLPQEVLNEN QEPVLGPVRGPFPIIV SQSKVLPVPQ VYPFPGPIPN VQVTSTAV LQPEVMGVSK LDQWLCEKL

Antihypertensive ACE inhibitor ACE inhibitor ACE inhibitor Antimicrobial Antihypertensive DPP-IV inhibitor

Skrzypczak et al. (2020)

LDAQSAPLR LKGYGGVSLPEW LKALPMH AASDISLLDAQSAPLR IIAEKTKIPAVF IDALNENK VLVLDTDYK LKALPMH LKPTPEGDLEIL LKGYGEKTKIPAVF DIS EVD VFK

ACE inhibitor ACE inhibitor ACE inhibitor Antimicrobial Antimicrobial Antimicrobial Antimicrobial DPP-IV inhibitor DPP-IV inhibitor DPP-IV inhibitor ACE inhibitor ACE inhibitor ACE inhibitor

Worsztynowicz et al. (2020)

Jia et al. (2020)

Guo et al. (2019)

ACE, Angiotensin-converting enzyme; API, atmospheric pressure ionization; DPP-IV, dipeptidyl peptidase-IV; ESI, electrospray; HPLC, high-performance liquid chromatography; IT, ion trap; MALDI, matrix-assisted laser desorption/ionization-time-of-flight; MS, mass spectrometry; QqQ, triple quadrupole; QTOF, quadrupoletime-of-flight; TOF, time-of-flight.

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MBPDB), or the peptides had previously been identified, generally using in vitro assessment approaches. For instance, some of the peptides identified using an HPLC-Orbitrap separation/analysis after hydrolysis of whey protein with trypsin or Protease M were previously reported in the databases as ACE inhibitory (β-lg 48LDAQSAPLR56, 92VLDTDYK100, and α-la 99 VGINYWLAHK108) and DPP-IV inhibitory and antimicrobial peptides (β-lg 141 TPEVDDEALEK151 and 92VLVLDTDYK100) (Hinnenkamp & Ismail, 2021). In addition to the identification of BAPs, several applications in the field of food authentication using chromatographic techniques alone or in combination with MS have been recently reviewed (Valletta et al., 2021).

8.4 Biological effects of whey-derived bioactive peptides 8.4.1

Antidiabetic peptides

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by progressive loss of insulin secretion (β-cell dysfunction) in parallel with insulin resistance in tissues. It has also been reported that oral administration of whey proteins and their hydrolysates can contribute to the control of blood glucose levels and insulinotropic responses in animal models (Gaudel et al., 2013) and in humans (Jakubowicz & Froy, 2013). An increase in insulin secretion was observed when whey hydrolysates or their gastrointestinal digests were present to isolated mouse Langerhans islets (Mortensen et al., 2012) as well as in a two-tiered model with Caco-2 cells in the apical and BRIN-DB11 in the basolateral chamber (Santos-Hernández et al., 2021). Inhibition of the activity of DPP-IV is one of the numerous therapeutic strategies used for the management of T2DM. DPP-IV is a serine protease involved in the inactivation of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose inhibitory polypeptide (GIP). These hormones act by stimulating glucose-dependent insulin secretion after meal ingestion in addition to their involvement in the satiety effect (see Section 8.4.9). Therefore its inhibition has been shown to result in a better regulation of glycemia in type 2 diabetic subjects (Scheen, 2012). Administration of a tryptic digest of β-lg to mice revealing that their DPP-IV inhibitory properties were associated with an induction of insulin secretion and a reduction in plasma glucose following an oral glucose tolerance test (Uchida et al., 2011). To date, several studies have identified peptides derived from WPHs as DPP-IV inhibitors using in vitro enzyme inhibition assays. For instance, the whey protein
derived peptides IPI, IPA, VAGTWY, IPAVF, IPAVFK and TPEVDDEALEK,

8.4 Biological effects of whey-derived bioactive peptides

VLVLDTDYK, LAHKALCSEKL, WLAHKAL were identified as DPP-IV inhibitors (Power et al., 2014; Silveira et al., 2013; Uchida et al., 2011). IPI is the most potent food protein
derived DPP-IV inhibitory peptide identified to date with an IC50 of 3.9 μM. Peptides 105LAHKALCSEKL115 and 95ILDKVGINY103 from α-la were also demonstrated to inhibit DPP-IV by direct interaction with the enzyme’s active site (Lacroix & Li-Chan, 2014). α-La-derived peptides, such as 1EQLTKCEVFR10, 98KILDKVGINYWLAHK108, 95ILDKVGINYWLAHK108, 99 VGINYWLAHK108, and 115LDQWLCEKL123, with DPP-IV inhibitory activity measured in vitro (IC50 values of 883, 930, 456, 765, and 131 μM, respectively) were identified (Jia et al., 2020). A peptide fraction enriched in cationic peptides produced DPP-IV inhibitory activity, probably due to the selective concentration in the cationic recovery fraction of β-lg 78IPAVFK83, 15VAGTWY20, and 1 LIVTQTMK8 (Geoffroy et al., 2022). However, the bioavailability and activity of whey protein
derived DPP-IV inhibitory peptides during digestion remain to be determined. While animal and human trials have demonstrated that the specific inhibition of DPP-IV (with synthetic inhibitory compounds) increases the half-life of total circulating GLP-1, decreases plasma glucose, and improves impaired glucose tolerance (Deacon et al., 2000; Mitani et al., 2002). Further in vivo with synthetic peptides corresponding to whey protein
derived sequences is required to confirm these in vitro results.

8.4.2

Antihypertensive peptides

Hypertension is a risk factor for cardiovascular diseases while blood pressure and vascular tone are controlled by the renin angiotensin system. ACE, which catalyzes the degradation of the inactive decapeptide angiotensin I to the potent vasoconstrictor octapeptide angiotensin II, is a key metabolic enzyme which is targeted during pharma-based interventions to control blood pressure. The majority of the whey protein
derived peptides reported to date have an ACE inhibitory activity. A large number of peptides from bovine β-lg have been identified as ACE inhibitors or are potential precursors for the generation of ACE inhibitory peptides, for example, 142ALPMHIR148, 11DIQK14, 40RVY42, 76TKIPA80, 77 KIPA80, 78IPAVFK83, 96DTDYK100, 122LVR124, 146HIR148, 148RLSFNP153, 15 VAGTWY20, 139ALK141, 4GLDIQK14, 125TPEVDDEALEK135, 10LDIQKVAGTW19, 12 IQKVAGTW19, 95LDTDY99, 140LKALPMH147, 149LSFNPTQ155, 24MAA26, and 15 VAGT18 (Hernández-Ledesma et al., 2004; Jiang & Zhao, 2014; Lacroix & LiChan, 2014; Mullally et al., 1997; O’Keeffe et al., 2017; Pan & Guo, 2010; Power et al., 2014). However, current knowledge highlights that a correlation between in vivo antihypertensive effects and an in vitro measured ACE inhibitory activity does not always exist. In order to exert an antihypertensive effect, it is

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necessary that peptides survive gastrointestinal digestion, are absorbed, and reach the target tissue in an active form. All of these processes are not taken into account during in vitro activity assays. The largest in vivo effects on blood pressure reduction have been observed for short peptide sequences. A limited number of studies have evaluated the bioactivity of ACE inhibitory peptides in vivo (Abubakar et al., 1998; Hernández-Ledesma, Miguel, et al., 2007; Hernández-Ledesma, Quirós, et al., 2007; Nurminen et al., 2000; Sipola et al., 2002). For instance, the antihypertensive effect of the whey protein
derived peptide 78IPA80 was reported in spontaneously hypertensive rats (SHR) (Abubakar et al., 1998), whereas β-lg-derived sequences 103LLF105 and 58LQKW61, which mediated a significant decrease in blood pressure in SHR, were also identified (Hernández-Ledesma, Miguel, et al., 2007). However, both fragments were susceptible to proteolytic degradation after incubation with pepsin and Corolase PP, with 58LQK60 being the active end product following simulated gastrointestinal digestion of 58LQKW61. The absorption, distribution, and clearance of IW and WL in human plasma as well as studying its pharmacokinetic behavior were monitored (Kaiser et al., 2016). These peptides are released during the enzymatic hydrolysis of bovine whey protein with pepsin and pancreatin and were identified as ACE inhibitors in vitro. The ACE activity in plasma after ingestion of a WPH containing IW and WL was determined (Martin et al., 2020). However, their results showed that although plasma ACE activity decreased in parallel to the increase of IW and WL plasma concentrations, the resulting peptide concentrations cannot fully explain the reduction of ACE activity in plasma due to a direct enzyme inhibition effect. Therefore further in vivo studies are required to understand the mechanism(s) involved.

8.4.3

Antimicrobial peptides

Antimicrobial peptides can prove beneficial in relation to food quality and safety as biopreservatives and to human health for their antibiotic effects. In addition, it has been reported that some antimicrobial peptides also promote an immediate defense response against pathogenic microbes. These peptides are thus considered to have a role in the adaptive immune system by enhancing monocytes, dendritic cells, and T cells (Mohanty et al., 2016). The antimicrobial activity can be reported as MIC and MBC values, which describes the bacteriostatic versus bactericidal activity of the tested peptides. Among the bovine whey proteins, lactoferrin (LF) is the most studied for its antimicrobial activity due to the presence of two antimicrobial clusters, including lactoferricin (Lfcin) (17FKCRRWQWRMKKLGAPSITCVRRAF41) and lactoferampin (268WKLLSKAQEKFGKNKSR284) within its primary

8.4 Biological effects of whey-derived bioactive peptides

sequence (Théolier et al., 2014). Digestive enzymes, such as trypsin, pepsin, and chymotrypsin, have been extensively used for the generation of whey protein
derived antimicrobial peptides (Brandelli et al., 2015; Dinika et al., 2020; Nongonierma, O Keeffe, et al., 2016). Bovine Lfcin is the best known antimicrobial peptide of dairy origin reported to date, it can be released from LF using pepsin or chymosin. Within this fragment, LF 20RRWQWRMKKLG30 was the first studied antimicrobial peptide, the activity of which has been linked to the Arg residues located at the N-terminal as well as the presence of the hydrophobic amino acids, Trp and Met (Mohanty et al., 2016; Théolier et al., 2014). Whey-derived antimicrobial peptides with activity against some pathogenic microorganisms (e.g., B. subtilis, E. coli, and Pseudomonas aeruginosa) have been previously reviewed elsewhere (Brandelli et al., 2015; Khan et al., 2018; Nongonierma, O Keeffe, et al., 2016). In addition, a milk protein
derived antimicrobial peptide database (MilkAMP) has been established (Théolier et al., 2014). The antimicrobial potency of peptides is influenced by their physicochemical characteristics, including their net charge, molecular mass, and conformational and hydrophobic properties. It was noted that potent antimicrobial peptides share common key features, that is, they contain cationic (have net positive charge between 1 2 and 1 9) and hydrophobic residues and have a low molecular mass (B10
50 amino acid residues) (Dullius et al., 2018; Khan et al., 2018; Mohanty et al., 2016; Théolier et al., 2014). This amphipathic characteristic plays a key role in promoting the disruption of bacterial cell membranes, which in turn leads to the formation of transient membrane pores affecting permeability or which result in the breakdown of the plasma membrane (Brandelli et al., 2015; Mohanty et al., 2016). This was evidenced in a study reporting that a peptide fraction eluted between 6.5% and 13.0% acetonitrile during semi-preparative RP-HPLC of bovine cheese whey following hydrolysis using immobilized Alcalase which exerted the most potent antifungal against Candida albicans ATCC 18804 (MIC: 10 mg/mL) (da Cruz et al., 2020). This attribute was linked to the intermediate hydrophilicity character of this peptide fraction. Furthermore, it has been reported that Gram-positive pathogens are more sensitive to whey-derived antimicrobial peptides. This was illustrated by the fact that tryptic peptides from β-lg, including 15VAGTWY20, 25 AASDISLLDAQSAPLR40, 78IPAVFK83, 92VLVLDTDYK100, only exerted antibacterial activity against Gram-positive bacteria (e.g., B. subtilis and Staphylococcus lentus) (Pellegrini et al., 2001). A number of BAPs derived from β-lg and α-la identified in bovine milk hydrolysates using a fungal protease from A. oryzae possessed both antibacterial and antifungal activities. The peptides identified therein were rich in Arg and Lys residues which may contribute to their antimicrobial properties (Zanutto-Elgui et al., 2019). The β-lg-derived peptides

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(92VLVLDTDYKKLYL103 and 14KVAGTWYSL22) and the LF peptide 20 RRWQWRMKKLG30, which were released from cheese whey following hydrolysis with calf rennet and pepsin, showed antibacterial activity against E. coli and B. subtilis having MIC and MBC values ranging between 75
300 and 150
600 μg/mL, respectively (Elbarbary et al., 2019). Among these three peptides, 92VLVLDTDYKKLYL103 and 20RRWQWRMKKLG30 exhibited similar antimicrobial potency against both pathogens.

8.4.4

Antioxidant peptides

Whey proteins are considered as a source of antioxidant peptides due to their amino acid composition which is rich in sulfur-containing [B1.7% (w/w) protein], branched-chain amino acids [26%, (w/w), protein] (Corrochano et al., 2018), and hydrophobic amino acids [particularly Trp present at 5.3% (w/w, protein)] in α-la (Nongonierma & FitzGerald, 2015). These features have been associated with the in vitro antioxidative properties of food proteins/peptides when assessed using the oxygen radical absorbance capacity (ORAC), the DPPH, and the ABTS1 scavenging activity along with the FRAP assays. These assays determine different mechanisms of antioxidant activity as previously described elsewhere (Lorenzo et al., 2018; Power et al., 2013). It is well documented that the choice of hydrolytic enzyme can have a major contribution to the antioxidative potency of the hydrolysate generated and the antioxidant peptides released. It was shown that WPC hydrolyzed with Alcalase gave higher Trolox equivalent antioxidant capacity (TEAC) values ranging from 15 to 60 μmol Trolox equivalent (TE)/g and contained a higher proportion of antioxidant peptides (20.7%
24.5%) than those obtained during Flavourzyme hydrolysis (giving TEAC values ranging from 8 to 30 μmol TE/g and containing 13.5%
16.9% antioxidant peptides) (García et al., 2022). The distribution of these peptides was also found to be hydrolysis condition dependent, that is, depending on the E:S, pH, and hydrolysis temperature and duration employed. Similarly, in the case of WPC hydrolyzed with Debitrase HYW20 and FlavourPro Whey, a higher number of previously reported β-lg-derived antioxidant peptides were found in the Debitrase HYW20 hydrolysates (17GTWYSL22, 23 AMAASDISLL32, 24MAASDISLL32, 45ELKPTPEGDLEIL57, 71IIAEKTKIPAVF82, 94 VLDTDYK100, 95LDTDYKK102, 123VRTPEVDDE131, 125TPEVDDEALEK135), while only TPEVDDEALEK was detected in the FlavourPro Whey generated hydrolysates (Kleekayai et al., 2020). In addition, the occurrence of these peptides in the hydrolysates was also dependent on the hydrolysis conditions employed, for example, whether using pH-stat control at pH 7.0 or free-fall pH starting at pH 7.0. The inhibition of intracellular reactive oxygen species (ROS) generation, as tested in oxidatively stressed HepG2 cells, by the hydrolysates (ranging between 19.7% and 78.2%) was influenced by the enzyme preparation used, but it did not appear to be impacted by the hydrolysis

8.4 Biological effects of whey-derived bioactive peptides

conditions employed. Two antioxidant peptides derived from BSA, that is, 416 DQFEKLGEYGFQNAL430 and 435TRKVPQVSTPTL446, which were obtained during WPC hydrolysis with a novel metalloprotease preparation isolated from Eupenicillium javanicum were reported (Neto et al., 2019). To the best of our knowledge, limited information regarding whey-derived peptides being synthesized and assessed for their in vitro antioxidant activity appears to have been reported to date. Three β-lg-derived peptides, 15VAGT18, 24MAA26, and 71IIAE74 identified in WPC hydrolyzed with Corolase PP, exerting ORAC activity ranging from 0.33 to 1.79 μmol TE/mmol peptide have been reported (O’Keeffe et al., 2017). 15VAGTWY20 identified in a tryptic digest of β-lg exhibited ORAC activity and had a value of 5.63 μmol TE/μmol peptide (Power et al., 2014). Another β-lg-derived peptide, 19WYSL22, exhibited DPPH and superoxide scavenging activities having half-maximal effective concentration (EC50) values of 273.63 and 558.42 μM, respectively (Hernández-Ledesma, Quirós, et al., 2007). Two α-la peptides, 115LDQW118 and 101INYW104, purified from a thermolysin hydrolysate of α-la, showing 100% ABTS1 scavenging activity when tested at 2.5 μM, were identified (Sadat et al., 2011). It should be noted that the antioxidative potency measured using in vitro assays may not directly correlate to cell-based antioxidant effects (Honzel et al., 2008; Kleekayai et al., 2020). These reports indicate that in vitro findings may not be readily translated to the more complex systems, that is, to in vivo. Therefore it is recommended that cell-based in situ models should be used to assess the potential bioavailability and intracellular bioactivity of antioxidant compounds. Furthermore, the key antioxidative enzymes expressed in response to oxidative stress, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), have also been assessed during in situ cell-based and in vivo studies. To date, the antioxidative activity assessed using cell-based and in vivo models has mainly focused on WPC, WPI, individual whey proteins, and their hydrolysates, as previously summarized elsewhere (Corrochano et al., 2018; Giblin et al., 2019). However, as already mentioned, studies on specific wheyderived peptides are limited. A number of whey-derived peptides (β-lg 142 ALPM145 and 52GDLE55, α-la 99VGIN102, and BSA 568AVEGPK573) when tested at 5 mM showed a reduction in intracellular ROS generation in 2,20 -azobis(2methylpropionamidine) dihydrochloride (ABAP)-treated murine myoblast (C2C12: 34.4%
53% ROS generation) and hepatocyte (HepG2: 35%
52.6% ROS generation) cells (Corrochano et al., 2019). Some recent in vivo studies reported on whey-derived peptides showing the ability to modulate oxidative stress biomarkers. For instance, a recent small animal study reported that a commercial whey peptide preparation (WHP8350) improved age-related oxidative stress following administration of 1.5 g/kg body weight when given to D-galactose-induced aging C57BL/6N male mice for 6 weeks. A significant (P , .05) increase in the activities of SOD in serum and GPx in serum and liver, when

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compared to control group fed with distilled water, was reported (Yu et al., 2021). Ingestion of WHP8350 at a similar dose also showed a protective effect against Cobalt-60 radiation (8 Gy) damage in female BALB/c mice, an extended survival time of 3
4 days was reported in the treatment group compared to the control group without WHP administration (Liu et al., 2021). This outcome was linked to significantly (P , .05) higher SOD and GPx activities in the serum and liver of the treatment group which had been administered with the WHP8350 for 14 days.

8.4.5

Anticancer peptides

Peptides having anticancer activity possess amphiphatic characteristics having a cationic charge (between 1 2 and 1 9) and have a high hydrophobicity (containing between 40% and 60% hydrophobic amino acids). They generally contain between 5 and 30 amino acid residues and share a similar membranolytic activity as in antimicrobial peptides (Chen et al., 2016; Dullius et al., 2018). Therefore, anticancer peptides are rich in Arg and Lys residues, while the presence of aromatic and hydrophobic residues (such as Pro, Leu, Gly, Ala, and Tyr) may promote interaction with cancer cell membrane bilayers and thereby exert selective and potent cytotoxicity effects against cancer cells. Furthermore, shorter peptides with a greater molecular mobility and diffusivity have been implicated in promoting better interaction with cancer cells (Chalamaiah et al., 2018; Dullius et al., 2018). In general, anticancer activity is associated with antiproliferation/cytotoxicity against a range of cancer cells. Various human cancer cell lines such as breast carcinoma (MCF-7), hepatic cells (HepG2), colon carcinoma (HT29, HCT15), the human monocytic leukemia cell line (THP-1), and leukemia (Jurkat T) cells have been used extensively for the in situ assessment of the potential anticancer properties of food protein and peptide preparations (Chalamaiah et al., 2018). Food proteins, including whey proteins, having anticancer properties have been extensively discussed previously (Leischner et al., 2021; Teixeira et al., 2019). Among the whey proteins, LF is one of the most studied source of whey-derived anticancer peptides to date, mainly focusing on LfcinB (17FKCRRWQWRMKKLGAPSITCVRRAF41). Pepsin hydrolysis has been demonstrated to be one of the most frequently used hydrolytic approaches for the generation of anticancer peptides (Chalamaiah et al., 2018). A peptic LF peptide preparation, when tested at 0.5
2.0 mg/mL, was reported to induce apoptotic cell death in the human oral squamous carcinoma (OECM-1) cell line, causing DNA fragmentation following 24- to 48-h incubation (Sakai et al., 2005). A peptide purified from pepsin hydrolysis of LF, 17 FKCRRWQWRMKKLGAPSITCVR38, showed antiproliferative activity through induction of apoptosis in human leukemia (HL-60) cells having an IC50 value

8.4 Biological effects of whey-derived bioactive peptides

of 2.22 μM (Roy et al., 2002). LfcinB showed cytotoxicity against neublastoma (NB) cell lines having IC50 values ranging between 15.5 and 37.0 μM. This effect was linked to an activation of mitochondrial apoptosis (Eliassen et al., 2006). LfcinB was also reported to induce apoptosis in Jurkat T leukemia cells by disrupting both the cell and the mitochondrial membranes (Mader et al., 2007, 2005). Simulated gastrointestinal digests from bovine colostrum, when tested at concentrations of 3, 10, and 30 mg/mL, showed an apoptosis-related cytotoxicity against human breast cancer (MDA-MB-231) cells and significantly (P , .05) increased proliferation of peripheral blood mononuclear cells (PBMCs) at a test concentration of 3 mg/mL (Fajardo-Espinoza et al., 2021).

8.4.6

Immunomodulatory peptides

The recent global pandemic related to SARS-CoV-2 infection has increased attention on the search for evidence of food components that may support immune function. Whey proteins are considered as potential sources of immunomodulatory and antiviral compounds, as recently reviewed (Gallo et al., 2022). The mechanisms of immunomodulatory effects of protein hydrolysates and peptides have been described previously (Kiewiet et al., 2018). Immunomodulatory effects are associated with the stimulation or inhibition of certain functions of the immune systems, for example, regulation of proinflammatory cytokine expression, promotion of antibody production, and proliferation of lymph nodes and spleen cells. Immunomodulatory peptides generally appear to have multifunctional biological activities (Chalamaiah et al., 2018; Kiewiet et al., 2018; Reyes-Díaz et al., 2018). Therefore they often share similar structural features with anticancer, antimicrobial, and antiinflammatory peptides. This includes the presence of a positively charge residue near the N-terminus, a high number of aromatic and aliphatic residues with an amphipathic character (Chalamaiah et al., 2018; Kleekayai & FitzGerald, 2022; Pavlicevic et al., 2022). As described elsewhere, a range of enzymatic hydrolysis parameters can influence the biological activity as a consequence of the types of BAPs released. Trypsin, pepsin, and Alcalase have been extensively used for the generation of immunomodulatory peptides (Chalamaiah et al., 2018). The hydrolysis conditions also showed an impact on immunomodulatory effects, for example, in the case of splenocyte proliferation by Alcalase WPC hydrolysates, which was influenced by the hydrolysis duration (Ma et al., 2014). It was noted that the molecular mass or peptide length can modulate immunomodulatory responses through different systems. For example, in the case of a tryptic digest of β-lg, a fraction enriched in peptides .5 kDa induced a Type 1 T helper (Th1) cell response by inducing the secretion of interferon (IFN)-γ in PBMCs. On the other hand, the ,1-kDa peptide fraction induced proliferation of PBMCs along

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with the production of tumor necrosis factor (TNFα) and transforming growth factor (TGF)-β cytokines (Rodríguez-Carrio et al., 2014). The upregulation of TGF-β by the ,1-kDa fraction has been linked to an increase in the regular T cell (Treg: CD41CD25highCD127-FOXP31) population. This was attributed to the positive charge and the high hydrophobicity of the β-lg peptides identified in this fraction, for example, 1LIVTQTMK8, 79IPAVFK83, and 142ALPMHIR148. Oral admiration of peptic digested LF (300 mg/kg/day for 7 days) during animal studies increased the population of CD41, CD81, and asialoGM11 cells in spleen and in the peripheral blood of tumor-bearing mice, as well as stimulated interleukin (IL)-8 production in intestinal epithelium on day 3 (Kuhara et al., 2000). Mice fed with a commercial semielemental diet containing whey di- and tripeptides (Peptino, Terumo, Japan) for 5 days showed an increase in Peyer’s patch lymphocytes, and in intestinal and respiratory IgA levels in the intestinal and respiratory tracts (Moriya et al., 2018). A number of studies have demonstrated the potential of WPHs and peptides in the reduction of cow’s milk allergy by modulating immune responses in animal models. In acute skin sensitized mice, oral admiration of a commercial WPH (20 mg/day) led to an increase in the regular B (Breg) and Treg cell populations in spleen and IgA1 B cells in the mesenteric lymph nodes (MLN) along with the activation of Th17 cells in the Peyer’s patches of the intestinal epithelium (Kiewiet et al., 2017). Among 31 β-lg-derived peptides tested, 47 LLDAQSAPLRVYVEELKP64 showed the most potent whey-induced allergy reduction in mice by suppression of the level of IgE and by increasing the percentage of Treg (CD251Foxp31) and plasmacytoid dendritic 1 1 (CD11b CD103 ) cells in the MLN (Meulenbroek et al., 2013). Similarly, an encapsulated nanoparticle containing 80 μg of β-lg-derived peptides (41AASDISLLDAQSAPLRVY58 or 47LLDAQSAPLRVYVEELK64) led to a suppression of TNFα release in splenocytes (Liu et al., 2022). Oral administration of a commercial WPH (50 mg/mouse) showed a reduction in acute allergic skin and mast cell degranulation in whey-induced allergic responses (van Esch et al., 2011). The reduction in allergic response was linked to an increase in Foxp31 Treg cell numbers in the MLN. Some examples of WPHs and wheyderived peptides having immunomodulatory effects against cow’s milk allergy have been summarized (Kiewiet et al., 2018).

8.4.7

Antiinflammatory peptides

Inflammation has been associated with a range of health conditions such as cancer, cardiovascular disease, obesity, and diabetes. Inflammation involves immune responses by recruiting leukocytes that can be triggered by various factors, for example, oxidative stress, tissue injury, and infection. Inflammatory stimuli also activate intracellular signaling pathways, including nuclear factor

8.4 Biological effects of whey-derived bioactive peptides

(NF)-κB, mitogen-activated protein kinases (MAPK), and Janus kinase (JAK)-signal transducer and activator of transcription. These mechanisms can in turn induce the production of inflammatory mediators such as proinflammatory cytokines [i.e., IL-1, IL-1β, IL-6, IL-8, IL-12, TNFα, interferon-γ (INFγ)], granulocyte-macrophage colony-stimulating factors, and inflammatory proteins and enzymes (Chen et al., 2018). Therefore inactivation of the NF-κB pathway by suppression of these biomarkers, modulating other intracellular pathways, and the upregulation of antiinflammatory cytokines (i.e., IL-4, IL-10, IL-11, and TGF-β) may prove beneficial in controlling inflammatory conditions (Chen et al., 2018; Yahfoufi et al., 2018). It has been reported that the antiinflammatory properties of peptides may be influenced by the presence of hydrophobic amino acid residues (such as Phe, Tyr, Pro, Val, Ile, and Leu) and positively charged amino acids (Arg, His, and Lys) (Ma et al., 2016; Nan et al., 2007). The role of whey proteins in the regulation of inflammation has been recently reviewed (Ali et al., 2021). Assessment of antiinflammatory properties can be carried out using in situ cell-based models by challenging cells with an inflammation stimulus, for example, lipopolysaccharide (LPS), concanavalin A (con A), etc. A number of studies have demonstrated the antiinflammatory potential of WPHs and peptides in both cell-based and animal studies. A high hydrostatic pressure pretreated Alcalase β-lg hydrolysate reduced nitric oxide (NO) production by 63% (at 1 mg/mL) and suppressed gene expression of TNFα and IL-1β (at 10 μg/mL) in LPS-stimulated RAW 264.7 macrophage cells (Bamdad et al., 2017). Similarly, it was reported that an Alcalase hydrolysate of WPC (0.25 mg/mL) also suppressed NO production and the TNFα, IL-1β, and IL-6 levels released in the supernatant of LPSinduced RAW 264.7 cells (Mansinhbhai et al., 2021). WPI hydrolysis using Pronase (tested at 5 mg/mL) and a peptide identified therein, β-lg 78IPAV81 (when tested at 200 μM) suppressed IL-8 gene expression and reduced phosphorylation of inflammatory signaling molecules [i.e., p65 NF-κB, extracellular signal-regulated protein kinase (ERK)1/2, p38 MAPK, c-Jun NH2-terminal kinase/stress-activated kinase (JNK)1/2, and spleen tyrosine kinase (Syk)] in TNFα-induced Caco-2 cells (Oyama et al., 2017). Eight whey-derived antiinflammatory peptides in WPI hydrolyzed with Alcalase were identified (Ma et al., 2016). Among these peptides, α-la 116DQWL119 displayed the most potent antiinflammatory effect by inhibiting mRNA expression of IL-1β (49.5% and 59.6%), cyclooxygenase (COX)-2 (62.1% and 69.7%), and TNFα (42.9% and 52.4%), and by reducing IL-1β (51.3% and 61.5%) and TNFα (48.7% and 66.6%) released in LPS-induced RAW 264.7 macrophages, when tested at 10 and 100 μg/mL, respectively. In addition, it was suggested that the suppression of these inflammatory markers was associated with modifications of the NF-κB and p38 MARK pathways.

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As already outlined, the commercial whey peptide preparation WHP8350 has been demonstrated to regulate immune responses in animal studies by the suppression of TNFα and IL-1β production in the brain tissue of Dgalactose aging induced C57BL/6N male mice (Yu et al., 2021) and in the downregulation of serum TNFα and IL-6 in radiation-induced oxidatively stressed female BALB/c mice (Liu et al., 2021). Oral administration of whey-derived peptides, β-lg 42YVEEL46 and 102YLLF105 (at 500 and 50 μg/ kg/day for 8 weeks), led to a reduction in serum TNFα (14% and 30%, respectively), while maintaining antiinflammatory TGF-β levels in ovariectomized osteoporotic mice (Pandey et al., 2018). In acute pancreatitis C57BL/6 mice, oral admiration of a commercial formula containing whey peptides (MEIN, Meiji Co., Ltd., Japan) and a fermented milk product resulted in a reduction in plasma monocyte chemoattractant protein (MCP)-1 levels and improved inflammation-related remote organ injuries including splenomegaly and hepatomegaly along with an elevation of hepatic enzymes (i.e., aminotransferase and alanine aminotransferase) (Nakamura et al., 2018).

8.4.8

Opioid-like peptides

Opioid receptors are located in the central and peripheral nervous systems, in the immune and endocrine systems, and throughout the intestinal epithelium. Some peptides may have affinity for opiate receptors and thereby display opiate-like effects. These opioid peptides are able to be recognized by specific receptors and therefore they may be regarded as exogenous supplements to the endogenous opioidergic systems of the human organism. Two studies demonstrated the stimulation of mucus metabolism in gastric mucosa after the administration of bovine α-la (Ushida et al., 2003, 2007). Furthermore, other reports showed the activity of α-la and its hydrolysates on gastric ulcers in in vivo rat models (Mezzaroba et al., 2006) and as inducers of mucin secretion in the jejunum of the rats ex vivo (Claustre et al., 2002). Of the 23 peptides identified from cow’s milk proteins with opioid effects, only 2 were from whey proteins (Nielsen et al., 2017). In similarity with endogenous opioid peptides, milk-derived opioid peptides contain a Tyr residue at the N-terminal and another aromatic residue (Tyr or Phe) in the third or fourth position. An opioid peptide derived from α-la was identified following hydrolysis with pepsin (50YGLF53, known as α-lactorphin), whereas the β-lg-derived opioid peptide (102YLLF105, known as β-lactorphin) was identified after digestion with pepsin and trypsin (Antila et al., 1991; Martínez-Maqueda et al., 2012; Pihlanto-Leppälä, 2000). It was subsequently shown that α-lactorphin can induce mucin secretion and MUC5AC expression following 4
24 h of exposure, although significance

8.4 Biological effects of whey-derived bioactive peptides

was only reached after 24 h (Martínez-Maqueda et al., 2012). The activity of a β-lg hydrolysate, containing β-lactorphin, on mucin secretion and gene expression in intestinal human goblet cells HT29-MTX was reported (Martínez-Maqueda et al., 2013). Moreover, a WPC hydrolysate containing β-lactorphin was able to stimulate mucin secretion and MUC5AC expression in human intestinal goblet HT29-MTX cells. Another peptide derived from β-lg 146HIRL149 has been shown to be a neurotensin agonist with high selectivity for the NT2 receptor (Yamauchi, Usui, et al., 2003). Different activities, such as ileum-contracting, antihypertensive, analgesic, and antinociceptive, have been reported for this peptide as a result of its binding to the neurotensin receptor (Yamauchi, Sonoda, et al., 2003).

8.4.9

Satiety hormone-inducing peptides

Anorexigenic hormones are secreted from specialized enteroendocrine cells located throughout the gastrointestinal tract (Gribble & Reimann, 2016) which in turn represents the main connection between the gastrointestinal system and the appetite center of the brain (Kaelberer & Bohórquez, 2018). Ingested nutrients which are directly in contact with the intestinal epithelium are involved in the release of hormones such as cholecystokinin (CCK), peptide YY (PYY), and the incretin hormones GLP-1 and GIP. These hormones participate in the inhibition of gastric emptying and contribute to an improvement in insulin secretion while also promoting pancreatic enzyme secretion and central nervous system signaling. An increasing interest in the study of milk protein
derived digestion products currently exists due to their potential to modulate satiety signals in the gut. Exposure to WPI increased GLP-1 secretion in the murine enteroendocrine cell line STC-1 (Power-Grant et al., 2015) and proglucagon mRNA levels in human colorectal (NCI-H716) cell line (Wazzan, 2018). An increase in GLP-1 secretion on exposure of STC-1 cells to α-la and β-lg was reported (Gillespie et al., 2015). Moreover, both groups reported a loss of bioactivity of the whey proteins after their incubation with gastrointestinal enzymes (Gillespie et al., 2015; Power-Grant et al., 2015). However, human jejunal samples taken after the ingestion of a whey protein solution, as well as the simulated gastrointestinal digests from whey protein, induced a comparable secretion and expression of the hormones CCK and GLP-1 in the murine enteroendocrine cells STC-1 (Santos-Hernández et al., 2018). Other studies performed in vivo with a liquid whey preload solution showed an increase in plasma CCK, insulin, glucagon, GIP, and GLP-1 in healthy men (around 75 years old) (Giezenaar et al., 2018) and an increase in plasma GLP-1 and PYY in obese women (Rigamonti et al., 2019).

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Most of the studies to date have evaluated hydrolysates or gastrointestinal digests as hormonal inducers. However, only a small number of peptides have been identified related to hormonal secretion or satiety effect, therefore there is a lack of scientific knowledge about the molecular structure and hormone signaling of specific food protein
derived peptides. Peptide 146 HIRL149, known as β-lactotensin derived from a chymosin digest of bovine β-lg, was intraperitoneally injected and orally administered in two different studies to fasted C57BL/6J mice. A significant decrease in food intake was observed in both cases (Hou et al., 2009). Peptide AFKAWAVAR, known as Albutensin A obtained after tryptic hydrolysis of serum albumin, delayed gastric emptying and decreased food intake in fasted dYY mice when intraperitoneally administrated (Ohinata et al., 2002). A peptide obtained following the tryptic and peptic digestion of β-lg, 142ALPMH146, increased CCK release in STC-1 cells. However, shorter forms of this peptide did not produce any effect, whereas a synthesized scrambled sequence of 142ALPMH146 (PHLMA) also increased CCK secretion (Tulipano et al., 2017). However, further studies are required to determine the relevance of the structural characteristics required for these hormone secretion inducer peptides.

8.5

Conclusion and future prospects

Several approaches can be employed for the generation of whey-derived BAPs, such as in vitro enzymatic hydrolysis, in vivo gastrointestinal digestion, microbial fermentation, the application of in silico databases, or combinations of these processes, in order to obtain peptides with beneficial effects in the human body. Since the development of advanced proteomic and peptidomic techniques, peptide sequences resistant to gastrointestinal digestion have been identified and these have been tested, at least in vitro in order to confirm their bioactive potential. Moreover, advances in peptidomic techniques allows identification of peptides subjected to in vitro, in situ, or in vivo digestion, which provides knowledge on the potential bioavailability of these peptides when digested under physiological conditions. However, there is still a scientific gap in fully understanding the changes that occur to food proteins during in vivo gastrointestinal digestion, and how protein molecules are modified by different enzymes and the impact on their subsequent intestinal absorption. Furthermore, there are gaps in the scientific knowledge on the impact of different food matrices on both the release and the stability, and the ultimate efficacy of BAPs. The use of in silico databases can help in the performance of a preliminary screening of possible BAPs, reducing the number of experiments required to be performed in vivo or in the laboratory. This tool may be considered as a novel green processing technology aiding in designing of whey-derived BAP release strategies, identifying the

References

structural requirements of peptides for a specific target bioactivity, and ultimately helping in the design of novel peptidomimetics.

Acknowledgments The authors acknowledge Enterprise Ireland under the Innovation Partnership Programme (IP 2020 0942), which is cofunded by the European Regional Development Fund (ERDF) under Ireland’s European Structural and Investment Funds Programmes 2014
2020 for T. Kleekayai. M. SantosHernández received funding from Enterprise Ireland for funding under Disruptive Technologies Innovation Fund (DTIF; grant no. DT20180088).

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CHAPTER 9

Bioactive peptides derived from camel milk proteins Priti Mudgil and Sajid Maqsood Department of Food Science, College of Agriculture and Veterinary Medicine, United Arab Emirates University, Al Ain, United Arab Emirates

9.1

Introduction

The shift in consumer perception from meat proteins is driving an increased demand for alternative sources such as dairy or plant-derived proteins (Lagrange et al., 2015). In addition to providing nutrition, proteins also provide innumerable health benefits. Within their parent structure, proteins conceal various encrypted biologically active peptide fractions which when released upon hydrolysis in gut by gastrointestinal or microbial enzymes impart a range of enhanced positive health benefits to the host in comparison to intact structure (Mudgil, Jobe, et al., 2019). These peptides derived from various food proteins have become an interesting area of research with more noticeable scientific evidence for their positive effects in management of various lifestyle-related disorders such as hypertension, obesity, hypercholesterolemia, and diabetes, along with other physiological effects such as antimicrobial, antiallergenic, immunomodulatory, antiviral, antiosteoporotic, anticancerous, and antihemolytic. Milk proteins either as whole milk proteins, caseins, or whey proteins from various domesticated animals have been explored for their potential for bioactive peptide production (Ali Redha et al., 2022; Chaudhari & Hati, 2022; Muthukumaran et al., 2022). To date, bioactive peptides from bovine milk proteins have been extensively studied, while non-bovine milk proteins remain an underexplored (Kilari et al., 2021). Therefore, in order to meet consumer expectations, alternative sources of milk proteins are being explored for the production of bioactive peptides (Izadi et al., 2019). Among non-bovine milk sources, camel milk has been extensively utilized for treatment of various ailments such as autoimmune diseases and metabolic disorders since ancient times (Mati et al., 2017). Conventionally, camel milk has been used as a local remedy for the treatment of diseases such as tuberculosis, asthma, dropsy, and jaundice (Ali Redha et al., 2022). Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00009-6 © 2023 Elsevier Inc. All rights reserved.

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Additionally, it is attributed to have better digestibility and nutritional value than bovine milk, making it one of the alternatives sources for human consumption (Muthukumaran et al., 2022). Published reports have claimed camel milk for their antimicrobial, immunomodulatory, antidiabetic, antiautistic, antihypertensive, and anticancerous properties (Al haj & Al Kanhal, 2010; Meena et al., 2016). Camel milk is consumed predominantly in Middle Eastern and North African countries such as Saudi Arabia, Oman, United Arab Emirates, Ethiopia, Somalia, South Sudan, Sudan, and Djibouti with some states in India, China, and Russia either as fresh or in its fermented forms (Konuspayeva & Faye, 2004; Panwar et al., 2015). Recently, camel milk is gaining importance as one of the most valuable food sources for people residing in arid- and semi-arid regions of the world and is lauded for its potential to garner revenue for these countries (Kamal-Eldin et al., 2022). Often termed as white gold of the dessert it is also attracting high demand in many European countries and North America (Faraz et al., 2020). The unique health benefits associated with the consumption of camel milk are largely attributed to the differences in their protein composition in comparison to their bovine counterparts (Khalesi et al., 2017). Camel milk is reportedly high in antimicrobial protein like lysozyme and have a higher component of vitamin C (Faye et al., 2019; Konuspayeva et al., 2009). In addition, similar to human milk, camel milk lacks β-lactoglobulin rendering it nonallergenic properties in comparison to bovine milk (Mudgil et al., 2022). Camel milk is differential characterized from milk of other domestic animals’ for the ratio of its casein fractions and size of casein micelle which is bigger than other species milk. Camel milk contains less of α-s caseins and more of β-casein, lactoferrin, and α-lactalbumin with minimal quantities of κ-casein (Mohamed et al., 2020). These facts illustrate the untapped potential of camel’s milk as a nutrient source and is being considered as a potential alternative to the bovine milk especially because of its better digestibility and low allergenicity (Mudgil et al., 2022). In the previous decade, various camel milk proteins have been explored for their potential towards the generation of bioactive peptides with various physiological benefits such as antimicrobial (Mahmoud Abdel-Hamid et al., 2020; Algboory & Muhialdin, 2021), antifungal (Mudgil, AlMazroui, et al., 2022), antihypertensive (Kumar et al., 2016a,b; Moslehishad et al., 2013), anticancerous (Murali et al., 2021), antidiabetic (Baba, Mudgil, Kamal, et al., 2021; Tagliazucchi et al., 2018), antiobesity (Baba, Mudgil, Baby, et al., 2021; Mudgil, Baba, et al., 2022; Mudgil, Baby, Ngoh, Vijayan, et al., 2019; Mudgil et al., 2018), and antihemolytic (Jafar et al., 2018).

9.2 Bioactive peptides from camel milk proteins

Various studies have also reported the generation of multivalent peptides having several bioactive properties and among both type of camel milk proteins, caseins are attributed to have better potential for generation of peptides with more promising bioactive properties than camel whey proteins (Mudgil, Baba, et al., 2022; Mudgil et al., 2021; Salami et al., 2009, 2010, 2011). However, the bioactive properties of generated peptides are largely dependent on their chain length, number of amino acids, amino acid composition, chain conformation, hydrophobicity, and specific amino acids at N and C terminals (Bo et al., 2021; Nongonierma & FitzGerald, 2016; Nongonierma & Fitzgerald, 2016). Attempts are being aimed toward the controlled release of specific peptides with explicit biofunctional attributes by employing microbial food-grade enzymes under controlled hydrolytic conditions (Peighambardoust et al., 2021). Thorough understanding on the enzyme specificity toward different cleavage positions and number of cleavage sites present within the target protein sequence can also help in specific peptides preparation. Further, regulation of certain process parameters such as time of hydrolysis, enzyme: substrate concentration, and optimum temperature can influence the hydrolysis reaction kinetics and in turn the peptide length and its compositions (Baba, Baby, et al., 2021; Baba, Mudgil, Baby, et al., 2021; Baba, Mudgil, Kamal, et al., 2021). In the past few years various reviews have been published on the bioactive properties of intact camel milk. However, to the best of our knowledge, summarized literature regarding peptides obtained from camel milk proteins are limited. Therefore this book chapter summarizes all the currently published literature related to bioactive peptides obtained from different camel milk proteins or its fractions under in silico, in vitro, and in vivo. Additionally, the role of structure and activity
related functions toward derivatization of camel milk peptides is also presented.

9.2

Bioactive peptides from camel milk proteins

The present chapter compiles all the reported biological activities reported in protein hydrolysates or identified peptides obtained from various camel milk proteins. Till today bioactive peptides obtained from camel milk proteins has been shown to exert multiple biological activities ranging from antioxidant, antihypertensive, antimicrobial, antiinflammatory, antihemolytic, antidiabetic, antiobesity, anticancerous, and antiaging properties. The derivatization of bioactive peptides from camel milk proteins has employed various strategies encompassing ultrasonication; enzymatic hydrolysis using digestive enzyme (trypsin, pepsin, pancreatin, chymotrypsin), microbial enzymes (alcalase, thermolysin, proteinase K, pronase E), and plant-derived enzymes

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(bromelain and papain); or microbial fermentation using various probiotics mainly from lactic acid bacteria group. Lactic acid bacteria are known to possess mainly two types of proteases: cell wall bound exopeptidases and intracellular peptidases such as endopeptidases, aminopeptidase, tripeptidase, and dipeptidase (Korhonen & Pihlanto, 2006).

9.2.1

Antioxidant

Antioxidant properties of camel milk protein hydrolysates remain the most widely studied biological functions for camel milk proteins (Table 9.1 and Table 9.2) (Ali Redha et al., 2022). Overall, it has been observed that various factors such as type of enzymes, enzyme:substrate ratio used, duration of Table 9.1 Antioxidant effects of protein hydrolysates obtained from camel milk via enzyme hydrolysis or fermentation. Substrate

Antioxidant assay

TEAC or IC50

References

Proteinase K, thermolysin, trypsin, and chymotrypsin Chymotrypsin, trypsin, pepsin

ABTS

2.5
20 μM

Salami et al. (2010)

ABTS

2
17 μM

Salami et al. (2011)

Trypsin

DPPH Lipid peroxidation inhibition ABTS

 80%  30%

Al-Saleh et al. (2014)

0.11
0.17 μM

Jrad et al. (2014)

IC50 5 6.8 μg/mL


Rahimi et al. (2016) Kumar et al. (2016)a,b

23
50.3 μM 17.3
21.9 μM 31.3
39.3 μM

Al-Shamsi et al. (2018)

1513.0 μM Vit C/g 11.9 μM Vit C/g

Tagliazucchi et al. (2018)

Production process

Enzymatic hydrolysis Whey Whole casein, β-casein Casein

Colostrum whey and casein Casein Casein

In vitro gastrointestinal digestion [simulated gastrointestinal digestion (SGID)] Proteinase K Alcalase, chymotrypsin, and papain

Skim milk

Alcalase, bromelain, and papain

Skim milk

SGID

αS-casein

Trypsin amd α-chymotrypsin

ABTS ABTS, DPPH, and FRAP ABTS, DPPH Metal chelating ABTS Hydroxyl radicals lipid peroxidation inhibition ABTS Hydroxyl radicals Ferrous ion chelation

78.2%

IC50 5 59.88 μg/mL

Addar et al. (2019)

Continued

9.2 Bioactive peptides from camel milk proteins

Table 9.1 Antioxidant effects of protein hydrolysates obtained from camel milk via enzyme hydrolysis or fermentation. Continued Substrate

Production process

Skim milk from different breeds

SGID

Casein Casein Whey

SGID SGID

Whey

Papain

Antioxidant assay

TEAC or IC50

References

ABTS DPPH FRAP ABTS DPPH, ABTS, and CUPRAC ABTS

663.6
1665 μM 693.6
1081.4 μM 207.1
463.1 μM 0.25 μM

Maqsood et al. (2019)

60.9 μg/mL

Osman et al. (2021)

Moslehishad et al. (2013) Shori and Baba (2014); Shori (2018)

Jrad et al. (2020) Akan (2021)

Microbial fermentations Milk

Lactobacillus (Lb.) rhamnosus PTCC 1637

ABTS

844.08
1737.88 μM

Soybean
camel milk yogurt Allium sativum Camel milk yogurt Milk

Streptococcus (S.) thermophilus, Lb. bulgaricus

DPPH




Lb. plantarum, Lb. paraplantarum, Lb. kefiri, Lb. gasseri, Lb. paracasei, Leu. lactis, Weissella (W.) cibaria, and Enterococcus (E.) faecium

DPPH and ABTS

57.90 μM 1484.35 μM

Soleymanzadeh et al. (2016)

ABTS

45.38%

Zahedi et al. (2016)




Abd Elhamid and Elbayoumi (2017) Ayyash, Al-Dhaheri, et al. (2018); Ayyash, Al-Nuaimi, et al. (2018) Soleymanzadeh et al. (2019) Jrad et al. (2020)

Fermented camel milk (Chal) Milk Milk

Lactobacillus spp. Lactococcus (Lc.) spp.

DPPH and FRAP ABTS and DPPH

β-casein

Leuconostoc (Leu.) lactis PTCC1899

ABTS




Casein Whey

Microbial and enzymatic hydrolysis (S. thermophilus, Lb. bulgaricus, pepsin, and pancreatin) Ultrasonication and Lb. delbrueckii subsp. lactis

ABTS and DPPH




DPPH




Gammoh et al. (2020)

Various Lactobacillus spp.

DPPH FRAP

 50%
80% 0.125-0.325 mg GAE/mL

El-Sayed et al. (2021)

Casein Whey Whole milk

S. thermophilus, Lb. bulgaricus

30%
70% Inhibition

hydrolysis, and temperature of hydrolysis affect the antioxidant potential of generated peptides. This is usually because these factors in turns affect the extent of hydrolysis and length and composition of peptides generated (Corrêa et al., 2011; Mudgil, Omar, et al., 2019).

237

Table 9.2 Antioxidant peptides identified from camel milk via enzyme hydrolysis or fermentation. Sequence

Parent protein

Antioxidant assay

Method for production

References

NEDNHPGALGEPV KVLPVPQQMVPYPRQ

Whole camel milk

Pepsin and pancreatin

Homayouni-Tabrizi et al. (2016)

LEEQQQTEDEQQDQL YLEELHRLNAGY RGLHPVPQ IY LK LY VY FIPYPNY QIPQCQALPNIDPPTVE FFQLGDYVA RPKYPLRY RPKYPLRY MDQGSSSEESINVSQQKF VRNIKEVESAEVPTENKISQ LHQGQIVMNPW TLTDLENLHL MVPYPQRAMPVQ PFQEPVPDPVRGLHPVPQ QDKIYTFPQ SISSSEESITHINKQKIEKF QPKVMDVPKTKETIIPKRKEMPLLQ KCLQDGAGDVAFVKDSTVF KADAVTLDGGL KFGRGKPSGFQL TVVSNNGNREYGL CGSIVPRREWRAL SSCAMRCLDPVTEDSF

Whole camel milk

DPPH radical scavenging, ABTS radical scavenging, superoxide dismutase (SOD), fatty acid peroxidation assays, hydroxyl radical scavenging assay DPPH radical scavenging, ABTS radical scavenging, and hydroxyl and superoxide free radical scavenging

Pepsin and pancreatin

Homayouni-Tabrizi et al. (2017)

Various proteins

ABTS radical scavenging, hydroxyl radical scavenging, and inhibition of lipid peroxidation

Simulated gastrointestinal digestion(SGID)

Tagliazucchi et al. (2018)

κ-casein

DPPH radical scavenging, superoxide free radical scavenging assay

Pepsin

Ibrahim et al. (2018)a

αs1-casein

αs2-casein

β-casein

Lactoferrin

Lactalbumin PGRP Cys to rich Protein

ENTMRETMDFLKSLF ATTLEGKLVEL ATTLEGKLVEL KCLQDGAGDVAFVKDSTVF KADAVTLDGGL KFGRGKPSGFQL TVVSNNGNREYGL CGSIVPRREWRAL SSCAMRCLDPVTEDSF ENTMRETMDFLKSLF FIPYPNY QIPQCQALPNIDPPTVE RPKYPLRY FFQLGDYVA RPKYPLRY VRNIKEVESAEVPTENKISQ MDQGSSSEESINVSQQKF LHQGQIVMNPW QDKIYTFPQ MVPYPQRAMPVQ PFQEPVPDPVRGLHPVPQ TLTDLENLHL SISSSEESITHINKQKIEKF QPKVMDVPKTKETIIPKRKEMPLLQ RLDGQGRPRVWLGR TPDNIDIWLGGIAEPQVKR VAYSDDGENWTEYRDQGAVEGK MVPYPQR VPYPQR

Lactophorin A Lactophorin B Lactophorin B Lactoferrin

Superoxide [xanthine oxidase (XOD)/ nitroblue tetrazolium(NBT)] reduction assay, DPPH- reduction assays Saccharomyces cerevisiae stress model

Pepsin

El-Sayed et al. (2021)

Whole bactrian camel milk

DPPH and ABTS radical scavenging

Trypsin

Wali et al. (2020)

β-casein

ABTS radical scavenging

β-casein (Bactrian camel)




Microbial fermentation (Leuconostoc lactis PTCC1899) Natural fermentation (Camelus bactrianus milk)

Soleymanzadeh et al. (2019) Ganzorig et al. (2020)

Lactalbumin (40
52) PGRP (7
19) Cys to rich Protein Lactophorin A κ-casein αs1-casein

αs2-casein

β-casein

Continued

Table 9.2 Antioxidant peptides identified from camel milk via enzyme hydrolysis or fermentation. Continued Sequence

Parent protein

Antioxidant assay

Method for production

References

LLILTC AVALARPK YPLR LSSHPYLEQLYR TQDK LAVP NEPTE VSSTTEQK LAVPIN KPVAIR LLNEK

Whole camel milk

Superoxide free radical scavenging assay (SFRSA), ABTS assay, hydroxyl free radical scavenging assay (HFRSA)

Microbial fermentation (L actobacillus plantarum KGL3A)

Dharmisthaben et al. (2021)

9.2 Bioactive peptides from camel milk proteins

In the previous few years many researchers have studied the antioxidant potential of peptides generated from whole milk proteins via hydrolysis or microbial fermentation. Variable degree of antioxidant potential has been observed in these reports as compiled in Table 9.1. In two different studies conducted by AlShamsi et al. (2018) and Tagliazucchi et al. (2018), hydrolysis of skim camel milk by alcalase, bromelain, papain, and simulated gastrointestinal digestion (SGID) indicated that overall alcalase-generated hydrolysates showed maximum ABTS radical scavenging potential. In these hydrolysates, it was observed that ABTS radical scavenging activity was more potent than DPPH radical scavenging while bromelain generated peptides showing higher DPPH radical scavenging than papain- and alcalase-generated hydrolysates. Interestingly, ferric reducing antioxidant power (FRAP) activity was found to decrease upon hydrolysates generation (Al-Shamsi et al., 2018). Unlike this, SGID of skim milk sample was found to improve the FRAP activity for generated peptides (Maqsood et al., 2019). It was also interesting to note that camel milk obtained from different breeds and their generated hydrolysates displayed wide variation in their antioxidant potential via DPPH, FRAP, and ABTS assays (Maqsood et al., 2019). In another study on camel colostrum milk proteins, Jrad et al. (2014) reported the antioxidant activities of the pepsin-generated colostrum whey and casein hydrolysates. Although non-significant variation between colostrum protein
based hydrolysates was found, however, comparison with literature suggested that these hydrolysates were the most potent antioxidant hydrolysates studies till date in terms of their antioxidant potential. Similarly, Ibrahim et al. (2018a) digested camel whey and casein proteins using pepsin and studied these hydrolysates for xanthine oxidase superoxide radical scavenging and DPPH radical scavenging. Results obtained suggested that digestion of whey protein was similar to their intact protein, whereas casein hydrolysates showed significant improvement in their antioxidant activity against both types of radicals. Salami et al. (2010) analyzed whey protein hydrolysates generated through chymotrypsin, proteinase K, thermolysin, and trypsin and observed that chymotrypsin-generated camel whey hydrolysates presented maximum antioxidant capacity followed by proteinase K (Table 9.1). Similarly, Sephadex G-25-based fraction from papain-generated camel whey protein hydrolysates reported significantly potent ABTS radical scavenging activity 60.9 μg/ mL (Osman et al., 2021). Similar observations were reported for the hydrolysis of camel caseins, where chymotrypsin-generated hydrolysates were more potent antioxidant than trypsin- and pepsin-generated hydrolysates. It was also reported that fractionation of hydrolysates had significant impact on ABTS radical scavenging effects of hydrolysates. Amongst whey-generated hydrolysates all the fraction below 5 kDa possessed stronger ABTS radical scavenging effects, while amongst casein-generated hydrolysate fractions above 5 kDa possessed stronger scavenging potential (Salami et al., 2011).

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Similar findings were reported by Addar et al. (2019), where chymotrypsingenerated camel αS-casein hydrolysates showed higher antioxidant activity in comparison to trypsin-generated hydrolysates. These findings suggested that antioxidant activity is affected by not only the enzyme but also the substrate. These findings were further supported by Kumar et al. (2016a, 2016b) as chymotrypsin-formed hydrolysates revealed superior antioxidant potential in comparison to trypsin-generated hydrolysates. However, according to Rahimi et al. (2016) even though no significant differences were seen in IC50 values, stronger scavenging was observed for peptides with size less than 3 kDa among fractionated proteinase K
derived camel casein hydrolysates. Overall, all these research investigations have suggested that these antioxidant capability of hydrolysates are largely dependent upon length, molecular mass, amino acid composition, and the sequence and positioning of amino acids in the peptide chains (Rahimi et al., 2016). In a very interesting study, impact of ultrasonication along with fermentation with Lactobacillus bulgaricus was studied for the generation of antioxidant peptides and it was reported that ultrasonication induces unfolding of protein structure making them more prone to microbial hydrolysis thus increasing antioxidant peptide generation (Gammoh et al., 2020). Further microbial fermentation is also considered as popular mean to generate potential bioactive peptides with myriad of bioactive properties including antioxidant. Many studies in this direction have been conducted to evaluate the potential of fermented camel milk proteins with strong ABTS and DPPH radical scavenging (Table 9.1). In most of the studies various Lactobacillus (Lb.) spp. remained widely used in comparison to other lactic acid bacteria groups for camel milk fermentation. In one such study, whole camel milk fermented using Lb. rhamnosus PTCC 1637 strain and later fractionated samples showed ABTS radical scavenging activity between 844.08 and 1737.88 μM (Moslehishad et al., 2013). Further, Lb. plantarum, Lb. paraplantarum, Lb. kefiri, Lb. gasseri, Lb. paracasei, Lb. delbrueckii subsp. lactis, and Lb. delbrueckii subsp. bulgaricus have been used for similar purpose by other researchers (Ayyash, Al-Dhaheri, et al., 2018; El-Sayed et al., 2021; Gammoh et al., 2020; Jrad et al., 2019; Abd Elhamid & Elbayoumi, 2017; Soleymanzadeh et al., 2016; Zahedi et al., 2016). Similarly, during an attempt to make enriched yogurts from camel milk via fermentation with Streptococcus (S.) thermophilus, Lb. bulgaricus plain camel milk yogurts extended better antioxidant profile than bovine milk yogurts indicating camel milk protein hydrolysis to peptides with stronger radical scavenging activities (Shori & Baba, 2014; Shori, 2018). Another study evaluated the potential of two Lactococcus strains, that is, Lactococcus lactis spp. cremoris and Lactocossus lactis spp. lactis for the generation of antioxidant peptides via camel milk fermentation (Singh et al., 2018).

9.2 Bioactive peptides from camel milk proteins

The results reported 13.84% and 11.53% inhibition of ABTS radical, whereas 5.25% and 4.56% inhibition for DPPH radicals by cremoris and lactis strains, respectively. Leuconostoc lactis PTCC1899 was employed by Soleymanzadeh et al. (2019) for camel milk β-casein fermentation and antioxidant activity was evaluated via ABTS and DPPH radical scavenging assay. This study found potent scavenging of both radicals in hydrolysates fraction with MW ,3 kDa. Further identification of the most potent peptides in this fraction revealed MVPYPQR peptide with an TEAC values of 8933.05 μM. One interesting findings in many of these studies was the impact of storage period on the antioxidant potential of peptides. Overall, observations suggested that upon prolonged storage antioxidant activities of protein hydrolysates were found to be substantially improved suggesting that further proteolysis of peptides generating more shorter chain peptides with higher antioxidant potential (Ayyash, Al-Dhaheri, et al., 2018; Ayyash, Al-Nuaimi, et al., 2018; Shori & Baba, 2014). Further investigations have been advanced toward the identification of peptide sequences with stronger antioxidant potential and the various sequence discovered from different findings are included in Table 9.2. In the quest to identify different antioxidant peptides within camel milk proteins, two different reports on the generation of peptides using pepsin pancreatin-based hydrolysis resulted in the identification of five different peptides with sequences NEDNHPGALGEPV, KVLPVPQQMVPYPRQ, LEEQQQTEDEQQDQL, YLEELHRLNAGY, and RGLHPVPQ (Homayouni-Tabrizi et al., 2017, 2016). Similarly, Ibrahim et al. (2018a) investigated the peptide sequences generated from different camel milk protein via gastric enzyme (pepsin) hydrolysis. Overall, two sequences each from κ-casein and αs2-casein; three from αs1-casein and lactoferrin; six from β-casein; and one each from lactalbumin, peptidoglycan recognition proteins (PGRP), Cys-rich protein, lactophorin A, and lactophorin B were identified (Table 9.2). Another study identified three peptides RLDGQGRPRVWLGR, TPDNIDIWLGGIAEPQVKR, and VAYSDDGENWTEYRDQGAVEGK from trypsingenerated hydrolysates (Wali et al., 2020). Further peptides MVPYPQR from β-casein of dromedary camel milk proteins and VPYPQR from β-casein of Bactrian camel milk obtained via microbial fermentation were identified by Soleymanzadeh et al. (2019) and Ganzorig et al. (2020).

9.2.1.1 Structural activity relationship of camel milk
derived antioxidant peptides Bioactive peptides usually exert their antioxidative effects by stabilization of free radical through different mechanisms, either via transfer of hydrogen atom or via donating their electron. In this regards, the type of amino acids

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inherent to peptide sequence plays a major role such as peptides containing tyrosine which quench free radical by hydrogen atom transfer, while those containing cysteine, histidine, or tryptophan employ electron transfer as their antioxidant mechanism (Esfandi et al., 2019). Further, the antioxidative activity of bioactive peptides also heavily depend upon several parameters such as amino acid composition, chain length, size (500
1500 Da), sequence of the residue/ functional side chain present, and procedure involved in production of peptides (Song et al., 2020). As reported previously, the presence of hydrophobic amino acids in the peptide chain enhances their lipid solubility which in turn improves their interaction with cell membranes giving them entry within the cytoplasm, a target sight for scavenging free radicals (Soleymanzadeh et al., 2019). Therefore amino acids such as cysteine, tryptophan, tyrosine, methionine, phenylalanine, histidine, isoleucine, leucine, and proline are known for their effective antioxidant capabilities (Salami et al., 2011). Not only hydrophobic peptides, the hydrophilic peptides also participate well in radical scavenging by acting as metal chelator, enhancing peptide solubility while easing interaction, and proton exchanges with radical species (Liu et al., 2016). Therefore, the comparative position in the sequence and interaction of amino acids with the environment can suggestively impact the antioxidant property of peptides. Additionally, higher electron-donating capability of some specific amino acids such as histidine, proline, methionine, lysine, tyrosine, and cysteine conveying them with high antioxidant activity (Moslehishad et al., 2013; Salami et al., 2009, 2010, 2011). For instance, histidine’s imidazole group, phenyl aniline’s aromatic group, tryptophan’s indolic group, and sulfur hydrogen group of methionine and cysteine correlate with the antioxidant properties of bioactive peptides containing higher number of these amino acids. Not only the amino acid content but also the type of employed enzyme influence the antioxidant properties of peptides. For example, trypsin and papain cleave at peptide bonds contributed by an adjacent amino acid lysine, arginine, and phenylalanine, while bromelain preferred amino acid next to lysine, arginine, phenylalanine, and tyrosine. Similarly, chymotrypsin can preferably cleave peptide bond next to tryptophan, tyrosine, and phenylalanine, although it exhibits low specificity for leucine, methionine and histidine. Pepsin preferentially cleaves at phenylalanine and leucine at P1 position with no cleavage actions for any other amino acids at this position. In addition, pH conditions are also determined to impact the specificity of enzyme for specific hydrolysis (Keil, 2012). According to Ibrahim et al. (2018a) and El-Sayed et al. (2021), the antioxidant effects of peptides with hydrophilic residue were more potent than

9.2 Bioactive peptides from camel milk proteins

peptides with hydrophobic peptides. As a general observation, it is well noted that peptides with leucine or phenylalanine at C-terminus have significant impact on their antioxidant properties, whereas hydrophobic residues at the C-terminal of peptides have been documented to be significant in radical scavenging activity specifically. Further, amino acid residues such as glutamine and aspartate due to the presence of extra electrons, and tyrosine’s phenolic hydroxyls groups will allow them to act as electron donor or release hydrogen thus supporting a high reducing potential. Even though extensive research toward the generation of camel milk derived peptides with antioxidant activities has been conducted, the main focus for determining antioxidant activity has been based on in-vitro assays mainly. Therefore the real antioxidant potential of these peptides under real condition warrants further investigations for utilization of in vivo or cell linebased models.

9.2.2

Antihypertensive

Antihypertensive properties of various camel milk protein hydrolysates have been explored in the previous decade using the inhibition ACE-I, a major predisposition to high blood pressure and subsequently hypertension. A wide variation in ACE inhibitory potential of hydrolysates generated by different researcher has been observed with inhibitory concentration (IC50) ranging from as low as 20 μg/mL (Salami et al., 2011) to 3.93 mg/mL (Moslehishad et al., 2013) (Table 9.3). As per the study conducted by Solanki et al. (2022), fermentation of skim milk from Indian breed of camel by Lb. acidophilus NCDC-15 strain produced hydrolysates with an 78.33% ACE inhibition, whereas fermentation of camel milk from middle eastern breeds by Lc. lactis KX881782 and Lactobacillus strains (Lr.K777, Lp.K779, and Lp.K772) resulted in inhibition values of 40%
80% (Ayyash, Al-Dhaheri, et al., 2018; Ayyash, Al-Nuaimi, et al., 2018). Further, fermentation of pure casein and whey proteins by Lb. delbrueckii subsp. lactis resulted in 42.81% and 60.85% inhibition, respectively. These wide variation in inhibitory percentages could be either attributed to variations in microbes used, protein substrate, or variation in milk originating from different breeds of camels. Similar observations were also made in studies originating from enzymatic hydrolysates of camel milk, For instance, Maqsood et al. (2019) evaluated the ACE-IC50 values of hydrolysates obtained from four breeds of camel after SGID. The results suggested that Saheli-SGID hydrolysates possessed more potent inhibitory potential against ACE (IC50 5 0.1 mg/mL) than Hozami, Pakistani (IC50 5 0.5 mg/mL), and Omani breeds (IC50 5 1.61 mg/mL) (Table 9.3).

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Table 9.3 Antihypertensive properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Source

Method of derivation

Assay

% Inhibition/IC50 values

Fermentation by Lactobacillus acidophilus NCDC-15 Hydrolysis by papain

HHL

78.33%

Solanki et al. (2022)

ABZ-Gly-Phe (NO2)-Pro HHL

179.9 μg/mL

Osman et al. (2021) Gammoh et al. (2020)

References

In vitro studies Indian breed skim milk Whey proteins Whey proteins Casein proteins Whey proteins Casein proteins Whey Pakistani breed skim milk Saheli breed skim milk Hozami breed skim milk Omani breed skim milk Skim milk

Ultrasonication Fermentation by Lactobacillus delbrueckii subsp. lactis Fermentation by Leuconostoc (Leu.) lactis PTCC1899 SGID

HHL

0.05 mg/mL 0.16 mg/mL Mudgil, Baby, Ngoh, Kamal, et al. (2019)

HHL

0.04
0.05 mg/mL 0.02
0.05 mg/mL 0.02 mg/mL 75.79%, 64.14%, and 86.30% 40%
80%

HHL

.80%

HHL

40%
80%

Ayyash, Al-Dhaheri, et al. (2018) Ayyash, Al-Nuaimi, et al. (2018)

HHL

1748.2 μg/mL

SGID

HHL

1171.4 μg/mL

Hydrolysis by proteinase K Fermentation by Lactobacillus rhamnosus PTCC 1637

FAGPP FAGPP

36 μg/mL 3.93 mg/mL

HHL

Skim milk

Trypsin, chymotrypsin, and pepsin Fermentation by Lactococcus lactis KX881782 Fermentation by Lactobacillus strains (Lr.K777, Lp.K779, and Lp.K772) SGID

Skim milk Casein proteins Whole milk

Whey proteins Skim milk Skim milk

0.05 mg/mL

Soleymanzadeh et al. (2019) Maqsood et al. (2019)

0.01 mg/mL

Alcalase (3,6, and 9 h) Bromelain (3,6, and 9 h) Papain (3,6, and 9 h) Pepsin, trypsin, and combined enzymes

Whole casein

49.88% 24.43% 60.85% 42.81% 1.61 mg/mL

FAGPP

Ugwu et al. (2019)

Jafar et al. (2018)

Tagliazucchi et al. (2018) Tagliazucchi et al. (2016) Rahimi et al. (2016) Moslehishad et al. (2013)

Continued

9.2 Bioactive peptides from camel milk proteins

Table 9.3 Antihypertensive properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Continued % Inhibition/IC50 values

Source

Method of derivation

Assay

Whole casein β-casein (β-CN) In vivo studies Skim milk

Pepsin, trypsin, chymotrypsin, and SGID

FAGPP

20
600 μg/mL 23
161 μg/mL

Salami et al. (2011)

Fermented camel milk

Fructoseinduced hypertensive rats Spontaneously hypertensive rats Spontaneously hypertensive rats

NA

Alshuniaber et al. (2021)

NA

Yahya et al. (2017)

NA

Kanso et al. (2016)

Skim milk

Casein

Lactobacillus helveticus (LMG11445) and Streptococcus thermophilus (ATCC 19258) Hydrolysis by trypsin

Further, more investigations into the antihypertensive effects of SGID-derived hydrolysates by Tagliazucchi et al. (2016) and Tagliazucchi et al. (2018) also reported similar level of ACE-IC50 values of 1171.4 and 1748.2 μg/mL, respectively. However, further fractionation of these crude hydrolysates led to eight fractions F1
F8 where fractions F1, F2, and F4 showed improved ACE-IC50 of 38.6, 107.9, and 37.2 μg/mL, respectively. These observations indicated that peptide size is a major determinant of antihypertensive properties. Similarly, variation between enzyme used for hydrolysis were also observed, such as hydrolysis of casein by proteinase K produced hydrolysates with ACE-IC50 of 36 μg/mL (Rahimi et al., 2016), while pepsin-, trypsin-, chymotrypsin-, and SGID-derived hydrolysis of caseins led to ACE-IC50 values between 20 and 600 μg/mL (Salami et al., 2011). Bromelain and alcalase produced hydrolysates showed ACE-IC50 values in the range of 0.02
0.05 mg/mL (Mudgil, Baby, Ngoh, Kamal, et al., 2019). Their investigation also reported that pepsin-generated hydrolysates were most potent inhibitors while trypsingenerated hydrolysates were found to be the weakest inhibitors. Hydrolysis of whey proteins by papain-produced hydrolysates showed an ACE-IC50 value of 179.9 μg/mL (Osman et al., 2016), whereas, in a similar study, the same hydrolysates displayed an ACE-IC50 values of 0.02 mg/mL (Mudgil, Baby, Ngoh, Kamal, et al., 2019). Similarly, whey protein digested by trypsin, chymotrypsin, and pepsin produced an inhibition of 40%
80%. However, no significant change in ACE inhibitory potential of colostrum proteins following pepsin digestion was noted by Jrad et al. (2014).

References

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Gammoh et al. (2020) studied the effect of ultrasonication of the ACE inhibitory activity of camel milk caseins and whey protein separately and observed a significant increase in ACE inhibitory activity of proteins establishing that high magnitude sound wave can induce structural modifications of proteins and subsequently breakage of proteins to peptides. Some workers have also studied the effect of refrigerated storage on the changes in ACE inhibitory properties of camel milk protein hydrolysates. In one such study, long-duration storage showed significant increase in antihypertensive potential (Ayyash, Al-Dhaheri, et al., 2018; Ayyash, Al-Nuaimi, et al., 2018). However, Alhaj et al. (2018) did not observe any significant change in ACE inhibitory potential of camel milk fermented by yogurt starter cultures over a 15-day storage. Some studies have also investigated the effect of camel milk protein hydrolysates on the blood pressure under in vivo conditions and the outcome of these investigations are briefly compiled in Table 9.3. Kanso et al. (2016) conducted experiments on camel casein tryptic hydrolysates in spontaneously hypertensive (SH) rats. Oral administration of camel casein tryptic hydrolysates at 800 mg/kg/day for 15 days decreased blood pressure and heart rate during the first week. As per the study, this relaxation was mediated through endothelium-dependent activation of the NO pathway. In other two studies, impact of fermented milk was investigated in fructoseinduced hypertensive and SH rats (Alshuniaber et al., 2021; Yahya et al., 2017). As per the findings from Alshuniaber et al. (2021), camel milk hydrolysates at a dose of 1200 mg for 3 weeks induced significant reductions in systolic and diastolic blood pressure and were comparable to commercial drug nifedipine. Moreover, levels of ACE, serum lipid, and serum glucose profile were observed. Additionally, significant improvement in insulin levels toward baseline were also observed (Alshuniaber et al., 2021). Similar results pertaining to the decreased activity and level of ACE in plasma of fermented skim camel milk administered SH rats were observed by Yahya et al. (2017). In this study, it was observed that short-term and long-term administrations of fermented camel milk improve blood pressure (systolic and diastolic) till at 4 and 8 h of post administration but did not continue untill 24 h post administration (Yahya et al., 2017). Although antihypertensive properties of peptides generated from camel milk has been extensively studies, majority of them are performed under in vivo conditions using enzymatic marker ACE-I. Studies under in vivo conditions are still limited and warrants further investigations toward using cell line and molecular marker-based models and validation in the in vivo (animal and human) models.

9.2 Bioactive peptides from camel milk proteins

9.2.2.1 Structural activity relationship (SAR) of camel milk
derived antihypertensive peptides Quantitative structural activity relationship (QSAR) modeling has revealed a definite association between molecular characteristics of peptides and their ACE inhibitory properties. Recent advances in QSAR and molecular docking studies has been fundamental in understanding the mechanistic evaluation of ACE inhibition by peptides. Overall, ACE contains three pockets, namely S1, S2, and S10 in its active site. Pocket S1 is characterized by amino acid alanine at 354 (Ala354), glutamic acid at 384 (Glu384), and tyrosine at 523 (Tyr523). Similarly, S2 pocket contains glutamine at 281 (Gln281), histidine at 353 and 513 (His353 and His513), lysine at 511 (Lys511), and tyrosine at 520 positions (Tyr520). Pocket S10 contains glutamic acid at 162 (Glu162). Peptides with capability to interact with these residues are generally characterized as peptides with strong ACE inhibitory potential. In addition, Zn2 1 ion, as a ligand, also plays a vital role in the ACE-I active site along with residues Glu 411, His383 and His387. His353, Ala354, Ser355, Glu384, His513, and Pro519 can form strong and stable complexes with peptides via van der Waals interactions or hydrogen bonding. Tyr360 have the capability to form conjugates interaction, whereas Glu384 and Arg522 can form noncovalent interactions providing additional stability to ACE
peptide complexes (Ma et al., 2019). Hence, interaction with these specific residues can be predicted as a characteristic of ACE inhibitory peptides. Together with ACE enzyme, inherent characters of peptides also affect their inhibitory potential. Major characteristics that affect the antihypertensive properties of bioactive peptides are molecular weight, chain length, amino acid composition, their isomeric forms, and position of specific amino acids at the N- and Cterminal of peptide chain (Baba, Baby, et al., 2021). In general, shorter peptides with an aromatic, hydrophobic, or basic residue at their C-terminus have shown the highest ACE-I inhibition activities (Jafar et al., 2018; Moslehishad et al., 2013; Mudgil, Baby, Ngoh, Kamal, et al., 2019). Therefore amino acids such as proline, histidine, lysine, valine, and arginine or proximity of phenylalanine, tryptophan, and tyrosine at the C-terminal may cause an upsurge in the ACE inhibitory potential of the peptides (Sagardia et al., 2013). Further studies have also indicated that peptides with hydrophobic amino acids at the positions adjoining the C-terminal isoleucine, leucine, tryptophan, phenylalanine, proline, and valine induce strong binding to ACE thus promoting their inhibition (Jahangiri et al., 2014). Further, existence of amino acids such as glycine, leucine, isoleucine, and valine at Nterminal has been reported as preferential interacting peptides for ACE-I (Iwaniak et al., 2014). In addition, amino acid residues present in the active site of ACE-I favors hydrophobic interactions with the c-terminal proline (Li et al., 2014). Similar sequences have been identified in camel milk protein hydrolysates where peptides with proline at C-terminal have shown potent inhibition. For example,

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IPP, LPP, and LPLP all containing proline and lysine at penultimate position provided them all with stronger inhibition of ACE. The peptide sequences were identified from camel milk protein via various methodologies and their IC50 values are provided in Table 9.4. Previous studies have also confirmed that the existence of a Pro-end residue promotes ACE inhibitory property, such as LVYSHTEPIP, VMVPFLQP, and VPDPVRGLHP peptides identified in β-casein hydrolysates derived via fermentation through S. thermophilus LMD-9 (ATCC BAA491) (El Hatmi et al., 2016). Moreover, PAGNFLP and FCCLGPVPP from whey protein derived via pepsin hydrolysis also indicated the presence of proline terminal (Baba, Baby, et al., 2021). LPP, VIP, VLP, ILP, PYP, LPLP, HLPLP, LHLPLP, VP, and IPP generated via SGID of camel milk have yielded potent inhibition of ACE as indicated with their lower value of ACE-IC50 (Tagliazucchi et al., 2018, 2016). Similarly, peptide MVPYPQR (IC50 5 30 μM), identified from β-casein hydrolysates of Leu. lactis PTCC1899 reported a strong bond between the major active site residue Glu162 (S’1 pocket of ACE) and the arginine present in the C-terminal that distorted tetrahedral geometry of ACE-I presenting its inhibition potential (Soleymanzadeh et al., 2019). Another mechanistic insight into the molecular binding and docking studies of camel milk
derived bioactive peptides from camel milk against ACE-I also confirmed the prime importance of last 3 amino acid at the C-terminal (Mudgil, Baby, Ngoh, Kamal, et al., 2019). In their studies it was shown that papainand alcalase-generated hydrolysates possessed 14 and 1 peptides, respectively, that were able to bind to up to 11 important active sites of ACE-I and were designated as competitive inhibitors for ACE-I. These peptides block the active sites of ACE-I, thereby obstructing the substrate.

9.2.3

Antimicrobial peptides from camel milk

Antimicrobial potential of camel milk is described in literature from centuries (El Agamy et al., 1992). However, in recent years an interest in understanding the antimicrobial potential of camel milk protein
derived hydrolysates has seen an expansion. The reports claim increased antimicrobial actions of camel milk protein hydrolysates than their intact proteins. Different investigations have targeted different modes of peptide generation and explored against a wide variety of pathogens (Gram positive and negative). Recent studies have even evaluated camel milk
derived peptides against drug-resistant microorganisms (Abdel-Hamid et al., 2016; Muhialdin & Algboory, 2018). The major pathogens tested against camel milk
derived peptides include Pseudomonas (P.) aeruginosa, Escherichia coli, Salmonella (Sal.) spp., Staphylococcus (Sta.) aureus, Listeria (Li.) monocytogenes, Bacillus (B.) cereus, Streptococcus (Str.) mutans, and Candida spp.

Table 9.4 Studies related to antihypertensive properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Bioactive peptides CCGM PAGNFLM NGLMHR MFE FM PAVACCL PPLPCHM YDFL LP LRPFL LRFPL LC HSGF MM PAGNFLP PVAAAPVM FCCLGPVPP MLPLML PFTMGY NQPFPKF WGLDPYK AVMPKWW AVMPQWW CLSPLQFR AHFPQFFQ AHMPQFFQ MEPLPVPFL MEPPLVPFL SHLPNLRYF FYKQWKFL AEWLHDWKL MGWVPKMFSPGG LSWPMLKPKVM AVASEDGGVACAVMPQWW

Protein source

Method of derivation

References

Whey

Hydrolysis by pepsin

Baba, Baby, et al. (2021)

Skim milk

Hydrolysis by papain

Mudgil, Baby, Ngoh, Kamal, et al. (2019)

Continued

Table 9.4 Studies related to antihypertensive properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Continued Bioactive peptides AY VK VKAYQIIPNL AYQIIPNLRYF AYQIIPNLR AYQIIPNL LNKIYQYYQTFLWPEY PWNHIKRYF IY QK LNK TVY IP IC50 5 150 μM PL IL VF IC50 5 9.1 μM TF IC50 5 18.0 μM YP IC50 5 658 μM IY IC50 5 2.1 μM LY IC50 5 18.0 μM YL LPP IC50 5 9.6 μM VIP VLP ILP PYP RPK LPLP HLPLP LHLPLP IC50 5 5.8 μM VP IC50 5 880 μM VY IC50 5 7.1 μM IPP IC50 5 5.0 μM

Protein source α-s2-Cnsv2

Method of derivation

References

In silico digestion of camel casein Isoform UP1, and UP2 by proteases

Ryskaliyeva et al. (2019)

SGID

Tagliazucchi et al. (2018)

α-s2-Cnsv1 α-s2-casein

Skim milk

GLx ALx IC50 5 3.4 μM VV SLx PLx LxP VLx LxV TLx LxT LxLx GGI DLx VM FS SF PF FT LH PGV SLG GLxV TVA GSQ LxY LxLxG PVV VPT IAI VVT SLxT PPL NPT SHT

Skim milk

SGID

Tagliazucchi et al. (2016)

Continued

Table 9.4 Studies related to antihypertensive properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Continued Bioactive peptides VLN DLT AYP PLxQ ILD QIT LxHP IC50 5 1.6 μM VYS LLQ ILE VPY FPQ HIM GVPK KPVA IAHP LQSP TVTH IPPK TEPI IIPK LQPK YPPQ VAHIPS TPVSPR SHTEPI SLNEPK VP IC50 5 880 μM VY IC50 5 7.1 μM IPP IC50 5 5.0 μM DLENLHLPLPL NLHLPLPLL LTDLENLHLPLPL TDLENLHLPLP TDLENLHLPLPL TLTDLENLHLPLPL SLSQFKVLPVPQQMVPYPQRAMPV

Protein source

Colostrum protein

Method of derivation

References

SGID

Zeineb Jrad et al. (2014)

HPVPQP QEPVPDPVR MVPYPQR (IC50 5 30 μm)

β-casein

Natural spontaneous fermentation

Ganzorig et al. (2020)

β-casein

Fermentation by Leuconostoc lactis PTCC1899

LSLSQFKVLPVPQ SLSQFKVLPVPQ SQFKVLPVPQ TDLENLHLPLPL DLENLHLPLPL LENLHLPLPL KVLPVPQQMVPYPQ VLPFQEPVPDPVRG FQEPVPDPVR VMVPFLQPK NPQYPPGNVQ MDTIEPVSVACIS PPPGSKSTGT GFFALIPGIE GFFALIPGIE GPSSGFFGMR XXLVGXQASD ELVASIPR MIQAEKNPPL TSIVIIGGGPGGYEAA LQYGPLADILGE QNVLDFHR MPRKGPAPK VPPAVWNSGNYNS VNPNTPIR G&FMRF DPPFAPRM QNPLDFHR GFFALIPGIE VNPNTPIR ITCLSDINSK CISSSTPPYDLNRFK ATVQGGIMYRMP

β-casein

Fermentation by Lactobacillus helveticus or Lactobacillus acidophilus.

Soleymanzadeh et al. (2019) Omar Amin Alhaj (2017)

Whole milk

Fermentation by Lactobacillus bulgaricus NCDC (09) and Lactobacillus fermentum TDS030603

Solanki et al. (2017)

Continued

Table 9.4 Studies related to antihypertensive properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Continued Bioactive peptides APGQDFMRF MAREHSKENTR AVHGQFATL GFKDLLKGAAKALVKTVLF ATVQGGIAYRMP PPPGSKSTGT VRTPVTVQTKVDNIKKY VIAGGSAAIIG DAMMNQAVRE QSAPGNEAIPP VNPNTPIR QNVLDFHR GPSSGFFGMR PPPGSKSTGT PPPGSKSTGT SRPYSFGL SMIGGVMSKG RIVLVGPPGAGKGTQAAYLAQNLSIPHIATGDLFR AVHGQFATL ELLSEINR AGFVLKGYTKTSQ QNVLDFHR SPPFAPRL SPPFAPRL MDFNISGIGNVS VIAGGSAAIIG LVYSHTEPIP VMVPFLQP LHLPLPL KVLPVPQ VPYPQR VPDPVRGLHP ALPPKKNQD (IC50 5 19.9 μmol/L)

Protein source

Method of derivation

References

β-casein

Fermentation by Streptococcus thermophilus LMD-9 (ATCC BAA-491)

El Hatmi et al. (2016)

κ-CN

Fermentation by Lb. helveticus 130B4

Quan et al. (2008)

9.2 Bioactive peptides from camel milk proteins

Foremost work toward antimicrobial peptides (AMPs) from camel milk protein has been conducted from whey and casein fractions generated by either microbial or enzymatic hydrolysates (Table 9.5). Camel milk
derived antibacterial peptides were first investigated by Salami et al. (2010), where hydrolysates of camel whey protein generated by four different enzymes such as proteinase K, thermolysin, chymotrypsin, and trypsin were tested against E. coli Dh1α and each hydrolysate showed inhibition of 47.3%, 26.4%, 20.0%, and 19.1%, respectively. Further, pepsin-derived colostrum whole and whey hydrolysates for their antimicrobial actions against E. coli XL1 blue and Li. innocua LRGIA01 were compared (Jrad et al., 2014). The results obtained suggested that colostrum whey protein hydrolysates were more potent antimicrobial than whole colostrum protein hydrolysates (Table 9.5). Thereafter, same researchers explored the antibacterial potential of camel casein hydrolysates toward Li. innocua, B. cereus, Sta. aureus, E. coli, and P. aeruginosa (Jrad et al., 2015). The results interestingly found out that the hydrolysates supported the growth of P. aeruginosa but weakened the growth of Li. innocua, B. cereus, Sta. aureus, and E. coli by 6.32%, 1.83%, 4.9%, and 19.73%, respectively. Recently antibacterial effects of camel milk lactoferrin-derived hydrolysates against two strains of Li. innocua were tested. Although most of the hydrolysate’s fractions were unable to inhibit the growth of Li. innocua, two fractions which showed significant inhibition. Later exploration into the sequence analysis of peptides from these two-fraction identified various peptides such as QLFGSPAGQKDL, GSPAGQKDLL, VLKGEADAL, LDCVHRPVKGY, WAKDLKL, RIDKVAHL, FSASCVPCVDGKEYPNLCQLCAGTGENKCACSSQEPYFGY, FKDSALGL, and IDKVAHL and all of which carried at least 50% sequence homology with AMP database suggesting their antimicrobial potential. However, further investigations into the mechanism of action of these peptides and their antimicrobial properties in pure form still need to be investigated (Jrad et al., 2019). Similarly, whey proteins hydrolyzed by papain indicated strong antibacterial activity against Sal. typhimurium, E. coli, B. cereus, and Sta. aureus (Abdel-Hamid et al., 2016). Results also suggested that low-molecular-weight peptides fractions were significantly antibacterial against all pathogens tested with minimum inhibitory concentrations of 0.01, 0.09, 0.09, and 0.39 mg/mL, respectively. Insights into mechanism of antibacterial actions of these peptides indicated that these peptides were capable of inducing strong ultrastructural changes that eventually led to loss in cell integrity causing cell lysis through leakage of cytoplasm by inducing pores in cell membranes (AbdelHamid et al., 2016). Casein hydrolysates derived by alcalase, α-chymotrypsin, and papain were also evaluated for their antibacterial potential against E. coli, B. cereus, Sta. aureus, and Li. monocytogenes (Kumar et al., 2016a). Different fraction indicated different level of inhibition against all pathogens tested with some fraction even showing lower inhibition than intact casein protein. Overall,

257

Table 9.5 Antimicrobial properties of camel milk protein hydrolysates obtained via fermentation or enzymatic hydrolysis. Substrate

Pathogens tested

Method of production

IC50 values or % inhibition

References

Whey

E. coli Dh1α

Proteinase K, thermolysin, chymotrypsin, trypsin

47.3%, 26.4%, 20.0%,19.1%

Salami et al. (2010)

Colostrum protein (CPH), colostrum

E. coli XL1 blue,

SGID

CPH—9%/10 μg/mL; CWPH—

Jrad et al. (2014)

29%/10 μg/mL

whey (CWPH)

CPH—11.10%/10 μg/mL; CWPH—

Li. innocua LRGIA01

15.4%/10 μg/mL Whey

Sal. typhimurium, E. coli, B. cereus, Sta.

Papain

23.3, 34.0,12.0, 49.3 mm

SGID

Stimulated the growth of PA

Jrad et al. (2015)

8.45
19.11, 10.20
17.93,

Kumar et al. (2016)a

aureus Casein

L. innocua LRGIA01, B. cereus ATCC

Abdel-Hamid et al. (2016)

11778, Sta. aureus nosoco 301, E. coli XL1 bleu, P. aeruginosa ATCC 15742 Casein

Sta. aureus (MTCC No.

Alcalase, α-Chymotrypsin Papain

8.67
16.32, 8.65
18.95 mm

7443), E. coli (MTCC No. 2991), Li. monocytogenes (MTCC No. 657), B. cereus (MTCC No. 6728) Whole milk Whole milk

Whey fractions

Sta. aureus, E. coli, Morganella

Str. thermophilus, Lactobacillus (Lb.) delbrueckii

morganii, P. aeruginosa

sp. bulgaricus

10, 24, 14, 16 mm

Algboory et al. (2017)

B. cereus, E. coli, Sal. typhimurium , and Sta. aureus,

Lb. acidophilus, Str. thermophilus, Lb.

11
13, ND, 11
13, 11
13 mm,

Alhaj (2017)

helveticus, and Str. thermophilus

11
13, 11
13,14
16, 14
16 mm

E. coli, Sta. aureus, Sal typhimurium,Str.

Trypsin

6.90
19.20, 4.03
16.30, Nil
14.95,

mutans Whey fractions

P. aeruginosa PAO1, methicillin-resistant

Papain

22.3 and 19 mm

Lb. plantarum IS10

5%
80%,15%
78%

Alcalase and protease




Sta. aureus Camel milk fractions

E. coli O157:H7, Sta. aureus subsp. Candida albicans,Candida krusei, Candida parapsilosis

Abdel-Hamid et al. (2020)

aureus Whey casein

Wang et al. (2020)

Nil
8.30 mm

Algboory and Muhialdin (2021) AlMazroui, et al. (2022)

9.2 Bioactive peptides from camel milk proteins

alcalase- and α-chymotrypsin-derived hydrolysates presented more antibacterial potential than papain-generated hydrolysates. In general, their results suggested that the inhibition of pathogens was mainly determined by the molecular weight of the peptides with higher molecular weight peptide fraction indicating more inhibition of pathogens. Although their findings are contrary to a general notion toward bioactive peptides which generally indicated that low-molecular-weight peptides are better in their biofunctional attributes. Some studies on other peptides has also indicated similar trend where larger peptides were found to carry more antimicrobial potential (Jrad et al., 2019). Water-soluble extracts of two fermented camel milk samples (Lb. acidophilus and Str. thermophilus as S1 and Lb. helveticus and Str. thermophilus as S2) were evaluated for their antibacterial activities against B. cereus, E. coli, Sal. typhimurium, and Sta. aureus. The effect of storage on antibacterial activities was also evaluated and indicated no significant differences. The results showed S1 was not able to inhibit E. coli growth; however, all other pathogens tested were inhibited by both samples, that is, S1 and S2. It was also observed that S2 was more antibacterial than S1 (Alhaj et al., 2018). Camel milk fermented with yogurt cultures also indicated antibacterial potential against E. coli, Sta. aureus, M. morganii, and P. aeruginosa (Algboory et al., 2017). Further these researchers investigated the antibacterial activity of Lb. plantarum IS10 fermented Iraqi camel milk against Sta. faecalis, Shigella dysenteriae, Sta. aureus, and E. coli and inhibition in the range of 15%
25 % against all tested pathogens was observed (Muhialdin & Algboory, 2018). Further, this fermented camel milk was fractionated and evaluated for their antibacterial activity against E. coli and Sta. aureus. The results suggested that Fraction 14 was most active which was then sequenced using MS/MS and resulted in identification of 30 peptides out of which 7 novel peptides FVVTPK, RGLVPL, ELLPDMPLNQ, APGPLVVPPVGPPPP, PLPASGLL, VMVSGVAGNPGA, and HPPGSGLL showed 50% similarity to AMPs in ADP database (Table 9.6) (Algboory & Muhialdin, 2021). These peptides exhibited similarity to peptides and bacteriocins isolated from different strains of bacteria indicating a possible correlation of peptide sequence with their antimicrobial action. Moreover, these peptides were more active against Sta. aureus than E.coli. Being a Gram-negative microorganism E. coli is understandably more resistant to antimicrobial action owing to presence of extra outer membrane of lipopolysaccharide that by acting as a barrier provides more protection to this group (Algboory & Muhialdin, 2021). Similarly, Ganzorig et al. (2020) identified HPVPQP, VPYPQR, RPKYPLR, and QEPVPDPVR from fermented Bactrian camel milk. In silico prediction and sequence homology match suggested their antimicrobial potential. In another study conducted by Khajeh et al. (2021) a peptide IAGKCGLVPVL

259

Table 9.6 Antimicrobial peptides derived from camel milk proteins. Sequence

Parent protein

Pathogen tested

Method of proteolysis

References

QLFGSPAGQKDL, GSPAGQKDLL, VLKGEADAL, LDCVH RPVKGY, WAKDLKL, RIDKVAHL, FSASCVPCVDGKEYPN LCQLCAGTGENKCACSSQEPYFGY, FKDSALGL, IDKVAHL

Lactoferrin

Li. innocua (ATCC 33090 and LRGIA 01)

Pepsin

Jrad et al. (2019)

FVVTPK, VLPGPAVPKLLCLA, LLSLNTFK, VPLPVGPPVVNFR, FATTVPVLPRPPPVVAGAFA, RGLVPL, DLPLHQVDSH, AMDF GEEHTTT, TNVGLAADQEYPCL, ELLPDMPLNQ, DLPLNTM, TL PVPEKMVPYDVVMHVV, MVDLVSGATPL, DLPLMT, LTVDVA HAGGP, DTVGGPPAAGGT, AATPLLTEPQVGA, PAGTDLLYSP GVPAPPQ, LTVDAHKGVPL, LPVPGH, GPVGGPAA, FQPVPLT M, AAFTPAG, APGPLVVPPVGPPPP, PLPASGLL, TLSAAH, LVYP TAQGP, VMVSGVAGNPGA, YETPAPVF, HPPGSGLL

Whole milk

E. coli O157:H7 Sta. aureus subsp. aureus

Lactobacillus (Lb.) plantarum IS10

Algboory and Muhialdin (2021)

Lactophorin

E. coli, Sta. aureus

Lb. plantarum

Muhialdin and Algboory (2018)

IYMESPQPTDTSPAQ, FRNTATQSEETKE, VIMSNHQVSPSED, SSFRNTATQSEETKE, LHPVPQESS, SSFRNTATQSEE IKEVESPAE, VPTENKISQ, AEVPTENKISQ, NIKEVESPAE, AVRNIKEVESPAE, VESPAEVPTENKISQ, VAIHPSKED

α-S2casein. β-casein

QMVPYPQR, SITHINKQKIEK, ITHINKQKIEK, VMDVPKTKET, LTDLEN, AMPVQA, VLPFQEPVPDPVRG, LHPVPQP,LVYSH α-S1casein ILKEDMP, IASEDGGKTDVMPQ, NEPDSIEE, QNEPDSIEE, IDELKDTRN, FQNEPDSIEE, IAHPSSYDTPE, IDELKDTR, IAHPSSYD, RPKYP IAGKCGLVPVL

Lactoferrin

Sta. aureus, P. aeruginosa, Acinetobacter baumannii

In silico design

Khajeh et al. (2021)

PFQEPVPDPVRG, PFQEP VPDPVRGLHPVPQPLV

Casein

Sta. aureus CNRZ 3, Li. innocua ATCC33090, and E. coli ATCC 25922

Pepsin

Almi-Sebbane et al. (2018)

HPVPQP, VPYPQR, RPKYPLR, QEPVPDPVR

Whole milk

In silico prediction and sequence homology match

Natural fermentation

Ganzorig et al. (2020)

9.2 Bioactive peptides from camel milk proteins

was identified from camel milk lactoferrin through in silico design and was active against Sta. aureus, P. aeruginosa, and Acinetobacter baumannii. Further PFQEPVPDPVRG and PFQEPVPDPVRGLHPVPQPLV were identified from pepsin hydrolysate of camel casein protein and showed antibacterial actions against Sta. aureus CNRZ 3, Li. innocua ATCC 33090, and E. coli ATCC 25922 (Almi-Sebbane et al., 2018). Further, camel whey hydrolysates generated by papain hydrolysis were fractioned into low- and high-molecular-weight fractions and tested for their action against drug-resistant pathogens and also for their antibiofilm activity (Abdel-Hamid et al., 2020). Pathogens used were P. aeruginosa (PAO1) as an indicator microorganism for biofilm generation and methicillin-resistant Sta. aureus (MRSA) strain, known for their chronic and life-threatening infections (Abdel-Hamid et al., 2020). Contrary to results by Kumar et al. (2016a), low-molecular-weight peptides were more active than high-molecular-weight ones. The minimum inhibitory concentration was found to be 0.156 and 0.3125 mg/mL against P. aeruginosa and MRSA, respectively. Electron microscopic studies revealed that peptides caused cell death by inducing cell leakage and cell elongations (AbdelHamid et al., 2016, 2020). Similar results were also observed by Wang et al. (2020), where fractionated tryptic hydrolysate of camel whey when evaluated against E. coli, Sal. typhimurium, Str. mutans, and Sta. aureus reported higher inhibition of bacterial growth by peptides with lower molecular weight. A recent study conducted on the potential of camel milk, camel whey and camel casein hydrolysates derived by alcalase and protease has evaluated for their antifungal effects using various Candida spp. (Mudgil, AlMazroui, et al., 2022). Results revealed that hydrolysates produced had higher inhibition than intact proteins. It was also revealed that for some hydrolysates inhibition was even more than commercial drug fluconazole. The effect of hydrolysates was stronger against Candida albicans and Candida krusei, while against Candida parapsilopsis only alcalase-derived camel casein hydrolysate was found to be effective (Mudgil, AlMazroui, et al., 2022).

9.2.3.1 Structural activity relationship of camel milk
derived antimicrobial peptides Although numerous studies on antimicrobial potential of peptides have been undertaken, the exact mechanism of their antimicrobial action still remains elusive. As a generic notion most potent AMPs share the features of being small, highly positive charged and contain well-defined hydrophobic and hydrophilic areas (Tian et al., 2021). Generally, AMPs are categorized into two categories depending upon their mechanism of action as membrane and nonmembrane disruptors. The peptides in first category follows specific

261

262

CHAPTER 9:

Bioactive peptides derived from camel milk proteins

chain of actions, as also outlined by Abdel-Hamid et al. (2020), for growth inhibition of pathogen. These AMPs must be able to diffuse through cell wall and then interact with cytoplasmic membranes. This is generally achieved through electrostatic interaction between the positive charged amino acids within the peptide with negatively charged cell membrane components. It is noteworthy that for Gram-positive pathogen, peptides just have to permeate through cell wall before making interaction with membrane; however, against Gram-negative pathogen they have to overcome an extra layer of outer membrane. This results in low activity of peptide against Gramnegative microorganisms in comparison to Gram-positive ones (Bhonsle et al., 2007). As a well-established fact that bacterial strains vary significantly in the composition of their membranes and, therefore, their interaction with peptides will also vary. Antimicrobial properties of AMPs vary with the protein source, structure, spatial structural helicity, and class of peptides. Together with the structural properties and amino acid composition, surrounding environmental characteristics can also regulate the functionality of AMPs. Major environmental factors affecting AMP’s action are pH, heavy metals, and presence of protease (Ali Redha et al., 2022). As indicated by Wang et al. (2020), the tryptic hydrolysates of camel whey had high level of hydrophobic amino acids (51.29%) indicating a positive relationship between hydrophobic amino acids and antimicrobial properties (Wang et al., 2020). Overall, AMPs can exert pathogen killing via four different models (carpet, barrel stave, toroidal pore, and aggregate model) for forming pores or channels in bacterial membranes, through the carpet model acts like a detergent AMPs and destroys the cell membrane by forming micelles and pores (Yan et al., 2021). As the name suggests, in barrel stave model all AMPs aggregate together and make barrel shape structure in the cell membrane causing the leakage of cytoplasmic fluid and eventually result in cell death. Through the toroidal pore model, AMPS affect the osmotic sustainability of cells by getting embedded within the membrane making it bend inward causing a distortion and then by creating a pore they cause the eruption of cellular contents. In the aggregate model, AMPs first make a lipid peptide complex and get embedded within cell membrane, thereafter this complex crosses the cell membrane and interacts with intracellular material such as DNA, RNA, and enzymes and alters the major cellular functions such as transcription and translation. It is also suggested that different peptides might also employ multiple of these model for causing cellular arrest (Yan et al., 2021). To conclude, most of the studies related to antimicrobial action of camel milk
derived peptides have concentrated on the production or identification of peptides. Very few studies have actually elucidated the mechanism of peptide actions. Moreover, in vivo trials have not been conducted till date and

9.2 Bioactive peptides from camel milk proteins

need further explorations in animals and cell line models before going for human trials. Further research in the area of SAR is needed. The gaps in knowledge present a huge scope for researchers to continue delving in AMPs derived from camel milk
derived proteins. Furthermore, as emergence of multidrug-resistant strains is putting a burden on healthcare system, this area needs further attention as well.

9.2.4 Antidiabetic peptides derived from camel milk proteins Camel milk has been long known for its antidiabetic potential with mechanism unknown, therefore a great deal of efforts has been directed in investigating the mechanism for its antidiabetic action. Numerous in vitro studies have been conducted toward the inhibition of key enzymatic markers of diabetes such as (1) carbohydrate digestive enzyme, namely α-amylase (AM) and α-glucosidase (AG) and (2) dipeptidyl peptidase-IV (DPP-IV), an enzyme responsible for degradation of incretins. Since AM and AG are involved in hydrolysis of 90% of dietary carbohydrates, their partial inhibition is considered as key strategies for controlling postprandial hyperglycemia (Mudgil, Jobe, et al., 2019). Further, DPP-IV catalyzes the degradation of insulinotropic hormone, that is, glucagon-like peptide-1 and glucose-dependent insulinotropic peptide (GIP) (Nongonierma et al., 2019; Nongonierma & FitzGerald, 2019; Power et al., 2014). These hormones regulate the secretion of insulin in postprandial phase, thereby regulating the glucose absorption. Ultimately, degradation of these hormones disturb glucose homeostasis (Ashraf et al., 2021). Therefore, inhibition of DPP-IV is a much pursued strategy for controlling postprandial hyperglycemia and thus management of diabetes (Liu et al., 2019). The major studies and peptides identified from camel milk protein hydrolysates are presented in Table 9.7 and Table 9.8.

9.2.4.1 Dipeptidyl peptidase-IV inhibitory peptides from camel milk proteins Inhibition of DPP-IV is the most extensively studied antidiabetic mechanism of camel milk
derived hydrolysates (Table 9.7). Various researchers have probed the camel milk proteins and their fraction for generation and identification of DPP-IV inhibitory peptides (Table 9.8). Whey proteins have been the preferred substrate for the generation of DPPIV inhibitory peptides. Trypsin-generated hydrolysates of camel whey were studied using response surface methodology for optimization of hydrolysis conditions such as temperature, hydrolysis duration, and enzyme:substrate ratio (Nongonierma et al., 2017). A significant correlation between all the

263

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Bioactive peptides derived from camel milk proteins

Table 9.7 In vitro studies related to antidiabetic properties of camel milk protein hydrolysates or fermentate. Source

% Inhibition/IC50 Values

Method of hydrolysate production

References

Shori and Baba (2014)

α-amylase Whole milk

 30%

Skim milk

.34%

Skim milk

.40%

Yogurt-mix powder (containing Lb. acidophilus, Lb. bulgaricus, Lb. casei, Bifidobacterium bifidus, and S. thermohilus) Lb. reuteri-KX881777, Lb. plantarumKX881772, Lb. plantarum-KX881779 and Lb. plantarum DSM2468 Lc. lactis KX881782 and Lb. acidophilus DSM9126

Skim milk

Whey

0.027 mg/mL 0.025
0.27 mg/mL 0.030
0.074 mg/mL 0.29
3.69 mg/mL

Alcalase [50 C, E:S (1%), @3, 6, and 9 h] Bromelain [50 C, E:S (1%), @3, 6 , and 9 h] Papain [50 C, E:S (1%), @3, 6 , and 9 h] Pepsin using response surface methodology (RSM)

Casein

0.59
1.36 mg/mL

Alcalase and pronase E [50 C, E:S (1%), 3 and 6 h] followed by in vitro SGID

Ayyash, Al-Dhaheri, et al. (2018) Ayyash, Al-Nuaimi, et al. (2018) Mudgil et al. (2018)

Baba, Mudgil, Kamal, et al. (2021) Mudgil et al. (2021)

α-glucosidase Whole milk

 8%
13%

Skim milk

 25%
40%

Skim milk

 30%
40%

Whey Whey

 20%
78.59% 0.64
2.16 mg/mL

Casein

0.59
1.47 mg/mL

DPP-4 Whey Whey

 60%
96% 0.52-1.26 mg/mL

Pepsin, trypsin, and chymotrypsin Trypsin (RSM)

17.2 mg/mL 0.55 mg/mL

In vitro SGID Trypsin (RSM)

Skim milk Enriched whey

Yogurt-mix powder (containing Lb acidophilus, bL. bulgaricus, bL. casei, Bifidobacterium bifidus, and S. thermohilus) Lb. reuteri-KX881777, Lb. plantarumKX881772, Lb. plantarum-KX881779 and Lb. plantarum DSM2468 Lc lactis KX881782 and Lb. acidophilus DSM9126 Hydrolysis by pepsin, trypsin, and chymotrypsin Hydrolysis by pepsin using response surface methodology Alcalase and pronase E [50 C, E:S (1%), 3 and 6 h] followed by in vitro SGID

Shori and Baba (2014)

Ayyash, Al-Dhaheri, et al. (2018) Ayyash, Al-Nuaimi, et al. (2018) Kamal et al. (2018) Baba, Mudgil, Kamal, et al. (2021) Mudgil et al. (2021)

Kamal et al. (2018) Nongonierma et al. (2017) Tagliazucchi et al. (2018) Nongonierma et al. (2019)

Continued

9.2 Bioactive peptides from camel milk proteins

Table 9.7 In vitro studies related to antidiabetic properties of camel milk protein hydrolysates or fermentate. Continued Source Skim milk

% Inhibition/IC50 Values

Casein

0.09
0.18 0.17
0.32 0.16
0.46 0.66
2.70

mg/mL mg/mL mg/mL mg/mL

Whey

0.44
8.26 mg/mL

Method of hydrolysate production 

Alcalase [50 C, E:S (1%), @3, 6, and 9 h] Bromelain [50 C, E:S (1%), @3, 6, and 9 h] Papain [50 C, E:S (1%), @3, 6, and 9 h] Alcalase and pronase E [50 C, E:S (1%), 3 and 6 h] followed by in vitro SGID Pepsin [30 C, E:S (2%), 6 h]

conditions was established toward their impact on DPP-IV inhibitory activity of generated peptides. Overall, 16 hydrolysates were generated and the DPPIV-IC50 values of these hydrolysates ranged between 0.52 and 1.26 mg/mL. The study identified 15 peptides out of which two novel peptides LPVPQ and WK were identified. Another study conducted on tryptic hydrolysates of whole camel milk protein further identified 9 novel camel (dromedary) milk peptides FLQY, FQLGASPY, ILDKEGIDY, ILELA,LLQLEAIR, LPVP, LQALHQGQIV, MPVQA, and SPVVPF out of which peptides LPVP and MPVQA possessed substantially high DPP-IV inhibition as indicated by their low DPP-IV IC50 of 87.0 and 93.3 μM, respectively (Nongonierma et al., 2018). Further studies conducted on camel whey protein hydrolysates generated by trypsin were reported to identify the second most potent peptide VPV till date with a DPP-IV-IC50 values of 6.6 μM after the commercial drug peptide diprotin (IPI) (Nongonierma et al., 2019). Furthermore, DPP-IV inhibition of camel whey protein hydrolysate
derived using pepsin, trypsin, and chymotrypsin was studied by Kamal et al., (2018). Comparative analysis revealed the superiority of pepsin-derived hydrolysates in inhibiting DPP-IV (Kamal et al., 2018). Overall, inhibition in the range of 90% was achieved by peptic hydrolysates. Later optimization of pepsin-derived hydrolysates for the generation of potent DPP-IV inhibitory peptides was undertaken by Ashraf et al. (2021) and mechanistic evaluation of peptides for their antidiabetic action were explored through inhibition of DPP-IV and human insulin receptor (hIR) activity. Generated hydrolysates displayed DPP-IV-IC50 values in the range of 0.44
8.26 mg/mL with hydrolysates produced at 30 C, with a E:S ratio of 1.0%
2.0% for 2 and 6 h having the least DPP-IV-IC50 values (Ashraf et al., 2021). This study also unveiled that peptic camel whey hydrolysates induced the phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and positively affected hIR activation and glucose uptake (Ashraf et al., 2021).

References Mudgil et al. (2018)

Mudgil et al. (2021) Ashraf et al. (2021)

265

Table 9.8 Camel milk
derived antidiabetic peptides with inhibitory properties against dipeptidyl peptidase-IV (DPP-IV), α-amylase, and α-glucosidase. Sequence

Protein source

Method for hydrolysis

Antidiabetic activity assay

LPVPQ VL LPQ IP LPL LPLPL YP LP VP AL EK FF FL HL LA LL LPLPL LPVPQ PP

β-casein Various proteins Various proteins Various proteins β-casein β-casein Various proteins Various proteins Various proteins Diverse Diverse Diverse Diverse Diverse Diverse Diverse β-CN (f136-140) β-CN (f172-177) Diverse

Trypsin

SL WK WL YL YP

Diverse αs2-CN (f69-70) Diverse Diverse Diverse

DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition μM DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition 3170 μM

Trypsin

IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50 IC50

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

44 μM 74 μM 82 μM 150 μM 241 μM 325 μM 658 μM 712 μM 880 μM 882.1 μM 3216.75 μM 546.84 μM 399.6 μM 143.2 μM 91; 300 μM 191.7 μM 325 μM 43.69 μM 4343.48; 5860

IC50 IC50 IC50 IC50 IC50

5 5 5 5 5

2517.08 μM 40.6 μM 43.6 μM 940.2 μM 658.1; 7564.02;

References Tagliazucchi et al. (2018)

Nongonierma et al. (2017)

AMPVQAVLP FHMSG FKIEEQQQTEDEQQDKI FKIEEQQQTEDEQQDKIY FLPPLQPAV FLQY FQLGASPY FR2 FY2 IAHPSSY ILDKEGIDY ILELA ILELAVVSPIQFR LE2/IE2 LEELHR LLQLEAIR LMY LPVP LQALHQGQIV LQLEAIR LR/IR2 LSSHP LT2/IT2 LV2/IV2/VL2/VI2 LVY LY2/IY MPVQA NFLPPLQP VAWYYPPQV VLP/VIP VLPVP VPYPQR YPLR YPPQVMQY YPSYGINYY

β-CN (f185-193) α-La (f31-35) β-CN (f34-50) β-CN (f34-51) β-CN (f76-84) αs2-CN (f71-74) αs1-CN (f168-175) αs1-CN (f39-40) αs2-CN (f65-66), κ-CN (f67-79) αs1-CN (f188-194) α-La (f95-103) αs1-CN (f28-32) αs1-CN (f28-40) diverse αs1-CN (f97-102) αs1-CN (f108-115) β-CN (f144-146) β-CN (f172-175) αs2-CN (f75-84) αs1-CN (f109-115) diverse αs1-CN (f125-129) diverse diverse β-CN (f59-61) diverse β-CN (f186-190) β-CN (f75-82) αs1-CN (f176-184) β-CN (f171-173), β-CN (f191-193), κ-CN (f124-126) β-CN (f171-175) β-CN (f179-184) αs1-CN (f4-7) αs1-CN (f180-187) κ-CN (f35-43)

Trypsin

DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition

Nongonierma et al. (2018)

5 .1000 μM 5 .1000 μM

5 347.8 6 42.8e μM

5 177.8 6 22.2c μM 5 87.0 6 3.2a μM 5 .1000 μM

5 93.3 6 8.0a μM

DPP-IV inhibition 5 .1000 μM DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition DPP-IV inhibition

Continued

Table 9.8 Camel milk
derived antidiabetic peptides with inhibitory properties against dipeptidyl peptidase-IV (DPP-IV), α-amylase, and α-glucosidase. Continued Sequence

Protein source

Method for hydrolysis

Antidiabetic activity assay

References

EPVK FLNK FYAPELL

αs1-CN (f147
150) αs2-CN (f141
144) in serum albumin from 6 mammalian species α-La (f29
31) α-La (f105
110) κ-CN (f17
21) αs1-CN (f109
111) β-CN (f89
92) β-CN (f86
88); β-CN (f118
120) β-CN (f213
215) β-CN (f69
71) αs1-CN (f4
6) Casein

Trypsin

DPP-IV IC50 5 330.1 6 55.9 μM DPP-IV IC50 5 1367.2 6 107.3 μM DPP-IV IC50 5 .2000.0 μM

Nongonierma et al. (2019)

Alcalase, pronase E, followed by SGID

DPP-IV IC50 5 ND μM DPP-IV IC50 5 239.7 6 24.7 μM DPP-IV IC50 5 1360.7 6 95.2 μM DPP-IV IC50 5 ND μM DPP-IV IC50 5 .2000.0 μM DPP-IV IC50 5 55.1 6 5.8 μM DPP-IV IC50 5 6.6 6 0.5 μM DPP-IV IC50 5 35.0 6 2.0 μM DPP-IV IC50 5 360.1 6 28.1 μM Peptides predicted to have multiple biological properties

Casein

Alcalase, pronase, and SGID

Potent α-amylase inhibitors

Casein

Alcalase, pronase , and SGID

Potent α-glucosidase inhibitors

Casein

Alcalase, pronase, and SGID

Potent DPP-IV inhibitors

IIF LAHKPL LLNEK LQL LQPK VPF VPV YPI YPLR MM AAGF DMML LP FFE FLWPEYGAL DPPP HSGF MFE LP LM MSNYF HSGF LP DGALHPPL LPTGWLM DEPYPLL LFLCCC QDNPPL HASWPLL DPPP FLWPEYGAL ACGP DGALHPPL LPTGWLM, MFE GPAHCLL HLPGRG QNVLPLH PLMLP

Mudgil et al. (2021)

PAGNFLM NGLMHR PAVACCL PPLPCHM PAGNFLP PVAAAPVM MLPLML PFTMGY AEWLHDWKL ALWGAGGGGLGLSSGR CFLPLPLLK DNLMPQFM FCLPLPLLK FMFFGPQ GMAGGPPLL HCPVPDPVRGL KDLWDDFKGL KFQWGY LLPAPPLL LTMPQWW MMHDFLTLCM MSKFLPLPLMFY SQDWSFY WGLWDDMQGL WNWGWLLWQL YWYPPK YWYPPQ TLMPQWW MPSKPPLL AVVSPLKPCC PAGNFLMNGLMHR PAVACCLPPLPCHM MLPLMLPFTMGY PAGNFLPPVAAAPVM CCGM MFE FCCLGPVPP

Whey

Pepsin

Potent DPP-IV inhibitors

Ashraf et al. (2021)

Skim milk

Alcalase

Potent DPP-IV and α-amylase inhibitors

Mudgil et al. (2018)

Potent binder and inhibitors for α-amylase and α-glucosidase

Baba, Mudgil, Kamal, et al. (2021)

Bromelain

Whey

Bromelain and alcalase Pepsin

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Further, skimmed camel milk was hydrolyzed using SGID (Tagliazucchi et al., 2018), alcalase, bromelain, and papain (Mudgil et al., 2018). The results suggested that alcalase-derived hydrolysates produced peptides with strongest DPP-IV inhibition as indicated by their low values of DPP-IV-IC50 of 0.09 mg/mL, whereas SGID-derived camel protein hydrolysates showed 17.2 mg/mL (Table 9.7). Tagliazucchi et al., (2018) further identified peptides in the most active fraction and reported three peptides LPVPQ, VL, and LPQ with markedly lower DPP-IV-IC50 of 44, 74, and 82 μM. As per the study of Mudgil et al. (2018), two peptides DNLMPQFM and WNWGWLLWQL in alcalase-derived hydrolysate A9 had maximum DPP-IV binding sites. In another recently conducted study, casein hydrolysates produced by alcalase and pronase E showed potent DPP-IV-IC50 values of 0.62 and 0.66 mg/mL. Moreover, peptides HLPGRG, QNVLPLH, and PLMLP were found to be most potent against DPP-IV (Mudgil et al., 2021).

9.2.4.2 α-Amylase (AM) inhibitory peptides from camel milk proteins

A number of studies on the α-amylase inhibitory properties of various camel milk protein hydrolysates have been performed over past decade and are compiled in Table 9.7. Amylase inhibitory potential of fermented camel milk was investigated via fermentation with a mixed yogurt culture by Shori and Baba (2014) and the results suggested that water-soluble extract obtained from fermented camel milk had higher AM inhibition than fermented bovine milk. Also in similar study, camel milk fermentation was carried out by various strains of lactic acid bacteria such as Lb. reuteri-KX881777, Lb. plantarum-KX881772, Lb. plantarum-KX881779, Lb. plantarum DSM2468, Lc. lactis KX881782, and Lb. acidophilus DSM9126 by Ayyash, Al-Dhaheri, et al. (2018) and Ayyash, AlNuaimi, et al. (2018). Overall, the AM inhibition varied in range of 34% till 60%. It was concluded that Lb. reuteri-KX881777 and Lc lactis KX881782 produced hydrolysates with more AM inhibitory potential than standard probiotic culture of Lb. acidophilus DSM9126. Whey protein hydrolysates produced upon hydrolysis with pepsin, trypsin, and chymotrypsin produce AM inhibition in the range of  30%
93% and further, chymotrypsin- and pepsin-generated hydrolysates displayed more inhibition than trypsin-generated ones. Time of hydrolysis was found to significantly impact the percentage of AM inhibition with 6-h hydrolysates displaying more inhibition than 3-h hydrolysates (Kamal et al., 2018). Further exploration into the pepsin-generated whey hydrolysates using response surface methodology yielded AM-IC50 between 0.29 and 3.69 mg/mL. The result also suggested that more potent AM inhibitory hydrolysates could be generated using low enzyme concentration (0.5%) in combination with different levels of time and temperature (Baba, Mudgil, Kamal, et al., 2021).

9.2 Bioactive peptides from camel milk proteins

Depending upon the number of binding sites and interaction with amino acids in the active site of AM, peptides with sequence PAGNFLMNGLMHR, PAVACCLPPLPCHM, MLPLMLPFTMGY, and PAGNFLPPVAAAPVM were identified as potent AM inhibitory peptides (Baba, Mudgil, Kamal, et al., 2021). Further, enzymatic hydrolysates of camel milk proteins as a whole or their whey and casein fractions have also been evaluated. Whole camel milk proteins digested with alcalase, bromelain, and papain yielded hydrolysates with AM-IC50 values in range of 0.027
0.074mg/mL, with alcalase and bromelain generating peptides with better amylase inhibitory actions (Mudgil et al., 2018). Pepsite 2-based interaction model between peptides and enzyme revealed that 10 peptides, AEWLHDWKL, DNLMPQFM, GMAGGPPLL, FMFFGPQ, KDLWDDFKGL, KFQWGY, MMHDFLTLCM, MSKFLPLPLMFY, SQDWSFY, and WGLWDDMQGL, from alcalase-derived hydrolysates were able to bind active site residues with the amylase binding site, whereas MPSKPPLL from bromelain-derived hydrolysates showed the most potent binding with amylase suggesting its potential in further development of antidiabetic nutraceutical (Table 9.8). However, before making any substantial claims, further investigations in animals and cell line models are warranted (Mudgil et al., 2018).

9.2.4.3 α-Glucosidase (AG) inhibitory peptides from camel milk proteins Although the studies on AG inhibitory potential are limited in comparison to DPP-IV and amylase, the obtained reports provide evidence on the glucosidase inhibitory potential of camel whey
and camel casein
derived peptides (Table 9.7) (Ali Redha et al., 2022). Together with AM inhibition, AG inhibition was also assessed in camel milk yogurt by Shori and Baba (2014). Similar to AM inhibition, AG inhibition was more prominent in camel milk yogurt than bovine milk yogurt. Similarly, fermentation of camel milk by different probiotic lactic acid bacteria isolated from camel milk produced an AG inhibition value of 25%
40% (Ayyash, Al-Dhaheri, et al., 2018; Ayyash, AlNuaimi, et al., 2018). AG inhibitory potential of whey protein hydrolysates produced upon hydrolysis with pepsin, trypsin, and chymotrypsin displayed the similar trend like AM where pepsin- and chymotrypsin-generated hydrolysate displayed significantly stronger inhibition than trypsin-generated hydrolysates. Overall, the inhibition values were more than the parent protein and varied in ranges of 24%
78%. A significant effect on time of hydrolysis and type of enzyme was noticed (Kamal et al., 2018). Therefore, in order to find out the optimum temperature, time, and enzyme:substrate ratio, Baba, Mudgil, Kamal, et al. (2021) utilized response surface methodology design and produced 27

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different hydrolysates with AG-IC50 between 0.64 and 2.16 mg/mL. Upon peptide identification in four most potent hydrolysates, H4, H7, H16, and H18, a total of 43, 48, 45, and 48 peptides were identified from which 15 were ranked as bioactive depending on their peptide ranking score. An insight into the sequence analysis of these 15 peptides revealed the presence of amino acid methionine at C-terminal which is a typical characteristic of peptides with AG inhibitory potential (Ibrahim et al., 2018b). The molecular interaction with enzyme using pepsite revealed that peptides PAGNFLMNGLMHR, MFE, PAVACCLPPLPCHM, and MLPLMLPFTMGY could bind 11 important residues of AG, while CCGM and PAGNFLPPVAAAPVM bound 10 residues on enzyme indicating their solid potential to inhibit AG. Recently, camel casein hydrolysates produced upon alcalase and pronase E digestion were also checked for their potency to inhibit AG under in vitro conditions. The hydrolysates were further digested to check the stability of peptide to SGID conditions. Results revealed that AG-IC50 values of all hydrolysates ranged between 0.59 and 1.47 mg/mL, with SGID further improving their AG inhibitory properties (Mudgil et al., 2021). It was also noted that in the study reported by Baba, Mudgil, Kamal, et al. (2021) peptides LPTGWLM, MFE and GPAHCLL could bind up to 11 of enzyme hotspots, indicating higher possibility of competitive and noncompetitive inhibition from these peptides. Overall, antidiabetic studies on camel milk
derived peptides show promising results; however, for further investigation and development of functional foods or nutraceuticals, their capability to extend these beneficial effects in vivo needs to be tested as well. Till date, two studies have illustrated the antidiabetic potential of camel milk
derived peptides in vivo. Ebaid et al. (2015) studied the wound healing capacity of trypsin-hydrolyzed camel whey peptides in diabetic rats. It was observed that oral feeding of trypsin-derived peptides at a dose of 25 mg/kg body weight restored the activities of major antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). Level of lipid peroxidation markers was also found to be normally regulated. Further analysis through gene expression in wounded skin tissue also suggested the normalization of inflammation markers such as TNF-α, nuclear factor kappa B, and macrophage migration inhibitory factor. In another study, alcalase-derived camel milk hydrolysate fed at 500 mg/kg body weight to streptozotocin-induced diabetic rats showed normalization of liver function markers, that is, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and significant positive impacts on various parameters such as blood glucose, oral glucose tolerance, lipid profile, liver function markers, and antioxidant enzymes. Significant improvements in oxidative stress markers such as superoxide dismutase, catalase, reduced glutathione, and malondialdehyde were also observed. Streptozotocin-induced hepatic damage was found to be significantly normalized and number and size of pancreatic islets

9.2 Bioactive peptides from camel milk proteins

were found to be increased. Overall, although these in vivo studies have again validated the superiority of camel milk as an antidiabetic agent, further research is still needed to target other risk factors of diabetes.

9.2.4.4 Structural activity relationship of camel milk
derived antidiabetic peptides

For the characteristics of α-amylase inhibitory peptides, it has been reported that peptides that can bind to the domains A and C (catalytic domain) or B (substrate binding domain) are deemed to be potent inhibitors of α-amylase (Ngoh & Gan, 2016, 2018). Additionally, this enzyme works through a substrate sliding mechanism and inhibition of its formation is considered key mechanism in inhibiting the resulting action of enzyme. Another mechanism that is proposed for AM inhibition is induction of rigidity in the region 303
309 and 351
359 due to peptide binding causing the distortion in conformation and subsequently substrate binding is halted (Mudgil et al., 2021). Furthermore, as the active site of α-amylase contains aromatic amino acids, peptides with aromatic amino acids have better possibility of making stronger interactions. Hence, the presence of amino acids such as tyrosine, phenylalanine, and tryptophan is the key characteristic of potent AM inhibitory peptides. Moreover, to reach to its maximum activity level, AM needs chloride ions, and therefore, amino acids such as phenylalanine, aspartic acid, arginine, glutamine, proline, and tryptophan that can likely bind to chloride-binding sites of AM can disrupts its activation (Siow & Gan, 2016). Similarly, calcium ion is also important in stabilizing the active site cleft of AM by formation of an ionic bridge in domains A and B. Therefore peptides containing amino acids with calcium binding affinity such as aspartic acid and glutamic acid are crucial for amylase inhibition (Ngoh & Gan, 2018). In addition, the importance of the presence of glycine or phenylalanine at the N-terminus position, and phenylalanine or leucine at the C-terminus of α-amylase inhibitor peptides (Ngoh & Gan, 2016) has also been reported. Presence of hydrophilic arginine at C-terminus is reported to create strong hydrogen bonds with active site residues of AG (Liu et al., 2021). These strong hydrogen bonds are involved in stabilizing the peptides in the active site, thereby prohibiting the interaction with actual substrate (Liu et al., 2021; Zhang et al., 2016). Moreover, the presence of glutamine, proline, and serine adjacent to N-terminal has been reported to promote AG inhibition (Zhang et al., 2016). Further, hydroxyl or basic side chain at the N-terminus, proline adjacent to C-terminus, and alanine and methionine interact with active site residues of AG through their hydrophobic interactions (Vilcacundo et al., 2017).

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The structural variations in identified DPP-IV inhibitory peptides are quite diverse. It was noted that instead of amino acid composition, it is the sequence and position of amino acids that contributes to the DPP-IV inhibitory activity of peptides (Neves et al., 2017). The various structural features of DPP-IV inhibitory peptides were analyzed for their alignment, location, and frequency of occurrence (Nongonierma & FitzGerald, 2019). Presence of tryptophan, threonine, methionine at N-terminus and alanine, leucine, and histidine at C-terminus have been a consistent feature of potent DPP-IV inhibitory peptides. Presence of hydrophobic amino acids such as alanine, glycine, isoleucine, leucine, proline, methionine, tryptophan, and valine is considered concomitant with higher potency of peptide for DPP-IV inhibition. It is believed that hydrophobic amino acids in the DPP-IV inhibitory peptides interact with hydrophobic pockets in S1 subsite of DPP-IV. Although the presence of hydrophilic amino acids such as threonine, histidine, glutamic acid, serine, lysine, and arginine in peptides has recently been correlated with their DPP-IV inhibitory activities, their actual role in determining the potency of DPP-IV inhibitory sequences has not particularly been established till now. The N-terminal amino acid residue for DPP-IV inhibitor peptides is preferably suggested to be a hydrophobic amino acid (such as tryptophan), and proline or alanine or a low-molecular-weight amino acid as second to last residues at the N-terminus .

9.2.5 Antiobesity peptides from camel milk proteins and their structural activity relationship Inhibition of enzymes involved in lipid metabolism has been targeted as a key approach in the management of obesity (Lunagariya et al., 2014). Pancreatic lipase (PL) and cholesteryl esterase (CE) are two key enzymes that have been central to the discovery of antiobesity agents (Birari & Bhutani, 2007). Althwab et al. (2020) studied the effect of whole camel milk and camel whey supplementation on the obesity parameters and lipid profile in rats fed on a high-fat diet. The finding suggested significant reductions in body weight gain, adipocyte size, and the weight of perirenal adipose tissue. Significant reductions for fasting glucose, low density lipoprotein (LDL), and total cholesterol were also noticed (Althwab et al., 2020). Research works related to antiobesity potential of food protein
derived peptides are limited. However, in past few years, some reports have emerged which indicated that milk proteins can be used as a substrate for the development of antiobesity nutraceuticals or drugs (Kumar, 2019). In vitro assays have also been employed by scientists to study the hypocholesterolemic properties of peptides derived from camel milk proteins as whole or its fractions such as casein and whey. Mudgil et al. (2018) used alcalase-, bromelain-, and papain-derived hydrolysates from camel skim milk and reported significant improvements in PL-IC50 values among various hydrolysates generated.

9.2 Bioactive peptides from camel milk proteins

The PL-IC50 values ranged between 0.025 and 0.079 mg/mL, with papaingenerated hydrolysates being most active inhibitor of PL. Investigation into the mechanism of PL inhibition by identified peptides revealed that the peptides such as FCLPLPLLK and KFQWGY were capable of binding to catalytic sites responsible for triglyceride hydrolysis (Ser153 and His264) and the substrate binding site Phe216. Peptides FMFFGPQ, MSKFLPLPLMFY, YWYPPK, and YWYPPQ showed binding affinity to another substrate binding site Phe78. Another set of peptides LTMPQWW showed binding to His152, TLMPQWW bound to Phe78, His152, Phe216, and His264, whereas MPSKPPLL to Phe78, Ser153, Phe216, and His264. As higher number of binding site reflects positively toward PL inhibitory potential, it was suggested that these peptides can further be explored for cell lines and in vivo trials in more details (Mudgil et al., 2018). Further, same set of hydrolysates were also investigated for their potential to inhibit CE and results showed similar trend to that of PL inhibition (Mudgil, Baby, Ngoh, Vijayan, et al., 2019). CE-IC50 values ranged between 32.43 and 9.10 μg/mL protein equivalent with papain hydrolysates again showing superior inhibition than alcalase and bromelain. The CE active site comprises a catalytic triad (Ser194, Asp320, and His435) and an oxyanion hole (Gly107, Ala108, and Ala195) vital for its catalytic function. Overall, peptides KFQWGY, SQDWSFY, and YWYPPQ from alcalase-derived hydrolysates A9; TLMPQWW in bromelain hydrolysate B9; and 16 peptides AVMPQWW, CYCKPQQFPMVKMYG, FNLPPLKPAVM, FPPMPQPQ, GGQPFPQM, LCLENLHLPLPLF, LPPLVPFLKNPLM, MYQQWKFL, NQPFPKF, RMPKWW, VLMPQWW, VPFVPFLQPKVM, WMPQWEG, WSPLQMR, and YWYPPQ in papain hydrolysates P9 displayed prominent binding to CE. Peptides FNLPPLKPAVM, FPPMPQPQ, LCLENLHLPLPLF, and LPPLVPFLKNPLM showed binding to the catalytic triad units. Further investigation into molecular mechanics with generalized Born and surface area binding energies revealed that peptides WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR showed effective binding. All of these peptides formed hydrogen bonds with catalytic triad residues. Further hydrogen and hydrophobic interactions were seen between these peptides and Ala108 and Ala195 amino acids are responsible for the coordination of oxyanion intermediate formation during reaction (Mudgil, Baby, Ngoh, Vijayan, et al., 2019). The study also corroborated the presence of hydrophobic amino acids with enhanced binding of peptides to the active site of CE. In a similar study from camel whey protein hydrolysates, pepsin-based hydrolysates showed prominent PL and CE inhibition (Jafar et al., 2018). Trypsin-generated hydrolysate T6 displayed better CE inhibition, while chymotrypsin-generated hydrolysate C3 shows better PL inhibition suggesting different enzymes produce peptides with different specificity against PL and CE. Further extension of the similar work by Baba, Mudgil, Baby,

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et al. (2021), for optimization of process parameters for the generation of CE and PL inhibitory peptides from pepsin hydrolysates of camel whey, identified peptides PAGNFLPPVAAAPVM, FCCLGPVPP, LP, and MLPLMLPFTMG that showed binding to interesting sites within CE enzyme. Similar to peptides obtained in study conducted by Mudgil, Baby, Ngoh, Vijayan, et al. (2019), peptide PAGNFLPPVAAAPVM showed binding to Ala108 suggesting disruption of oxyanion intermediate formation. FCCLGVPP and LP binding affinity to Ser194 and His435 of active site also suggested their ability as potent CE inhibitor. Similarly, PAGNFLPPVAAAPVM, MLPLMLPFTMGY, and LRFP showed effective interaction with 11-12 residues on PL with potent interaction with amino acids Ser153, His264, His152, Phe78, and Phe216 from the active sites of PL. It was also reported that peptides with more hydrophobic residues in their sequence showed better binding than other peptides suggesting a positive correlation between hydrophobic amino acids and PL inhibition. On the similar lines, hydrolysis of camel casein by alcalase and pronase E and subsequent hydrolysis also showed effective PL and CE inhibition with PL-IC50 values in mg/mL and CE-IC50 values between 0.3 and 0.63 mg/mL. Among the identified peptides, MSNFYF, AAGF, and FLWPEYGAL showed the potential to be competitive inhibitors of PL, preventing access of dietary triacylglycerols to reach enzyme and thereby possible reduction in the formation of fatty acids.

9.2.6 Other biological properties of camel milk
derived hydrolysates The other underexplored bioactive properties of camel milk peptides include anticancerous, antiinflammatory, antihaemolytic, urease inhibitory, hepatoprotective, and antiaging properties. Recently, two different studies have evaluated the anticancerous properties of camel whey hydrolysates. In the first study, whey hydrolysates obtained from trypsin, chymotrypsin, and pepsin were tested against liver cancer cell line (HepG2). Results showed that strong antiproliferative activities in chymotrypsin hydrolysates with an approximate inhibition of 93.5%
95.5% in a dose-dependent manner. Other camel whey hydrolysates generated using pepsin and trypsin also showed significant decline in proliferation of cancerous cells. Similarly, LEEQQQTEDEQQDQL, YLEELHRLNAGY, and RGLHPVPQ peptides derived from camel milk via pepsin were also predicted to act against HepG2 cells through molecular docking interaction. Although low antiproliferative activity was seen against HepG2 cells, significant improvements in the level of superoxide dismutase and catalase gene expression were observed (Homayouni-Tabrizi et al., 2017). In another study of whey protein hydrolysates generated via pepsin hydrolysis tested against colon cancer cell line HCT116, it was observed that

9.2 Bioactive peptides from camel milk proteins

hydrolysates generated at different time, temperature, and enzyme:substrate ratio showed wide variation in their antiproliferative activities (Murali et al., 2021). Hydrolysate generated at 45 C, 1% E:S ratio, and digested for 6 h displayed the maximum inhibition of HCT cells with an IC50 value of 221 μg/ mL. Later investigations also revealed the arrest of G2/M cell cycle and change in the expression profile of Cdk1, p-Cdk1, Cyclin B1, p-histone H3, p21, and p53. Two peptides were identified and docking suggested an inhibition of enzyme Polo-Like Kinase-1 (PLK1) that plays a definitive role in cell cycle progression, the centrosome cycle, mitosis, and cellular responses to DNA damage (Murali et al., 2021). Another area of studies from camel milk peptides included their potential antiinflammatory and antihemolytic properties. Different researchers have explored various enzymes for the generation of hydrolysates from camel whey, camel milk, and casein for their antiinflammatory and antihemolytic properties in comparison to standard antiinflammatory drug diclofenac. As indicated by the results obtained, antiinflammatory activity of alcalase-derived camel milk hydrolysates were 3-4 times higher than those obtained from bromelain and papain hydrolysis. Positive correlation between time and antiinflammatory activity was found for hydrolysate from all three enzymes (Mudgil, Baby, Ngoh, Kamal, et al., 2019). In another such study, hydrolysis of whey proteins using different condition of temperature, time, and pepsin concentration hydrolysates prepared at 30 C with 0.5% enzyme concentration after 6 h of hydrolysis showed maximum antiinflammatory potential as indicated by their diclofenac sodium equivalent capacity values (Murali et al., 2021). Similarly, upon evaluation of three enzymes trypsin, chymotrypsin, and pepsin toward the generation of antiinflammatory peptides, pepsin-generated hydrolysates showed maximum activity (Kamal et al., 2018). Oxidation in cells induces erythrocytes hemolysis and therefore its control is considered of biological significance (Jafar et al., 2018). It is proposed that peptides or proteins exert antihemolytic action through the creation of surface protective covering around erythrocytes. Till today only one such study has determined the antihemolytic properties of camel whey hydrolysates generated via three different enzymes (pepsin, trypsin, and chymotrypsin) (Jafar et al., 2018). Pepsin- and trypsin-based hydrolysates showed better hemolytic activities in comparison to chymotrypsin hydrolysates. As outlined above that peptides from camel milk have shown excellent antimicrobial properties, their potential in the treatment of urinary tract infection has also been studied through inhibition of enzyme urease. Camel casein
derived peptides via trypsin and chymotrypsin hydrolysates have shown significant inhibition of urease by low-molecular-weight as well as highmolecular-weight fractions (Addar et al., 2019). Further, peptides from papain-based hydrolysis of skimmed camel milk has also shown that oral

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feeding of peptides at 200 mg/kg body weight effectively reduced thioacetamide-induced hepatorenal toxicity in rats together with normalization of other biological parameters such as lipid profile and normalization of enzyme involved in renal and liver functions. The hepatorenal protective effects of papain camel milk whey hydrolysates on thioacetamide (TAA)-induced toxicity in male Wistar albino rats were explored (Osman et al., 2021). Rats were treated with 50, 100, and 200 mg/kg body weight per day of camel whey protein hydrolysates, orally. Hydrolysates, especially at higher doses, were effective in reducing the effect of TAA-induced oxidative tissue damage and enhancing the activity of antioxidant enzymes; lipid profile (total lipid, triglyceride, total cholesterol, highdensity lipids, low-density lipids, and very-low-density lipids); and renal and liver functions as compared to untreated rats.

9.3

Future perceptions

Camel milk protein
derived peptides have shown a range of bioactive properties both in vitro and in vivo conditions. However, extent of in vivo studies is still limited before any substantial claims into health-promoting effects of these peptides can be made. Overall, it was noticed that selection of enzyme and hydrolysis conditions have a significant effect in controlling the biological functionality of these peptides. Moreover, further studies using clinical interventions are needed to validate the findings of in vitro studies and provide evidence toward the pharmacological and therapeutical values of camel milk
derived peptides. Further studies using synthetic peptides are also warranted to understand their safety, mechanism, and exact biological actions. Moreover, studies such as antiinflammatory, anticancerous, and hepatoprotective effects are scarce and require further explorations. Although some evaluation of antiinflammatory, renin inhibitory properties of peptides identified has been done using in silico tools, direct evidence using pure peptides or using molecular docking tools are still lacking. Therefore this should be considered as the next approach and further investigations through in vivo and cell line model should be undertaken. Also, enzymatic hydrolysate remains the dominant method for peptide generation while use of fermentation as a tool for producing cost-effective peptides remains underexplored, therefore more studies should be conducted in this direction. Furthermore, most of the studies from camel milk revolve around the use of dromedary camel milk with limited reports from Bactrian camel milk that could also be proven to be an important source of bioactive peptides.

References

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73. Available from https://doi.org/10.1016/j.phanu.2018.02.002. Murali, C., Mudgil, P., Gan, C. Y., Tarazi, H., El-Awady, R., Abdalla, Y., Amin, A., & Maqsood, S. (2021). Camel whey protein hydrolysates induced G2/M cellcycle arrest in human colorectal carcinoma. Scientific Reports, 11(1). Available from https://doi.org/10.1038/s41598-02186391-z. Muthukumaran, M. S., Mudgil, P., Baba, W. N., Ayoub, M. A., & Maqsood, S. (2022). A comprehensive review on health benefits, nutritional composition and processed products of camel milk. Food Reviews International. Available from https://doi.org/10.1080/87559129.2021.2008953. Neves, A. C., Harnedy, P. A., O’Keeffe, M. B., Alashi, M. A., Aluko, R. E., & FitzGerald, R. J. (2017). Peptide identification in a salmon gelatin hydrolysate with antihypertensive, dipeptidyl peptidase IV inhibitory and antioxidant activities. Food Research International, 100, 112
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131. Available from https://doi. org/10.1016/j.foodchem.2017.04.166. Nongonierma, A. B., Cadamuro, C., Le Gouic, A., Mudgil, P., Maqsood, S., & FitzGerald, R. J. (2019). Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of a camel whey protein enriched hydrolysate preparation. Food Chemistry, 279, 70
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43. Available from https://doi.org/10.1016/j.tifs.2016.01.022. Nongonierma, A. B., & Fitzgerald, R. J. (2016). Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Peptides, 79, 1
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Nongonierma, A. B., Paolella, S., Mudgil, P., Maqsood, S., & FitzGerald, R. J. (2017). Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of camel milk protein hydrolysates generated with trypsin. Journal of Functional Foods, 34, 49
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348. Available from https://doi.org/10.1016/j. foodchem.2017.10.033. Osman, A., El-Hadary, A., Korish, A. A., AlNafea, H. M., Alhakbany, M. A., Awad, A. A., & AbdelHamid, M. (2021). Angiotensin-I converting enzyme inhibition and antioxidant activity of papain-hydrolyzed camel whey protein and its hepato-renal protective effects in thioacetamideinduced toxicity. Foods, 10(2), 1
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10. Peighambardoust, S. H., Karami, Z., Pateiro, M., & Lorenzo, J. M. (2021). A review on healthpromoting, biological, and functional aspects of bioactive peptides in food applications. Biomolecules, 11(5), 631. Available from https://doi.org/10.3390/biom11050631. Power, O., Nongonierma, A. B., Jakeman, P., & Fitzgerald, R. J. (2014). Food protein hydrolysates as a source of dipeptidyl peptidase IV inhibitory peptides for the management of type 2 diabetes. Proceedings of the Nutrition Society, 73(1), 34
46. Available from https://doi.org/ 10.1017/S0029665113003601. Quan, S., Tsuda, H., & Miyamoto, T. (2008). Angiotensin I-converting enzyme inhibitory peptides in skim milk fermented with Lactobacillus helveticus 130B4 from camel milk in Inner China Mongolia. Journal of the Science of Food and Agriculture, 88(15), 2688
2692. Available from https://doi.org/10.1002/jsfa.3394. Rahimi, M., Ghaffari, S. M., Salami, M., Mousavy, S. J., Niasari-Naslaji, A., Jahanbani, R., Yousefinejad, S., Khalesi, M., & Moosavi-Movahedi, A. A. (2016). ACE- inhibitory and radical scavenging activities of bioactive peptides obtained from camel milk casein hydrolysis with proteinase K. Dairy Science & Technology, 96(4), 489
499. Available from https://doi.org/ 10.1007/s13594-016-0283-4. Ryskaliyeva, A., Henry, C., Miranda, G., Faye, B., Konuspayeva, G., & Martin, P. (2019). Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides. Scientific Reports, 9(1). Available from https://doi. org/10.1038/s41598-019-41649-5. Sagardia, I., Roa-Ureta, R. H., & Bald, C. (2013). A new QSAR model, for angiotensin Iconverting enzyme inhibitory oligopeptides. Food Chemistry, 136(3
4), 1370
1376. Available from https://doi.org/10.1016/j.foodchem.2012.09.092. Salami, M., Moosavi-Movahedi, A. A., Ehsani, M. R., Yousefi, R., Haertlé, T., Chobert, J. M., Razavi, S. H., Henrich, R., Balalaie, S., Ebadi, S. A., Pourtakdoost, S., & Niasari-Naslaji, A. (2010). Improvement of the antimicrobial and antioxidant activities of camel and bovine whey proteins by limited proteolysis. Journal of Agricultural and Food Chemistry, 58(6), 3297
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CHAPTER 10

Bioactive peptides from fermented milk products D.E. Cruz-Casas1, S.N. Chávez-García1, L.A. García-Flores1, G.A. Martínez-Medina1, R. Ramos-González2, L.A. Prado-Barragán3 and A.C. Flores-Gallegos1,* 1

Bioprocesses and Bioproducts Research Group. Food Research Department, School of ´ Chemistry, Universidad Autonoma de Coahuila. Saltillo, Coahuila, Mexico, 2 ´ CONACYT Universidad Autonoma de Coahuila. Saltillo, Coahuila, Mexico, 3 Biotechnology Department, Biological and Health Sciences Division, Metropolitan Autonomous University, Ciudad de Mexico, Mexico *Corresponding author. e-mail address: [email protected]

10.1

Introduction

Fermented milk products are made from liquid milk; cow’s milk is commonly used. However, several regions make traditional fermented milk products from goat, camel, sheep, mare, buffalo, or donkey milk (Ashaolu, 2019). Microbial strains such as lactic acid bacteria (LAB) and/or yeasts are added to the milk (George Kerry et al., 2018). These microorganisms grow well in milk because they use lactose as a carbon source and release lactic acid. This process reduces the milk’s pH and increases its acidity (GarcíaBurgos et al., 2020; Mallappa et al., 2021). The best-known fermented milk products are yogurt, dahi, lassi, koumiss, skyr, kefir, cheese, sour cream, and buttermilk (Chen et al., 2019; Mallappa et al., 2021). Fermented products of different kinds of milk are shown in Fig. 10.1. Each of these products uses a unique mixture of microorganisms, as well as specific conditions of the fermentation process (García-Burgos et al., 2020). In addition, the fermentation medium is based on liquid milk, which differs in composition depending on milch animal species. These differences provide fermented milk products a unique sensory, rheological, nutritious, and biological properties (Mallappa et al., 2021). In recent years, the consumption of fermented milk products has increased because these products have several advantages due to the fermentation process. For example, at the market level, these products have a longer shelf life, Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00010-2 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 10.1 Fermented products obtained from milk of different animal species.

which facilitates their distribution, and their taste is acceptable, which helps in their sale and consumption (Akdeniz & Akalın, 2019). Other advantages are that fermented milk products are recommended for lactose-intolerant people; these also prevent gastrointestinal infections such as diarrhea, eliminate antinutritional factors, and increase the bioavailability of nutrients (García-Burgos et al., 2020; Shetty & Sarkar, 2020). According to several reports, fermentation of dairy products has been identified to release bioactive compounds, such as bioactive peptides (García-Burgos et al., 2020). Bioactive peptides are protein fragments with potential health benefits for the consumer (Sosalagere et al., 2022). These exhibit antioxidant, antihypertensive, antimicrobial, antithrombotic, immunomodulatory, opioid, and mineral-binding effects. The sequence, composition, size, and charge of the amino acids in bioactive peptide influence their biological activities (Ahmed & Hammami, 2019; Sánchez & Vázquez, 2017). Bioactive peptides are inactive in the native protein, and these need to be released to exhibit activity through proteolysis using processes such as fermentation, enzymatic hydrolysis, in vitro digestion, and others (Kehinde & Sharma, 2020). Microorganisms in fermented milk products produce proteases and peptidases responsible for cleaving milk proteins, thus releasing bioactive peptides (Ross et al., 2017). For example, bioactive peptides with antimicrobial, antioxidant, anticancer, antiinflammatory, and angiotensin I-converting enzyme (ACE-I) inhibitory activity have been found in products such as kefir (Vieira et al., 2021). In addition, ACE-I inhibitory and antidiabetic peptides have been identified in koumiss, a fermented mare’s milk product (Song et al., 2017). However, the bioactivities of the peptides found

10.2 Bioactive peptides from fermented bovine milk products

in fermented milk products may vary as the milk, microorganisms, and fermenting conditions during fermentation are different. This work aims to analyze the different bioactive peptides that have been obtained from fermented milk products based on the animal species from which they are produced.

10.2 Bioactive peptides from fermented bovine milk products Bovine milk is consumed worldwide and generates the most extensive production (Claeys et al., 2014). Bovine milk is recognized as nourishing food, with significant quantities of proteins, amino acids, and fatty acids, vitamins, and minerals (Pereira, 2014). It also contains other specialized components such as immunoglobulins, hormones, growth factors, cytokines, nucleotides, peptides, polyamines, and enzymes, which try to guarantee and support the growth and development of calf (Haug et al., 2007). Usually, bovine milk contains substantial quantities of excellent quality proteins, with great digestibility and a significant proportion of essential amino acids (Szwajkowska et al., 2011). Bovine milk has been reported with a protein content of 30 39 g/L (Claeys et al., 2014). Values could vary due to diverse factors such as cow breed, feeding, stage of lactation, season, age, energy balance, and health of the udder (Le et al., 2017). Fermented bovine milk products have been developed to preserve nutrients; nevertheless, their physicochemical and biological properties also change. Multiple milk-derived fermented foods, including yogurt and fermented milks, cultured buttermilk, acidophilus milk, sour creams, kefir, and others, are reported with numerous health benefits (Shiby & Mishra, 2013). Multiple fermented products are produced in different parts of the world. Fermented products vary in the class of milk and microorganism involved in their production, generating a broad type of textures, flavors, and functionalities and making the accessibility of this class of functional compounds feasible to many cultures. The fermented bovine milk could be manufactured by the use of a wide variety of microorganisms such as Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Pediococcus acidilactici, or even with yeast strains such as Kluyveromyces fragilis or Candida kefir. Some fermented products could be fermented by molds (Chandan et al., 2017). The fermentation process of bovine milk is described in Fig. 10.2.

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FIGURE 10.2 General scheme for production of fermented bovine milk products.

During the fermentative process, some encrypted peptide sequences could be released, enhancing the functionality of the fresh bovine milk. Bovine milk proteins have been reported as a significant source of bioactive peptides (Korhonen & Pihlanto, 2006). Ayyash et al. (2018) have analyzed the potential bioactivities in bovine fermented milk employing two different bacterial strains, L. lactis KX881782 and L. acidophilus DSM9126 and compared camel and bovine milk fermentation with each strain separately, and all extracts show antioxidant, antiproliferative, antidiabetic, and ACE-I inhibitory activities. This study points out the importance of milk origin and the fermentative microorganism employed.

10.2.1

Soful

The functionality of bovine fermented products is not limited to laboratorydesigned experiments. A fermented product labeled as Soful and commercialized in Mexico has been studied in detail (Domínguez-González et al., 2014). Analysis of peptide fractions from this milk fermented by L. casei Shirota and S. thermophilus revealed that eight fractions showed ACE-I inhibitory effect; six more fractions offered antithrombotic activities, and two fractions showed both activities. Furthermore, two fractions did not lose their activities after simulated gastrointestinal digestion.

10.2 Bioactive peptides from fermented bovine milk products

10.2.2

Yogurt

Yogurt is defined as a fermented milk product containing live bacteria such as L. delbrueckii subsp. bulgaricus and S. thermophilus, with at least 10 million active bacteria per gram in the final product (Donovan & Hutkins, 2018) and is a widely used fermented product. The composition of yogurt is closely related to the milk class employed for their production. Nevertheless, the content of some nutrients such as vitamins, minerals, and proteins increases due to evaporation phenomena during their production (Baspinar & Gülda¸s, 2021). Microbial metabolism also produces essential biological molecules, including vitamins, conjugated linoleic acid, γ-aminobutyric acid, and bioactive peptides, among others (Donovan & Hutkins, 2018). The LAB used for the production of yogurt possess an important proteolytic system. Allowing to obtain a set of amino acids from media components, especially those amino acids for which LAB have a low potential for their production. The proteolytic system includes different exo- and endopeptidases, acting in a successive procedure and generating important biological active protein fragments (Mann et al., 2017). Bovine-milk yogurt shows antioxidant activity. It also downregulates the inflammatory process in ovine monocytes mediated by IL-1B and IL-12B cytokines and upregulates antiinflammatory molecules IL-10 in neutrophils (Moschopoulou et al., 2018). Yogurts employing skimmed milk using classic starter culture (S. thermophilus, and L. bulgaricus) or alternative bacterial consortium (L. acidophilus, L. casei, and L. paracasei) generate peptide fractions with antimicrobial effects against Escherichia coli and Staphylococcus aureus. All samples inhibit the proliferation of HT-29 colon cancer cells (Sah et al., 2016). Sah et al. (2016) isolated and identified two peptides from a yogurt combined with pineapple powder. The peptides have the sequence: YQEPVLGPVRGPFPIIV and SLPQNIPPPLTQTPVVVPPF. Both peptides are derived from β-casein and exhibit antioxidant and antiproliferative activities.

10.2.3

Koumiss

Another class of fermented products is the Koumiss (Kumys, kumis). This product is a traditional fermented beverage usually prepared with mare’s milk and consumed in central Asian regions; nevertheless, in Europe and North America, it is manufactured with bovine milk. Koumiss is a sour, bubbly, and mildly alcoholic beverage, the native microorganisms of which are composed of LAB (L. bulgaricus and L. acidophilus), and lactose-, nonlactose-, and noncarbohydrate-fermenting yeasts (Kluyveromyces marxianus var marxianus, Candida koumiss, Saccharomyces cartilaginous and Mycoderma spp.) (Kirdar, 2021). When cow’s milk is employed, 2.5% of

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sugars must be added to the production (Akuzawa et al., 2011).

bulk

mixture

for

Koumiss

It has been reported that in southwest Colombia, Koumiss is traditionally consumed. This beverage is produced using bovine milk and a successive/ coculture between LAB and yeasts. Also, sugarcane and cinnamon are added ´ ´ to the mixture (Chaves-Lopez et al., 2014). Chaves-Lopez et al. (2012) isolated multiple yeast strains from southwest Colombian kumis, where the most prominent genera were Galactomyces geotrichum, Pichia kudriavzevii, Clavispora lusitaniae, and Candida tropicalis. From 93 strains isolated from the fermented product, 18 were identified as ACE-I inhibitory peptide producers derived from milk, with inhibitory activities between 8% and 88%. After in vitro gastrointestinal digestion, C. lusitaniae was the best strain producer for antihypertensive peptides. This study could help to identify the correct strains to generate a large-scale product with specific health benefits (Chaves´ Lopez et al., 2012).

10.2.4

Kefir

This product is an ancient beverage, typically produced in Russia, East of Europe, and part of Asia. Kefir constitutes an alcoholic-lactic fermentation with complex and characteristic yeast flavors and aromas, including alcohol, acetaldehyde, diacetyl, lactic acid, and acetone. Kefir produces a fizz sensation in the tongue by microbial carbon dioxide generated. This product is manufactured by immersion of “kefir grains,” a semihard component with a cauliflower shape. Kefir grains are composed of an exopolysaccharide matrix containing a wide microbial consortium that could include not only lactic bacteria such as L. kefir, L. kefirgranum, and some species leuconostocs, lactococci, and other lactobacilli but also yeast as S. kefir, C. kefir, and Torula spp. (Guneser et al., 2019). Ebner et al. (2015) have reported the presence of a significant quantity of bioactive peptides from kefir produced from bovine milk using the classic kefir grains and a commercial starter culture (L. lactis ssp., Leuconostoc spp., S. thermophilus, Lactobacillus spp., kefir yeast, and kefir grain microflora). After kefir production, peptide fraction was extracted and analyzed by chromatographic techniques such as MALDI-TOF-MS. After that, the spectrum was compared in the MASCOT database to elucidate their amino acidic structure. The authors identified that both, classic kefir grains and kefir fermented with a commercial starter culture produced about 257 peptides: 230 with the commercial starter culture and 124 from the classic kefir grains. Between the two types of beverages, 97 sequences were shared. These peptides belong specially to the casein proteins mainly to β-casein , αS1-casein , κcasein , αS2-casein. The identified peptide sequences are previously reported

10.2 Bioactive peptides from fermented bovine milk products

with bioactivities as ACE-I inhibitory effect, antioxidant, antimicrobial, antithrombotic, mineral binding, opioid, and immunomodulating activities.

10.2.5

Others

Some other traditional fermented products have been reported as bioactive peptide sources as Jammed, a Bedouin cuisine product generated by the same starter culture as yogurt but with a thicker texture, salted, and sundried. The functional activities reported for this fermented paste were antioxidant, ACE-I, and α-amylase inhibitory activities. Viili is a Scandinavian fermented product, which is produced yeasts (K. marxianus, S. unisporus, and P. fermentans), and mold candidum). Viili is popular in Finland, and bioactive peptides reported previously, especially with antioxidant and ACE-I activities (Bakry & Campelo, 2018).

from LAB, (Geotricum have been inhibitory

The fermented bovine milk products are highly diversified due to their easy access to this class of food and beverage. However, bioactive peptide remains low explored, especially in native or traditional fermented products, making it a great field to explore. The different fermented bovine milk products and the biological activities of the other peptides identified are shown in Fig. 10.3.

FIGURE 10.3 Biological activities of peptides found in different fermented bovine milk products.

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10.3 Bioactive peptides from fermented goat milk products Fermented goat milk products have begun to arouse the interest of today’s society, which seeks to consume foods that, in addition to fulfilling their primary function (nutrition), also provide beneficial health effects. These characteristics have been identified in fermented goat milk products since they have a high nutritional value and healthy properties (Haenlein, 2017). Compared to bovine milk, goat milk contains smaller fat globules, abundant ˙ proteins, minerals, and vitamins (Dolatowska-Zebrowska et al., 2019). In addition, this milk has excellent digestibility, but processes such as fermentation improve this characteristic and increase its therapeutic potential by releasing compounds such as bioactive peptides (Ranadheera et al., 2019). The most important fermented goat milk products are yogurt, Kefir, and dahi.

10.3.1

Yogurt

One of the most important fermented milk products globally is yogurt (Ibrahim et al., 2021). Yogurt from goat milk is fermented with LAB such as L. delbrueckii spp. bulgaricus, L. rhamnosus, and S. thermophilus (Jia et al., 2016). It has been identified that this product contains biomolecules such as bioactive peptides that contribute to the physiological effects of yogurt when consumed (Nguyen et al., 2020). According to Rahmawati and Suntornsuk (2016), in a study evaluating the different bioactivities of yogurts produced from goat, cow, and sheep milk, it was determined that goat milk yogurt has antioxidant and antimicrobial activity peptides. Furthermore, these peptides’ effect was more significant than the bioactivities shown by yogurt made from cow’s milk. Other reports mention that peptides with ACE-I enzyme inhibitory and antiinflammatory activity have been found in goat milk yogurt (Nguyen et al., 2020; Wuragil et al., 2019).

10.3.2

Kefir

Kefir is made by mixing kefir grains and milk, in this case, goat’s milk (Farag et al., 2020). The microorganisms involved in the fermentation are in a symbiotic association and consist of LAB, acetic acid bacteria, yeasts, and molds (Puruto˘glu et al., 2020). Kefir, made from goat milk, is recognized to have health benefits for the consumer, and according to several reports, bioactive peptides are partially responsible for this activity (Amorim et al., 2019).

10.3 Bioactive peptides from fermented goat milk products

As many as 11 bioactive peptides with antibacterial, antioxidant, ACE-I inhibitory, antithrombotic, and dipeptidyl peptidase IV inhibitory effects have been identified in studies by Izquierdo-González et al. (2019) four of which belonged to β-casein, three to κ-casein, two to αs2-casein, one to αs1-casein, and the other to β-lactoglobulin. In another investigation by Wang et al. (2021), the sequences EMPFPK and VLPVPQK, corresponding to peptides with ACE-I inhibitory and antioxidant activity, respectively, were found. It is essential to continue studies on the bioactive peptides found in Kefir made from goat’s milk since they are scarce.

10.3.3

Dahi

It is one of the oldest fermented milk products in India. It is traditionally made from cow, buffalo, or goat milk and adds artisanal cultures. According to the year’s season, it is incubated overnight or for 2 4 days. Commercially, the milk is pasteurized and fermented using defined mesophilic and thermophilic microorganisms (Mallappa et al., 2021). So far, there are no studies on the bioactive peptides found in Dahi made from goat’s milk. Still, it is known that it has biological effects that benefit the consumer’s health because it has been tested in a model of immune suppression in mice that stimulate the mucosal immune system and improve the defense system against intestinal infections (Babaee et al., 2011). These biological activities may be due to antimicrobial and immunomodulatory peptides, which needs to be confirmed. The different fermented goat milk products and the biological activities of the other peptides identified are shown in Fig. 10.4.

FIGURE 10.4 Biological activities of peptides found in different fermented goat milk products.

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10.4 Bioactive peptides from fermented camel milk products 10.4.1

Camel milk

Although, camel milk in past was only produced for self-consumption by shepherds, it is now commercialized in the national and international market (Konuspayeva & Faye, 2021). Its production is concentrated in African countries, such as Somalia, Nigeria, and Ethiopia whcih accounts for 90% world camel milk production. The remaining 10% is produced in Asian countries, such as China, United Arab Emirates, India, and Afghanistan (FAO, 2019). The nutritional value of camel milk is higher than cow’s milk and similar to human milk (Ho et al., 2022). It has a low content of saturated fatty acids, lactose, and casein and a high content of minerals (calcium, copper, iron, magnesium, phosphorus, potassium) and vitamins, such as vitamin C (Swelum et al., 2021). It also has an excellent balance of essential amino acids. Protein concentration varies between 2.15% and 4.9% and mainly composed of caseins such as αS1-, αS2-, β-, and κ-casein. In a smaller percentage, camel milk contains whey proteins such as α-lactalbumin, serum albumin, lactoferrin, and immunoglobulin G (Mati et al., 2017). In the regions where camel milk is produced and consumed for food, it is also used for medicinal purposes, such as treating diseases such as asthma, tuberculosis, jaundice, dropsy, diabetes, and various infections (Maqsood et al., 2019). In addition, it is also reported to have anticytotoxic and immunomodulatory properties. These beneficial effects are attributed to various compounds, such as some bioactive peptides generated during milk digestion in the gastrointestinal tract (Mati et al., 2017). In recent years, interest in bioactive peptides from camel milk has grown. According to Ali Redha et al. (2022), it has been identified that the biological activities of the peptides found in this food are antioxidant, antiinflammatory, antihypertensive, antibacterial, antidiabetic, anticancer, antiobesity, antibiofilm, and antihemolytic activity. These peptides were released from camel milk using enzymatic hydrolysis or microbial fermentation (Ali Redha et al., 2022).

10.4.2

Bioactive peptides from fermented camel’s milk

Since ancient times, shepherds have been consuming fermented camel milk because of nonavailablity of cold chain for its preservation in the desert (Marsh et al., 2014). Fermentation of camel milk causes beneficial changes, such as improving taste, odor, and color (Rahman et al., 2009). Generally, previously fermented milk is used to inoculate the raw milk and

10.4 Bioactive peptides from fermented camel milk products

this causes the microflora of the product to be more diverse (Konuspayeva & Faye, 2021). Camel milk fermentation is considered a traditional ancestral method worldwide. It involves the conversion of lactose into lactic acid by the action of the milk’s natural microflora, which includes LAB and, in some cases, yeasts (Konuspayeva & Faye, 2021). LAB such as L. helveticus, L. plantarum, L. lactis spp. cremoris, L. lactis spp. lactis, S. thermophilus, L. delbrueckii sp. bulgaricus, Leuconostoc mesenteroides, and Enterococcus faecium are reported (Abdou et al., 2018; Ali Redha et al., 2022). The dominant yeasts are Trichosporon asahii, Pichia fermentans, and Rhodotorula mucilaginosa (Ider et al., 2019). Camel fermented milk has even significantly higher biological activities than cow’s milk, and these are correlated with the bioactive peptides released due to the fermentation process (Ayyash et al., 2018). A peptide AIGPVADLHI exhibiting ACE-I inhibitory activity (Solanki, 2022) and antioxidant activity in new sequences such as LLILTC, YPLR, TQDK has been obtained (Dharmisthaben et al., 2021). Antimicrobial activities in 30 new peptides have also been reported (Algboory & Muhialdin, 2021). Camel milk also has several endogenous peptides with diverse bioactivities (Zhang et al., 2022). There have not been enough studies on the characterization of the biological activities of camel milk peptides obtained by fermentation.

10.4.3 Bioactive peptides from fermented camel milk products Fermented camel milk products are traditional in each region and have a specific taste, flavor, and texture. They are mostly consumed by the population of the area where they are produced (Konuspayeva & Faye, 2021). Shubat in Kazakhstan and China (Zhadyra et al., 2021), garris in Sudan (Ahmed et al., 2012), laven in the Arab countries (Algruin & Konuspayeva, 2015), khoormog in Mongolia (Guo et al., 2021), susac in Kenya (Maitha et al., 2019), and ititu and dhanaan in Ethiopia (Berhe et al., 2019; Seifu et al., 2012) are the best known fermented camel milk products in the literature. So far, no report has been found describing the bioactive peptides that can be identified in these fermented milk products. Therefore it is necessary to conduct more research on these products. Although their consumption and production are not yet significant worldwide, new bioactive peptides can be found since it is known that the population also consumes them for their beneficial health properties. Fig. 10.5 shows the different fermented camel milk products and the biological activities reported.

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FIGURE 10.5 Biological activities of peptides found in fermented camel milk and different fermented camel milk products.

10.5 Bioactive peptides from fermented mare milk products Mare’s milk has been consumed throughout history, mainly in Central Asia. Its consumption has been limited due to the commercialization and industrialization of other types of milk. However, its consumption has been observed due to its benefits to consumers’ health. Horses are used as dairy animals in the former USSR, Mongolia, and some places in China (Salimei & Park, 2017). Mare’s milk is considered an essential source of nutrients and is food with more excellent digestibility than other milk. In terms of its nutritional content, it is very similar to that of human milk (Kushugulova et al., 2018). Mare’s milk has more albumin-type proteins than human milk, thus improving its digestibility. In addition, it provides a high tryptophan content, thus enhancing cognitive performance in people susceptible to stress. It has lowfat content, resulting in a drink with a lower energy intake than cow’s milk. This type of milk has fat globules of a smaller diameter within its lipid composition, thus contributing to more efficient digestion. Its main lipid content comprises 80% triglycerides, 9% 10% free fatty acids, and 5% 19% phospholipids. In contrast, human and cow’s milk provide mostly triglycerides (97% 98%). It is also considered a beverage with a much lower atherogenic index than cow’s milk, which means that the lipids in mare’s milk are less likely to adhere to the endothelial wall of our circulatory system, thus preventing atherosclerosis. Regarding carbohydrates, mare’s milk mainly contains lactose and, to a lesser extent, monosaccharides and oligosaccharides (Karav et al., 2018). Mare’s milk provides between 58 and 70 g/kg of lactose,

10.5 Bioactive peptides from fermented mare milk products

15 and 28 g/kg of protein, and 3 and 30 g/kg of lipids (Barreto et al., 2019; Jastrze˛bska et al., 2017). A series of peptides derived from whey protein have been identified, inhibiting the dipeptidyl peptidase-IV enzyme involved in developing type 2 diabetes (Song et al., 2017). On the other hand, a product derived from mare’s milk known as kumis has the presence of bioactive peptides with inhibitory activity on the ACE (Chen et al., 2010). In Europe, many products are made from mare’s milk, such as yogurt, curd, and kumis. However, the main product consumed from mare’s milk is kumis (Yakunin et al., 2017).

10.5.1

Koumiss

The main product obtained from the fermentation of milk bears the name qymyz, kumis, or koumiss. The koumiss is a slightly alcoholic drink in which LAB and yeasts are involved in its fermentation. It is a traditional drink of the people of Central Asia (Real, 2020). After subjecting the mare’s milk to lactic acid fermentation and alcoholic fermentation, this product is obtained. There are two types of koumiss: traditional koumiss, fermented from starters with unspecified microorganisms, and koumiss fermented with pure cultures (Rahimov et al., 2018). Traditional koumiss is prepared by inoculating mare’s milk and other supplements such as boiled millet, malt, and sour cow’s milk. Three groups of fermentation initiators have been described in traditional koumiss: spontaneous fermentation caused by microorganisms present in fresh mare’s milk, fermentation caused by fermented dairy products, or the combination of both types of initiators (Hou et al., 2019). The microorganisms present in the traditional koumiss have not yet been defined precisely. However, several studies report the growth of Acetobacter aceti, L. bulgaricum, and S. thermophilus bacteria and yeasts such as Saccharomyces lactis and S. cartilaginous, together with yeasts of the Mycoderma genus. Another study in Mongolia showed that L. helveticus was the bacteria with the most significant presence in the drink and the yeasts K. marxianus, K. unispora, and S. cerevisiae. Kumis fermented with pure cultures takes a series of steps for its production. In this case, a starter culture is prepared with L. bulgaricus and S. lactis. The milk does not undergo any heating process; it must reach particular acidity and fat levels. A second bulk starter culture is then prepared based on the first (Kondybayev et al., 2021). In the literature, it has been proven that there are different peptides present in koumiss. The results suggest that koumiss is rich in ACE-I inhibitor

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FIGURE 10.6 Biological activities of peptides found in fermented camel milk products.

peptides (Fig. 10.6). This indicates that regular koumiss consumption can benefit cardiovascular health (Reba, 2020).

10.6 Bioactive peptides from fermented sheep milk products Sheep’s milk is considered a functionally active food with high nutritional content. Thanks to its content of fatty acids, immunoglobulins, and nonimmune proteins, these characteristics are attributed. The consumption of sheep’s milk has increased due to the greater demand for cheese and other dairy products such as infant formulas and nutraceutical products (Pulina et al., 2018). The animal’s feeding is a critical factor since it affects the quality of the milk obtained and other factors such as breed, individual, parity, season, and stage of lactation (Mohapatra et al., 2019). The functional properties of sheep’s milk come from the number of bioactive peptides with antioxidant, antimicrobial, antihypertensive, immunomodulatory, and antithrombotic functions. In addition, it has physicochemical and biochemical characteristics that are not distinguished in other types of milk, such as the presence of prebiotics. The nutritional composition differs between the different seasons of the year (Altomonte et al., 2019). The values corresponding to sheep’s milk are as follows: 18.3% total solids, 6.0% fat, 12.3% nonfat solids, 4.9% lactose, 0.94% ash, and 5.2% protein (Moatsou & Sakkas, 2019; Nudda et al., 2020). The average fat in sheep’s milk is higher than that of cow’s milk, which makes it ideal for manufacturing cheeses and fermented milk (Fig. 10.7) (Pazzola, 2019). Its lipid fraction comprises a high percentage of short- and medium-chain fatty acids and a high conjugated linoleic acid (Bernard et al., 2018; Chikwanha et al., 2018). The latter has been linked to different types of biofunctionality, such as inhibiting different types of cancer due to the immune or inflammatory modulation it presents and the inhibition of osteoporosis, reducing triglycerides, and cholesterol in the blood (Burrow et al., 2018). In addition to this, it has a low omega-6:omega-3 ratio, high sphingomyelin content, and

10.6 Bioactive peptides from fermented sheep milk products

FIGURE 10.7 Average lipids profile of sheep and cow milk. CHOL, Cholesterol; DAG, diglycerides; FFA, free fatty acids; SM, sphingomyelin; TAG, triglycerides.

its fat globules are smaller in diameter, making it more digestible (Lordan et al., 2018). On the other hand, the micelles of sheep’s milk are similar to those of goat’s milk, reporting higher mineralization levels with more calcium and phosphorus (Amalfitano et al., 2019). Regarding carbohydrates, lactose is relatively less than fat and protein. The percentages of minerals are higher than those found in cow’s milk (Giorgio et al., 2018). Sheep’s milk has recently been considered an important source of ACE-inhibiting peptides, making it a food of excellent nutritional quality due to its functional properties and nutritional content (Lorenzo et al., 2018). Due to its composition, sheep’s milk is used mainly to produce cheeses and fermented beverages such as yogurt in the countries of the Mediterranean basin (Pazzola, 2019).

10.6.1

Koopeh

Koopeh is a traditional Iranian cheese made from raw sheep’s milk and left to mature for 4 months. Although starter cultures are not applied for the fermentation process, the bacteria present in the milk become more predominant during maturation, which provides the characteristic taste and smell. Koopeh is a high-fat cheese with a semihard texture with a buttery and spicy

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flavor. Recent studies have shown that this type of cheese is a potential source of antioxidant peptides. In addition to antioxidant activity, peptides that inhibit ACE with an antihypertensive effect have also been reported (Banihashemi et al., 2020; Sagardia et al., 2010; Tribst et al., 2020).

10.6.2

Yogurt

Fermented sheep’s milk has been shown to provide various health benefits with regular consumption, including a decrease in acute inflammation and a reduction in type 2 diabetes and hypertension. These benefits are attributed to the interaction of the probiotic bacteria with the intestinal microbiota and the bioactive peptides generated during the fermentation process, and those released during the digestion of the fermented product. For example, Nguyen et al. (2020) reported a series of bioactive peptides with antihypertensive activity in yogurt (Papadimitriou et al., 2007; Silva et al., 2019).

10.7

Conclusion

There are several fermented products made from different types of milk. These products have been identified as having beneficial effects on consumers’ health. This is related to the fermentation process, which releases compounds such as bioactive peptides. It has been found that fermented milk products have bioactive peptides. However, there are still not enough studies of all the possible bioactivities they could present in vivo. Furthermore, there is no research on the bioactive peptides that could be found in traditional fermented milk products, even though many of these foods are used for medicinal purposes.

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Downstream processing of therapeutic bioactive peptide Pourahmad Rezvan1 and Hosseini Elahesadat2 1

Department of Food Science and Technology, Varamin- Pishva Branch, Islamic Azad University, Varamin, Iran, 2Department of Food Science and Technology, Science and Research Branch, Islamic Azad University, Tehran, Iran

11.1

Introduction

Over the last two decades, it has been acknowledged that dietary proteins are not only essential sources of amino acids but also contribute to several physiological activities beyond nutritional value by biologically active peptides which may ultimately influence health. Bioactive peptides have been defined as sequences of 2 20 amino acid residues that can be found in many naturally occurring proteins (Meisel & FitzGerald, 2003). These peptides may remain inactive in the native protein sequence until being liberated by enzymatic proteolysis during gastrointestinal digestion either by fermentation or ripening during food processing or manufacturing (acids, alkali, heating, etc.). In this regard, dairy products are considered as rich sources of bioactive peptides with various healthpromoting properties such as antithrombotic, antimicrobial, antioxidant, antihypertensive, and immunomodulatory, and sometimes these peptides have multifunctional activity in order to provide health benefits to humans (Kim & Wijesekara, 2010; Maestri et al., 2016; Park & Nam, 2015; Sánchez & Vázquez, 2017; Shaik & Sarbon, 2020). Milk as lacteal secretion from healthy mammalian species contains several nutritional constituents including proteins, carbohydrates, fats, minerals, and vitamins and possesses a wide variety of chemical and functional properties. Milk is also indispensable nutritional food in the human diet (Kumar et al., 2016). Among various nutrients, milk protein components alone can be considered as the most valuable sources of bioactive peptides which provide these functionalities (Korhonen & Pihlanto, 2006, 2007; Meisel, 2005). 313 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00011-4 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 11.1 Major bioactive functional compounds derived from milk.

Caseins and whey proteins account for 80% and 20% of milk proteins, respectively. Bioactive peptides are derived from these proteins (Fig. 11.1). Casein as a heterogeneous protein has been categorized as four major forms: αs1-casein, αs2-casein, β-casein, and κ-casein and whey proteins contain β-lactoglobulin, α-lactalbumin, bovine serum albumin, lactoferrin, and immunoglobulins. It is worth noting that most of the produced bioactive peptides in the different types of milk are derived from caseins (Hodgkinson et al., 2019; Nielsen et al., 2017). Milk-derived bioactive molecules have been classified into four main areas: (1) gastrointestinal development, activity, and function; (2) immunological development and function; (3) infant development; and (4) antimicrobial activity, including antibiotic and probiotic action (Gobbetti et al., 2007). Since milk-derived peptides possess various physiological and physicochemical properties, these have emerged as a new profitable sector for the dairy and specialized biological industries. These are also regarded as important, new, and functional components in the formulation of both dairy and nondairy foods, dietary supplements, and even pharmaceuticals with the aim of targeting diseases (Krissansen, 2007; Playne et al., 2003; Shah, 2000). Bioactive peptides derived from cows’ milk proteins are depicted in Table 11.1 In most case, the amino acid sequence of milk-derived bioactive peptides is encrypted within a parent protein and these can be released in three different ways, namely (1) enzymatic hydrolysis during gastrointestinal digestion

11.2 Production mechanisms of bioactive peptides

Table 11.1 Bioactive peptides derived from cows' milk proteins (Saxelin et al., 2003). Bioactive peptides

Protein precursor

Peptide sequence (†)

Bioactivity

Casomorphins α-Lactorphin β-Lactorphin Lactoferroxin Casoxins Casokinins Casoplatelins Casecidin Isracidin Immunopeptides Phosphopeptides Lactoferricin

α- and β- casein α-lactalbumin β-lactoglobulin Lactoferrin κ-casein α- and β-casein κ-casein, transferrin α- and β-casein α-casein α- and β-casein α- and β-casein Lactoferrin

YPFPGPIPNSL YGLF
NH2 YLLF
NH 2 FKCRRWNRMKKLGAPSIT-CVRRAF SRYPSY
OCH 3 FFVAP MAIPPKKNQDK YQEPVLGPVRGPFPIIV RPKHPIKHQGLPQEVLNENLLRF PGPIPN RELEELNVPGEIVES*LS*S*S*EESITR FKCRRWNRMKKLGAPSIT-CVRRAF

Opioid agonists Opioid agonists Opioid agonists Opioid agonists Opioid agonists Antihypertensive Antithrombotic Antimicrobial Antimicrobial Immunostimulants Mineral carriers Antimicrobial

in vivo through digestive or microbial enzymes, (2) microbial fermentation during milk fermentation with proteolytic starter cultures (dairy starter cultures viz., Lactobacillus helveticus, Lactobacillus acidophilus and probiotic viz., Lactobacillus casei, Bifidobacterium lactis) and cheese ripening; and (3) proteolysis by proteolytic enzymes including alcalase, chymotrypsin, pepsin, thermolysin, papain, pancreatin, and trypsin which are derived from bacteria, molds, and yeast cells (Aspergillus oryzae)(Korhonen & Pihlanto, 2003, 2006). In addition, chemical synthesis and recombinant DNA technology can be employed to produce small and large sequence of peptides (Möller et al., 2008). Fig. 11.2 shows possible mechanisms for the release of milk bioactive peptides. In order to produce a bioactive peptide, the most appropriate technology strongly depends on the molecular size of the target molecule (Guzmán et al., 2007). Despite the selectivity of most of these methods, all production methods generate mixtures where the target peptide is present along with a series of other compounds (impurities). Therefore peptide of interest must be separated from all impurities which are produced during the manufacturing process as every impurity could potentially have adverse impact on the human body. In this regard, the current chapter focuses on the different processes used for fractionation and purification of the bioactive peptides derived from milk and milk products.

11.2

Production mechanisms of bioactive peptides

As previously stated, there are three different ways to release inactive peptides from native proteins in order to obtain bioactive peptides.

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FIGURE 11.2 Possible mechanisms for the release of bioactive peptides from dietary milk proteins.

11.2.1

Enzymatic hydrolysis

The most common method for the production of bioactive peptides such as angiotensin I-converting enzyme (ACE) inhibitory peptides, caseinophosphopeptides (CPP), and antihypertensive, antibacterial, and immunomodulatory peptides from casein and whey proteins involves enzymatic hydrolysis using digestive enzymes, namely pepsin, trypsin, and chymotrypsin (Gobbetti et al., 2007; Korhonen, 2009). Additionally, specific or nonspecific proteases can be used either singly or in combination in order to obtain defined peptide sequence with specific molecular weight (MW) (Singh et al., 2014). Table 11.2 represents milk- and milk product-derived bioactive peptides by using different enzymes.

11.2.2

Fermentation

Dairy products such as yogurt, sour milk, and cheese can release bioactive peptides when exposed to the action of specific bacteria which trigger the fermentation of this kind of foods. Lactic acid bacteria (LAB), for instance, a large group of beneficial bacteria which are widespread in nature and in our digestive systems, are commonly used to break down milk proteins mainly αs1- and β-caseins for the production of bioactive peptide. Among LAB,

11.2 Production mechanisms of bioactive peptides

Table 11.2 Examples of bioactive peptides released from milk proteins by various enzymes.

Enzymes used Chymotrypsin, pepsin, and trypsin Alcalase

Trypsin

Precursor protein/ protein source

Amino acid segment

Whey from cheese Hydrolysate of commercial cheese β-lactoglobulin, α-lactalbumin

Peptide sequence

Bioactivity

References

f104 108

WLAHK, TLAHL

ACE inhibitory

Didelot et al. (2006)

f156 160, f136 140

PPEIN, PLPLL

ACE inhibitory

Mao et al. (2007)

f22-25, f32-40, f81-83, f94100, f106-111, f142-146f5052, f99-108, f104-108 f192 196

LDAQSAPLR, VFK, VGINXW, LAHK LYQQP

ACE inhibitory ACE inhibitory

PihlantoLeppälä et al. (2000) Otte et al. (2007)

f133 138

LHLPLP

ACE inhibitory

Quirós et al. (2007) HernandezLedesma et al. (2002) Miguel et al. (2009) Phelan et al. (2009)

Pepsin, trypsin K-proteinase, thermolysin Proteinase from Enterococcus faecalis Thermolysin

β-casein, β- lactoglobulin, α-lactalbumin β-casein β-lactoglobulin

f46 53, f58 61, f103 105, f122 125

VTP, VTPPP, LLP

ACE inhibitory

Pepsin

β-casein

Not mentioned

Enzyme culture of bacterium and plants Alcalase

Na-casein

Not mentioned

Not mentioned Not mentioned

ACE inhibitory Antioxidative

Na-casein

f156 160, f136 140

PPGIA, PLPLL

N-proteinase

β-lactoglobulin

Not mentioned

SAPLTVT

ACE inhibitory antioxidative ACE inhibitory

L. helveticus had the highest extracellular proteinase activity and is also able to release the largest number of peptides in the fermented milk and form antihypertensive peptides (ACE inhibitory activity) from milk proteins (Aihara et al., 2005; Chen et al., 2007; Jauhiainen et al., 2005; Korhonen, 2009; Seppo et al., 2003). The different bioactive peptides derived by fermentation process by using various microorganisms are shown in Table 11.3.

11.2.3

Enzymes derived from proteolytic microorganisms

Bioactive peptides have also been derived from milk protein using a combination of microorganisms and proteolytic enzymes. Microorganisms used are

Mao et al. (2007) Ortiz-Chao et al. (2009)

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Table 11.3 Bioactive peptides released from milk proteins by various microorganisms.

Microorganisms Used

Precursor Protein/ Protein Source

Amino acid segment

Lb.helveticus, Saccharomyces cerevisiae

β-casein, κ-casein

Not mentioned

Lb. helveticus CP90 proteinase

β-casein

Lb. helveticus CPN 4

Caseins

Lb. delbrueckii subsp. bulgaricus SS1, Lactococcus lactis subsp. cremoris FT4 Lb. delbrueckii subsp. bulgaricusStreptococcus thermophilus 1 Lc. Lactis subsp. lactis biovar. diacetylactis Lb. helveticus CM4& Saccharomices cerevisae Lb. helveticus LBK16H

β-casein, κ-casein

Lb. helveticus NCC2765 Lb. delbrueckii ssp. bulgaricus SS1

Peptide Sequence

Bioactivity

References

VPPIPP

ACE inhibitory, antihypertensive

f169 175

LYVLPVPE

ACE inhibitory

f146 14, f114 115, f58 59 Not mentioned

YP

ACE inhibitory

Nakamura et al. (1995a, 1995b) Maeno et al. (1996) Yamamoto et al. (1999)

Many fragments

ACE inhibitory

Gobbetti et al. (2000)

β-casein

Not mentioned

SKVYP

ACE inhibitory

Ashar and Chand (2004)

β -andκ-casein β-casein, κ-casein, αS1- casein β-casein β-casein

Not mentioned Not mentioned

VPPIPP

Hypotensive

Vasilje and Shah (2008)

VPPIPP

Hypotensive ACE inhibition

NLHLPLPLL LNVPGEIVE

ACE inhibition ACE inhibition

NIPPLTETPV

ACE inhibition

VPP, IPP

Lc. Lactis ssp. cremoris FT4

β-casein

Lb. helveticus ICM 1004 cellfree extract

β-casein

Streptococcus thermophilus

κ-β casein

f147 155 f6 14, f7 14, f73 82, f74 82, f75 82. f7 14, f47 52, f169 175 f84 86, 74 76, 108 110 f58 61

Enterococcus faecalis CECT 5727 L. acidophilus L10, L. casei L26 L. delbrueckii ssp. bulgaricusIFO 13953

β-casein

f133 138

LHLPLP

Yogurt from bovine milk κ-casein

Not mentioned f96 106

Not mentioned

ACE inhibitionanti, hypertensive ACE inhibitory antihypertensive ACE inhibitory antihypertensive ACE inhibitory

AAHPHPHLSSPM

Antioxidative

TPTT

Pan et al. (2005) Tsai et al. (2008) Quirós et al. (2007) Donkor et al. (2007) Kudoh et al. (2001)

11.3 Downstream processing of bioactive peptides

Table 11.4 Examples of bioactive peptides released from milk proteins by various mixture of microorganisms and enzymes. Precursor protein/ protein source

Amino acid segment

Lactobacillus GG enzymes 1 pepsin and trypsin L. rhamnosus 1 digestion with pepsin and corolase PP Commercial products 1 digestion Commercial products 1 digestion Commercial products 1 digestion L. rhamnosus, pepsin and corolase PP

β-casein, αs1-casein

Not mentioned

YPFPAVPYPQR, TTMPLW

β-casein

f(49 52), f(193 201), f(98 105) Not mentioned

DKIHPF, YQEPVL, VKEAMAPK FPEVFEK

ACE inhibition

β-casein

f169 175

KVLPVPE

Antioxidative

αs1-casein

Not mentioned

KTTMPLW

β-lactoglobulin

ALIHPP, TGGVL, VLGAMAPL

Proteinase from E. faecalis

β-casein

f46 53, f58 61, f103 105, f122 125 f133 138

Possible Immunomodulation ACE inhibitory antioxidative

LHLPLP

ACE inhibitory

Combination of microorganisms and enzymes used

α

s1-casein

Peptide sequence

Bioactivity

References

Opioid, ACE inhibitory, immunostimulatory ACE inhibitory antioxidative

Rokka et al. (1997)

usually Lactobacillus GG or L. rhamnosus, while proteolytic enzymes include digestive enzymes such as chymotrypsin, pepsin, and trypsin; bacterial protease such as thermolysin and alcalase; and fungal protease such as corolase PP. Some bioactive peptides derived from milk proteins are shown in Table 11.4.

11.3 Downstream processing of bioactive peptides (isolation, purification, and characterization) There is a growing demand for the production and isolation of bioactive peptides derived from milk and milk-based products as these are involved in regulator activity of the gastrointestinal system, metabolic regulation, and regulation of the immune system. In this regard, these are being proposed as effective nutraceuticals and functional food ingredients in order to reduce the risk of chronic diseases and promote body health. Therefore much attention has been paid to the characterization of bioactive peptides which are isolated or prepared via hydrolysis from milk and dairy products. In order to isolate

HernandezLedesma et al. (2004) Vasiljevic and Shah (2008)

HernandezLedesma et al. (2002) Quirós et al. (2007)

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and fractionate bioactive peptides from milk and dairy products, generally techniques such as selective precipitation, membrane filtration, gel filtration, ion exchange, liquid chromatography, and combination of one or two techniques are used. The fractionation methods that can be applied to bioactive peptides derived from milk include traditional batch membrane reactors, continuous membrane reactors, ion-exchange chromatography, ultrafiltration (UF), isoelectric focusing, electrofiltration, electrodialysis, simulated moving bed, and sizeexclusion chromatography (SEC). Although, these methods are highly suitable for the production and concentration of bioactive peptides, there are certain concerns associated with the production techniques as most of the production methods produce not only peptide of interest but also other peptides and further series of impurities are present in reaction mixture. Consequently, other peptides and impurities must be removed during the downstream step of manufacturing. There are various purification techniques that have their own advantages and disadvantages. Additionally, the purification strategy should be considered for each case as the imposed requirements vary depending on the application (Carta & Jungbauer, 2010).

11.3.1

Fractionation methods

In fractionation methods, precipitation steps are achieved by means of the addition of organic solvents (ethanol, methanol, or acetone); acids [trichloroacetic acid (TCA), sulfosalicylic acid, phosphotungstic acid (PPTA)]; and salts (ammonium sulfate) or just by shifting of pH to the isoelectric point. Precipitation of proteins often leads to a selective fractionation of peptides by their solubility into the precipitating agent (Korhonen, 2002); However, in some cases, the addition of chemical compounds caused peptide degradation and changes in the biological activity and physical properties of bioactive peptide. Plaisancié et al. (2013) used 2% TCA to isolate peptide fraction [(β-CN (94-123)] from yogurt.

11.3.2

Membrane separation techniques

Membrane separation processes as one of the best technologies are accessible for the peptide fractionation with a specific range of MW (Korhonen & Pihlanto, 2003). These are an integral part of the downstream processing of the bioactive products and separation of protein hydrolysates from complex matrices (Amorim et al., 2016; Nielsen & Olsen, 2002). This preparation mechanism is based on selective permeability of components in a solution through the membrane according to the driving forces. The main

11.3 Downstream processing of bioactive peptides

FIGURE 11.3 Pressure-driving membrane separation mode depending on the size of separation targets. MF, Microfiltration; NF, nanofiltration; RO, reverse osmosis; UF, ultrafiltration.

features of pressure-driven membrane techniques are summarized in Fig. 11.3. Amongst the membrane separation techniques, UF and nanofiltration (NF) are the main methods used to isolate bioactive peptides from protein hydrolysates due to the MW of the bioactive peptides falling within normal pore size range of these membranes. UF is generally applied to fractionate bioactive peptide from milk protein hydrolysates with aim of enhancing their functional properties. Since the separation of peptides by means of UF is primarily determined by the MW cutoff of the membrane, the UF process can separate peptides with MW of less than 7 KDa (Vermeirssen et al., 2005). Membrane separation processes are sometimes applied before enzymatic hydrolysis with the aim of performing hydrolysis of specific fractions. Esmaeilpour et al. (2016) ultrafiltered the hydrolysates produced from caseins after enzymatic hydrolysis by trypsin and ficin. The hydrolysate obtained by ficin (MW , 3 kDa) showed the highest antimicrobial activity. Karimi et al. (2021) have used UF membranes (10, 5, and 3 kDa), for the isolation of bioactive peptides from yogurt whey which was subjected to hydrolysis by pepsin and trypsin. Based on the results, the hydrolysate fraction with the highest antioxidant and antibacterial activity was obtained from trypsin with an MW of 5 10 kDa. Additionally, the combination of different types of membrane processing (such as UF and NF) can be utilized successfully for concentration of bioactive peptides (Groleau et al., 2004). Notably, UF membranes not only separate out small peptides but also are useful to remove enzymes which are used for hydrolysis of proteins during manufacturing. In this regards, Pihlanto-Leppälä et al. (1996) employed selective UF membranes (30 kDa) for the isolation of opioid peptides (α- and β-lactorphin), which required prior hydrolysis of α-lactalbumin and β-lactoglobulin by pepsin and mixtures of pepsin and trypsin, respectively. Moreover, enzymatic

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membrane reactors which consist of membrane separation process with enzymatic reaction are widely applied for continuous production of bioactive peptides in order to solve problems such as high cost of the enzymes and inefficiency that exist in traditional membrane batch method (Korhonen & Pihlanto, 2003; Pihlanto-Leppälä et al., 1996). Bordenave et al. (1999) and Sannier et al. (2000) succeeded in producing lactorphin through continuous hydrolysis of whey in an UF reactor. Bouhallab and TouzéTouzé (1995) obtained antithrombotic peptides which were derived from hydrolyzed caseinomacropeptide (CMP) through continuous extraction technique coupling UF membrane. Although membrane filtration technology has shown a good potential application in the separation of bioactive products, membrane fouling is main problem and this shortens the membrane life and increases cost as well. Therefore optimizing the structure and properties of membranes and developing new membrane systems with low fouling and high selectivity could promote the development of the membrane separation techniques

11.3.3

Chromatographic methods

Chromatography is the most appropriate method for the purification of valuable therapeutic products, where high resolution and selectivity are essentials (Sainio & Kaspereit, 2009). This technique is flexible as a wide variety of stationary and mobile phases is available to choose. Additionally, it is highly efficient in the separation of complex mixtures, where the components have similar chemical properties. The disadvantage of this method is that it is quite labor-intensive and time-consuming. Moreover, it is difficult to handle viscous mixtures, which cause increase in the back pressure and also requirement of organic solvents as mobile phase which renders these methods complicated and harmful to human and environment (Agyei et al., 2016). Several well-established chromatographic methods such as high-performance liquid chromatography (HPLC), reversed-phase high-performance liquid chromatography (RP-HPLC), fast protein liquid chromatography, ion-exchange chromatography (IEC), hydrophilic interaction chromatography, SEC, and liquid chromatography with tandem mass spectrometry (LC MS/MS) have already been developed for separation and purification of bioactive peptides (Carta & Jungbauer, 2010; Chavan et al., 2015).

11.3.3.1 Size-exclusion chromatography The partially purified extract obtained from membrane filtration technology is usually subjected to SEC and ion-exchange chromatography along with reversed-phase C18HPLC as the final purification step.

11.3 Downstream processing of bioactive peptides

SEC, also known as gel filtration chromatography, is commonly used for isolation, desalting, and MW estimation of peptides and proteins based on sieving which ultimately yields more purified hydrolysate by means of UF membranes (Wang et al., 2017). Therefore separation mechanism is based on differences in particle size in which some smaller molecules enter the pores of the gel and travel a longer distance, while larger molecules show much shorter retention times. The MW range of SEC medium impacts on the selectivity of the medium. SEC media cover an MW range from 100 to 8 3 107 Da for separation of different biomolecules from peptides to protein mixtures (Wang et al., 2017). There are many SEC media such as Superdex, Sephacryl, and DEAE Sepharose with special properties. In this technique, elution conditions can be varied in accordance with the sample and requirements for further purification or analysis without affecting separation. In addition, SEC is an important step in a purification scheme as it can provide high selectivity and high resolution. However, the resolution can be affected by many factors, such as the particle size, particle uniformity, bed height, column packing quality, flow rate, sample concentration, and volume (Wang et al., 2017). Bayram et al. (2008) evaluated superoxide radical scavenging activity of whey proteins which were purified by sephadex G200 chromatography. Zhang et al. (2013) obtained the antioxidant peptides derived from whey protein through purification with a Sephadex G-15 column. Liu et al. (2020) performed the study on the purification of antioxidant peptides from yak casein hydrolysate using Sephadex G-25. Based on above mentioned properties, this technology is appropriate to various separation and purification fields.

11.3.3.2 Ion-exchange chromatography IEC mode is commonly used technique which is based on the electro static interactions between the opposite charges on analytes (like biological molecules such as proteins, peptides, amino acids, or nucleotides) and stationary phase. This technique is mostly suitable for separation and detection of proteins and peptides differing in isoelectric points. These molecules acquire net charge which depends on pH of the mobile phase. In addition, IEC is implemented for high-resolution purification as an intermediate or final step for capturing the target peptides or removing impurities. Based on the target, type of samples, and resolution, different IEC media such as amorphous phosphated titanium oxide (https://pubs.rsc.org/en/content/articlehtml/2020/ en/c9en01466g) (APTO), MacroCap, MicroBeads, MonoBeads, Sephadex, Sepharose, and SOURCE can be used to purify the target product (Wang et al., 2017). For example, Fee and Chand (2006) have used cation-exchange chromatography packed with SP Sepharose to capture lactoferrin and lactoperoxidase from raw whole milk. Purification of α-lactalbumin from whey protein concentrate with Q Sepharose anionic exchanger has been

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achieved (Geng et al., 2015). Lactoferrin has been isolated from acid whey using monolithic ion-exchange chromatography (Matijaˇsi´c et al., 2020). In comparison with SEC method, this method could save time and improve accuracy by implementation of mass separation. The IEC also has some disadvantages such as being expensive, complex, and not applicable for biomolecules that are sensitive to pH, metal ions, and other factors. However, to separate different biological matrixes within a higher resolution and cheaper materials in order to define a more affordable and applicable methods, perhaps it is necessary to conduct a scholar on this method.

11.3.3.3 Reversed-phase liquid chromatography Reversed-phase liquid chromatography (RP-HPLC) mode is the most widely used technique for purification of peptides and protein mixtures (Åsberg et al., 2017). In this technique, the separation of peptides depends on their hydrophobic properties (Shaik & Sarbon, 2020) and the most employed stationary phases, typically for the small or low hydrophobic proteins (#10 kDa), is C18 column. Additionally, C8 or even C4 columns are preferred for intact protein separation, as these have better retentive characteristics, particularly in case of very hydrophobic peptides (Boone & Adamec, 2016). The great advantages of this technology include the ease of operation and high performance and needs short time to get the elution spectra compared to the SEC and IEC. Karimi et al. (2021) developed a method in which through enzymatic hydrolysis, UF and RP-HPLC on C18 column could isolate and purify antibacterial and antioxidative peptides (with an MW of 5 10 kDa) from yogurt whey. Liu et al. (2020) used semipreparative RPHPLC on C18 column for purification of antioxidant peptides from yak casein hydrolysate. Geng et al. (2015) developed a method for producing pure α-lactalbumin in amounts above 1 kg from whey protein concentrate (αWPC) which was firstly precipitated by acid and then target peptides were separated and purified by anion-exchange chromatography (AIEC), followed by purification by RP-HPLC on a PLRP-S 300Å column. ACE inhibitory peptide from sour milk fermented by L. helveticus LB10 has been purified on Sephadex G-15 gel filtration chromatography followed by RP-HPLC (Pan & Guo, 2010). In recent years, electrospray ionization, matrix-assisted laser desorption ionization (MALDI), and mass spectrometry (MS) have opened a new era for protein and peptides identification and characterization (Léonil et al., 2000). Liquid chromatography followed by tandem mass spectrometry (LC MS/MS) detection is generally applied to recognize peptide sequences (Vijaykrishnaraj & Prabhasankar, 2015). In addition, MALDI-time-of-flight (MALDI-TOF) mass spectrometric analysis is useful for producing peptide profiles of protein hydrolysates (Alomirah et al., 2000; Singh et al., 2014).

References

In summary, scientists are committed to find better methods by replacing the complex, time-consuming, and costly separation technologies with simple, fast, and cheap ones.

11.4

Conclusion

Milk and milk-based products have been identified as excellent resources for the extraction of functional bioactive compounds. A broad range of bioactive peptides have been isolated from these products and these exhibit various health-promoting properties such as antithrombotic, antimicrobial, antioxidant, antihypertensive, and immunomodulatory activities. Hence, these can be used as food or pharmaceutical ingredients with the aim of targeting diseases; however, their application as pharmaceutical and food ingredients to human health might be a problem as most of the researches remain in the stages of in vitro or animal experiments due to the time and cost shortcomings. Furthermore, applying them as therapeutic peptides needs to pass through a very strict purity specifications and, thus, choosing appropriate purification processes is one of the most important parts in manufacturing process. Although the purification techniques are developing rapidly and several media have been studied and improved, low efficiency and high cost are still limiting factors. As a result, improving the selectivity and resolution of the separation and purification technology is surely a difficult and important task.

References Agyei, D., Ongkudon, C. M., Wei, C. Y., Chan, A. S., & Danquah, M. K. (2016). Bioprocess challenges to the isolation and purification of bioactive peptides. Food and Bioproducts Processing, 98, 244 256. Available from https://doi.org/10.1016/j.fbp.2016.02.003. Aihara, K., Nakamura, Y., Kajimoto, O., Hirata, H., & Takahashi, R. (2005). Effect of powdered fermented milk with Lactobacillus helveticus on subjects with high-normal blood pressure or mild hypertension. Journal of the American College of Nutrition, 24(4), 257 265. Available from https://doi.org/10.1080/07315724.2005.10719473. Alomirah, H. F., Alli, I., & Konishi, Y. (2000). Applications of mass spectrometry to food proteins and peptides. Journal of Chromatography A, 893(1), 1 21. Available from https://doi.org/ 10.1016/S0021-9673(00)00745-7. Amorim, M., Pereira, J.O., Gomes, D., Pereira, C.D., Pinheiro, H., & Pintado, M. (2016). Nutritional ingredients from spent brewer’s yeast obtained by hydrolysis and selective membrane filtration integrated in a pilot process. Journal of Food Engineering, 185, 42 47. Available from https://doi.org/10.1016/j.jfoodeng.2016.03.032. Åsberg, D., Langborg Weinmann, A., Leek, T., Lewis, R. J., Klarqvist, M., Le´sko, M., Kaczmarski, K., Samuelsson, J., & Fornstedt, T. (2017). The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography. Journal of Chromatography A, 1496, 80 91. Available from https://doi.org/10.1016/j.chroma.2017.03.041.

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Ashar, M. N., & Chand, R. (2004). Fermented milk containing ACE inhibitory peptides reduces blood pressure in middle aged hypertensive subjects. Milchwissenschaft, 59(7), 363 366. Bayram, T., Pekmez, M., Arda, N., & Yalçin, A. S. (2008). Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment. Talanta, 75(3), 705 709. Available from https://doi.org/10.1016/j.talanta.2007.12.007. Boone, C., & Adamec, J. (2016). Top-down proteomics. Proteomic profiling and analytical chemistry: The crossroads (2, pp. 175 191). Elsevier Inc. Available from https://doi.org/10.1016/B978-0444-63688-1.00010-0. Bordenave, S., Sannier, F., Ricart, G., & Piot, J. M. (1999). Continuous hydrolysis of goat whey in an ultrafiltration reactor: Generation of alpha-lactorphin. Preparative Biochemistry and Biotechnology, 29(2), 189 202. Available from https://doi.org/10.1080/10826069908544890. Bouhallab, S., & Touzé, C. (1995). Continuous hydrolysis of caseinomacropeptide in a membrane reactor: kinetic study and gram-scale production of antithrombotic peptides. Le Lait, 75(3), 251 258. Available from https://doi.org/10.1051/lait:1995317. Carta, G., & Jungbauer, A. (2010). Protein chromatography. Process development and scale-up. WILEY-VCH. Chavan, R., Nema, P., & Mishra, V. (2015). Downstream processing of bioactive compounds from milk and whey. Current Biochemical Engineering, 2(1), 56 64. Available from https:// doi.org/10.2174/2212711902666150224234315. Chen, G. W., Tsai, J. S., & Sun Pan, B. (2007). Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation. International Dairy Journal, 17(6), 641 647. Available from https://doi.org/ 10.1016/j.idairyj.2006.07.004. Didelot, S., Bordenave, J. S., Rosenfeld, E., Fruitier, A. I., Piot, J. M., & Sannier, F. (2006). Preparation of angiotensin-I-converting enzyme inhibitory hydrolysates from unsupplemented caprine whey fermentation by various cheese microflora. International Dairy Journal, 16(9), 976 983. Available from https://doi.org/10.1016/j.idairyj.2005.09.009. Donkor, O. N., Henriksson, A., Singh, K. T., Vasiljevic, T., & Shah, P. N. (2007). ACE-inhibitory activity of probiotic yoghurt. International Dairy Journal, 17(11), 1321 1331. Available from https://doi.org/10.1016/j.idairyj.2007.02.009. Esmaeilpour, M., Ehsani, M. R., Aminlari, M., Shekarforoush, S., & Hoseini, E. (2016). Antimicrobial activity of peptides derived from enzymatic hydrolysis of goat milk caseins. Comparative Clinical Pathology, 25(3), 599 605. Available from https://doi.org/10.1007/s00580016-2237-x. Fee, C. J., & Chand, A. (2006). Capture of lactoferrin and lactoperoxidase from raw whole milk by cation exchange chromatography. Separation and Purification Technology, 48(2), 143 149. Available from https://doi.org/10.1016/j.seppur.2005.07.011. Geng, X. L., Tolkach, A., Otte, J., & Ipsen, R. (2015). Pilot-scale purification of α-lactalbumin from enriched whey protein concentrate by anion-exchange chromatography and ultrafiltration. Dairy Science & Technology, 95(3), 353 368. Available from https://doi.org/10.1007/s13594-015-0215-8. Gobbetti, M., Ferranti, P., Smacchi, E., Goffredi, F., & Addeo, F. (2000). Production of angiotensin-I-converting-enzyme inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4. Applied and Environmental Microbiology, 66(9), 3898 3904. Available from https://doi.org/10.3168/ jds.S0022-0302(95)76689-9. Gobbetti, M., Minervini, F., & Rizzello, C. G. (2007). Handbook of food products manufacturing (pp. 489 517). John Wiley & Sons. Groleau, P. E., Lapointe, J. F., Gauthier, S. F., & Pouliot, Y. (2004). Effect of aggregating peptides on the fractionation of β-LG tryptic hydrolysate by nanofiltration membrane. Journal of Membrane Science, 234(1 2), 121 129. Available from https://doi.org/10.1016/j.memsci.2004.01.016.

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CHAPTER 12

Enzyme actions during cheese ripening and production of bioactive compounds R. Vázquez-García and Sandra T. Martín-del-Campo Tecnologico De Monterrey, School of Engineering and Sciences, Queretaro, Mexico

12.1

Introduction

Cheese ripening is a very complex phenomenon, including a wide variety of reactions happening simultaneously. The main biochemical phenomena could be grouped into three main groups: proteolysis, lipolysis, and glycolysis. Those phenomena are carried out by the action of a large number of enzymes of different origins, such as milk, coagulants, and starter as well as nonstarter microorganisms. During cheese making and ripening, a wide variety of biologically active compounds are produced. Among the bioactive compounds produced in cheese ripening, health-promoting compounds are particularly interesting and are getting more attention. For most of these bioactive compounds, starters, ripening time, and conditions are determinant for their production and concentration and their degradation to other bioactive or nonbioactive compounds. Protein breakdown during primary and secondary proteolysis produces a wide variety of peptides, showing different sizes, structures, functional properties, and biological activities. Additionally, free amino acids released could be used as substrates to produce aroma compounds and other bioactive compounds such as γ-aminobutyric acid or ornithine. On the other hand, through lipolysis, compounds such as free fatty acids and phospholipids are released and become available for other biochemical reactions such as the production of some important fatty acids, carotenoids, or vitamins, depending on the starters. This chapter will discuss the main bioactive compounds in relation to the enzymes involved in their synthesis. 331 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00012-6 © 2023 Elsevier Inc. All rights reserved.

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12.2 12.2.1

Bioactive compounds Peptides

Milk contains many proteins, which are transformed during the technological processes subjected during cheese manufacturing. Caseins are proteins with a globular structure. The casein in milk is present in the form of structural units called micelles which are highly stable structures that undergo important transformations when the κ-caseins present on the outside of micelles are affected by enzymatic activities and such enzymes can be of natural, technological, or microbiological origin (Darewicz et al., 2011; Spinnler, 2017). In the cheese, proteolysis supports the development of its texture mainly due to the protein hydrolysis and also due to the decrease in water activity. Proteins are consumed when the new ionized carboxylic acid molecules and amino groups are released due to peptide hydrolysis. The flavor of cheese depends almost entirely on the type of amino acids present after the proteolytic hydrolysis process and can vary taking into account the amino acid profile, also the production of free amino acids due to the development of microorganisms since they take them as precursors of diverse metabolites to carry out their metabolic processes (Ardö et al., 2017; Cantor et al., 2017; Haque et al., 2008; Mounier et al., 2005). The main sources of proteolytic enzymes, known as proteinases and peptidases, are enzymes native to milk to produce large peptides; exogenous enzymes in cheese production promoting medium peptides; enzymes from starter bacteria; and enzymes produced by ripening microorganisms that hydrolyze casein structure into short peptides and free amino acids. On the other hand, ripened cheeses contain adventitious flora that also can produce peptidases. These microorganisms are mostly added intentionally considering the organoleptic characteristics expected in the final product (Ardö et al., 2017; Gobbetti et al., 2002). The most important exogenous enzyme is chymosin, which is considered the main protease traditionally used in precipitation in cheese productions. It hydrolyzes F105-M106 of κ-casein, the micelle stabilizer, causing its destruction. Generally, the enzyme is eliminated through the whey, but steadily it remains in the coagulum, promoting protein hydrolysis. If this enzyme stays in cheese, it can hydrolyze F23-F24, L-A, L-F, F-Y, and L-D bonds in αs1casein (Ardö et al., 2017; Carles & Dumas, 1985). These effects are related to the appearance of peptides with certain biological activities. In αs2-casein, some resistance to this enzyme has been observed since the cleavage site is generally restricted to hydrophobic molecule areas. It has been reported to have better affinity if external conditions are present during ripening, such as pH variation and sodium chloride concentration since the peptide bonds are

12.2 Bioactive compounds

hydrolyzed depending on the ionic strength, pH, and milk type (Jaros & Rohm, 2017; McSweeney et al., 1995; Uniacke-Lowe & Fox, 2017). Another external enzyme widely used as a coagulant is pepsin, usually accompanied by chymosin. This enzyme is more sensitive to pH. However, it is not widely used during the ripening process. Its action is less specific in the case of bovine milk protein and thus hydrolysis protein at different sites leads to the release of diverse peptides (Ardö et al., 2017; Linhares Carreira et al., 2003; O’Sullivan et al., 2005; Uniacke-Lowe & Fox, 2017). On the other hand, there are native milk enzymes. The best known is plasmin, a protease directly related to the ripening of cheese. Its main function is to destroy fibrin clots and release plasminogens associated with milk micelles. This enzyme is highly specific for tyrosine, and its action is direct on α-casein and β-casein. In high-temperature cooked cheeses, such as Emmental, pepsin is an important caseinolytic agent since it is heat resistant (Grappin et al., 1999). Its optimum pH is 7.5, therefore it is also active in cheeses with high pH values after lactic acid catabolism and protein deamination, stimulating the action of the enzyme, facilitating proteolysis, and influencing the quality of the cheese. Its activity promotes the release of medium-sized peptides and, together with the enzymes released by lactic acid bacteria, the production of free amino acids is promoted. There are other proteinases from milk; however, there is no evidence of their activity in cheese proteolysis (Ardö et al., 2017; O’Sullivan et al., 2005). Once the cheese has been produced using external enzymes, it goes to the cheese ripening, where it is subjected to a refining process during which external conditions such as temperature, humidity, and time promote the appearance of certain characteristics such as smell, texture, and flavor. These characteristics are modified by the type of microorganisms that develop in the cheese matrix, and considering the type of microorganism to develop, the desired organoleptic characteristics are produced, thus having different types of cheeses (Calzada et al., 2014; Chamba & Irlinger, 2004; McSweeney et al., 2006). It has been observed that the proteolysis of caseins results in the formation of long peptides that are generally insoluble in water and medium-sized peptides that tend to be soluble in water. Water-soluble extracts are those that can present much more synergy with various physiological functions and those that have shown biological functions (McSweeney et al., 2006, 2017). Lactic acid bacteria are microorganisms that require a significant amount of free amino acids for their growth. Whole milk does not contain free amino acids; however, its proteolytic system promotes the conversion of caseins into small peptides and free amino acids, which contributes to modifying the flavor of the cheese. One of the most used microorganisms is those from Lactococcus, Lactobacillus, and Streptococcus genera, all of them are cheese

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starter bacteria. Amino acid release is largely catalyzed by intracellular peptidases and is related to bitterness in the final product in some cheeses such as Cheddar (Broadbent et al., 2002, 2011; Bütikofer et al., 2008; Chopard et al., 2001; Courtin et al., 2002; Hynes et al., 2001; McAuliffe, 2017). One of the most studied bacterial origin enzyme is lactocepin which is found in the lactic acid bacteria wall. Its release occurs due to the effect of free calcium ions after casein precipitation and produces oligopeptides of different sizes (Boutrou et al., 2001; Parente & Cogan, 2004). Concerning intracellular peptidases, they are the ones that promote the release of free amino acids after membrane destruction and are released into the cheese matrix (De Dea Lindner et al., 2008; De Freitas et al., 2007). The released amino acids can be utlized by other microorganisms for their development and reproduction. Arthrobacter genus bacteria produce lactocepin and can be found on the surface of externally crusted ripened cheeses such as Brie and Camembert (Fox & McSweeney, 1996; Irlinger et al., 2017). Some cheeses are ripened with the help of molds and yeasts, such as Camembert and Roquefort cheese. Penicillium spp. is one of the microbiota's major components, and its enzymes are of great importance in ripening processes. Penicillium camemberti and Penicillium roqueforti are considered important microorganisms in dairy technology. Both fungi synthesize an aspartyl proteinase capable of hydrolyzing αs1-casein much faster than other caseins. Also, both yeasts produce acid proteinases that hydrolyze β-caseins. The relationship among the presence of Penicillium species, their proteinases activity, and bioactivity peptide release has been widely demonstrated. These enzymes prefer some linkages such as K-V, K-E, or K-I, although there is a preference for caseins with these amino acids. Production of metalloproteins is also common in this genus and has similar properties in both species, with a pH range between 5.5 and 6. Another enzyme 'aspartic proteinase' shows a specificity on hydrophobic and aromatic peptides at a much lower pH than other enzymes (Chen et al., 2012; Fox & McSweeney, 1996; Irlinger et al., 2017; Lavoie et al., 2012). In the case of yeasts of the genus Geotrichum, which is found on the surface of molded cheeses, the production of extracellular and intracellular proteinases has also been extensively studied. However, it is known that the supply of free amino acids and peptides is lower in comparison with other microorganisms (Spinnler, 2017). Other yeasts widely used in the production of aged cheeses such as Cheddar are Debaryomyces hansenii and Yarrowia lipolytica. Although they are microorganisms that are more related to lipolytic activities, there is evidence that describes the release of peptidases that, to a lesser extent, promote the appearance of flavors and sensorial characteristics commercially relevant (De Wit et al., 2005). The presence of peptidases and the production of peptides leads to the production of certain bioactivities that have been widely disseminated both in the whole milk of cow, goat, sheep, buffalo, and camel and, in consequence, in

12.2 Bioactive compounds

cheese and dairy products. There is evidence of the presence of peptides with activities that include antihypertensive effects, inhibitors of the angiotensininhibiting enzyme, antioxidant, immunomodulatory, antibacterial, and antithrombotic effects, among others (Abdel-Hamid et al., 2017; Choi et al., 2012; Clare & Swaisgood, 2000; El-Salam & El-Shibiny, 2013; Haque & Chand, 2008). Bioactive peptides released during cheese ripening exhibit different activities depending on the structure, as shown in Table 12.1 and Table 12.2.

Table 12.1 Bioactive peptides released by the action of microbial origin enzymes. Bioactivity

Peptide information

Cheese type

Milk of origin

Angiotensinconverting enzyme inhibition

Ethanol-soluble fraction

Cotija

Cow milk

Water-soluble extract

Asiago d’allevo Gouda

Cow milk

Unidentified

Unidentified TQTPVVVPPFLQPEIM

Antimicrobial Antioxidant

Cow milk

Fresh cheese Fresh cheese

Cow milk Ewe milk

αS2-CN f(31-40) PRKEKLCTTS β-CN f (191-201) YQEPVLGPVRG αS2-CN f(116132) NAGPFTPTVNREQLSTS YQEP, VPLVL, YQEPVLGP from β-casein NPL, NPLHPILH from αs1-casein Water-soluble fraction No protein nitrogen fraction

Idiazabal type

Sheep milk

Fresh cheese Coalho Cotija

Sheep and goat milk Cow milk Cow milk

Water-soluble fraction Unidentified

White Caciocavallo

Cow milk Cow milk

Unidentified

Paneer soft

Buffalo milk

Water-soluble fraction αs1-CN (110 114) (EIVPN) and β-CN (176 182) (KAVPYPQ) αs1-CN (180-199) SDIPNPIGSENSEKTTMPLW β-CN (193209) YQQPVLGPVRGPFPIIV β-CN (191209) LLYQQPVLGPVRGPFPIIV

Coalho Fresh cheese Burgos

Cow milk Cow milk Cow milk

References Hernández-Galán et al. (2016) Lignitto et al. (2010) Nilsen, Pripp, Høstmark, Haug, and Skeie (2014) Paul and Van Hekken (2011) Pisano, Scano, Murgia, Cosentino, and Caboni (2016) Sagardia, Iloro, Elortza, and Bald (2013) Silva, Pihlanto, and Malcata (2006) Silva et al. (2012) Hernández-Galán et al. (2016) Barac´ et al. (2016) Perna, Intaglietta, Simonetti, and Gambacorta (2015) Qureshi, Amjad, Nadeem, Murtaza, and Munir (2019) Silva et al. (2012) Timon, Andres, Otte, and Petron (2019) Timón, Parra, Otte, Broncano, and Petrón (2014)

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Table 12.2 Bioactive peptides released by the action of exogenous or milk-origin enzymes. Bioactivity

Peptide information

Cheese type

Milk of origin

References

Angiotensinconverting enzyme inhibition

VPP; IPP in an ethanol-soluble fraction

Gamalost, Pultost, Norvegia, Castello, French Brie, Port Salut, and Kasam Cheddar

Not specified

Bütikofer et al. (2008)

Cow milk

Gupta, Mann, Kumar, and Sangwan (2013) Bernabucci, Catalani, Basiricò, Morera, and Nardone (2014) Dimitrov, Chorbadjiyska, Gotova, Pashova, and Ilieva (2015) Erkaya and Sengul ¸ (2015) Gómez-Ruiz, Ramos, and Recio (2002) Meyer, Bütikofer, Walther, Wechsler, and Sieber (2009)

Water-extract solution Water-soluble fraction

Parmigiano Reggiano

Cow milk

SLSSSSPE (fraction 15-20 β-casein)

White brined

Cow milk

Water-soluble extracion VRGPFP, VVAPFPE, KKYNVPQL, GPVRGPFP VPP and IPP

White Manchego

Cow milk Ewe milk

Berner, Emmental, Gruyère, Appenzeller, and Tilsiter Cheddar

Not Specified

Cow milk

Cheddar

Cow milk

Gamalost and Pultost Norwegian Gamalost

Not identified

Cheddar cheese

Buffalo and cow milk

“Festivo” low-fat cheese White-brined

Not specified

DKIHPF k-CN (f 96-102), αs1-CN (f 19), αs1-CN (f 1-7), αs1-CN (f 1-6), αs1-CN (f 24-32), and β-CN (f 193-209) Ethanol (70%) soluble fraction At least 42 peptides were directly related to the biological activity Peptides observed in a watersoluble extract with the existence of VPP and IPP αs1-casein N-terminal peptides, f(1-9), f(1-7), and f(1-6) Water-soluble extract APLHPILHQ [αs1-casein (CN)] YPFPGPIPN (β-CN, A2-5P; f 60 68) β-CN f58-72 (LVTPFPGPIHNSLPE) β-CN YQEPVLGPVRGPFPIIV (f193 209) αS1-CN EVLNENLLRF (f14 23)

Gouda, Emmental, Blue, Camembert, Edam, and Havarti Crescenza, Italico, Gorgonzola, and Mozzarella Mexican fresh cheese

Goat milk

Cow milk Not specified

Not specified

Cow milk

Ong, Henriksson, and Shah (2007) Ong and Shah (2008)

Pripp, Sørensen, Stepaniak, and Sørhaug (2006) Qureshi, Vegarud, Abrahamsen, and Skeie (2012) Rafiq, Huma, Pasha, Shahid, and Xiao (2017) Ryhänen, Pihlanto-Leppälä, and Pahkala (2001) Sahingil, Hayaloglu, Kirmaci, Özer, and Simsek (2014) Saito, Nakamura, Kitazawa, Kawai, and Itoh (2000) Smacchi and Gobbetti (1998) Torres-Llanez, GonzálezCórdova, HernandezMendoza, Garcia, and Vallejo-Cordoba (2011)

Continued

12.2 Bioactive compounds

Table 12.2 Bioactive peptides released by the action of exogenous or milk-origin enzymes. Continued Bioactivity

Peptide information

Cheese type

Milk of origin

References

Antimicrobial

Unidentified RPKHPIKHQ, RPKHPIKHQG, RPKHPIKHQGLPQ, RPKHPIKHQGLPQE, HQPHQPLPPT, MHQPHQPLPPT, PKHPIKHQ, RPKHPIKHQG, RPKHPIKHQGLPQ, and RPKHPIKHQGLPQE were originated from αs1-casein Unidentified Water-soluble extract Unidentified

Cottage Canastra artisanal minas

Cow milk Cow milk

Dal Bello (2012) Fialho et al. (2018)

Cow milk Cow milk Not specified

García-Cano et al. (2014) Lignitto et al. (2012) Théolier, Hammami, Fliss, and Jean (2014) Bottesini et al. (2013)

Unidentified

Cotija Asiago d’allevo Mozzarella, Gouda, Swiss, and Guda Parmigiano Reggiano Fresh cheese

Water-soluble fraction

Cheddar

Water-soluble fraction

Feta, Roquefort, Pecorino Mexican goat cheese

Antioxidant

Water-soluble fraction

Acid-soluble fraction

12.2.2

Cow milk Cow, sheep, and goat mixed milk Buffalo and cow milk Ovine milk Goat milk

Revilla et al. (2016)

Huma, Rafiq, Sameen, Pasha, and Issa Khan (2018) Meira et al. (2012) Vázquez-García, Cardador-Martínez, Orihuela-López, RamosHernández, and Martíndel-Campo (2021)

Conjugated linoleic acid

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of the linoleic acid (Z,Z-9,12-octadecadienoic acid, C18:2), including the 9,11- and 10,12-octadecenoic acids presenting both cis and trans configurations (Aydin, 2005; Domagała et al., 2010). The most abundant CLA in cheese is the cis 9, trans 11 isomer or rumenic acid (Akuzawa et al., 2009). CLA is naturally present in milk fat. The original concentration in milk depends on the mammal species (Bergamo et al., 2003), season, as well as to the animal diet (Abilleira et al., 2009; Addis et al., 2005; Bergamo et al., 2003). Higher concentrations of CLA in buffalo milk than in cow milk have been reported and thus relatively more CLA in related cheeses has been

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reported (Bergamo et al., 2003). In both cases, organic products presented higher concentrations of CLA than conventional cheeses. In general, cheeses present higher CLA concentrations than the original milk. CLA could be synthesized during fermentation by microorganisms such as lactic acid bacteria, bifidobacterias, propionic acid bacteria, and Enterococcus (Akuzawa et al., 2009; Beermann & Hartung, 2013). It has been reported that propionic and lactic acid bacteria produce CLA using oleic acid, while lactic acid bacteria and Bifidobacterium longum use linoleic acid (Akuzawa et al., 2009). The concentration of CLA in cheese depends on the kind of cheese (Akuzawa et al., 2009), species (Bergamo et al., 2003), milk production season (Abilleira et al., 2009) or practices (Bergamo et al., 2003), milk treatment (Chamba & Perreard, 2002; Gnädig et al., 2004), the cheese making process, strains (Beermann & Hartung, 2013; Chamba & Perreard, 2002), and ripening time (Chamba & Perreard, 2002). Increase in free CLA has been reported during ripening of Emmental cheese (Chamba & Perreard, 2002). Researchers have identified 14 different CLA isomers in Idiazabal Ewe cheeses produced in spring and winter (Abilleira et al., 2009). However, only six of the evaluated isomers presented have higher concentrations in winter than in spring. A 50% higher CLA concentrations in organic Mozzarella (from buffalo or cow milk) and Parmigiano cheeses than in conventional cheeses has been reported (Bergamo et al., 2003). CLA biological activity includes atherosclerosis prevention, immune response increasing, anticarcinogenic effects (Aydin, 2005; Beermann & Hartung, 2013)

12.2.3

Gama aminobutyric acid and L-ornithine

Gama aminobutyric acid (GABA) and L-ornithine, nonprotein amino acids, are present in small quantities in raw foods such as vegetables but in higher amounts in fermented products such as cheese. GABA shows important activity on brain function, and it could prevent anxiety, sleeplessness, and depression and act as an inhibitory transmitter in the central nervous system (Carafa et al., 2019). Moreover, GABA could prevent inflammation, stimulate the immune system, and positively affect diabetes and hypertension prevention (Carafa et al., 2019). On the other hand, L-ornithine has shown hypnotic and sedative effects that could attenuate fatigue, increasing the energy consumption efficiency (Diana et al., 2014). Starter microorganisms produce GABA and ornithine from the L-Glutamic acid released during cheese proteolysis. GABA is produced through the action of the glutamic acid decarboxylase (GAD), a pyridoxal 5’-phosphate (PLP)dependent enzyme, from L-glutamic acid (Fig. 12.1). The GABA synthesis pathway was described in details earlier (Fenalti et al., 2007); briefly, GAD will be an active holoenzyme and GABA, but also, in a side reaction, produce

12.2 Bioactive compounds

FIGURE 12.1 Synthesis of GABA from L-glutamic acid. GABA, Gama aminobutyric acid.

succinic semialdehyde (SSA), pyridoxamine 5’-phosphate (PMP), and the inactive apoenzyme. GABA concentration in cheeses depends on the milk origin, ripening microorganisms, climate, cheese making process, and the ripening time (Carafa et al., 2019; Diana et al., 2014; Freitas et al., 1998; Redruello et al., 2020). A maximum GABA concentration of 91 6 22 mg/kg after 20-day ripening of experimental raw cow’s milk mini cheeses inoculated with Streptococcus thermophilus 84C has been reported (Carafa et al., 2019). Diana et al. (2014) have evaluated GABA concentration in different Spanish cheeses from farms and artisanal markets (Diana et al., 2014). They reported a mean concentration of 0.33 6 0.05 g/kg. Picante cheese reached a GABA concentration of 13.33 6 0.46 mg/100 g dry matter after 180 ripening days (Freitas et al., 1998). GABA concentration in cheeses from a different origin, climate, ripening time, among other factors has been evaluated (Redruello et al., 2020). These authors found that the evaluated factors are important in GABA accumulation in the cheeses. Studies have been carried out to identify the strains responsible for GABA synthesis in different cheeses. Franciosi et al. (2015) isolated different microorganisms from Nostrano-cheeses and evaluated their GABA production capacity (Franciosi et al., 2015). They observed that from the isolated species, S. thermophilus, Lactobacillus paracasei, and Lactobacillus rhamnosus presented the highest GABA productions (80.0 6 2.7, 14.8 6 5.3, and 11.3 6 0.72 g/kg, respectively). An increase in GABA concentration in ripened cheeses has been reported (Bütikofer & Fuchs, 1997). These authors mention an increase of GABA concentration after 150 days of ripening of Appenzeller (1.0 mg/kg/day), Gruyère (1.1 mg kg/day), Raclette (3.7 mg kg/day), Sbrinz (1.3 mg kg/day), and Tilsiter (1.7 mg/kg/day) cheeses. Ornithine mean concentration of 0.45 6 0.06 g/kg in different Spanish cheeses from farms and artisanal markets has been reported (Diana et al., 2014).

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Cheeses made with GABA-producer strains of L. lactis presented higher amounts of ornithine than cheeses made without GABA-producer L. lactis (Hagi et al., 2019).

12.2.4

Carotenoids

Carotenoids are a group of compounds occurring in nature produced by photosynthetic organisms and some nonphotosynthetic bacteria. They are present in some colored cheeses due to the presence of nonphotosynthetic bacteria, such as the orange pigmented Brevibacterium linens. Three important carotenoids have been described in B. linens (isorenieratene, 3-hydroxy-isorenieratene, and 3,3’-di-hydroxy-isorenieratene) as well as the complex synthetic pathway (Dufossé & de Echanove, 2005). The presence of these three carotenes and their isomers has been reported for Vieux-Pané cheese (Galaup et al., 2015). Authors associated the presence of these red pigments to the activity of the Brevibacterium linens group (B. linens and B. aurantiacum sp. nov.).

12.3

Conclusion

During cheese ripening, enzymes play an important role in producing a wide variety of bioactive compounds. Even though there is important knowledge about the kind and origin of the enzymes involved in their production, the information is not well described in most cases. Advances in analytical methodologies made it possible to understand the bioactive compounds synthesis mechanisms better, to characterize the enzymatic mechanisms and enzymes structures, but also to discover more bioactive compounds.

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Dimitrov, Z., Chorbadjiyska, E., Gotova, I., Pashova, K., & Ilieva, S. (2015). Selected adjunct cultures remarkably increase the content of bioactive peptides in Bulgarian white brined cheese. Biotechnology & Biotechnological Equipment, 29(1), 78 83. Available from https://doi.org/ 10.1080/13102818.2014.969918. Domagała, J., Sady, M., Grega, T., Pustkowiak, H., & Florkiewicz, A. (2010). The influence of cheese type and fat extraction method on the content of conjugated linoleic acid. Journal of Food Composition and Analysis, 23(3), 238 243. El-Salam, MHA, & El-Shibiny, S. (2013). Bioactive peptides of buffalo, camel, goat, sheep, mare, and yak milks and milk products. Food Reviews International, 29(1), 1 23. Available from: https://doi.org/10.1080/87559129.2012.692137. Erkaya, T., & S¸ engul, M. (2015). Bioactivity of water soluble extracts and some characteristics of white cheese during the ripening period as effected by packaging type and probiotic adjunct cultures. Journal of Dairy Research, 82(1), 47 55. Available from https://doi.org/10.1017/ S0022029914000703. Fenalti, G., Law, RHP, Buckle, A. M., Langendorf, C., Tuck, K., Rosado, C. J., Faux, N. G., Mahmood, K., Hampe, C. S., Banga, J. P., Wilce, M., Schmidberger, J., Rossjohn, J., ElKabbani, O., Pike, R. N., Smith, A. I., Mackay, I. R., Rowley, M. J., & Whisstock, J. C. (2007). GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nature Structural and Molecular Biology, 14(4), 280 286. Available from: https://doi.org/ 10.1038/nsmb1228. Fialho, T. L., Carrijo, L. C., Júnior, M. J. M., Baracat-Pereira, M. C., Piccoli, R. H., & de Abreu, L. R. (2018). Extraction and identification of antimicrobial peptides from the Canastra artisanal minas cheese. Food Research International, 107, 406 413. Available from https://doi. org/10.1016/j.foodres.2018.02.009. Fox, P. F., & McSweeney, PLH (1996). Proteolysis in cheese during ripening. Food Reviews International, 12(4), 457 509. Available from: https://doi.org/10.1080/87559129609541091. Franciosi, E., Carafa, I., Nardin, T., Schiavon, S., Poznanski, E., Cavazza, A., Larcher, R., & Tuohy, K. M. (2015). Biodiversity and γ -aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow’s milk cheeses. BioMed Research International. Available from: https://doi.org/10.1155/2015/625740. Freitas, A. C., Fresno, J. M., Prieto, B., Franco, I., Malcata, F. X., & Carballo, J. (1998). Influence of milk source and ripening time on free amino acid profile of Picante cheese. Food Control, 9(4), 187 194. Available from: https://doi.org/10.1016/S0956-7135(97)00073-X. Galaup, P., Sutthiwong, N., Leclercq-Perlat, M. N., Valla, A., Caro, Y., Fouillaud, M., Guérard, F., & Dufossé, L. (2015). First isolation of Brevibacterium sp. pigments in the rind of an industrial red-smear-ripened soft cheese. International Journal of Dairy Technology, 68(1), 144 147. Available from: https://doi.org/10.1111/1471-0307.12211. García-Cano, I., Serrano-Maldonado, C. E., Olvera-García, M., Delgado-Arciniega, E., PeñaMontes, C., Mendoza-Hernández, G., & Quirasco, M. (2014). Antibacterial activity produced by Enterococcus spp. isolated from an artisanal Mexican dairy product, Cotija cheese. LWT Food Science and Technology, 59(1), 26 34. Available from https://doi.org/10.1016/j. lwt.2014.04.059. Gnädig, S., Chamba, J. F., Perreard, E., Chappaz, S., Chardigny, J. M., Rickert, R., Steinhart, H., & Sébédio, J. L. (2004). Influence of manufacturing conditions on the conjugated linoleic acid content and the isomer composition in ripened French Emmental chesse. Journal of Dairy Research, 71(3), 367 371. Available from: https://doi.org/10.1017/S0022029904000226. Gobbetti, M., Stepaniak, L., De Angelis, M., Corsetti, A., & Di Cagno, R. (2002). Latent bioactive peptides in milk proteins: Proteolytic activation and significance in dairy processing. Critical Reviews in Food Science and Nutrition, 42(3), 223 239. Available from: https://doi.org/10.1080/ 10408690290825538.

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Gómez-Ruiz, J. Á., Ramos, M., & Recio, I. (2002). Angiotensin-converting enzyme-inhibitory peptides in Manchego cheeses manufactured with different starter cultures. International Dairy Journal, 12(8), 697 706. Available from https://doi.org/10.1016/S0958-6946(02)00059-6. Grappin, R., Beuvier, E., Bouton, Y., & Pochet, S. (1999). Advances in the biochemistry and microbiology of Swiss-type cheeses. Lait, 79(1), 3 22. Available from: https://doi.org/10.1051/lait:199911. Gupta, A., Mann, B., Kumar, R., & Sangwan, R. B. (2013). ACE-Inhibitory Activity of Cheddar Cheeses Made with Adjunct Cultures at Different Stages of Ripening. Advances in Dairy Research, 1(1), 102. Available from https://doi.org/10.4172/2329-888X.1000102. Hagi, T., Nakagawa, H., Ohmori, H., Sasaki, K., Kobayashi, M., Narita, T., & Nomura, M. (2019). Characterization of unique metabolites in γ-aminobutyric acid-rich cheese by metabolome analysis using liquid chromatography-mass spectrometry. Journal of Food Biochemistry, 43(11). Available from: https://doi.org/10.1111/jfbc.13039. Haque, E., & Chand, R. (2008). Antihypertensive and antimicrobial bioactive peptides from milk proteins. European Food Research and Technology, 227(1), 7 15. Available from: https://doi. org/10.1007/s00217-007-0689-6. Haque, E., Chand, R., & Kapila, S. (2008). Biofunctional properties of bioactive peptides of milk origin. Food Reviews International, 25(1), 28 43. Available from: https://doi.org/10.1080/ 87559120802458198. Hernández Galán, L., Cardador Martínez, A., Picque, D., Spinnler, H. E., López del Castillo Lozano, M., & Martín del Campo Barba, S. T. (2016). Angiotensin converting enzyme inhibitors and antioxidant peptides release during ripening of Mexican Cotija hard cheese. Journal of Food Research, 5(3), 85 91. Available from https://doi.org/10.5539/jfr.v5n3p85. Huma, N., Rafiq, S., Sameen, A., Pasha, I., & Issa Khan, M. (2018). Antioxidant potential of buffalo and cow milk Cheddar cheeses to tackle human colon adenocarcinoma (Caco-2) cells. Asian-Australasian Journal of Animal Sciences, 31(2), 287 292. Available from https://doi.org/ 10.5713/ajas.17.0031. Hynes, E., Ogier, J. C., & Delacroix-Buchet, A. (2001). Proteolysis during ripening of miniature washed-curd cheeses manufactured with different strains of starter bacteria and a Lactobacillus plantarum adjunct culture. International Dairy Journal, 11(8), 587 597. Available from: https://doi.org/10.1016/S0958-6946(01)00094-2. Irlinger, F., Helinc, S., & Jany, JL, (2017). Secondary and adjunct cultures. Cheese: Chemistry, physics and microbiology, . (4, pp. 273 300). (1, pp. 273 300). Elsevier Inc. Available from: https:// doi.org/10.1016/B978-0-12-417012-4.00011-9. Jaros, D., & Rohm, H. ,. (2017). Rennets: Applied aspects. Cheese: Chemistry, physics and microbiology: (1, pp. 53 67). Elsevier Inc., https://doi.org/10.1016/B978-0-12-417012-4.00003-X. Lavoie, K., Touchette, M., St-Gelais, D., & Labrie, S. (2012). Characterization of the fungal microflora in raw milk and specialty cheeses of the province of Quebec. Dairy Science & Technology, 92(5), 455 468. Available from: https://doi.org/10.1007/s13594-011-0051-4. Lignitto, L., Cavatorta, V., Balzan, S., Gabai, G., Galaverna, G., Novelli, E., Sforza, S., & Segato, S. (2010). Angiotensin-converting enzyme inhibitory activity of water-soluble extracts of Asiago d'allevo cheese. International Dairy Journal, 20(1), 11 17. Lignitto, L., Segato, S., Balzan, S., Cavatorta, V., Oulahal, N., Sforza, S., Degraeve, P., Galaverna, G., & Novelli, E. (2012). Preliminary investigation on the presence of peptides inhibiting the growth of Listeria innocua and Listeria monocytogenes in Asiago d’Allevo cheese. Dairy science & technology, 92(3), 297 308. Available from https://doi.org/10.1007/s13594-012-0057-6. Linhares Carreira, R., Dias Medeiros Silva, V., Morais, H. A., Da Motta, S., Gonçalves Junqueira, R., & Pinto Coelho Silvestre, M. (2003). Otimização Da Hidrólise Da Caseína Para Elevar O Teor De Pequenos Peptídeos: Emprego Da Pepsina. Ciência e Agrotecnologia, 27(3), 625 634. Available from: https://doi.org/10.1590/S1413-70542003000300018.

References

McAuliffe, O. (2017). Genetics of lactic acid bacteria. Cheese: Chemistry, physics and microbiology. 4 (1, pp. 227 247). Elsevier, https://doi.org/10.1016/B978-0-12-417012-4.00009-0. McSweeney, PLH, Fox, P. F., & Olson, N. F. (1995). Proteolysis of bovine caseins by cathepsin D: Preliminary observations and comparison with chymosin. International Dairy Journal, 5(4), 321 336. Available from: https://doi.org/10.1016/0958-6946(94)00010-M. McSweeney, PLH, Hayaloglu, A. A., O’Mahony, J. A., & Bansal, N. (2006). Perspectives on cheese ripening. Australian Journal of Dairy Technology, 61(2), 69 77. McSweene, PLH, Ottogalli, G., & Fox, PF, (2017). (4, pp. 781 808). Diversity and classification of cheese varieties: An overview. Cheese: Chemistry, physics and microbiology, (1, pp. 781 808). Elsevier Inc. Available from: https://doi.org/10.1016/B978-0-12-417012-4.00031-4. Meira, S. M. M., Daroit, D. J., Helfer, V. E., Corrêa, A. P. F., Segalin, J., Carro, S., & Brandelli, A. (2012). Bioactive peptides in water-soluble extracts of ovine cheeses from Southern Brazil and Uruguay. Food Research International, 48(1), 322 329. Available from https://doi.org/ 10.1016/j.foodres.2012.05.009. Meyer, J., Bütikofer, U., Walther, B., Wechsler, D., & Sieber, R. (2009). Hot topic: Changes in angiotensin-converting enzyme inhibition and concentrations of the tripeptides Val-Pro-Pro and Ile-Pro-Pro during ripening of different Swiss cheese varieties. Journal of Dairy Science, 92(3), 826 836. Mounier, J., Gelsomino, R., Goerges, S., Vancanneyt, M., Vandemeulebroecke, K., Hoste, B., Scherer, S., Swings, J., Fitzgerald, G. F., & Cogan, T. M. (2005). Surface microflora of four smear-ripened cheeses. Applied and Environmental Microbiology, 71(11), 6489 6500. Available from: https://doi.org/10.1128/AEM.71.11.6489-6500.2005. Nilsen, R., Pripp, A. H., Høstmark, A. T., Haug, A., & Skeie, S. (2014). Short communication: Is consumption of a cheese rich in angiotensin-converting enzyme-inhibiting peptides, such as the Norwegian cheese Gamalost, associated with reduced blood pressure? Journal of Dairy Science, 97(5), 2662 2668. Available from https://doi.org/10.3168/jds.2013-7479. Ong, L., Henriksson, A., & Shah, N. , P. (2007). Angiotensin converting enzyme-inhibitory activity in Cheddar cheeses made with the addition of probiotic Lactobacillus casei sp. [original article]. Lait, 87, 149 165. Available from https://doi.org/10.1051/lait:2007004. Ong, L., & Shah, N. P. (2008). Release and identification of angiotensin-converting enzymeinhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses. LWT-Food Science and Technology, 41(9), 1555 1566. O’Sullivan, N. A., Sousa, M. J., O’Grady-Walsh, D., Uniacke, T., Kelly, A. L., & McSweeney, PLH (2005). Ripening of Camembert-type cheese made from caprine milk using calf rennet or kid rennet as coagulant. International Journal of Dairy Technology, 58(1), 13 18. Available from: https://doi.org/10.1111/j.1471-0307.2005.00166.x. Parente, E., & Cogan, TM, (2004). Starter cultures: General aspects. Cheese: Chemistry, physics and microbiology (1, pp. 123 147). Elsevier Ltd. Paul, M., & Van Hekken, D. (2011). Assessing antihypertensive activity in native and model Queso Fresco cheeses. Journal of Dairy Science, 94(5), 2280 2284. Available from https://doi. org/10.3168/jds.2010-3852. Perna, A., Intaglietta, I., Simonetti, A., & Gambacorta, E. (2015). Short communication: Effect of genetic type on antioxidant activity of Caciocavallo cheese during ripening. Journal of Dairy Science, 98, 3690 3694. Available from https://doi.org/10.3168/jds.2014-9097. Pisano, M. B., Scano, P., Murgia, A., Cosentino, S., & Caboni, P. (2016). Metabolomics and microbiological profile of Italian mozzarella cheese produced with buffalo and cow milk. Food Chem, 192, 618 624. Available from https://doi.org/10.1016/j.foodchem.2015.07.061. Pripp, A. H., Sørensen, R., Stepaniak, L., & Sørhaug, T. (2006). Relationship between proteolysis and angiotensin-I-converting enzyme inhibition in different cheeses. LWT-Food Science and Technology, 39(6), 677 683.

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Qureshi, T. M., Vegarud, G. E., Abrahamsen, R. K., & Skeie, S. (2012). Characterization of the Norwegian autochthonous cheese Gamalost and its angiotensin I-converting enzyme (ACE) inhibitory activity during ripening. Dairy science & technology, 92(6), 613 625. Available from https://doi.org/10.1007/s13594-012-0078-1. Qureshi, T. M., Amjad, A., Nadeem, M., Murtaza, M. A., & Munir, M. (2019). Antioxidant potential of a soft cheese (paneer) supplemented with the extracts of date (Phoenix dactylifera L.) cultivars and its whey. Asian-Australas J Anim Sci, 32(10), 1591 1602. Available from https:// doi.org/10.5713/ajas.18.0750. Rafiq, S., Huma, N., Pasha, I., Shahid, M., & Xiao, H. (2017). Angiotensin-converting enzymeinhibitory and antithrombotic activities of soluble peptide extracts from buffalo and cow milk Cheddar cheeses. International Journal of Dairy Technology, 70(3), 380 388. Available from https://doi.org/10.1111/1471-0307.12373. Redruello, B., Szwengiel, A., Ladero, V., del Rio, B., & Alvare, MA, (2020). Identification of technological/metabolic/environmental profiles of cheeses with high GABA contents. LWT, 130, https://doi.org/10.1016/j.lwt.2020.109603. Revilla, I., González-Martín, M. I., Vivar-Quintana, A. M., Blanco-López, M. A., Lobos-Ortega, I. A., & Hernández-Hierro, J. M. (2016). Antioxidant capacity of different cheeses: Affecting factors and prediction by near infrared spectroscopy. Journal of Dairy Science, 99(7), 5074 5082. Available from https://doi.org/10.3168/jds.2015-10564. Ryhänen, E.-L., Pihlanto-Leppälä, A., & Pahkala, E. (2001). A new type of ripened, low-fat cheese with bioactive properties. International Dairy Journal, 11(4-7), 441 447. Available from https://doi.org/10.1016/s0958-6946(01)00079-6. Sagardia, I., Iloro, I., Elortza, F., & Bald, C. (2013). Quantitative structure activity relationship based screening of bioactive peptides identified in ripened cheese. International Dairy Journal, 33(2), 184 190. Available from https://doi.org/10.1016/j.idairyj.2012.12.006. Sahingil, D., Hayaloglu, A. A., Kirmaci, H. A., Özer, B., & Simsek, O. (2014). Changes of proteolysis and angiotensin-I converting enzyme-inhibitory activity in white-brined cheese as affected by adjunct culture and ripening temperature. Journal of Dairy Research, 81(4), 394 402. Available from https://doi.org/10.1017/S0022029914000326. Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y., & Itoh, T. (2000). Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese. Journal of Dairy Science, 83(7), 1434 1440. Silva, S., Pihlanto, A., & Malcata, F. (2006). Bioactive peptides in ovine and caprine cheeselike systems prepared with proteases from Cynara cardunculus. Journal of Dairy Science, 89(9), 3336 3344. Silva, R. A., Lima, M. S. F., Viana, J. B. M., Bezerra, V. S., Pimentel, M. C. B., Porto, A. L. F., Cavalcanti, M. T. H., & Lima Filho, J. L. (2012). Can artisanal “Coalho” cheese from Northeastern Brazil be used as a functional food? Food Chemistry, 135(3), 1533 1538. Available from https://doi.org/10.1016/j.foodchem.2012.06.058. Smacchi, E., & Gobbetti, M. (1998). Peptides from several Italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria, Pseudomonas fluorescens ATCC 948 and to the angiotensin I-converting enzyme. Enzyme and microbial technology, 22(8), 687 694. Spinnler, H. E. (2017). (4, pp. 911 928). Surface mold-ripened cheeses. Cheese: chemistry, physics and microbiology., (1, pp. 911 928). Elsevier Inc. Available from: https://doi.org/10.1016/ B978-0-12-417012-4.00036-3. Théolier, J., Hammami, R., Fliss, I., & Jean, J. (2014). Antibacterial and antifungal activity of water-soluble extracts from Mozzarella, Gouda, Swiss, and Cheddar commercial cheeses produced in Canada. Dairy science & technology, 94(5), 427 438. Available from https://doi.org/ 10.1007/s13594-014-0170-9.

References

Timon, M. L., Andres, A. I., Otte, J., & Petron, M. J. (2019). Antioxidant peptides ( , 3kDa) identified on hard cow milk cheese with rennet from different origin. Food Res Int, 120, 643 649. Available from https://doi.org/10.1016/j.foodres.2018.11.019. Timón, M. L., Parra, V., Otte, J., Broncano, J. M., & Petrón, M. J. (2014). Identification of radical scavenging peptides ( , 3 kDa) from Burgos-type cheese. LWT-Food Science and Technology, 57(1), 359 365. Torres-Llanez, M. J., González-Córdova, A. F., Hernandez-Mendoza, A., Garcia, H. S., & VallejoCordoba, B. (2011). Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese. Journal of Dairy Science, 94(8), 3794 3800. Available from https://doi.org/10.3168/ jds.2011-4237. Uniacke-Lowe, T., & Fox, P. F. (2017). (4, pp. 69 113). Chymosin, pepsins and other aspartyl proteinases: Structures, functions, catalytic mechanism and milk-clotting properties. Cheese: Chemistry, physics and microbiology., (1, pp. 69 113). Elsevier Inc. Available from: https://doi.org/ 10.1016/B978-0-12-417012-4.00004-1. Vázquez-García, R., Cardador-Martínez, A., Orihuela-López, M. A., Ramos-Hernández, L. S., & Martín-del-Campo, S. T. (2021). Preliminary Study of Extended Ripening Effects on Peptides Evolution and DPPH Radical Scavenging Activity in Mexican Goat Cheese. Catalysts, 11(8), 967. Available from https://doi.org/10.3390/catal11080967.

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Immobilization of β-galactosidases R. Hemamalini1, Sumit Kumar2 and Sunil Kumar Khare1 1

Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, India, 2Amity Institute of Biotechnology. Amity University, Noida, Uttar Pradesh, India

13.1

Introduction

Most enzymes are proteins synthesized by living cells and catalyze complex, lifesustaining biochemical reactions. The hallmarks of enzyme action are very specific toward the substrate(s) on which these act. While enzymes regulate the rate at which chemical reactions proceed, these do not alter the reaction’s equilibrium (Robinson, 2015). Besides catalyzing reactions in situ, the enormous industrial potential of enzymes was discovered in the 19th century when the first successful cell-free fermentation was demonstrated (Heckmann & Paradisi, 2020). Nowadays, enzymes are an integral part of a wide range of industrial processes where seemingly impossible reactions are made feasible by enzymes in an efficient and eco-friendly manner (Ureta et al., 2021). β-Galactosidase (EC 3.2.1.23), also known as lactase, is an essential enzyme in carbohydrate metabolism (Fig. 13.1). A deficiency of the enzyme in the small intestine of the human body is known to cause lactose intolerance. This requires the pretreatment of milk products with the lactase enzyme before consumption by the lactose-intolerant population and paves the way for a significant industrial application of lactase. This and other industrial applications of the enzyme have necessitated the efficient immobilization of the enzyme (Khare & Gupta, 1987; Saqib et al., 2017). This chapter looks briefly at the various aspects of β-galactosidase, such as its sources, structure, and reactions that need consideration for planning application and immobilization of the enzyme. Immobilization of the enzyme has been dealt with at length, covering strategies employed for immobilization, challenges, latest trends, and prospects for immobilization and use of the enzyme. 351 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00013-8 © 2023 Elsevier Inc. All rights reserved.

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FIGURE 13.1 Activities of β-galactosidase. Source: From Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Science, 21(12), 1792 1807. https://doi.org/10.1002/ pro.2165.

13.2

Sources of β-galactosidase

A study of the available literature shows that commercially used β-galactosidases are commonly sourced from microorganisms and plants (Fig. 13.2). Escherichia coli is used in cloning experiments in molecular biology (Juers et al., 2012). Bacterial and yeast enzymes are preferred for lactose hydrolysis and galactooligosaccharide (GOS) production, while the fungal enzyme is used for whey utilization. The enzyme from Kluyveromyces lactis (Kl-β-gal) is well studied (de Albuquerque et al., 2021). β-Galactosidases from plants have been implicated in ripening of fruits such as papaya, strawberry, and tomatoes, where they have been purified from apple, muskmelon, coffee, and mango (Saqib et al., 2017) (Fig. 13.2). Among animals, β-galactosidase present on the brush borders of the small intestine in the eutherian mammals converts lactose to glucose and galactose.

13.4 Classification of β-galactosidases

Bacteria

Plants

Sources of β-galactosidase

Fungi

Yeasts

FIGURE 13.2 Common sources of β-galactosidase. Source: From Saqib, S., Akram, A., Halim, S. A. & Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. 3 Biotech, 7(1). https://doi.org/10.1007/s13205-017-0645-5.

13.3

Structure of β-galactosidase

β-Galactosidase from E. coli and the yeast K. lactis has been well characterized. The E. coli β-galactosidase is known to have an average diameter of 12 nm and a tetrameric structure of four identical subunits of 1023 amino acids of 135,000 Da each. These four subunits interconnect to form five welldefined structural domains, one of which is the catalytic domain. The third domain residues mainly form the active site. It is revealed through several studies on β-galactosidases that catalytic site consists of two glutamic acid residues. One of these acts as a nucleophile and another as a Brønsted proton donor. Consequently, it can be surmised that the mechanism should be the same for all (de Albuquerque et al., 2021; Ureta et al., 2021). The K. lactis enzyme, Kl-β-gal, consists of 119 kDa monomers which form active dimers or tetramers. The active site has been located between the first, third, and fifth domains (de Albuquerque et al., 2021).

13.4

Classification of β-galactosidases

As per the Carbohydrate-Active enZYmes (CAZy) database (http://www.cazy. org/), β-galactosidases are classified into six different glycosyl hydrolase (GH) families (Fig. 13.3) (Lu et al., 2020). Although β-galactosidases have been classified into six families, all belong to the superfamily, Clan A. Most known β-galactosidases belong to GH2, GH35, and GH42 families. The β-galactosidases from bacteria and yeast belong to the GH2 family, including the cold-active enzyme from

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Arthrobacter sp. The GH35 family includes β-galactosidases sourced from bacteria, fungi, plants, and animals, while the GH42 family includes β-galactosidases sourced from thermophilic bacteria. On the other hand, the other GH families contain few representatives. For instance, GH1 contains β-galactosidases derived from archaea, while GH59 and GH147 have one representative each (Lu et al., 2020) (Fig. 13.3).

13.5

Reactions of β-galactosidase

The reaction catalyzed by β-galactosidase can be broadly understood as a three-step process as shown in Fig. 13.4. When the nucleophilic acceptor of the galactosyl residue in the last step is water, galactose and glucose are formed (Ureta et al., 2021).

FIGURE 13.3 Classification of β-galactosidases based on sequence similarity. Source: From Lu, L., Guo, L., Wang, K., Liu, Y., & Xiao, M. (2020). β-Galactosidases: A great tool for synthesizing galactose-containing carbohydrates. Biotechnology Advances, 39, 107465. https://doi. org/10.1016/j.biotechadv.2019.107465.

First

Formaon of Lactose enzyme complex

Second

Formaon of Galactosylenzyme complex and release of Glucose

Last

Transfer of Galactose to a hydroxylcontaining nucleophilic acceptor

FIGURE 13.4 Steps involved in the reaction of β-galactosidase. Source: From Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Science, 21(12), 1792 1807. https://doi.org/10.1002/pro.2165.

13.6 Immobilization of β-galactosidase

Thus the β-galactosidase enzyme, also known as lactase, is generally employed to get glucose and galactose from lactose or allolactose (Nath et al., 2014). The reaction is occasionally also utilized as a technological approach to conquer the possibility of lactose deposition and achieve a higher degree of sweetness, because of better solubility and sweetening property of glucose and galactose as compared to lactose (Panesar et al., 2018; Ureta et al., 2021). Besides hydrolysis, the enzyme also catalyzes transgalactosylation of lactose to form allolactose (Juers et al., 2012).

13.6

Immobilization of β-galactosidase

Immobilization involves attaching or incorporating enzymes onto a support material to form an insoluble, recyclable, and robust enzyme preparation. The immobilized enzyme form can take part in chemical reactions with various process conditions, preferably with no considerable loss of stability or activity. Broadly, the underlying chemistry that has been tapped while immobilizing enzymes on material supports includes adsorption, covalent binding, cross-linking, and entrapment. While not many advancements have happened in the underlying chemistry, all the latest developments in β-galactosidase immobilization have taken place concerning support materials used for the enzyme’s immobilization. These include nanomaterials fabricated as nanoparticles, nanofibers, nanotubes, nanoporous, nanocomposites, and nanosheets. As these substances deliver a better surface-area-to-volume ratio, the loading of the enzyme is improved, leading to better enzyme immobilization and stabilization (Ureta et al., 2021). Immobilization ensures the recovery of expensive enzymes from industrial processes. Being proteins, enzymes are also highly sensitive to process conditions such as temperature, pH, and trace-level inhibitors. Immobilization also helps build more robust biocatalysts for industrial applications (Homaei et al., 2013; Van De Velde et al., 2002). In the ensuing discussion, the conventional as well as novel support materials used in β-galactosidase immobilization have been discussed.

13.6.1

Functional enzyme aggregates

Commercial E. coli β-galactosidase was immobilized by chemical cross-linking using glutaraldehyde into functional insoluble aggregates (Khare & Gupta, 1990). The enzyme was also conjugated with Concanavalin-A using glutaraldehyde as a cross-linker to form ConA-β-galactosidase conjugate (Khare & Gupta, 1988). Cross-linked derivatives of β-galactosidase were prepared by treating the

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enzyme with either of the following chemicals—glutaraldehyde, bisimidoesters, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS) (Khare & Gupta, 1987).

13.6.2

Gel beads and lattices

Alginate/H0 (acrylamide-co-acrylic acid) gel beads were produced and altered by subsequent soaking with polyethyleneimine (PEI) and glutaraldehyde (GA) as a usable carrier appropriate for enzyme immobilization (Hassan et al., 2018). β-Galactosidase of K. lactis was immobilized in calcium alginate spheres and gelatin for cheese whey lactose hydrolysis (Mörschbächer et al., 2016). Apricot (Prunus armeniaca Kaisa) β-galactosidase was immobilized on concanavalin-A-layered cellulose-alginate hybrid gel (Ansari et al., 2014). Commercial β-galactosidase from Aspergillus oryzae was immobilized on activated κ-carrageenan beads by covalent binding (Elnashar et al., 2014).

13.6.3

Chitosan

Adduct formation between the chitosan-carboxymethyl chitosan polymers and the enzyme was used to immobilize β-galactosidase from Bacillus circulans (Franco & Mesa, 2021). β-Galactosidase from K. lactis was immobilized on chitosan activated with glutaraldehyde to achieve very active and stable catalysts for lactulose production utilizing cheese whey and fructose as the substrate (de Albuquerque et al., 2018). Two β-galactosidases from Lactobacillus reuteri L103 and Lactobacillus bulgaricus DSM 20081 were immobilized on chitin using a chitin-binding domain, ChBD on these enzymes (Pham et al., 2017). β-Galactosidase from Aspergillus niger was immobilized in chitosan/xanthan multilayers (PEMs) deposited by dip-coating method on corona polylactice pads (Iliev et al., 2016). β-Galactosidase from A. oryzae was adsorbed on celite, covalently coupled to chitosan, and aggregated by cross-linking (Gaur et al., 2006). The enzyme from the same organism was also immobilized on fibers composed of alginate and gelatin hardened with glutaraldehyde (Tanriseven & Do˘gan, 2002). A commercial Kluyveromyces fragilis β-galactosidase, Lactozym 3000, was immobilized on chitosan (Carrara & Rubiolo, 1994).

13.6.4

Nanoparticles

Polymer nanoparticles were used to anchor the enzyme molecules through affinity binding and then encapsulated inside hydrogel microparticles using a microfluidic device paired with UV-LEDs (Suvarli et al., 2022). Commercial β-galactosidase sourced from Bifidobacterium bifidum was immobilized by physical adsorption on halloysite, an aluminosilicate nanomaterial (Tizchang et al., 2021). β-Galactosidase from A. oryzae was immobilized covalently on functionalized silica nanoparticles for lactose and whey hydrolysis (Goel et al., 2017),

13.7 Conclusion

while immobilization of β-galactosidase from Lactobacillus plantarum HF571129 was accomplished on ZnO nanoparticles (Selvarajan et al., 2015).

13.6.5

Metal affinity columns

p(AAm-HEMA) Cryogel disks prepared by free radical polymerization were chelated with Fe21 ions to produce an immobilized metal affinity chromatography (IMAC) material used to immobilize lactase (Inanan, 2022). Recombinant β-galactosidase fusion protein containing an N-terminal hexahistidine peptide was immobilized on Ni21-nitrilotriacetic acid (NTA) chelate column through adsorption (Piesecki et al., 1993).

13.6.6

Methacrylate and its variants

Crude K. lactis enzyme preparation Lactozym 3000 L was successfully immobilized onto activated carrier Lifetech ECR8309, a methacrylate polymer, via covalent binding of the enzyme, which is a favorable procedure to minimize very high enzyme utilization in its free form (Hollá et al., 2021). The UV-cured epoxy-based polymeric film was made from glycidyl methacrylate, trimethylolpropane triacrylate, and poly (ethylene glycol) methyl ether acrylate. 2-Hydroxy2-methylpropiophenone was employed as a photoinitiator. Covalent binding through epoxy groups was used to immobilize β-galactosidase from E. coli onto this film, and immobilization conditions were improved by the response surface methodology (Beyler-Çigil et al., 2021). Campello et al. (2012) also immobilized commercial β-galactosidase from K. lactis available as Lactozym on Eupergit C and studied the properties of the biocatalyst. Enzyme treated with glutaraldehyde or bisimidoesters was entrapped in polyacrylamide gel lattice (Khare & Gupta, 1988). β-D-Galactosidase from L. bulgaricus (1373) was immobilized by polyacrylamide gel lattice entrapment (Makkar et al., 1981). Novel magnetic beads prepared from glycidyl methacrylate (GMA) and styrene through suspension polymerization with porogenic agents such as cyclohexanol and lauryl alcohol were applied to immobilize commercial β-galactosidase from A. oryzae (Sun et al., 2013). Commercial β-galactosidase from E. coli was immobilized on polypropylene (PP) membranes that had been subjected to hydrophilic modification using chemical treatment prior to enzyme immobilization (Vasileva et al., 2012). K. lactis β-galactosidase was immobilized in polyvinyl alcohol gel (Hronská et al., 2009).

13.7

Conclusion

Despite many advantages in using enzymes compared to conventional catalysts, challenges associated with their industrial applications demand the

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employment of suitable strategies. β-Galactosidases are no exception. Immobilization is the most cost-effective strategy for their optimal industrial applications. It ensures the recovery of enzymes that are often expensive and available in limited quantities besides conferring other significant advantages for industrial catalysis.

Acknowledgments The financial support by the Indian Institute of Technology Delhi, and MHRD, Government of India is gratefully acknowledged.

References Ansari, S. A., Satar, R., Zaidi, S. K., Khan, M. J., Naseer, M. I., Al-Qahtani, M. H., & Maskat, M. Y. (2014). Future applications of apricot (Prunus Armeniaca Kaisa) ß galactosidase in dairy industry. Polish Journal of Chemical Technology, 16(3), 74 79. Available from https://doi.org/ 10.2478/pjct-2014-0054. Beyler-Çigil, A., Danis, O., Sarsar, O., Kahraman, M. V., Ogan, A., & Demir, S. (2021). Optimizing the immobilization conditions of β-galactosidase on UV-cured epoxy-based polymeric film using response surface methodology. Journal of Food Biochemistry, 45(4). Available from https://doi.org/10.1111/jfbc.13699. Campello da, G. S., Trindade, R. A., Rêgo, T. V., Burkert de, JFM, & Burkert, CAV (2012). Immobilization of ß-galactosidase from Kluyveromyces lactis on Eupergits C and properties of the biocatalyst. International Journal of Food Engineering, 8(3). Available from https://doi. org/10.1515/1556-3758.2760. Carrara, C. R., & Rubiolo, A. C. (1994). Immobilization of β-Galactosidase on chitosan. Biotechnology Progress, 10(2), 220 224. Available from https://doi.org/10.1021/ bp00026a012. de Albuquerque, T. L., de Sousa, M., Gomes e Silva, N. C., Girão Neto, CAC, Gonçalves, LRB, Fernandez-Lafuente, R., & Rocha, MVP (2021). β-Galactosidase from Kluyveromyces lactis: Characterization, production, immobilization and applications - A review. International Journal of Biological Macromolecules, 191, 881 898. Available from https://doi.org/10.1016/j. ijbiomac.2021.09.133. de Albuquerque, T. L., Gomes, SDL, D’Almeida, A. P., Fernandez-Lafuente, R., Gonçalves, LRB, & Rocha, MVP (2018). Immobilization of β-galactosidase in glutaraldehyde-chitosan and its application to the synthesis of lactulose using cheese whey as feedstock. Process Biochemistry, 73, 65 73. Available from https://doi.org/10.1016/j.procbio.2018.08.010. Elnashar, M. M., Awad, G. E., Hassan, M. E., Mohy Eldin, M. S., Haroun, B. M., & El-Diwany, A. I. (2014). Optimal immobilization of β -galactosidase onto κ-carrageenan gel beads using response surface methodology and its applications. The Scientific World Journal, 2014. Available from https://doi.org/10.1155/2014/571682. Franco, Y. N., & Mesa, M. (2021). Complementary experimental/docking approach for determining chitosan and carboxymethylchitosan ability for the formation of active polymer-β-galactosidase adducts. International Journal of Biological Macromolecules, 192, 736 744. Available from https://doi.org/10.1016/j.ijbiomac.2021.10.020. Gaur, R., Pant, H., Jain, R., & Khare, S. K. (2006). Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae β-galactosidase. Food Chemistry, 97(3), 426 430. Available from https://doi.org/10.1016/j.foodchem.2005.05.020.

References

Goel, A., Sinha, R., & Khare, S. K. (2017). Immobilization of A. oryzae 1 on silica nanoparticles: Development of an effective biosensor for determination of lactose in milk whey. Recent advances in applied microbiology (pp. 3 18). Springer Singapore. Available from https://doi. org/10.1007/978-981-10-5275-0_1. Hassan, M. E., Ibrahim, A. G., El-Wahab, A. H., Hai, F. A., Mahmoud, H., & Awad, G. E. (2018). Covalent immobilization of β-Galactosidase enzyme onto modified alginate gel beads. Journal of Materials and Environmental Sciences, 9, 2483 2492. Heckmann, C. M., & Paradisi, F. (2020). Looking back: A short history of the discovery of enzymes and how they became powerful chemical tools. ChemCatChem, 12(24), 6082 6102. Available from https://doi.org/10.1002/cctc.202001107. Hollá, V., Karkeszová, K., Antoˇsová, M., & Polakoviˇc, M. (2021). Transglycosylation properties of a Kluyveromyces lactis enzyme preparation: Production of tyrosol β-fructoside using free and immobilized enzyme. Process Biochemistry, 110, 168 175. Available from https://doi.org/ 10.1016/j.procbio.2021.08.016. Homaei, A. A., Sariri, R., Vianello, F., & Stevanato, R. (2013). Enzyme immobilization: An update. Journal of Chemical Biology, 6(4), 185 205. Available from https://doi.org/10.1007/ s12154-013-0102-9. Hronská, H., Grosová, Z., & Rosenberg, M. (2009). Hydrolysis of lactose in milk by Kluyveromyces lactis β-galactosidase immobilized in polyvinylalcohol gel. Journal of Food and Nutrition Research, 48(2), 87 91. Iliev, I. N., Marudova, M. G., Cholev, D. S., Vasileva, T. A., Bivolarski, V. P., Viraneva, A. P., Bodurov, I. P., & Yovcheva, T. A. (2016). Kinetic studies of β-galactosidase immobilized in chitosan/xanthan multilayers. Bulgarian Chemical Communications, 48, 354 358. Inanan, T. (2022). Cryogel disks for lactase immobilization and lactose-free milk production. LWT, 154(112608). Available from https://doi.org/10.1016/j.lwt.2021.112608. Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Science, 21(12), 1792 1807. Available from https://doi.org/10.1002/pro.2165. Khare, S. K., & Gupta, M. N. (1987). A crosslinked preparation of E. coli β-D-galactosidase. Applied Biochemistry and Biotechnology, 16(1), 1 13. Available from https://doi.org/10.1007/BF02798351. Khare, S. K., & Gupta, M. N. (1990). An active insoluble aggregate of E. coli β-galactosidase. Biotechnology and Bioengineering, 35(1), 94 98. Available from https://doi.org/10.1002/ bit.260350113. Khare, S. K., & Gupta, M. N. (1988). Immobilization of E. coli β-galactosidase and its derivatives by polyacrylamide gel. Biotechnology and Bioengineering, 31(8), 829 833. Available from https://doi.org/10.1002/bit.260310810. Lu, L., Guo, L., Wang, K., Liu, Y., & Xiao, M. (2020). β-Galactosidases: A great tool for synthesizing galactose-containing carbohydrates. Biotechnology Advances, 39(107465). Available from https://doi.org/10.1016/j.biotechadv.2019.107465. Makkar, HPS, Sharma, O. P., & Negi, S. S. (1981). Immobilization and properties of β-D-galactosidase from Lactobacillus bulgaricus. Journal of Biosciences, 3(1), 7 16. Available from https:// doi.org/10.1007/BF02703341. Mörschbächer, A. P., Volpato, G., & Souza de, CFV (2016). Kluyveromyces lactis β-galactosidase immobilization in calcium alginate spheres and gelatin for hydrolysis of cheese whey lactose. Ciência Rural, 46(5), 921 926. Available from https://doi.org/10.1590/0103-8478cr20150833. Nath, A., Mondal, S., Chakraborty, S., Bhattacharjee, C., & Chowdhury, R. (2014). Production, purification, characterization, immobilization, and application of β-galactosidase: a review. Asia-Pacific Journal of Chemical Engineering, 9(3), 330 348. Available from https://doi.org/ 10.1002/apj.1801.

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Panesar, P. S., Kaur, R., Singh, R. S., & Kennedy, J. F. (2018). Biocatalytic strategies in the production of galacto-oligosaccharides and its global status. International Journal of Biological Macromolecules, 111, 667 679. Available from https://doi.org/10.1016/j.ijbiomac.2018.01.062. Pham, M. L., Leister, T., Nguyen, H. A., Do, B. C., Pham, A. T., Haltrich, D., Yamabhai, M., Nguyen, T. H., & Nguyen, T. T. (2017). Immobilization of β-Galactosidases from Lactobacillus on Chitin Using a Chitin-Binding Domain. Journal of Agricultural and Food Chemistry, 65(14), 2965 2976. Available from https://doi.org/10.1021/acs.jafc.6b04982. Piesecki, S., Teng, W., & Hochuli, E. (1993). Immobilization of β-galactosidase for application in organic chemistry using a chelating peptide. Biotechnology and Bioengineering, 42(2), 178 184. Available from https://doi.org/10.1002/bit.260420205. Robinson, P. K. (2015). Enzymes: principles and biotechnological applications. Essays in Biochemistry, 59, 1 41. Available from https://doi.org/10.1042/BSE0590001. Saqib, S., Akram, A., Halim, S. A., & Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. 3 Biotech, 7(1). Available from https://doi.org/10.1007/s13205-017-0645-5. Selvarajan, E., Mohanasrinivasan, V., Subathra Devi, C., & George Priya Doss, C. (2015). Immobilization of β-galactosidase from Lactobacillus plantarum HF571129 on ZnO nanoparticles: Characterization and lactose hydrolysis. Bioprocess and Biosystems Engineering, 38(9), 1655 1669. Available from https://doi.org/10.1007/s00449-015-1407-6. Sun, S., Zhao, X., & Xu, X. (2013). Immobilization of β-Galactosidase from Aspergillus oryzae on magnetic poly(GMA-ST) beads. Asian Journal of Chemistry, 25(1), 381 384. Available from https://doi.org/10.14233/ajchem.2013.13092. Suvarli, N., Wenger, L., Serra, C., Perner-Nochta, I., Hubbuch, J., & Wörner, M. (2022). Immobilization of β-Galactosidase by encapsulation of enzyme-conjugated polymer nanoparticles inside hydrogel microparticles. Frontiers in Bioengineering and Biotechnology, 9. Available from https://doi.org/10.3389/fbioe.2021.818053. Tanriseven, A., & Do˘gan, s¸ (2002). A novel method for the immobilization of β-galactosidase. Process Biochemistry, 38(1), 27 30. Available from https://doi.org/10.1016/s0032-9592(02) 00049-3. Tizchang, S., Khiabani, M. S., Mokarram, R. R., Hamishehkar, H., Mohammadi, N. S., & Chisti, Y. (2021). Immobilization of β-galactosidase by halloysite-adsorption and entrapment in a cellulose nanocrystals matrix. Biochimica et Biophysica Acta - General Subjects, 1865(6). Available from https://doi.org/10.1016/j.bbagen.2021.129896. Ureta, M. M., Martins, G. N., Figueira, O., Pires, P. F., Castilho, P. C., & Gomez-Zavaglia, A. (2021). Recent advances in β-galactosidase and fructosyltransferase immobilization technology. Critical Reviews in Food Science and Nutrition, 61(16), 2659 2690. Available from https://doi.org/10.1080/10408398.2020.1783639. Van De Velde, F., Lourenço, N. D., Pinheiro, H. M., & Bakker, M. (2002). Carrageenan: A foodgrade and biocompatible support for immobilisation techniques. Advanced Synthesis and Catalysis, 344(8), 815 835. Vasileva, N., Iotov, V., Ivanov, Y., Godjevargova, T., & Kotia, N. (2012). Immobilization of β-galactosidase on modified polypropilene membranes. International Journal of Biological Macromolecules, 51(5), 710 719. Available from https://doi.org/10.1016/j.ijbiomac.2012.07.032.

CHAPTER 14

Low-lactose milk production using β-galactosidases Priscilla Romina De Gregorio1,2,3, Adriano Gennari1,2, Cathy Verônica Nied1, Giandra Volpato4 and Claucia Fernanda Volken de Souza1,2 1

Food Biotechnology Laboratory, University of Vale do Taquari - Univates, Lajeado, RS, Brazil, 2Biotechnology Graduate Program, University of Vale do Taquari - Univates, Lajeado, RS, Brazil, 3Reference Center for Lactobacilli - National Council for Scientific ´ Tucuman, ´ and Technological Research (CERELA-CONICET), San Miguel de Tucuman, Argentina, 4Federal Institute of Education, Science and Technology of Rio Grande do Sul, Campus Porto Alegre, Porto Alegre, RS, Brazil

14.1

Introduction

Lactose is the main source of calories in milk. It is usually hydrolyzed into glucose and galactose by lactase, a type of β-galactosidase found in the small intestinal mucosa. Lactose intolerance (LI) is a clinical syndrome manifested by symptoms such as abdominal pain, bloating, and diarrhea following lactose ingestion. Individuals with LI have lactase deficiency (LD), which is the inability to express lactase to hydrolyze lactose into absorbable glucose and galactose components (Misselwitz et al., 2019). There are three types of LD, including primary, secondary, and congenital deficiency. The most common cause is primary LD or lactase non-persistence (LNP), which relates to a gradual decline in lactase activity with increasing age. It is a hereditary condition that begins with decreased enzyme activity in childhood and manifests in adolescence or early adulthood. Injuries, infections, or inflammatory diseases of the intestinal mucosa can cause secondary LD. The rarest cause of LI is congenital LD, which results in a lack of lactase expression and severe symptoms immediately after birth (Misselwitz et al., 2019). LI prevalence varies across ethnicities, being more common in Africans, Asians, Hispanics/Latinos, and Americans. It is less prevalent in Nordic countries. Ethnic groups with a higher prevalence of LI are more likely to have LNP. Overall, 68% of the world’s population has problems in lactose digestion (Misselwitz et al., 2019; Oak & Jha, 2019). This forces the dairy industry to develop lactose-reduced or lactose-free dairy products by subjecting them to enzymatic hydrolysis with lactase (Vera et al., 2020). 361 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00014-X © 2023 Elsevier Inc. All rights reserved.

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The lactose-free product market is the fastest-growing sector of the dairy industry. The progress in sales has manifested in the high number of new lactose-free or lactose-reduced products launched on the market, such as pasteurized and ultrahigh-temperature (UHT)-treated milk, yogurt, cheese, and ice cream, which have increased considerably in recent years. The increased availability of these products, both on shelves and in refrigerated sections, has encouraged consumers to choose lactose-free dairy products over regular dairy products (Dekker et al., 2019). The low-lactose product market was estimated at USD 12.1 billion in 2020 and is expected to reach USD 18.4 billion by 2025, registering a compound annual growth rate of 8.7% in terms of value (Markets and Markets, n.d.). The biggest and fastest-growing lactosefree markets are Western Europe and North America, followed by Latin America (Dekker et al., 2019). Milk is the main product consumed among lactose-free and reduced dairy products, accounting for two-thirds of the market. Using low-lactose milk (LLM) in the manufacture of different dairy products has several advantages over using whole milk: (1) reduction of sucrose addition in flavored milk, because the hydrolysis of lactose can increase sweetening power by generating sugars such as glucose and galactose, which have higher solubility and sweetness than lactose (Dekker et al., 2019; Dorau et al., 2021); (2) prevention of lactose crystallization, thus avoiding the appearance of a sandy texture in low dairy products (“dulce de leche”, ice cream, condensed milk, and frozen milk) (Abbasi & Saeedabadian, 2015; Vera et al., 2020); and (3) an increased production rate of fermented products, such as yogurt and cheese, as lactose hydrolysis is often the limiting step (Vera et al., 2020). β-Galactosidase is one of the most important enzymes used in the dairy industry. Lactose degradation by β-galactosidase generates glucose and galactose monosaccharides by hydrolysis reaction or galactooligosaccharides (GOS) by transgalactosylation reaction. In the food industry, β-galactosidase is used for producing LLM, as well as for producing GOS, which have sweetening and prebiotic properties (Vera et al., 2020). Currently, the most common process used to produce LLM industrially involves dosing the soluble β-galactosidase enzyme right before packaging and allowing it to act during milk storage (Dekker et al., 2019; Schulz & Rizvi, 2021). However, scientific research has paid great attention to the immobilization process using packed-bed reactors to recycle enzymes and develop value-added products with higher yields (Di Cosimo et al., 2013; Dorau et al., 2021; Schulz & Rizvi, 2021; Ureta et al., 2021; Vasileva et al., 2016). Further studies on the development of immobilized enzyme bioreactors on a commercial scale are needed for more cost-effective, environmentally attractive low-lactose products (Schulz & Rizvi, 2021).

14.2 Characteristics of β-galactosidases

This chapter focuses on the methods of enzymatic hydrolysis with β-galactosidase for the production of LLM. It mainly covers the advantages and disadvantages of lactose hydrolysis techniques. The sources and catalytic reactions of β-galactosidase, its recombinant production, and its industrial use by companies are described. In addition, scalability issues with specific procedures and future research needed for generating commercially useful LLM are discussed.

14.2 14.2.1

Characteristics of β-galactosidases Sources of β-galactosidases

β-Galactosidase is a fairly ubiquitous enzyme found in plants, microorganisms, and the intestinal tract of mammals. However, its properties differ markedly depending on its origin. Plant and mammalian β-galactosidases have little commercial value, whereas β-galactosidases from microorganisms are of significant technological interest and are industrially applicable. Microorganisms as a source of β-galactosidases offer several advantages, such as easy handling, high proliferation rate, easy fermentation, and highproductivity yield (Movahedpour et al., 2021; Saqib et al., 2017). In this way, a wide variety of bacteria, yeast, and fungi have been evaluated as potential sources of β-galactosidases (Table 14.1). The most widely used β-galactosidases in the industry derive from yeasts and fungi such as Kluyveromyces spp. and Aspergillus spp. This is due to their ease of production, acceptable yields and productivities from microbial cultures, and high efficiency and resistance to pH and temperature. In addition, the products of these microorganisms are generally recognized as safe (GRAS) for human consumption, which is essential for food-related applications (Dekker et al., 2019; Movahedpour et al., 2021; Saqib et al., 2017). Escherichia coli β-galactosidases have been the most studied, but they are only commercially available for analytical rather than industrial purposes, as their use is not considered safe for food Table 14.1 Microbial genera assessed as potential sources of β-galactosidases. Source

Microbial genera

Bacteria

Alicyclobacillus, Alkalilactibacillus, Antarctic, Arthrobacter, Bacillus, Bacteriodes, Bifidobacterium, Caldicellulosiruptor, Clostridium, Corynebacterium, Enterobacter, Escherichia, Klebsiella, Lactobacillus, Leuconostoc,Paenibacillus, Pediococcus, Propioionibacterium, Pseudomonas, Pseudoalteromonas, Pyrococcus, Rhizobium, Streptococcus, Sulfolobus, Thermoanaerobacter,Thermotoga, Thermus, Trichoderma, Vibrio, Xanthomonas Bullera, Candida, Saccharomyces, Kluyveromyces Alternaria, Aspergillus, Auerobasidium, Curvularia, Fusarium, Mucor, Neurospora, Penicillum, Saccharopolyspora, Scopulariapsis, Streptomyces

Yeast Fungi

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applications. Bifidobacterium and lactic acid bacteria (LAB) (including the genera Lactococcus, Lactobacillus, and Streptococcus), which are GRAS, have also been considered good sources of β-galactosidases, mainly for functional food applications (Movahedpour et al., 2021; Saqib et al., 2017). In addition, β-galactosidases from other sources with relevant biotechnological properties have attracted increasing interest. Particular attention has been given to thermotolerant or coldactive enzymes from yeast and bacteria (Movahedpour et al., 2021; Oliveira et al., 2011). Recombinant DNA technology is currently being used to improve the expression and production of β-galactosidases from various sources. Microbial hosts recognized for their high, efficient production of heterologous proteins are used for this purpose. Heterologous expression and production of the most widely used β-galactosidases in the food industry (from the filamentous fungus Aspergillus niger and the yeast Kluyveromyces lactis) is carried out in recombinant yeast hosts such as Saccharomyces cerevisiae and Pichia pastoris (Movahedpour et al., 2021). In addition, engineering strategies such as site-directed mutagenesis, truncation, site saturation mutagenesis, random mutagenesis, DNA shuffling, and monobody modifications have been applied in search of β-galactosidases with improved features. Increased hydrolytic activity, improved substrate specificity, increased product yield, reduced product inhibition, and improved thermal stability or secretion signals are some of the desirable features introduced by bioengineering tools (Movahedpour et al., 2021; Oliveira et al., 2011).

14.2.2

Reactions catalyzed by β-galactosidases

β-Galactosidase (β-D-galactoside-galactohydrolase, E.C. 3.2.1.23) is an enzyme that catalyzes the hydrolysis of nonreducing terminal β-D-galactose residues from β-D-galactosides. The most common β-galactoside is lactose, a disaccharide between galactose and glucose (β 1,4 galactose-glucose) (Vera et al., 2020). Thus the main application of β-galactosidase has been lactose hydrolysis in milk for the production of LLM. The catalytic mechanism of β-galactosidase can be described in two steps, as shown in Fig. 14.1. First, the β-galactoside-enzyme complex is formed: the glycosidic bond is broken, and the galactose moiety is transferred to the active site of the enzyme. This step is assisted by the carboxylic chains of two glutamic acid residues present in the enzyme. As a result, a galactosyl-enzyme complex is formed while releasing glucose. In the second step, the enzyme transfers galactose residue to a nucleophilic acceptor containing a hydroxyl group that causes the product to release from the

14.2 Characteristics of β-galactosidases

FIGURE 14.1 Catalytic mechanism of β-galactosidase for hydrolysis and transgalactosylation reactions.

enzyme’s active site. When the nucleophilic acceptor is water, galactose is formed by hydrolysis reaction, whereas when the acceptor is another sugar, GOS are formed by transgalactosylation (Vera et al., 2020). Currently, the production of GOS from the transgalactosylation activity of β-galactosidase has attracted considerable attention by the dairy industry for the synthesis of high value-added compounds from lactose. GOS are nondigestible oligosaccharides with prebiotic properties, which are selectively utilized by host intestinal microorganisms conferring health benefits (Gibson et al., 2017). In the food industry, GOS are used as low-calorie sweeteners in fermented dairy products, soft drinks, confectionery, beverages, and cerealbased foods (Movahedpour et al., 2021; Vera et al., 2020).

14.2.3

Optimal reaction conditions for β-galactosidases

Depending on extraction sources, the properties of β-galactosidases vary. Their applications depend on their operating pH range. According to pH, enzymes can be divided into two types: acidic enzymes from fungi and neutral enzymes from bacteria and yeasts (Movahedpour et al., 2021; Nivetha & Mohanasrinivasan, 2017; Saqib et al., 2017). Bacterial β-galactosidases have

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an optimum action pH of 6.5 7.5 and an optimum temperature of 50 C 60 C. In addition, thermostable β-galactosidases can be obtained from thermophilic bacteria. Yeast β-galactosidases are generally used for lactose hydrolysis in milk, as they require an optimal pH range of 6.0 7.0, similar to that of milk. These enzymes have low heat stability, so temperatures above 55 C rapidly inactivate the enzyme. Fungal β-galactosidases have an optimum pH in the range of 3.0 5.0, so these enzymes are used in the hydrolysis of acid whey and permeate. In addition, fungal enzymes have a high optimum temperature of 55 C 60 C. However, cold-active acidic β-galactosidases can also be produced from some fungi, such as Neurospora crassa. Compared with yeast enzymes, fungal β-galactosidases are not pure, as they may also contain other enzymes, for example, amylase, lipase, and protease. This limits their application to high-acid products and pharmaceutical preparations (Nivetha & Mohanasrinivasan, 2017; Saqib et al., 2017). Table 14.2 shows properties of bacterial, yeast, and fungal β-galactosidases. The activities of β-galactosidases can be influenced by the presence of ions. For example, cations such as magnesium (Mg21), manganese (Mn21), and sodium (Na1) are required in yeast β-galactosidases such as Kluyveromyces lactis and Kluyveromyces fragilis, whereas exposure to heavy metals and calcium (Ca21) inhibits these enzymes. However, since calcium is not free in milk (it is bound to casein), calcium does not inhibit the activity of β-galactosidase in the production of LLM (Nivetha & Mohanasrinivasan, 2017; Saqib et al., 2017). The action of β-galactosidase can be inhibited by the products of lactose hydrolysis. Galactose is often a competitive inhibitor, whereas glucose only produces noncompetitive inhibition at high concentrations. Therefore it is very difficult to achieve complete hydrolysis unless high concentrations of the enzyme are used. Yeast enzymes are usually competitively inhibited by Table 14.2 Properties of bacterial, yeast, and fungal β-galactosidases. Source

Microorganisms

Enzyme location

pH

Temperature ( C)

Bacterial

Bacillus circulans Escherichia coli Lactobacillus thermophilus Leuconostoc citrovorum Kluyveromyces fragilis Kluyveromyces lactis Aspergillus foetidus Aspergillus niger Aspergillus oryzae Neurospora crassa

Intracellular Intracellular Intracellular Intracellular Intracellular Intracellular Extracellular Extracellular Extracellular Extracellular

6.5 7.2 6.2 6.0 6.0 6.5 3.5 3.5 4.5 -

56 40 55 65 30 30 66 55 55 4

Yeast Fungal

7.0 4.0 4.5 5.0

35 35 67 60

14.2 Characteristics of β-galactosidases

galactose and noncompetitively inhibited by glucose. The β-galactosidase from A. niger is strongly inhibited by galactose, whereas the β-galactosidase from Aspergillus oryzae is less so (Panesar et al., 2006).

14.2.4

Production and purification of β-galactosidases

An estimated 5.75 million tons of β-galactosidases are produced per year (Xavier, Ramana, & Sharma, 2018). Enzyme cost depends on production and purification levels (Panesar et al., 2010). On an industrial scale, β-galactosidase is produced with GRAS microorganisms, yeast (mainly from K. lactis), and fungal (mainly from A. niger and A. oryzae) consortia. Depending on the type of consortium, different substrates (e.g., cheese whey; chemically defined lactose-based medium; combination of skimmed milk powder, glucose, and yeast extract; lactose medium) are used in bioreactors with different modes of operation (fed batch, batch, batch and fed batch, or continuous) (Nath et al., 2014). Since most industrial β-galactosidases are intracellular enzymes (Table 14.2). One must disrupt cells completely to obtain high enzyme yields. Yeast cells are moderately easy to disintegrate. However, bacterial cells, especially Gram-positive bacteria such as LAB, which are smaller and have a robust cell wall, need more severe methods. This is one of the reasons why LAB enzymes are difficult to commercialize compared with successfully marketed yeast enzymes. Several methods of cell disruption exist, involving physical, chemical, or enzymatic means, or combinations thereof. Physical methods include bead milling, sonication, or high-pressure homogenization. Chemical treatments apply organic solvents or detergents to permeabilize the cell membrane, and enzymatic treatments use lytic enzymes such as lysozyme or mutanolysin (Dorau et al., 2021). In contrast, for the purification of β-galactosidase from crude extract, different separation techniques have already been tried, such as membrane separation, gel permeation chromatography, ion-exchange membrane chromatography, and zinc chloride, protamine sulfate, and ammonium sulfate precipitation (Nath et al., 2014). The β-galactosidase of A. niger is secreted into the extracellular medium, thus facilitating enzyme recovery. K. lactis can produce larger amounts of enzyme units than A. niger, which is an advantage. However, enzyme from K. lactis is intracellular, so the production costs associated with its extraction and subsequent processing are high. Two strategies can be applied to secrete β-galactosidase from K. lactis, using recombinant yeast strains with spontaneous lysis capacity or with heterologous secretion signal sequences for extracellular production. For this purpose, episomal plasmids were used to clone and express recombinant β-galactosidase from K. lactis (Oliveira et al., 2011).

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14.2.5

Sources of industrial β-galactosidase

Two types of β-galactosidases are currently available on the market: neutral and acid β-galactosidases. Neutral β-galactosidases are mainly used in the dairy industry for producing lactose-free products, although this type of enzyme is also available in some countries for consumers to treat milk at home. Acid β-galactosidases are available as a nutritional supplement to be consumed with regular dairy products (Dekker et al., 2019). Different companies market neutral β-galactosidases from K. lactis yeasts for lactose hydrolysis. The main manufacturers of these enzymes are the Japanese, who market their products through second suppliers such as Dupont (USA), Chr. Hansen (Denmark), and Novozymes (Denmark). Although these enzymes’ biochemistry is the same (lactose hydrolysis), they are sold at different potencies and purity grades, which may affect the final product differently. Table 14.3 shows a list of sources and suppliers of commercial β-galactosidases. Fungal enzymes from A. oryzae are mainly marketed under the trade names Lactase F and Tolerase, by Japanese and European companies, respectively, for Table 14.3 Sources and suppliers of commercial β-galactosidases. Sources

Microorganims

Trade name

Manufacturer/supplier

Yeast

Kluyveromyces lactis

Godo YNL2

Godo (Japan)/Dupont (USA) Amano Enzyme, Inc. (Japan)/Chr. Hansen (Denmark) Nagase (Japan)/ Novozymes (Denmark) DSM Food Specialties (Netherlands) Biocon Española, S.A. (Spain) Kerry (Ireland) DSM Food Specialties (Netherlands) Amano Enzyme, Inc. (Japan) Biocon Ltd. (Japan) Daiwa Fine Chemicals Co., Ltd (Japan) DSM Food Specialties (Netherlands) Chr. Hansen (Denmark) Novozymes (Denmark)

Halactase

Lactozyme Pure Maxilact Biolactase NL

Fungal

Aspergillus oryzae

Biolactase Tolerase Lactase F

Bacteria

Bacillus circulans

Lactoles L3 Biolacta FN5

Recombinant microorganisms

Self-cloned version of the regular K. lactis β-galactosidases β-galactosidase from Bifidobacterium bifidum expressed in Bacillus licheniformis host strain

Maxilact Smart Nola-Fit Saphera

14.2 Characteristics of β-galactosidases

producing GOS or as a nutritional enzyme. The different pH or temperature optima of these β-galactosidases make them less suitable for producing lactosefree products in most dairy matrices. Similarly, bacterial β-galactosidases from Bacillus circulans, supplied by Japanese companies, are used in the industry for their transglycosylase activity (Dekker et al., 2019; Dorau et al., 2021). Recently, the market has been supplying β-galactosidases from genetically modified hosts. Maxilact Smart, manufactured and supplied by DSM Food Specialties, is a self-cloned version of regular K. lactis β-galactosidases (Maxilact) that reduces hydrolysis time by 33% and requires 50% less dosage to achieve the same amount of product. A β-galactosidase from Bifidobacterium bifidum recombinantly produced in Bacillus licheniformis has also been introduced on the market under the trade names Nola Fit and Saphera, which allegedly have higher pH tolerance (Dekker et al., 2019; Dorau et al., 2021). However, the acceptance of these enzymes may be limited because, in several countries, the demand for genetic modification (GM)-free products has been gaining traction in the population. Several companies that manufacture GM-free products do not accept the Nola Fit and Saphera enzymes, though European legislation does not require GM labeling of the resulting products (Dorau et al., 2021).

14.2.6

Technologies for producing low-lactose milk

Two processes using soluble β-galactosidase are commonly used to industrially develop LLM: the batch process and the aseptic process. In reference to the sterilization time of milk, these are called prehydrolysis and posthydrolysis processes, respectively (Fig. 14.2) (Dekker et al., 2019; Dorau et al., 2021). In the batch process, raw or thermite milk is incubated with β-galactosidase for approximately 24 h at 4 C 8 C to prevent microbial growth. Subsequently, the milk is pasteurized, homogenized, packaged, and stored for a couple of weeks at 4 C. Some UHT milk producers also use this process; however, the aseptic process is more common for this type of milk. The advantage of the batch process is that no residual enzyme activity remains in the final product, as the enzyme is inactivated during pasteurization. However, the following should be noted: (a) Process control is quite long (between 24 and 30 h), as the enzyme does not work at its optimum temperature, so higher doses of the enzyme are often required. (b) Incubating milk in a tank for 1 day to hydrolyze lactose can create problems, especially in high-productivity factories. Production time can be shortened by using β-galactosidases with higher specific activity under these conditions (Dekker et al., 2019; Dekker & Daamen, 2011; Dorau et al., 2021).

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FIGURE 14.2 Schematic representation of the production of LLM using the aseptic or batch process. LLM, Low-lactose milk.

In the aseptic process, sterile β-galactosidase is added immediately after sterilization of the milk by UHT treatment, followed by packaging. Lactose hydrolysis takes place at room temperature during the quarantine period, usually required for UHT milk prior to shipment to the retailer. Thus enzyme dosage can be much lower than in the batch process, as both incubation time and temperature are higher. However, the following aspects must be considered: (a) there is no control of the process, since the enzyme is active in the final package of milk. The dairy producer must take storage temperature into account when dosing the enzyme, since temperature can vary from summer to winter in nonthermostatized warehouses.

14.2 Characteristics of β-galactosidases

(b) Sterile β-galactosidase can be obtained by two different procedures: (1) the enzyme is presterilized by the manufacturer, so the factory requires special dosing equipment for sterile injection; or (2) the nonsterile enzyme is sterilized only by filtration before being added to sterilized milk at the dairy factory. Therefore the aseptic process requires special equipment and highly trained operators to avoid microbial contamination of milk during enzyme injection. Nevertheless, the full process can run continuously when properly organized, which is a great advantage for factories requiring high throughput. (c) The enzyme remains active in the final product, which makes this process vulnerable to secondary activities caused by enzymatic activities, such as arylsulfatase activity. This can introduce off-flavors due to the formation of p-cresol from the sulfonated cresol naturally present in milk. Using high-quality β-galactosidase for this process avoids shelf life problems; and (d) Storing milk at high temperatures can cause off-flavors and browning of LLM compared to regular milk, reducing nutritional value. This is because lactose hydrolysis in milk increases the presence of monosaccharides that can trigger the Maillard reaction by proteases present in the milk or β-galactosidase preparation. Therefore storage temperatures and the choice of β-galactosidase affect the shelf life of LLM (Dekker et al., 2019; Dekker & Daamen, 2011; Dorau et al., 2021). Prior to the processes described above, raw milk is usually treated by chromatography or membrane filtration. This aims to remove a significant amount of lactose ( . 50%) and thus avoid doubling the sweetness of milk caused by lactose hydrolysis (Dekker et al., 2019; Dorau et al., 2021).

14.2.7

Future scope

The two main industrial applications of β-galactosidases are the “free enzyme” route using β-galactosidase in solution and the use of immobilized enzyme bioreactors. The free enzyme route is technically simpler but has the disadvantage that the soluble enzyme can hardly be reused. Enzyme immobilization is technically more complicated. However, it should be exploited for the advantages it offers: enzyme recycling, the possibility of continuous operation, and, in some cases, increased enzyme stability (Schulz & Rizvi, 2021). The methods of β-galactosidase immobilization, as well as their applications and the different bioreactor systems proposed, are discussed in the next section. Another important area under study includes the use of whole cells for β-galactosidase activity (Movahedpour et al., 2021). The main drawback is the low permeability of cell membranes to lactose, although this can be increased by treatment with chemical agents, such as detergents or solvents (Dorau et al., 2021).

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The use of recombinant expression systems is also vital for large-scale production of already identified enzymes and new enzymes with properties of interest, such as thermophilic and cold-active enzymes. In this area, recombinant production systems are crucial, as microbial sources of these enzymes are often not GRAS and can be tedious to grow and produce. In addition, recombinant systems are essential for generating enzyme-enhanced versions, such as those with higher substrate specificity, lower product inhibition, and higher transgalactosylation activity for GOS production (Movahedpour et al., 2021).

14.3

Immobilized β-galactosidases

The main industrial applications of β-galactosidases include food and beverage processing, pharmaceutical production, and wastewater treatment. Most of these biocatalysts are applied in their soluble form. This forbids their reuse (S. K. Sharma & Leblanc, 2017; Xavier et al., 2018) and raises the cost of industrial processes using β-galactosidases (Velasco-Lozano, 2020). In this context, an available strategy has been the immobilization of β-galactosidase. By definition, immobilized enzymes are biocatalysts that are physically confined or bound to supports, which can be reused and applied in continuous processes (Hackenhaar et al., 2021; Suresh et al., 2021). Immobilization can also improve enzymatic stability under different operating conditions, such as pH, temperature, and mineral salts. Immobilized β-galactosidases have greater applicability than their soluble counterparts. They can be used in bioaffinity chromatography, biosensors, and clinical diagnosis (Hanauer et al., 2021; Wolf et al., 2021). Considering that several factors can alter enzymatic activity and stability after immobilization, developing an immobilized biocatalyst with high operational performance poses several challenges. Fig. 14.3 shows the main methods for immobilizing β-galactosidases (Guerrero et al., 2017; Tizchang et al., 2021). Enzymes are attached to the support through physical adsorption, ionic bonds, and covalent bonds. They can also be trapped by encapsulation. Immobilization through adsorption and ionic bonds are the simplest methods, based on weak physical interactions. An advantage of these methods is that they maintain enzyme conformation. However, they require a strict control of process conditions during the use of the immobilized enzyme, to preserve the interactions between the protein and the immobilization matrix (Hackenhaar et al., 2021). The covalent bond method is the most widely used for immobilizing β-galactosidases, due to the greater stability of the bond formed. This technique’s main disadvantage is that the conformation of the enzyme’s active site may be altered, potentially compromising enzyme activity

14.3 Immobilized β-galactosidases

FIGURE 14.3 Schematic representation of the main methods used in enzyme immobilization.

(Gonz´alez-Delgado et al., 2018). In immobilization by encapsulation, enzymes are trapped within the internal structure of polymeric materials, thus forming gels, microcapsules, fibers, films, or membranes. Although encapsulation allows for easy separation, a drawback is the slow permeation of the substrate through the structure of the immobilization matrix (Aburto et al., 2018; Henriques et al., 2018; Ricardi et al., 2021, 2018). All immobilization methods have specific advantages and disadvantages, and their choice depends on the characteristics of β-galactosidase, the material used as support, and the form of application. The materials used for enzyme immobilization are called carrier matrices or supports. Below are matrix characteristics that determine the properties of the immobilized biocatalyst. Most support materials do not have all properties mentioned. Therefore, when selecting the immobilization matrix, one should consider the positive and negative characteristics associated with the process of immobilizing the enzyme of interest and its application (Henriques et al., 2018; Ricardi et al., 2021, 2018): (i) being cost-effective and sustainable, thus reducing the economic and environmental impact of the process;

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(ii) (iii) (iv) (v) (vi)

being inert after immobilization; possessing thermal and mechanical resistance; being able to immobilize high enzyme concentrations; avoiding adsorption of unwanted proteins; having chemical groups for the formation of bonds and interactions with the enzyme without inactivating it; (vii) having large surface area and high porosity. Based on their chemical composition, support matrices fall under two main categories: (1) inorganic support material and (2) organic support material. Organic support material is subdivided into natural and synthetic supports (Zahirinejad et al., 2021). Among the matrices for enzyme immobilization, biopolymers have the advantage of possessing “bio” properties, such as biofunctionality, biocompatibility, bioactivity, biostability, bioinertia, biodegradability, besides safety status (GRAS) (Molina et al., 2021; A. Sharma et al., 2021). In addition, using industrial waste as biomaterial for immobilization aligns with reuse strategies. This reduces environmental contamination and the costs of industrial processes by converting biopolymer waste into value-added products (Rafiee & Karder, 2020; A. Sharma et al., 2021; Xu et al., 2020). Table 14.4 presents each method of immobilization, the support used, and the main application for microbial β-galactosidases of different origins. Support features influence the reaction kinetics of immobilized β-galactosidases. When β-galactosidase is employed in its free form, hydrolysis reactions occur in a homogeneous mixture. In contrast, in its immobilized form, the diffusion of the enzyme may be compromised depending on the size and mass of the support. This problem can be circumvented through mechanical stirring, thus enabling the immobilized enzyme to reach maximum speed values close to or higher than those of the free form (Neto et al., 2021; Tizchang et al., 2021). The affinity of β-galactosidase to its substrate, lactose, or ortho-nitrophenyl β-D-galactopyranoside (ONPG) may also vary after immobilization. The enzyme typically binds to the substrate at random, thus hindering or preventing the substrate from binding to the active site of β-galactosidase. Solutions to this problem include chemical modifications to the support, modifications to the molecular structure of the enzyme, and immobilization processes in the presence of the substrate, to avoid reducing the affinity of catalysis (Henriques et al., 2018; Ricardi et al., 2021). Enzyme thermal stability is another relevant factor in immobilization processes. The industrial use of a thermally stable β-galactosidase can enable jointly running two unit operations, the enzymatic reaction of lactose hydrolysis, and a

14.3 Immobilized β-galactosidases

Table 14.4 Main features of immobilization studies employing different microbial β-galactosidases of distinct origins. β-galactosidase source

Support

Immobilization method

Bacillus circulans

Chitosan beads

Covalent bonds

Kluyveromyces lactis K. lactis

Polysaccharidebased hydrogels Pectin-based biohydrogels Agarose

Aspergillus oryzae Bifidobacterium bifidum Aspergillus aculeatus Aspergillus oryzae K. lactis K. lactis K. lactis A. oryzae

Halloysite nanotubes and cellulose Silica

Application

References

Encapsulation

Galactooligosaccharides synthesis Lactose hydrolysis

Covalent bonds

Lactose hydrolysis

Covalent bonds

Lactulose synthesis

Adsorption and entrapment

Lactose hydrolysis

Hackenhaar et al. (2021) Wolf, Tambourgi, and Paulino (2021) Hanauer et al. (2021) Guerrero et al. (2017) Tizchang et al. (2021)

Covalent bonds

Galactooligosaccharides synthesis Galactooligosaccharides synthesis Lactose hydrolysis and oligosaccharides synthesis Lactose hydrolysis lactulose synthesis Whey lactose hydrolysis

Calcium alginate gel Iron nanoparticles

Entrapment Ion exchange

Silica/chitosan Chitosan Calcium gelled gellan gum

Covalent bonds Covalent bonds Covalent bonds and entrapment

heat treatment of milk, for example. The enzyme’s bonds to the support can prevent conformational changes in the biocatalyst, which result in loss of activity. The ability to bond usually improves with the use of inorganic materials because their structure is more rigid. However, this effect can also be achieved with organic supports (Hackenhaar et al., 2021; Suresh et al., 2021). The mineral salts present in milk and dairy products may affect the β-galactosidase reaction; their effects may be enhanced or suppressed in the immobilized enzyme (Wahba, 2020). Therefore the process of immobilizing β-galactosidase should target improving the efficiency of milk lactose hydrolysis reactions. Small structural changes and greater rigidity of enzyme conformation after immobilization reduce inhibition, thus increasing the activity of immobilized β-galactosidase (de Albuquerque et al., 2021; Wahba, 2020). The main advantage of enzyme immobilization is the possibility of reusing the biocatalyst, both in batch processes and in continuous processes. The operational stability of β-galactosidase immobilized in hydrolysis or

´ Gonzalez-Delgado et al. (2018) Aburto et al. (2018) Henriques et al. (2018) Ricardi et al. (2018) Neto et al. (2021) Wahba (2020)

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galactotransylation reactions has shown great potential for application in industrial processes. This stability depends on the features of the support and the immobilization method. In addition to batch applications, immobilized β-galactosidases can be used in fixed- or fluidized-bed column reactors in continuous reaction modes(Gonz´alez-Delgado et al., 2018; Hanauer et al., 2021; Tizchang et al., 2021). Although immobilization offers benefits in relation to the use of β-galactosidase, several difficulties need to be overcome for the application of the immobilized biocatalyst in industrial processes (Basso & Serban, 2019).

14.4 Column reactors with immobilized β-galactosidases Developing processes that simulate industrial operating conditions is important for the diffusion of large-scale application of immobilized β-galactosidases. Therefore a key aspect of the study of immobilization is the sizing of enzymatic reactors. Fig. 14.4 illustrates ways of applying immobilized enzymes using stirred-tank and column reactors.

FIGURE 14.4 Models of enzymatic reactors: stirred-tank reactor operating in batch, stirred-tank reactor operating continuously, fixed-bed column reactor, and fluidized-bed column reactor.

14.4 Column reactors with immobilized β-galactosidases

Compared with the batch system, the continuous system offers greater efficiency in the control of reaction parameters, lower cost in the optimization of reaction conditions, and higher productivity. In addition, this form of operation prevents the reaction from being inhibited by accumulated metabolites. It also has fewer steps to product isolation and can be performed for extended periods of time (Wang et al., 2021). The fixed-bed (or packed-bed) reactor consists of two-phase systems in which the reaction mixture continuously passes through a stationary bed composed of the active material. These reactors have diverse industrial applications and can be used alone or in series (Gennari et al., 2019; Su et al., 2021). However, for use with immobilized enzymes, fluidized-bed column reactors stand out due to the possibility of variation in flow conditions, the nonformation of preferential pathways, and the better transfer of heat and mass intrinsic to the reaction (Eskandarloo & Abbaspourrad, 2018). The choice of fixed or fluidized bed depends mainly on the method of immobilization used. Because they are more resistant to desorption of the support surface, β-galactosidases immobilized by covalent bonds can be operated by both processes. Systems immobilized by interactions such as adsorption and electrostatic bonds tend to be less stable and so are preferably used in fixed-bed (de Albuquerque et al., 2021). Table 14.5 shows the support used, type of reactor, operating time, and the main application of immobilized β-galactosidases. The main limitation of using column reactors with β-galactosidases immobilized in large-scale bioprocesses is flow control, to keep the reactor under fluidization (Kirthiga et al., 2018).

Table 14.5 Main characteristics of column reactors with immobilized β-galactosidases. Support

Type of reactor

Glyoxyl agarose Cellulose Glass beads

Packed-bed Packed-bed Packed-bed

Macroporous amino resin Calcium alginate Duolite A568

Fluidizedbed Packed-bed Packed-bed

Application Synthesis of lactulose Lactose hydrolysis Production of galactooligosaccharides Production of galactooligosaccharides Lactose hydrolysis Lactose hydrolysis

Operating time 48 h 48 h 10 days

References

14 h

Guerrero et al. (2017) Gennari et al. (2019) Eskandarloo and Abbaspourrad (2018) Carevic´ et al. (2016)

60 days 30 days

Haider and Husain (2009) Fischer et al. (2013)

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14.5

Conclusions and perspectives

LLM and dairy products will be increasingly in demand due to the growing number of lactose-intolerant individuals. The production of LLM from enzymatic hydrolysis using β-galactosidases is a well-established technology. However, enhancing such production is necessary, considering the expansion of promising biotechnological possibilities.

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Low-lactose milk production using β-galactosidases

Neto, CACG, Silva, N. C. G., de Oliveira Costa, T., de Albuquerque, T. L., Gonçalves, L. R. B., Fernandez-Lafuente, R., & Rocha, M. V. P. (2021). The β-galactosidase immobilization protocol determines its performance as catalysts in the kinetically controlled synthesis of lactulose. International Journal of Biological Macromolecules, 176, 468 478. Available from https://doi. org/10.1016/j.ijbiomac.2021.02.078. Nivetha, A., & Mohanasrinivasan, V. (2017). Mini review on role of β-galactosidase in lactose intolerance. In IOP Conference Series: Materials Science and Engineering (Vol. 263, Issue 2). Institute of Physics Publishing. Available from https://doi.org/10.1088/1757-899X/263/2/022046. Oak, S. J., & Jha, R. (2019). The effects of probiotics in lactose intolerance: A systematic review. Critical Reviews in Food Science and Nutrition, 59(11), 1675 1683. Available from https://doi. org/10.1080/10408398.2018.1425977. Oliveira, C., Guimarães, P. M. R., & Domingues, L. (2011). Recombinant microbial systems for improved β-galactosidase production and biotechnological applications. Biotechnology Advances, 29(6), 600 609. Available from https://doi.org/10.1016/j.biotechadv.2011.03.008. Panesar, P. S., Panesar, R., Singh, R. S., Kennedy, J. F., & Kumar, H. (2006). Microbial production, immobilization and applications of β-D-galactosidase. Journal of Chemical Technology & Biotechnology, 81(4), 530 543. Available from https://doi.org/10.1002/JCTB.1453. Panesar, P. S., Kumari, S., & Panesar, R. (2010). Potential applications of immobilized β-galactosidase in food processing industries. Enzyme Research, 2010, 16. https://doi.org/ 10.4061/2010/473137. Rafiee, F., & Karder, F. R. (2020). Synthesis and characterization of magnetic glycocyaminemodified chitosan as a biosupport for the copper immobilization and its catalytic activity investigation. Reactive and Functional Polymers, 146, 104434. Available from https://doi.org/ 10.1016/j.reactfunctpolym.2019.104434. Ricardi, N. C., Arenas, L. T., Benvenutti, E. V., Hinrichs, R., Flores, E. E. E., Hertz, P. F., & Costa, T. M. H. (2021). High performance biocatalyst based on β-D-galactosidase immobilized on mesoporous silica/titania/chitosan material. Food Chemistry, 359. Available from https://doi. org/10.1016/j.foodchem.2021.129890. Ricardi, N. C., de Menezes, E. W., Valmir Benvenutti, E., da Natividade Schöffer, J., Hackenhaar, C. R., Hertz, P. F., & Costa, T. M. H. (2018). Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology. Food Chemistry, 246, 343 350. Available from https://doi.org/10.1016/j.foodchem.2017.11.026. Saqib, S., Akram, A., Halim, S. A., & Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. 3 Biotech, 7(1). Available from https://doi.org/10.1007/s13205-017-0645-5. Schulz, P., & Rizvi, S. S. H. (2021). Hydrolysis of lactose in milk: Current status and future products. Food Reviews International. Available from https://doi.org/10.1080/87559129.2021.1983590. Sharma, A., Thatai, K. S., Kuthiala, T., Singh, G., & Arya, S. K. (2021). Employment of polysaccharides in enzyme immobilization. Reactive and Functional Polymers, 167. Available from https://doi.org/10.1016/j.reactfunctpolym.2021.105005. Sharma, S. K., & Leblanc, R. M. (2017). Biosensors based on β-galactosidase enzyme: Recent advances and perspectives. Analytical Biochemistry, 535, 1 11. Available from https://doi.org/ 10.1016/j.ab.2017.07.019. Su, Z., Luo, J., Sigurdardóttir, S. B., Manferrari, T., Jankowska, K., & Pinelo, M. (2021). An enzymatic membrane reactor for oligodextran production: Effects of enzyme immobilization strategies on dextranase activity. Carbohydrate Polymers, 271, 118430. Available from https:// doi.org/10.1016/j.carbpol.2021.118430. Suresh, A., Shravan Ramgopal, D., Panchamoorthy Gopinath, K., Arun, J., SundarRajan, P., & Bhatnagar, A. (2021). Recent advancements in the synthesis of novel thermostable biocatalysts

References

and their applications in commercially important chemoenzymatic conversion processes. Bioresource Technology, 323. Available from https://doi.org/10.1016/j.biortech.2020.124558. Tizchang, S., Khiabani, M. S., Mokarram, R. R., Hamishehkar, H., Mohammadi, N. S., & Chisti, Y. (2021). Immobilization of β-galactosidase by halloysite-adsorption and entrapment in a cellulose nanocrystals matrix. Biochimica et Biophysica Acta - General Subjects, 1865(6). Available from https://doi.org/10.1016/j.bbagen.2021.129896. Ureta, M. M., Martins, G. N., Figueira, O., Pires, P. F., Castilho, P. C., & Gomez-Zavaglia, A. (2021). Recent advances in β-galactosidase and fructosyltransferase immobilization technology. Critical Reviews in Food Science and Nutrition, 61(16), 2659 2690. Available from https://doi.org/10.1080/10408398.2020.1783639. Vasileva, N., Ivanov, Y., Damyanova, S., Kostova, I., & Godjevargova, T. (2016). Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane. International Journal of Biological Macromolecules, 82, 339 346. Available from https:// doi.org/10.1016/j.ijbiomac.2015.11.025. Velasco-Lozano, S. (2020). Immobilization of enzymes as cross-linked enzyme aggregates: General strategy to obtain robust biocatalysts. Methods in Molecular Biology (2100, pp. 345 361). Humana Press Inc. Available from https://doi.org/10.1007/978-1-0716-0215-7_23. Vera, C., Guerrero, C., Aburto, C., Cordova, A., & Illanes, A. (2020). Conventional and nonconventional applications of β-galactosidases. Biochimica et Biophysica Acta - Proteins and Proteomics, 1868(1). Available from https://doi.org/10.1016/j.bbapap.2019.140271. Wahba, M. I. (2020). Mechanically stable egg white protein based immobilization carrier for β-Dgalactosidase: Thermodynamics and application in whey lactose hydrolysis. Reactive and Functional Polymers, 155. Available from https://doi.org/10.1016/j.reactfunctpolym.2020.104696. Wang, G., Wang, H., Chen, Y., Pei, X., Sun, W., Liu, L., Wang, F., Umar Yaqoob, M., Tao, W., Xiao, Z., Jin, Y., Yang, S-T, Lin, D., & Wang, M. (2021). Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae β-galactosidase, followed by purification using silica gel. Food Chemistry, 362, 130195. Available from https://doi.org/10.1016/j.foodchem.2021.130195. Wolf, M., Tambourgi, E. B., & Paulino, A. T. (2021). Stability of β-D-galactosidase immobilized in polysaccharide-based hydrogels. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 609. Available from https://doi.org/10.1016/j.colsurfa.2020.125679. Xavier, J. R., Ramana, K. V., & Sharma, R. K. (2018). β-galactosidase: Biotechnological applications in food processing. Journal of Food Biochemistry, 42(5). Available from https://doi.org/ 10.1111/jfbc.12564. Xu, J., Zhang, R., Han, Z., Wang, Z., Wang, F., Deng, L., & Nie, K. (2020). The highly-stable immobilization of enzymes on a waste mycelium carrier. Journal of Environmental Management, 271, 111032. Available from https://doi.org/10.1016/j.jenvman.2020.111032. Zahirinejad, S., Hemmati, R., Homaei, A., Dinari, A., Hosseinkhani, S., Mohammadi, S., & Vianello, F. (2021). Nano-organic supports for enzyme immobilization: Scopes and perspectives. Colloids and Surfaces B: Biointerfaces, 204, 111774. Available from https://doi.org/ 10.1016/j.colsurfb.2021.111774.

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Production of oligosaccharides, a prebiotic from lactose, using β-galactosidase Priscilla Romina De Gregorio1,2,3, Adriano Gennari1,2, Cathy Verônica Nied1, Giandra Volpato4 and Claucia Fernanda Volken de Souza1,2 1

Food Biotechnology Laboratory, University of Vale do Taquari - Univates, Lajeado, RS, Brazil, 2Biotechnology Graduate Program, University of Vale do Taquari - Univates, Lajeado, RS, Brazil, 3Reference Center for Lactobacilli - National Council for Scientific ´ Tucuman, ´ and Technological Research (CERELA-CONICET), San Miguel de Tucuman, Argentina, 4Federal Institute of Education, Science and Technology of Rio Grande do Sul, Campus Porto Alegre, Porto Alegre, RS, Brazil

15.1

Introduction

Oligosaccharides are polymers with 2 10 monosaccharide residues that have promising sensory characteristics for application in food, such as sweetening properties, as well as physiological effects on human health (Mano et al., 2018). These monosaccharide polymers can be classified as digestible or nondigestible (Patel & Goyal, 2012). In the nondigestible ones, the glycosidic bonds between the monosaccharide units (glucose, fructose, galactose, and xylose) are not susceptible to hydrolytic enzymes in the gastrointestinal tract because of their anomeric carbon atoms (C1 or C2). These oligosaccharides can be found in a free or bound form and can be obtained from natural sources or produced by physical, chemical, or enzymatic processes (Neri-Numa et al., 2016; Talens-Perales et al., 2015). Compared to chemical and physical methods, the use of enzymes for the production of oligosaccharides is a feasible, economical, and environment-friendly method (Panesar et al., 2018). β-Galactosidase is one of the most important industrial enzymes, which has been used for many decades in the dairy industry. The main application of β-galactosidase is related to the production of low-lactose dairy products, which are increasingly consumed and therefore commonly found on supermarket shelves (Dekker et al., 2019). Nowadays, the industrial use of β-galactosidase has been boosted, as this enzyme is able to catalyze the synthesis of oligosaccharide compounds, such as galactooligosaccharides (GOS), through the controlled kinetic reaction of lactose transgalactosylation. The transgalactosylation activity 383 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00015-1 © 2023 Elsevier Inc. All rights reserved.

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of β-galactosidase has a promising role in the synthesis of high value-added compounds from lactose, which have gained much attention in the dairy industry (Lu et al., 2020; Movahedpour et al., 2021; Vera et al., 2020). GOS can be defined as a carbohydrate-based functional food ingredient synthesized from lactose and composed of 2 8 or more monosaccharide residues; the terminal is glucose, while the remaining units involve galactose associated by glycosidic bonds (Panesar et al., 2018). GOS are nondigestible prebiotics that participate in the modulation of intestinal bacterial microbiota by favoring the growth of beneficial bacteria, such as Lactobacillus and Bifidobacterium species, which in turn produce short-chain fatty acids (SCFAs) as acetate, propionate, and butyrate, providing health benefits to the host (Mano et al., 2018). SCFAs can reduce luminal pH, which inhibits acid-sensitive enteropathogens. Acetate is involved in the butyrate formation, which is a primary substrate for colonic epithelial growth inducing the immunomodulatory cytokine production and promoting the excretion of ammonia and amines (Wilson & Whelan, 2017). Thus several biologically important applications have been attributed to GOS, such as their role in the immune system, protection against cancer, and prevention of cardiovascular and metabolic problems (Mano et al., 2018; Movahedpour et al., 2021; Saqib et al., 2017). In addition, GOS can bind to pathogens by preventing them from attaching to the host cell surface, thus acting as pathogen trap receptors and preventing their access into the gastrointestinal tract (Searle et al., 2010; Sinclair et al., 2009). Due to their beneficial effects, GOS are used as low-calorie sweeteners in fermented dairy products, confectionery, cereals, soft drinks, bread, beverages, and animal feed (Mano et al., 2018; Movahedpour et al., 2021; Vera et al., 2020). Therefore the growing global demand for the development of low-cost oligosaccharides and GOS is evident. The global market size for prebiotics exceeded USD 2.9 billion in 2015 and the commercial production of GOS is expected to generate a profit of USD 10.55 billion in the global market by 2025 (Markets, 2017). Different biotechnological processes are being studied for the development of new strategies for oligosaccharide synthesis. These include the use of whole cells in lactose biotransformation, the synthesis of microbial enzymes, especially β-galactosidases, and the development of enzymatic processes such as permeabilization, immobilization, and gene expression (Mano et al., 2018; Panesar et al., 2018). Additionally, other current trends have aimed to reduce production cost by using agro-industrial residues or by-products such as whey, molasses, corn cobs, wheat straw, sugar cane bagasse, and copra meal for the GOS production processes (Mano et al., 2018). In this context, the present chapter describes the applications of the enzyme β-galactosidase

15.2 Characteristics of β-galactosidases for the production of galactooligosaccharides

based on the transgalactosylation activity in the synthesis of high added-value oligosaccharides from lactose, as well as aspects that need further research for future large-scale applications.

15.2 Characteristics of β-galactosidases for the production of galactooligosaccharides 15.2.1

Sources of β-galactosidases

β-Galactosidases from a variety of microorganisms, including bacteria, archaea, yeasts, and fungi have reported GOS production in overall yields of 20% 50% (Lu et al., 2020; Mano et al., 2018; Panesar et al., 2018; Saqib et al., 2017). The most common microbial sources of β-galactosidase include the microorganisms Aspergillus oryzae, Aspergillus niger, Bacillus circulans, Kluyveromyces fragilis, and Kluyveromyces lactis (Panesar et al., 2018). The enzyme origin has a strong influence on the type of reaction catalyzed and on the amount and type of transgalactosyl product formed. Studies have revealed that β-galactosidases from A. oryzae and B. circulans presented a higher affinity to the transgalactosylation reaction, resulting in a maximum yield of GOS, while β-galactosidase from Kluyveromyces showed higher rates of hydrolytic activity. In contrast, β-galactosidases from A. oryzae fungus produced mainly trisaccharides with small amounts of tetrasaccharides, β-galactosidase from B. circulans bacteria produced mainly tetra- and pentasaccharides, and β-galactosidases from Kluyveromyces sp. yeasts synthesized tri- and tetrasaccharides (Panesar et al., 2018). Recombinant thermostable β-galactosidases have also been studied in the production of GOS, as the transgalactosylation reaction is favored by temperature, which increases the solubility of lactose and thereby increases the yield (Ansari & Satar, 2012). Site-directed mutagenesis has also been carried out to obtain mutant β-galactosidases that were subsequently expressed in Escherichia coli to increase the production of GOS. Although the use of E. coli is not permitted in the food industry, the process of mutagenesis and expression of mutants in generally recognized as safe microorganisms for the improved production of GOS may be a suitable strategy for large-scale use (Wu et al., 2013). Protein engineering has also been used as a newer procedure to improve enzyme stability and to achieve higher GOS yields. In this case, different techniques, such as the directed evolution method (DNA shuffling, error prone PCR), semirational method (CASTing), or rational method, can be

385

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carried out depending on the structure of the enzyme (Dalby, 2011; Hibbert & Dalby, 2005; Rico-Díaz et al., 2017; Yang et al., 2014).

15.2.2 Reactions catalyzed by β-galactosidases for the production of galactooligosaccharides GOS are produced by β-galactosidases using lactose as a substrate. The conversion of lactose to GOS by the enzyme results in mixtures containing GOS of different degrees of polymerization, unreacted lactose, and monomeric sugars (glucose and galactose) (Torres et al., 2010). In the first step, the enzyme binds with lactose by means of a covalent binding via galactosyl linkage, starting the catalysis of the reaction (Fig. 15.1). At this point, the reaction can follow different pathways depending on the degree of selectivity between the enzyme and the concentration of lactose, directly influencing the galactosyl acceptor identity. If the acceptor is a water molecule, a hydrolysis reaction will occur, generating a galactose-free molecule. But if the acceptor is another sugar molecule (glucose, lactose, galactose, or GOS), it acts as both donor and acceptor of the galactosyl moiety. This way, a new GOS is produced, where the degree of polymerization depends on the enzyme affinity to the acceptor (Mano et al., 2018; Vera et al., 2020).

CH2OH O OH OH OH OH

Lactose O

OH

CH2OH OH

OH

H 2O

O O OH

O-

O OH

OH

O OH

H

O

CH2OH

Glucose

CH2OH

O

OH

OH

O

+

O

H

O

Galactose

O-

OH

β-galactosidase

O

O-

β-galactosidase

CH2OH OH

O

OH

Galactose

OH

O O

β-galactosidase

CH2OH O OH CH2OH

CH2OH O H

O

+ O-

CH2OH

OH

CH2OH

OH

OH

O OH

OH

O O

OH

CH2OH

CH2OH

O O

OH OH

β-galactosidase

O O

O OH

O

Trisaccharide GOS - 3

OH

CH2OH

OH

GOS - 3

OH

OH

CH2OH

OH

OH

Lactose

FIGURE 15.1 Transgalactosylation reaction for the synthesis of galactooligosaccharides (GOS).

OH

OH

O O

OH

O O

OH

OH

O O

OH

OH OH

Tetrasaccharide GOS - 4

+

O

O

H

O

β-galactosidase

O-

15.2 Characteristics of β-galactosidases for the production of galactooligosaccharides

15.2.3 Optimal reaction conditions for the production of galactooligosaccharides The origin of the enzyme, the lactose concentration inherent in the process, the reaction time, and the temperature affect the variability, the GOS structure with respect to the saccharide composition, glycoside linkages, and the degree of polymerization (Gosling et al., 2010; Panesar et al., 2018; Saqib et al., 2017). At the beginning of the reaction, high lactose concentrations favor the transgalactosylation reaction, producing higher GOS yields (Mano et al., 2018; Muñiz-Marquez et al., 2016; Panesar et al., 2018; Vera et al., 2016). Subsequently, the hydrolytic reaction is favored and it decreases the yield of GOS. Thus reaction time is an important and critical factor; the reaction must be stopped at the point where the production of GOS is high (Torres et al., 2010). Another factor affecting reaction rate, enzyme stability, and lactose solubility is temperature. In general, the production of GOS increases with increasing temperature due to an increase in the reaction rate as well as lactose solubility. However, the increasing temperature can also increase the rate of enzyme inactivation (Vera et al., 2012). In contrast, the production of GOS is also affected by the use of unconventional media and reactor configuration (Vera et al., 2016). Low water activity conditions favor the transgalactosylation reaction, so two-phase systems, such as cyclohexane and water (95:5) or sodium dicotyl-sulfosuccinate/isooctane reverse micelles, have been used to increase the yields of GOS (Bednarski & Kulikowska, 2007; Chen et al., 2001; Shin & Yang, 1994). The aqueous twophase system can separate the desired product, enzyme, and inhibitors between the two phases of the system, increasing the GOS yield. However, although the production of GOS is increased with the use of nonconventional media, large-scale production of GOS using these systems can be hindered by the decreased reaction rate and the decreased solubility of lactose. In addition, organic solvents reduce the enzyme stability and have to be separated from the product, thus adding to the cost of production (Dong et al., 2015). The production of GOS is mostly carried out in the batch reactor, where, after a certain time, the enzyme is inactivated by heat to stop the reaction. In this system, inhibition of GOS production by glucose and galactose produced during the hydrolysis reaction is a limiting factor. With the use of a continuous reactor, product inhibition is less pronounced as glucose and galactose are concisely separated from the medium, thus increasing the yield of GOS (Das et al., 2011). Although continuous bioreactors are economically viable, reactor residence time, feed flow rate, and permeate collection rate are critical factors that must be considered during bioreactor design (Panesar et al., 2018).

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15.2.4

Production and purification of galactooligosaccharides

Different approaches such as whole and permeabilized cells, crude, purified, and engineered enzymes have been carried out for the production of GOS, as shown in Table 15.1. Whole cells have been used to carry out biochemical reactions for the production of GOS. The easy availability, the low cost required during production, and the stability of whole cells in the production of GOS offer advantages compared to purified enzymes, which also require purification steps. However, the use of whole cells presents the disadvantage of a slow reaction rate due to the permeability barrier of the cell envelope for substrate and product. To avoid these problems, permeabilization of the cells can be carried out (Chockchaisawasdee et al., 2005; Gobinath & Prapulla, 2015). Surface display technology for the production of GOS has also been reported as an alternative approach. For this, β-galactosidase from A. oryzae was immobilized on the surface of Yarrowia lipolytica, an erythritol-producing yeast. The technique was carried out by combining the β-galactosidase gene from A. oryzae to the YlPir1 gene encoding the cell wall protein of Y. lipolytica. This method is a promising approach, as it resulted in the production of about 160 g/L of GOS from 500 g/L of lactose at pH 5.5 and 60 C (An et al., 2016). Regarding the production of GOS using free β-galactosidase, the continuous production of GOS from an ultrafiltration membrane gives higher yields compared to the batch process under the same reaction conditions (Chockchaisawasdee et al., 2005). Ultrafiltration membrane reactors have also been applied jointly with a nanofiltration system using the commercial enzyme Lactozym Pure 6500. This proved to be a process with potential for industrial use as GOS of high purity and yield (80.1%) were obtained in a reaction time of 4 h (Ren et al., 2015). Likewise, a continuous process employing β-galactosidase from A. oryzae in an ultrafiltration membrane reactor for the production of GOS at a high initial lactose concentration of 470 g/L showed a 2.44-fold increase in productivity per unit mass of catalyst compared to the batch system. The stability of the enzyme, the large amount of substrate that can be processed, and the continuous removal of inhibiting products provide the better performance of this process compared to the conventional batch process. However, membrane fouling is one of the limitations of this process, which can be improved by optimizing the ratio of membrane area to the reaction volume (Córdova et al., 2016). Efficient, simple, and cost-effective techniques are applied for the separation of GOS from the mixture of residual sugars such as glucose, galactose, and nonreactive lactose, which are present in the reaction mixture as end

Table 15.1 Enzymatic methods for the production of galactooligosaccharides. Biocatalyst type

Source of β-galactosidase

Initial lactose (g/L)

Yield (g/ L)

GOS (%)

Whole cells

Lactobacillus plantarum

400

340

34

Kluyveromyces marxianus NCIM 3551 Pichia kluyveri Pseudozyma tsukubaensis Aspergillus aculeatus Aspergillus oryzae A. oryzae

300

-

36

400 400 400 400 400

56.04 62.96 135

14.01 15.71 24

A. oryzae A. oryzae Bacillus circulans

25.7 500 28.1

4.5 7.6

27 16.5

B. circulans Kluyveromyces lactis K. lactis K. lactis

400 400 300 2.1

119.8 102 7

41 29.9 33.4 15.2

K. lactis Sulfobus solfataricus Thermotoga naphthophila RKU10 Yarrowia lipolytica

400 -

100 -

51 61.7 59.22

500

160

SDS permeabilization Ethanol permeabilization Free enzymes

Engineered enzymes

References Gobinath and Prapulla (2015) Srivastava et al. (2015) Fai et al. (2014) Frenzel et al. (2015) Rodriguez-Colinas et al. (2014) Guerrero et al. (2015) Rodriguez-Colinas et al. (2014) Frenzel et al. (2015) Lisboa et al. (2012) Ren et al. (2015) Rodriguez-Colinas et al. (2014) Frenzel et al. (2015) Wu (2013) Yang et al. (2017) An et al. (2016)

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products (Panesar et al., 2018). GOS extraction and purification techniques studied include the following: Activated charcoal treatment: The GOS extraction from the sugar mixture has been carried out by eluting the oligosaccharide fractions adsorbed on the charcoal with ethanol. This method has proven to be simple, inexpensive, and fast for the purification of GOS. However, sufficient loss of trisaccharides and partial removal of disaccharides from the reaction mixture are major concerns when adopting this treatment method (Panesar et al., 2018). Size-exclusion chromatography: Different columns such as Bio-Gel P2, 26/60 Superdex 30 prep-grade, and Sephadex G-10 have allowed the separation of GOS according to molecular weight and using water or ethanol in Milli-Q water as eluents (Herna´ ndez et al., 2009; Panesar et al., 2018; Van Laere et al., 2000). This technique is capable of extracting GOS with a high degree of purity; however, the size-exclusion chromatography is generally expensive and time-consuming (Herna´ ndez et al., 2009). Nanofiltration: Separation of GOS from the reaction mixture into GOS-2, GOS-3, galactose, and glucose using different cellulose acetate membranes, such as CA77.5 and CA-80, has been achieved by this technique. Nanofiltration requires little energy and low installation and maintenance costs (Michelon et al., 2014). Because of its simplicity, this technique has gained a wider acceptance for the purification of oligosaccharides, but only in those cases where a higher degree of purity is not mandatory (Pruksasri et al., 2015). Simulated moving bed chromatography: Cation-exchange resins have been used to separate different oligosaccharide fractions by moving bed chromatography. The advantage of this technique is the continuous mode of production ˇ using water as eluent (Vankov´ a & Polakoviˇc, 2010). In addition, it also allows efficient separation of oligosaccharides in terms of productivity and purity (Mueller et al., 2021; Rajendran et al., 2009). Cation-exchange chromatography: Cationic resins have been extensively employed for the separation of carbohydrates such as oligosaccharides, hexoses, pentoses, and others (Sangwan & Tomar, 2011). A large number of low-cost molecules can be separated by this technique (Panesar et al., 2018). Supercritical fluid extraction: The continuous flow of carbon dioxide using ethanol and water as solvents has been employed to separate GOS from the sugar mixture using supercritical fluid technology. This technology has a potential application in the fractionation of GOS based on the degree of polymerization (Montañés et al., 2010). Microbial fermentation: The yeast Saccharomyces cerevisiae has been used to purify GOS allowing the complete removal of monosaccharides by converting them to

15.2 Characteristics of β-galactosidases for the production of galactooligosaccharides

ethanol and CO2 (Hern´andez et al., 2009). The purification of GOS with K. lactis and Lactobacillus helveticus has also been carried out, resulting in the reduction of monosaccharides and disaccharides (Sangwan et al., 2014). Unlike the other purification techniques, microbial fermentation produces a higher concentration of tri- and tetrasaccharides, but the disadvantage of this technique is the removal of cells and end products such as glycerol or trehalose from the sample at the end of fermentation (Ruiz-Matute et al., 2007). Furthermore, different analytical techniques have been employed to determine the yield and/or productivity of GOS in the reaction mixture. These include matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), MALDI coupled to time-of-flight MS (MALDI-TOF MS), high-performance liquid chromatography, nuclear magnetic resonance, electrospray ionization (ESI-MS), high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gas chromatography-MS (GC MS), thin-layer chromatography, capillary electrophoresis, and ultraviolet spectrophotometric method (Panesar et al., 2018).

15.2.5 Sources of industrial β-galactosidase and galactooligosaccharides Among the commercially available β-galactosidases, Lactozym 6500 L (Novozymes, Denmark) demonstrated the highest potential for GOS formation, followed by Biolactase NTL-CONC (Biocon Española, S.A., Spain) and Lactase F (Amano Enzyme, Inc., Japan). Lactozym is an active enzyme capable of completing the reaction in 1.5 h (Panesar et al., 2018). The incorporation of GOS in different food products such as yogurt, flavored milk, and others has been carried out by different markets. The main companies manufacturing GOS, in the form of powder and/or syrups, are Japanese, including Yakult Honsha (Tokyo), Snow Brand Milk Products (Tokyo), and Nissin Sugar Manufacturing Company (Tokyo). In addition, Friesland Foods Domo (formerly Borculo Domo ingredients) in the Netherlands and Clasado Ltd in the United Kingdom (UK) also stand out (Panesar et al., 2018). Also, several industries have emerged in the field of infant nutrition incorporating GOS as an ingredient into infant food; for example, Friesland Campina (manufacturer of Vivinal GOS and US-based Glycosyn) (Campina, 2016), Dairy Crest’s Functional Ingredient Business (manufacturer of GOS under the brand name “SureStart” in collaboration with Fonterra), and Nestle and Loblaw (manufacturer of Nestle Good Start 2) (Nestlé, 2017) and Omega 1 Infant Powder Formula (Omega, 2017). Moreover, GOS also have been manufactured for human and animal nutrition, such as the products under the brand name Promovita and Nutrabiotic, respectively (Ingredients, 2017). Other industries have also incorporated GOS in food supplements available as sachets or powders, for example,

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Clasado Biosciences in the United Kingdom (manufacturer of Bimuno powders containing GOS with lactose, glucose, and galactose) (Bimuno, 2017) and Klaire Labs in the United States (manufacturer of Galactomune, consisting of galactooligosaccharides and β-glucan) (Klaire, 2017). Table 15.2 shows the commercially available galactooligosaccharides, their trade name, manufacturer, form, and GOS concentration.

15.2.6

Future scope

GOS are normally synthesized by the enzyme β-galactosidase from bacteria, yeasts, and fungi using lactose as a substrate. The use of efficient catalysts for Table 15.2 Commercially available galactooligosaccharides Trade name

Manufacturer

Form

GOS concentration (%)

Bimuno

Clasado Biosciences Ltd, UK

BIOLIGO GL 5700 IMF Cup-Oligo H70 Cup-Oligo P Euoligo

Ingredion Inc., Korea Nissin Sugar Co. Ltd., Japan

Powder Syrup Syrup Liquid Powder -

. 80 . 57 57 70 70 -

Powder and Syrup Powder Syrup Syrup Powder Syrup Powder Syrup

100 57 55 55 90 59 69 72

Powder

29

Powder

29

GOS GOS GOS-270-P GOS-700-P GOS-800-P GOS-900-P GOS-1000-P GOS-570-S Oligomate 55 N Oligomate 55 P Profile GOS Promovita Purimune Vivinal GOS Vivinal GOS Easy drying syrup Vivinal GOS Powder Maltodextrin Vivinal GOS Powder WPC

Quantum Hi-Tech Biological Co. Ltd., China Lactose India Ltd., India Baoling Bao Biology, China New Franscisco Biotechnology Corporation, China

Yakult Honsha Co. Ltd., Japan Kerry Group, Ireland Dairy Crest limited, UK Corn Products International Inc., USA Friesland Campina Domo, Netherlands

15.3 Immobilized β-galactosidase for the production of galactooligosaccharides

the transgalactosylation reaction such as thermotolerant enzymes can increase the productivity of GOS. Also, the use of enzyme engineering and the development of efficient separation techniques for the recovery of GOS from other carbohydrates can further increase the yield of GOS, which improves the economics of the process and the utilization of GOS in the global market (Panesar et al., 2018). In contrast, enzyme immobilization for GOS production has gained great importance, as it has many advantages over free enzymes in terms of reusability and stability, thus affecting the economics of the process. Moreover, the immobilized enzyme facilitates the continuous mode of production due to the higher stability of the enzyme, which further improves the easy purification, the quantity, and the quality of the final product (Gonza´ lez-Cataño et al., 2017; Panesar et al., 2010).

15.3 Immobilized β-galactosidase for the production of galactooligosaccharides β-galactosidase immobilization has been advantageous in several applications, such as in the development of products with reduced levels of lactose, in improving the technological characteristics of dairy products, and in the production of GOS (Narisetty et al., 2022). In addition to their reusability potential and compared to their free form, immobilized enzymes may present better kinetic parameters and greater stability to the reaction conditions employed, such as pH and temperature (de Albuquerque et al., 2021; Hanauer et al., 2021). In general, higher concentrations of lactose and higher reaction temperatures provide higher yields of GOS (Urrutia et al., 2018; Wahba, 2017). Table 15.3 shows examples of immobilized microbial β-galactosidases used to produce GOS. The two main immobilization supports present high porosity and micrometric sizes, resulting in higher concentrations of immobilized enzymes and easy separation from the reaction medium, respectively. Within this category, the use of chitosan and sodium alginate spheres is highlighted, which also provide different methodologies for immobilization, such as cross-linking, covalent bonding, and adsorption (de Freitas et al., 2020; Todea et al., 2021). Microbial β-Galactosidases immobilized for the GOS production have been used in combination with ultrafiltration operations. Hackenhaar et al. (2021) and Wang et al. (2021) employed this technology to retain the immobilized enzyme, allowing the passage of substrate and products generated in the reaction. Thus the continuous removal of saccharides from the reaction medium and the addition of substrate enable the production of products on large scale. Ultrafiltration membranes also enable greater control

393

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Table 15.3 Microbial β-galactosidase immobilized on different supports for the synthesis of galactooligosaccharides. β-galactosidase Source

Immobilization method

Aspergillus aculeatus Bacillus circulans

Covalent bonding Covalent bonding

Aspergillus oryzae Kluyveromyces lactis K. lactis A. oryzae A. aculeatus

Covalent bonding Covalent bonding Covalent bonding Adsorption Covalent bonding

B. circulans

Covalent bonding

A. oryzae

Covalent bonding

Support

References

Glyoxyl-functionalized silica Aldehyde-activated agarose beads Chitosan Polysiloxane polyvinyl alcohol Chitosan Cotton cloth Chitosan-coated magnetic nanoparticles Glutaraldehyde-activated chitosan beads Glass beads

´ Gonzalez-Delgado et al. (2018) Rodriguez-Colinas et al. (2016) Urrutia et al. (2018) ´ Gonzalez-Cataño et al. (2017) De Freitas et al. (2020) Wang et al. (2021) Nguyen et al. (2019) Hackenhaar et al. (2021) Eskandarloo and Abbaspourrad (2018)

over the contact time of lactose with β-galactosidase, which is an important parameter since it affects the molecular structure, the yield, and the cost of GOS production (Wang et al., 2021). The synthesis of GOS using immobilized β-galactosidases can be done in different types of reactors, such as stirred tank reactors, packed bed reactors, and fluidized bed reactors, which allow continuous operation for long periods, in addition to batch and fed-batch modes (Panesar et al., 2018; Warmerdam et al., 2014). Due to the stability of immobilized β-galactosidases, GOS is mostly produced in the continuous mode of operation in fixed or fluidized bed column reactors, enabling purification and increasing the concentration of the final product (Guerrero et al., 2018). This process also favors the availability of the enzyme to the substrate, increasing the turnover number due to the constant removal of products and the simultaneous addition of lactose (Bezerra et al., 2020). Furthermore, β-galactosidase activity does not decrease due to inhibition by galactose, resulting in increased GOS productivity. β-galactosidases must be immobilized on macro- and micro-supports to apply the continuous mode of operation in fixed or fluidized bed column reactors, avoiding clogging and compaction problems (Yu & O’Sullivan, 2018). Even though packed bed reactors have a low cost and wide applicability, improvements are still needed for large-scale production of GOS using

References

immobilized β-galactosidases to become economically viable (Todea et al., 2021). Fluidized bed reactors are easy to operate and provide high mass transfer, presenting good results in continuous mode of operation for the production of GOS using immobilized β-galactosidases (Warmerdam et al., 2014). Both processes allow production on a large scale, operating continuously for long periods. In addition, they can be applied in series, from 2 to 5 reactors, maximizing GOS productivity (Chatre et al., 2021).

15.4

Conclusions and perspectives

The increasing consumption of prebiotic ingredients has led to the development of new products for application in the dairy industry. Among them, GOS produced from lactose using β-galactosidases stand out. There has been an increase in studies on transgalactosylation activity of these biocatalysts, especially in their immobilized form. To use these enzymes on an industrial scale, choosing the appropriate support and reactor is essential for maximizing the yield and productivity of the GOS production processes. It is necessary to improve studies by applying by-products and agro-industrial residues rich in lactose to establish adequate strategies to adapt the fermentation.

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CHAPTER 15:

Production of oligosaccharides, a prebiotic from lactose

Torres, D. P. M., Gonçalves, MDPF, Teixeira, J. A., & Rodrigues, L. R. (2010). Galacto-oligosaccharides: Production, properties, applications, and significance as prebiotics. Comprehensive Reviews in Food Science and Food Safety, 9(5), 438 454. Available from https://doi.org/ 10.1111/j.1541-4337.2010.00119.x. Tomar, S. K., Ali, B., Singh, R. R. B., Singh, A. K., & Mandal, S. (2014). Galactooligosaccharides purification using microbial fermentation and assessment of its prebiotic potential by in vitro method. International Journal of Current Microbiology and Applied Sciences, 3(4), 573 585. Urrutia, P., Bernal, C., Wilson, L., & Illanes, A. (2018). Use of chitosan heterofunctionality for enzyme immobilization: β-galactosidase immobilization for galacto-oligosaccharide synthesis. International Journal of Biological Macromolecules, 116, 182 193. Available from https:// doi.org/10.1016/j.ijbiomac.2018.04.112. Van Laere, K. M. J., Abee, T., Schols, H. A., Beldman, G., & Voragen, A. G. J. (2000). Characterization of a novel β-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides. Applied and Environmental Microbiology, 66(4), 1379 1384. Available from https://doi.org/10.1128/AEM.66.4.1379-1384.2000. ˇ Vankov´ a, K., & Polakoviˇc, M. (2010). Optimization of single-column chromatographic separation of fructooligosaccharides. Process Biochemistry, 45(8), 1325 1329. Available from https://doi.org/10.1016/j.procbio.2010.04.025. Vera, C., Córdova, A., Aburto, C., Guerrero, C., Su´arez, S., & Illanes, A. (2016). Synthesis and purification of galacto-oligosaccharides: state of the art. World Journal of Microbiology and Biotechnology, 32(12). Available from https://doi.org/10.1007/s11274-016-2159-4. Vera, C., Guerrero, C., Aburto, C., Cordova, A., & Illanes, A. (2020). Conventional and nonconventional applications of β-galactosidases. Biochimica et Biophysica Acta - Proteins and Proteomics, 1868(1). Available from https://doi.org/10.1016/j.bbapap.2019.140271. Vera, C., Guerrero, C., Conejeros, R., & Illanes, A. (2012). Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose. Enzyme and Microbial Technology, 50(3), 188 194. Available from https://doi. org/10.1016/j.enzmictec.2011.12.003. Wahba, M. I. (2017). Porous chitosan beads of superior mechanical properties for the covalent immobilization of enzymes. International Journal of Biological Macromolecules, 105, 894 904. Available from https://doi.org/10.1016/j.ijbiomac.2017.07.102. Wang, G., Wang, H., Chen, Y., Pei, X., Sun, W., Liu, L., Wang, F., Umar Yaqoob, M., Tao, W., Xiao, Z., Jin, Y., Yang, S. T., Lin, D., & Wang, M. (2021). Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae β-galactosidase, followed by purification using silica gel. Food Chemistry, 362, 130195. Available from https://doi.org/10.1016/j.foodchem.2021.130195. Warmerdam, A., Benjamins, E., De Leeuw, T. F., Broekhuis, T. A., Boom, R. M., & Janssen, A. E. M. (2014). Galacto-oligosaccharide production withimmobilized -galactosidase in a packed-bedreactor vs. free -galactosidase in a batch reactor. Food and Bioproducts Processing, 92 (4), 383 392. Available from https://doi.org/10.1016/j.fbp.2013.08.014. Wilson, B., & Whelan, K. (2017). Prebiotic inulin-type fructans and galacto-oligosaccharides: Definition, specificity, function, and application in gastrointestinal disorders. Journal of Gastroenterology and Hepatology, 32, 64 68. Available from https://doi.org/10.1111/jgh.13700. Wu, Y., Yuan, S., Chen, S., Wu, D., Chen, J., & Wu, J. (2013). Enhancing the production of galactooligosaccharides by mutagenesis of Sulfolobus solfataricus β-galactosidase. Food Chemistry, 138(23), 1588 1595. Available from https://doi.org/10.1016/j.foodchem.2012.11.052. Yang, H., Li, J., Shin, H. D., Du, G., Liu, L., & Chen, J. (2014). Molecular engineering of industrial enzymes: Recent advances and future prospects. Applied Microbiology and Biotechnology, 98 (1), 23 29. Available from https://doi.org/10.1007/s00253-013-5370-3.

References

Yang, J., Wang, Q., Zhou, Y., Li, J., Gao, R., & Guo, Z. (2017). Engineering T. naphthophila β-glucosidase for enhanced synthesis of galactooligosaccharides by site-directed mutagenesis. Biochemical Engineering Journal, 127, 1 8. Available from https://doi.org/10.1016/J. BEJ.2017.07.008. Yu, L., & O’Sullivan, D. J. (2018). Immobilization of whole cells of Lactococcus lactis containing high levels of a hyperthermostable β-galactosidase enzyme in chitosan beads for efficient galacto-oligosaccharide production. Journal of Dairy Science, 101(4), 2974 2983. Available from https://doi.org/10.3168/jds.2017-13770.

401

CHAPTER 16

Production of lactulose from cheese whey Azis Boing Sitanggang IPB University, Department of Food Science and Technology, Bogor, Indonesia

16.1

Introduction

The major by-product in the dairy industry is whey. Whey is formed during the production of cheese and casein when milk is coagulated. In the process of producing 1 2 kg of cheese using 10 L of milk, whey is produced approximately as much as 8 9 L. Annually, 160 million tons of whey are produced worldwide with a growth rate of around 2%. Whey has a high biological oxygen demand at about 30,000 50,000 ppm, and chemical oxygen demand at about 60,000 80,000 ppm. This can cause whey to pollute the environment (Kareb & Aïder, 2019; Karim & Aider, 2022a). About 95% of whey is made out of the water, while the remaining 5% comprises solids. On a dry basis, the solids in whey are composed of lactose as the main component at around 70% and 14% of whey proteins (such as α-lactalbumin and β-lactoglobulin, peptides, and free amino acids), while the rest comprises minerals and some residual fats (Kareb & Aïder, 2019; Karim & Aider, 2022a; Rocha & Guerra, 2020). According to the manufacturing process, whey can be divided into two types: (1) sweet whey, which is formed by rennet coagulation during the production process of hard and soft cheese, and (2) acid whey, which is formed by the acid coagulation of fresh milk in the production process of cheese and Greek yogurt. Acid whey has a higher concentration of minerals due to the dissolution of colloidal calcium phosphate of casein micelles during the acidification process. Sweet whey contains glycomacropeptide fraction produced by the enzymatic hydrolysis of κ-casein. The free amino acid content of sweet whey is higher than that of acid whey (Kareb & Aïder, 2019). Out of all the whey produced worldwide, only about 50% 70% is utilized, meanwhile, the remaining 30% 50% is discarded as effluent. Therefore the valorization of whey to produce substances with added value is important (Sitanggang et al., 2016b). At present, whey valorization is done in terms of whey protein isolate (WPI) or concentrate (WPC), or whey powder. WPC and WPI can be manufactured by using a Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00016-3 © 2023 Elsevier Inc. All rights reserved.

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membrane process (i.e., ultrafiltration), while whey powder is produced by spray drying the liquid whey. These forms of whey are currently used for various purposes in the food and beverage industries (Momen et al., 2022; Padilla et al., 2015). Lactose (4-O-β-galactopyranosyl-D-glucopyranose, C12H22O11) (see Fig. 16.1) is a disaccharide composed of D-glucose and D-galactose. Lactose has a relative sweetness of 0.16 (about 20% 30% of sucrose), lower than glucose and fructose (with a relative sweetness of 0.74 0.80 and 1.17 1.75, respectively) (Kareb & Aïder, 2019; Rocha & Guerra, 2020). Lactose also has low solubility compared to other disaccharides. With a concentration of approximately 4.8 g/100 mL, lactose is the main component of cheese whey. As a “conditional” prebiotic, gut microorganisms can utilize lactose as a carbon source by fermentation. The fermentation of lactose produces lactic and butyric acids that can lower the pH of the gut, creating a condition in which pathogenic bacteria cannot survive/reproduce, and therefore promoting gut health. The acidic condition of the intestinal tract can also enhance the absorption of minerals (e.g., calcium and magnesium). However, lactose intolerance hinders the usage of lactose in nutritional supplements (Kareb & Aïder, 2019). Lactose has numerous functional properties and is used in various food products. However, the food industry only uses a small fraction of the daily output of lactose because of its less preferred properties, such as low digestibility and a reduced solubility compared to sucrose. Therefore lactose generated by the industry is much higher than real needs (Rocha & Guerra, 2020). Prebiotics are substrates selectively utilized/fermented that can benefit the host’s health by inducing changes in the composition of or the intestinal microorganism itself. Prebiotics from carbohydrates are mostly known, such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, and

FIGURE 16.1 Structure of (A) lactose and (B) lactulose.

16.2 Lactulose production

FIGURE 16.2 Derivatives of lactose. Source: Modified from Nath, A., Verasztó, B., Basak, S., Koris, A., Kovács, Z., & Vatai, G. (2016). Synthesis of lactose-derived nutraceuticals from dairy waste whey—A review. Food and Bioprocess Technology, 9(1), 16 48. https://doi.org/10.1007/s11947-015-1572-2.

lactulose. These saccharides can pass through the digestive system intact and can be fermented by beneficial gut microflora such as Bifidobacteria and Lactobacilli (Kareb & Aïder, 2019). Lactose (in which whey utilization is considered) can be used to produce various prebiotics, nutraceuticals, and other fine chemicals such as lactobionic acid, lactulose, lactosucrose, tagatose, gluconic acid lactone, and galactooligosaccharides, using various methods, as shown in Fig. 16.2. Several approaches adopted for lactose utilization are microbial fermentation, reduction oxidation reaction, isomerization, hydrolysis, and transgalactosylation. Currently, prebiotic lactulose synthesis receives a higher interest and is being studied intensively due to its functional characteristics and is also part of whey or lactose valorization. Therefore upgrading whey or lactose is mainly discussed in terms of lactulose production within this work.

16.2

Lactulose production

Lactulose (4-O-β-D-galactopyranosyl-D-fructose, see Fig. 16.1) is a disaccharide composed of galactose and fructose bound by a 1-4β- glycosidic

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linkage (Kareb & Aïder, 2019). As an isomer of lactose, lactulose also has the empiric formula of C12H22O11. The molecular weight of lactulose is 342.30 g/mol. Lactulose in syrup form is an odorless yellow clear syrup, and in its powder form, lactulose is white-colored, odorless and has a sweet taste. Lactulose is soluble in water, has partial solubility in methanol, and is insoluble in ether. Lactulose is commercially available in the form of anhydride and trihydrate structure in the form of white crystalline and pure lactulose (Kumari et al., 2022; Nooshkam et al., 2018). The mammalian digestive system cannot hydrolyze lactulose, thus lactulose passes through the digestive system without undergoing degradation. Therefore lactulose acts as a prebiotic or bifidogenic agent (Kareb & Aïder, 2019). With a sweetness level lower than that of sucrose (0.48 0.62 times the sweetness of sucrose), lactulose is used in fermented milk products and other food products as a sweetener substituent and as a functional ingredient. Lactulose is also used as a laxative and growth promoter for pigs and calves (Kareb & Aïder, 2019; Rocha & Guerra, 2020). As a nutraceutical, lactulose is used in infant food intended to develop Bifidobacteria in the infant’s intestinal tract and to eliminate pathogenic bacteria (Rocha & Guerra, 2020). Lactulose may form as a by-product of the ultra high temperature or sterilization process of milk. The high temperature (.100 C) induces chemical reactions in milk, including the isomerization of lactose into lactulose. However, only a small amount of lactulose is detected in heated milk because the neutral pH (pH- 6.7) of milk is not optimum for the formation of lactulose. The isomerization process of lactose into lactulose is favorable in alkaline conditions (Kareb & Aïder, 2019; Schuster-Wolff-Bühring et al., 2010). Production of lactose is gaining more attention as shown in Fig. 16.3. It can be seen that there is an upward trend in the interest in lactulose synthesis. Production of lactulose can be done by isomerization of lactose or transglycosylation of lactose. Isomerization of lactose into lactulose can be done by chemical, electro-activation (EA), or enzyme method, while transglycosylation is done by using an enzyme as a biocatalyst (Nooshkam et al., 2018). Multiple approaches have been reported to optimize the operating or reacting conditions in order to improve the reaction selectivity and yield (Sitanggang et al., 2016b).

16.2.1

Isomerization-based lactulose synthesis

16.2.1.1 Chemical method The chemical synthesis of lactulose is mainly carried out by isomerizing the lactose in alkaline conditions. Under the alkaline conditions, lactose undergoes the Lobry de Bruyn-Alberda van Ekenstein (LA) rearrangement with the help of various catalysts where the glucose is converted to fructose (Panesar & Kumari, 2011) (see Fig. 16.4). In LA arrangement, protonation of carbon

16.2 Lactulose production

FIGURE 16.3 Number of publications about lactulose production from 1946 to 2022. Source: Data obtained from https://app.dimensions.ai/discover/ publication

FIGURE 16.4 Lobry de Bruyn-Alberda van Ekenstein (LA) rearrangement. Source: With permission from Sitanggang, A. B., Drews, A., & Kraume, M. (2016). Recent advances on prebiotic lactulose production. World Journal of Microbiology and Biotechnology, 32(9). https://doi.org/10.1007/s11274-016-2103-7.

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number 2 (C2) yields C1QC2. This leads to the degradation of the C1QO bond, thus leaving reactive O species. As a consequence, the neighbor H atom (aOH from C2) is attracted by the reactive O atom, leaving the O atom at C2 in a reactive state. Eventually, the fructose molecule is produced through the rearrangement of C1QC2 into C1aC2, giving a double bond between C2 and O (Sitanggang et al., 2016b). The catalysts commonly used in the chemical synthesis of lactulose can be divided into homogeneous, heterogeneous, and complex catalysts. Homogenous catalysts include Ca(OH)2, KOH, K2CO3, MgO, tertiary amines, and NaAlO2. Heterogeneous catalysts can be zeolites, sepiolites, eggshell powder, and oyster shell powder (Kareb & Aïder, 2019). Chemical synthesis of lactulose using homogenous catalysts yields a low level of lactulose. Therefore it needs a large amount of catalyst to increase the reaction yield. The product that resulted from the chemical synthesis method also needs to be separated and purified from the catalyst and any unwanted by-products formed during the reaction. This separation/purification process, alongside the large amount of catalyst needed for the process, increases the cost of production. Heterogeneous catalysts are used in place of homogenous catalysts to mitigate the disadvantages of using homogenous catalysts. The removal of heterogeneous catalysts can easily be done by centrifugation. The isomerization reaction using homogenous catalysts is carried out at 70 C 100 C. At this temperature, 20% 33% of lactulose is produced within a few hours. However, with the increase in the reaction time, there is a significant reduction in the concentration of lactulose due to the degradation of lactulose and lactose, forming by-products, such as epilactose, and glucose, galactose, and isosaccharinic acid. A brownish color also develops at a high temperature. The microwave heating method can be effective in reducing the reaction time (Kareb & Aïder, 2019; Kumari et al., 2022). The limitation of using heterogeneous catalysts is their low reaction yields, which can be overcome by the addition of complexing reagents (e.g., borates and aluminates) in the reacting media. These reagents can form stable complexes with lactulose in alkaline conditions, therefore increasing the yield by shifting the reaction equilibrium to the synthesis of lactulose. However, these complexing reagents have high toxicities even at a small amount. Therefore they are not suited for the production of lactulose that is food-grade, as their process of removal is quite complex (Karim & Aider, 2022b; Kumari et al., 2022). Another method of chemical synthesis of lactulose can be done via the Amadori rearrangement reaction by using amines or ammonia. In this reaction, the synthesized lactosylamine undergoes the Amadori rearrangement reaction to form the complex lactulosylamine which is subsequently hydrolyzed. However, this process is less commonly found in practice (Nath et al., 2016). Several studies on the chemical isomerization-based lactulose synthesis are presented in Table 16.1. The promising strategies that may be followed to have higher reaction yields are using catalyst sodium aluminate, microwave-

16.2 Lactulose production

Table 16.1 Production of lactulose through different isomerization and transgalactosylation approaches. Reacting conditions Chemical isomerization Cheese whey; [Na2CO3] 5 0.51%; T 5 90 C; t 5 20.41 min Sweet whey lactose 5 4.2%; sodium aluminate 3 M; T 5 70 C; t 5 1h Cheese whey; [(NH4)2CO3] 5 0.76%; T 5 97 C; t 5 28.44 min Microwave-assisted isomerization; lactose 5 40 mg/mL; Irradiated at 2450 MHz; 600 W, t 5 60 s Whey permeate 5 200 g/L; molar ratio boron-to-lactose 5 1:1; pH 5 12; T 5 70 C; t 5 30 min Sweet whey permeate; catalyst: reduced ruthenium supported on activated carbon (5% Ru/C) in continuous stirred-tank reactor; P 5 60 bar; 600 rpm; T 5 60 C Acid whey permeate; catalyst: reduced ruthenium supported on activated carbon (5% Ru/C) in continuous stirred-tank reactor; ; P 5 60 bar; 600 rpm; T 5 60 C EA-based isomerization Feed solution 5 7% (w/v); T 5 10 C; 400 mA; t 5 40 min Whey permeate 5 6% (w/w); 330 mA; KCl; t 5 21 min

Lactulose yield (%) or concentration (g/L)

29.6% 66%

Seo et al. (2015) Nahla and Musa (2015)

29.6%

Seo et al. (2016)

10%

Nooshkam and Madadlou (2016) Sabater et al. (2017)

155.5 g/L 23%

15.3%

35% 35.1%

Pure lactose 5 5%, (w/w); 330 mA; KCl; t 5 14 min

38.7%

Whey permeate; 330 mA; t 5 35 min

39.8%

Lactose 5 10%; 900 mA; t 5 40 min

38%

Whey solution 5 7% (w/v); 900 mA; t 5 60 min

32%

Enzyme-based isomerization Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus; Lactose 5 700 g/L; pH 5 7.5; [E] 5 150 u/mL Cellobiose 2-epimerase from Caldicellulosiruptor bescii (CsCE); pH 5 7.0; T 5 80  C; [CsCE] 5 7.5 U/mL CbCEP in stirred fermenters (5 L); cheese whey 5 50 g/L; T 5 70  C Recombinant cellobiose 2-epimerase in permeabilized Escherichia coli cells; batch operation; [E] 5 12.5 U/mL; substrate 5 600 g/L; borate addition 5 120 g/L Batch operation; [E] 5 3.0 mg/mL; Substrate 5 500 g/L Enzyme-based transgalactosylation

References

Enteshari and Martínez-Monteagudo (2020) Enteshari and Martínez-Monteagudo (2020) Kareb et al. (2016) Djouab and Aïder (2019) Djouab and Aïder (2019) Djouab and Aïder (2019) Karim and Aider (2020a) Karim and Aider (2020b)

58%

Kim and Oh (2012)

42.4%

Wu et al. (2017)

30% (12.8 g/L)

Jameson et al. (2021)

65.1%

M. Wang et al. (2015)

76.0%

Shen et al. (2016)

Continued

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Table 16.1 Production of lactulose through different isomerization and transgalactosylation approaches. Continued Reacting conditions β-galactosidase from Kluyveromyces lactis immobilized on silica gels; flow rate 5 0.5 mL/min; lactose 5 20% (w/v) β-galactosidase obtained from K. lactis; [E] 5 300 U; [Sugars] 5 500 g/L; pH 5 6.8; T 5 40 C; agitation speed 5 200 rpm; mL/mF 5 1/2 β-galactosidase obtained from K. lactis; agitation speed 5 350 rpm; pH 6.8; mL/mF 5 1/2; HRT 5 9h Aspergillus oryzae β-galactosidase with UFX membrane (MWCO 10 kDa), continuous EMR’ [E] 5 10 U/mL; mL/mF 5 1/4; HRT 5 9h; T 5 40  C β-galactosidase from K. lactis; T 5 35-37 C; t 5 180 min β-galactosidase obtained from K. lactis; lactose/fructose 5 15/30% (w/v); substrate 5 200 g/L; T 5 50  C; pH 5 7 Immobilized A. oryzae β-galactosidase in packed bed reactor (continuous); T 5 50  C; pH 5 4.5; 50% w/w total sugars; flow rate 5 15 mL/min; mL/mF 5 12, MB/MIM 5 1/8 g/g Enzeco fungal lactase; lactose 5 5.2 g/ 100 g; pH 5 6; enzyme 5 200 nkat Immobilized enzyme with chitosan (2% w/v) cross-linked with glutaraldehyde (0.8% v/v) as support; T 5 40  C; pH 5 7 Enzyme immobilized on Aga_PEI; T 5 50  C, substrate 5 200 g/L; pH 5 7, [E] 5 4 U/mL; 120 rpm

Lactulose yield (%) or concentration (g/L)

References

19.1 g/L

Song et al. (2013)

6.9% (16.7 g/L)

Sitanggang et al. (2014)

14.5 g/L

Sitanggang et al. (2015) Sitanggang et al. (2016a)

8.7 g/L

55.6% 22.6 g/L 60%

Zimmer et al. (2017) de Albuquerque et al. (2018) Guerrero et al. (2019)

27%

Schmidt et al. (2020)

26.7% (17.3 g/L)

de Freitas et al. (2020)

11 g/L

Neto et al. (2021)

assisted isomerization (after purified by a methanolic procedure), and utilization of boron. The reaction yields were 66% 78% (Nahla & Musa, 2015; Nooshkam & Madadlou, 2016; Sabater et al., 2017). In a batch reactor, there was a directly proportional relationship between lactulose formation and temperature and the period of reaction up to a certain time (Nahla & Musa, 2015). The maximum amounts of lactulose produced using 1 M sodium aluminate were obtained after 4 h, approximately 62.50% and 63.63% at 30 C and 70 C, respectively. After 4 h, a gradual decrease in lactulose percentage was observed. This degradation corresponded with the temperature and reaction period. This study also showed that there was a link between the formation of galactose and fructose and the reaction time and temperature. Seo et al. utilized sodium carbonate to isomerize lactose to lactulose. The optimum reaction conditions were obtained by using the response surface methodology with Box-Behnken Design: reaction time of 20.4 min; the temperature at 89.8 C, and Na2CO3 concentration at 0.51% (Seo et al., 2015). Under the feed-batch reactor system, 16 g/L of lactulose was obtained in the

16.2 Lactulose production

first hour, and the galactose yield was at 10%. This condition was better than the batch reactor system where the maximum lactulose production was 8 g/L and galactose yield of around 45%. Nooshkam and Madadlou investigated the isomerization of lactulose with microwave assistance. The yield is increased alongside the increase of the heating time. Microwave heating done for 60 s converted approximately 11% lactose into lactulose. Lactulose yield at 50 and 60 s showed no significant difference (Nooshkam & Madadlou, 2016). This may be caused by the decrease in pH of the isomerized product. The result of microwave-assisted lactulose synthesis that has been purified by a methanolic procedure was on par with the yield obtained by conventional isomerization methods which require longer durations.

16.2.1.2 Electro-activation-based isomerization The EA is an alternative approach to overcome the limitations of chemical isomerization-based lactulose synthesis. This method is based on applied electrochemistry and aims to modify the physicochemical properties and reactivity of a solution by the work of an external electric field inside an ion-exchange membrane-modulated reactor. The conversion of lactose into lactulose is through the electrolysis of water at the cathode solution interface within the EA reactor. In an EA system, an alkaline condition is achieved without the usage of catalysts or alkalinizing substances. The electrolysis reaction of water and the disposition of the ion-exchange membrane creates the alkaline condition that is required for the isomerization process (Kareb & Aïder, 2019; Karim & Aider, 2022a). The substrate used in this method can be in the form of pure lactose or whey. The isomerization of lactose happens according to the LA rearrangements. The EA process can be carried out in a milder condition than the chemical method, at a temperature of between 0 C and 30 C. Although the operating temperature is lower, the EA process has a comparably considered yield to the chemical method which is normally carried out in harsher conditions with a temperature between 70 C and 130 C and in the presence of strong bases at the pH of B11.0. This is due to the lower activation energy owned by the EA process than that of the chemical method. The basic principle of the EA process is reduction oxidation reactions that happen at the electrodes. In the electrolysis reactor, there are two electrodes placed in an electrolytic solution. The lactose solution is placed on the cathodic side, while the electrolyte solution is placed on the anodic and central side. The central part is connected by using cation- and anion-exchange membranes to other compartments and the reaction is performed on the cathodic side (Nooshkam et al., 2018). As indicated above, the reaction involved in the EA process is the isomerization of lactose into lactulose via the LA rearrangement. This process needs proton acceptors that is the hydroxyl ions at the cathode side, caused by the electrolysis of water under the effect of an electric field. By using the

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adequate reactor configuration and appropriate ion-exchange membranes, the control of the pH of the electrolyte solution is possible. On the anode side, an acidification reaction happens and on the cathode side, a basification reaction happens that leads to a pH change in the electrolyte solution (Karim & Aider, 2022a). The reaction that happens on the cathode side is the reduction reaction, whilst on the anode side is the oxidation reaction. The advantages the EA method over other chemical methods for lactulose synthesis are that it is clean, safe, eco-friendly, and energy-saving. EA method is a more selective process than the chemical process. As indicated previously, EA may decrease energy consumption (Aider & Gimenez-Vidal, 2012; Karim & Aider, 2022b). The Aider group from the Université Laval, Canada has been researching most of the EA-based lactulose synthesis (see Table 16.1). The reaction conversions reported are in the range of 32% 40%. Karim and Aïder (2022b) mentioned that lactulose synthesis using the EA method was more efficient than the chemical method at an equal processing condition. The lactulose yield for the EA method was approximately 37% while the chemical method only yielded a maximum of 25% lactulose using a 6% WP solution. Aider and Gimenez-Vidal (2012) reported that in the isomerization of lactose, there was a high significance on the lactulose yield of the electric field intensity applied in lactulose production, while the influence of lactose concentration was not significant to the lactulose yield. At the electric current density of 9.30 mA/cm2 (200 mA total current), a higher isomerization yield was achieved. The type of feed used in the isomerization reaction and the reaction time were significant factors in the isomerization process. At an electric current density of 4.65 mA/cm2 (100 mA), no lactulose was detected until 30 min of reaction. However, when the reaction was extended to 40 min, a yield of 4.21% was obtained. At a higher electric current density of 9.30 mA/cm2 (200 mA), lactulose was detected after 20 min. The final product of the EA process comprised lactulose besides the unreacted lactose. Galactose and fructose were found in some of the samples (not systemically) in negligible amounts, giving lactulose of 96.28%. In accordance with a previous study (Aider & Gimenez-Vidal, 2012), Kareb et al. (2016) showed that the current intensity was also an important parameter to achieving a pH increase up to 10 as well as to increasing lactulose yield. A rapid increase in pH at around 20 min of the EA process happened because of the generation of hydroxyl ions. The conversion process of lactose into lactulose was faster by using lactose solution as substrate compared to whey solution. This was caused by the buffering effect of the various compounds found in the whey solution (e.g., whey proteins, citrate, and phosphate). Lactulose was not detected when the pH was acidic in the case of using whey as the substrate. The formation of by-products is minimal at a lower temperature, with only a limited amount of galactose formed. The maximum yield (i.e., B35%) with a low galactose production was achieved

16.2 Lactulose production

at 10 C after 40 min (Kareb et al., 2016). It is worth noting that the gradual decrease of lactulose due to the degradation of lactulose into galactose was observed at a higher current than 400 mA due to the Joule heating effect.

16.2.1.3 Enzyme-based isomerization Enzymatic isomerization of lactose into lactulose is a process where the glucose molecule in lactose is changed directly into a fructose molecule using the biocatalyst cellobiose 2-epimerase. This enzyme is capable of isomerizing the glucose molecule in cellobiose (disaccharide) into a molecule of fructose or mannose. Enzymatic isomerization of lactose can form a by-product in the form of epilactose (Karim & Aider, 2022b). The mechanisms of enzyme-based isomerization in lactulose synthesis are as follows: (1) ring breakage of the lactose molecule into its open-chain form, the keto group at C1, (2) the formation of a double bond between C1 and C2 of the open-chain form of lactose, and (3) isomerization of a glucose molecule into fructose molecule facilitated by the enzyme cellobiose 2-epimerase (H. Wang et al., 2013). The first report on the utilization of cellobiose 2-epimerase for isomerizing lactose into lactulose could obtain a reaction yield of 58% (Kim & Oh, 2012). With this yield, approximately 400 g/L lactulose was obtained from the isomerization of 700 g/L lactose. As the by-product, epilactose was found at the level of 107 g/L. Similar to the chemical isomerization method, the addition of borate as a complexing agent may increase the reaction yield (Kim & Oh, 2012). Jameson et al. (2021) studied lactulose generation using cellobiose 2-epimerase enzyme obtained from the microorganisms Caldicellulosiruptor bescii and Roseburia faecis (CbCEP and RfCEP). Maximum activity of CbCEP was observed at pH 7.5 and temperature of 70 C, while RfCEP was at pH 8 and temperature of 50 C. A proportional increase in lactulose and epilactose production was observed with the increase in incubation time. At the incubation period of 48 h, CbCEP converted 51% of lactose into 29.8% lactulose and 21.6% epilactose. RfCEP converted lactose into 30 g/L epilactose (19.3% conversion) at 37 C and 48 h of incubation. According to the study done by Kim and Oh, using the enzyme obtained from Caldicellulosiruptor saccharolyticus, the conversion of lactose into lactulose and epilactose increased alongside the increasing enzyme concentration. The optimum conditions were pH 7.5, the temperature of 8 C, lactose of 700 g/L, and enzyme activity of 150 u/mL (Kim & Oh, 2012). M. Wang et al. (2015) investigated lactulose production using recombinant cellobiose 2-epimerase in permeabilized Escherichia coli cells. Free enzyme and the enzyme permeabilized in the cells had an optimum pH of 7.5 in 50 mM Tris HCl buffer and an optimum temperature of 80 C. With the increasing addition of the biocatalyst, lactulose production also increased with the maximum biocatalyst concentration at 12.5 U/mL. After this point, the productivity of lactulose decreased due to

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the steric hindrance of the cell mass. Longer reaction times at a higher temperature also generated by-products colored by nonenzymatic browning. The addition of borate could enhance the lactulose yield by up to 66% at a borate-lactose molar ratio of 1:1. This phenomenon was caused by the formation of the borate lactulose complex, avoiding lactulose degradation, thus allowing the continuation of lactulose production.

16.2.2

Transgalactosylation-based lactulose synthesis

Other than enzymatic isomerization, enzymatic synthesis of lactulose can be done via transgalactosylation or transglycosylation. Transgalactosylation toward lactulose uses two substrates, such as lactose or whey and fructose (see Fig. 16.5). Fructose is considered a galactosyl acceptor. First, lactose is hydrolyzed, then the galactosyl moiety and the enzyme interact to form a galactosyl enzyme complex. In the presence of fructose, the enzyme transfers the galactosyl moiety to fructose. Herein, the galactosyl moiety is linked to the primary C-4 hydroxyl group of the fructose by O-glycosidic linkage. In the case of water as the galactosyl acceptor, the reaction yields galactose; this is considered a primary hydrolysis reaction. Transgalactosylation of lactose into lactulose is performed using enzymes belonging to the

FIGURE 16.5 Lactose transgalactosylation with β-galactosidase as a catalyst. Source: With permission from Sitanggang, A. B., Drews, A., & Kraume, M. (2016). Recent advances on prebiotic lactulose production. World Journal of Microbiology and Biotechnology, 32(9). https://doi.org/ 10.1007/s11274-016-2103-7.

16.2 Lactulose production

glycosyltransferase and glycosidase class as catalysts (Karim & Aider, 2022b; Sitanggang et al., 2016b). Enzymes in the galactosidase class (e.g., β-galactosidase) are more relevant in the transgalactosylation process because these are relatively inexpensive, available commercially, and are widely applied in food industry. These can be obtained from various sources, plant/animalbased or microbial sources, although enzymes obtained from each source have different characteristics or effectivity. Enzymes that are obtained from microbial sources have more commercial value than the enzymes derived from animal or plant sources due to their higher multiplication rate and high yield (Kumari et al., 2022; Panesar & Kumari, 2011). The enzyme for transgalactosylation can be used either in native or immobilized form. de Freitas et al. (2020) reported lactulose production using β- galactosidase from Kluyveromyces lactis NRRL Y1564. Using both immobilized (on chitosan-glutaraldehyde) and free enzymes, 40% lactose conversion was achieved. Both the free and immobilized enzymes showed higher activity at pH around 6.5 and 7.0, at the temperature of 40 C (de Freitas et al., 2020). During transgalactosylation, as the synthesis is carried out under an aqueous environment, digalactose and GOS are also produced alongside lactulose (Nath et al., 2016; Sitanggang et al., 2015; H. Wang et al., 2013). Herein, tuning the reaction conditions to have a higher reaction selectivity toward lactulose remains a challenge for this approach. The drawbacks of lactulose synthesis via transgalactosylation are the low yields compared to other methods (5% 15%) and the lack of reaction selectivity (Karim & Aider, 2022b). Multiple reaction factors, such as fructose-to-lactose ratio, enzyme concentration, and reacting temperature, have to be considered (Sitanggang et al., 2015) (refer to Table 16.1). Additionally to this, lactulose synthesis through transgalactosylation is a kinetically controlled reaction. Therefore the reaction goes backward as the lactulose concentration peaks; the same biocatalyst hydrolyzes lactulose back to galactose and fructose. This condition is called secondary hydrolysis which is encountered in the production of lactulose batch-wise. In a batch system, after the highest specific yield of 5 mglactulose/Uenzyme was obtained, at 5 h reaction, the reaction had to be stopped to circumvent secondary hydrolysis (Sitanggang et al., 2014). The ratio of lactose to fructose (mL/mF) in the substrate affects transgalactosylation selectivity. At a lower mL/mF ratio, hydrolysis and thus the production of GOS were suppressed and lactulose yield was higher. At an mL/mF ratio of 0.5, the maximum yield of lactulose was 6.85% (16.70 g/L) compared to 2.18% (8.64 g/L) and 4.30% (14.10 6 0.23 g/L) yield at mL/mF ratio of 2 and 1, respectively (Sitanggang et al., 2014). Due to the limitation of batch-wise synthesis, transgalactosylation reaction is more preferred under continuous operation, where the application of an enzymatic membrane reactor (EMR) may be a potent alternative (Sitanggang et al., 2014, 2015, 2016a). The use of a size-exclusion membrane reactor facilitates

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concomitantly reaction and separation process. The produced lactulose can be withdrawn continuously, leading to a reduced possibility of experiencing secondary hydrolysis (Sitanggang et al., 2015). This approach can be considered a cost-efficient technology. However, one must consider the electrostatic interaction between the enzyme molecules and the membrane surface that may lead to membrane fouling, thus reducing the overall productivity (Sitanggang et al., 2016a). During the continuous reaction, a lower agitation (i.e., 250 rpm) might lead to a lower lactulose yield while higher agitation ( . 500 rpm) causes enzyme deactivation (i.e., breakage of the tertiary structure of the protein) (Sitanggang et al., 2015). In addition to this, the optimization of flux and/or residence time has to be done in order to minimize membrane fouling.

16.3

Separation of lactulose

The lactulose synthesis process typically generated various unwanted byproducts alongside lactulose, thus lowering the purity of the final product. The unwanted substances are lactose, glucose, galactose, epilactose, tagatose, and formic acid. These substances are formed because lactose and lactulose undergo degradation during the synthesis. This can be avoided by lowering the pH and temperature of the process (Panesar & Kumari, 2011; SchusterWolff-Bühring et al., 2010). In the case of chemical isomerization-based lactulose synthesis, the reaction mixture is processed to separate lactulose from lactose, allow the remaining catalysts to crystallize, and recycle unreacted lactose. The amount of purified lactose obtained is affected by physical and chemical treatments involved during the purification process (Panesar & Kumari, 2011). The addition of strong acid, circulation over base ionexclusion resin, or pressurized by mixing with CO2 gas can be used to lower the pH of the mixture. The alkaline ions can be precipitated by the addition of acid and salts. Strong acids and a low temperature are needed to release lactulose from the complex and precipitate borates and aluminates as boric acid and aluminum hydroxide. Further separation can be done by using twocolumn liquid chromatography, column-packed resins, or distillation using methanol (Schuster-Wolff-Bühring et al., 2010). A successful continuous synthesis of lactulose has been reported for more than 25 days using EMR equipped with an enzyme dosing feature (Sitanggang et al., 2016a). A combination of this reactor design and the use of cellobiose 2-epimerase-based lactose isomerization may obtain higher productivity and a relatively simple separation procedure. Following this, such industrial preparative chromatography is the only main unit operation to separate lactose and lactulose (Sitanggang et al., 2016b). The complete strategy of how to separate

16.4 Health benefits of lactulose

lactulose from the reacting mixture has been reviewed elsewhere (Karim & Aider, 2022b; Sitanggang et al., 2016b).

16.4

intensively

Health benefits of lactulose

Lactulose has been used to improve sensory characteristics and the browning features of food products. Along with these functional benefits, lactulose also has multiple health benefits. Lactulose can act as a prebiotic because lactulose is not digested by the human digestive system, thus passing through the stomach and the small intestine without undergoing degradation. In the colon, Bifidobacterium and lactic acid bacteria utilize lactulose by metabolizing it into lactic, formic, and acetic acids. Alongside the formation of those acids, carbon dioxide gas is also produced (Nooshkam et al., 2018). As a prebiotic, lactulose could enhance the absorption of minerals such as Ca, Mg, Zn, Cu, and Fe. This is, presumably, due to an increase in mineral solubility and the permeability of intestinal mucosa (Aït-Aissa & Aïder, 2014; SchusterWolff-Bühring et al., 2010). The formation of acidic substances with low molecular weights also acts as a laxative, softening the stool by changing the osmolarity in the colon and drawing water into the colon, and also lowering the transit time in the colon (Schuster-Wolff-Bühring et al., 2010). Table 16.2 shows several studies reported to demonstrate the health benefits of lactulose. Lactulose can be used as Salmonella carriers, to treat constipation and hepatic encephalopathy, as an antiendotoxin, and antidiabetic agent, to treat inflammatory bowel diseases, reduce the risk of colonic carcinoma, prevent tumor formation, and identify colonic disorders (Kumari et al., 2022). As Salmonella carriers, lactulose causes a drop in the pH of the colon because of the formation of short-chain fatty acids by gut microflora, causing a condition in which Salmonella can not survive. Salmonella then is excreted and removed from the body (Aït-Aissa & Aïder, 2014; Panesar & Kumari, 2011). Lactulose can prevent endotoxemia in obstructive jaundice due to its antiendotoxin characteristics (Aït-Aissa & Aïder, 2014). It can also prevent endotoxin-related complications in operations, and in metabolic diseases (e.g., diabetes mellitus, hypercholesterolemia) (Panesar & Kumari, 2011). The antiendotoxin characteristic of lactulose is connected to its ability to treat inflammatory bowel disease as endotoxins produced by bacteria are linked to the pathogenesis of inflammatory bowel disease. Oral ingestion of lactulose causes a drop in pH in the intestine, creating an environment where pathogens such as Salmonella and Shigella growth are inhibited (Aït-Aissa & Aïder, 2014; Panesar & Kumari, 2011). The microflora found in the colon produces metabolism products that can influence colon carcinogenesis. The biochemical process may happen in the area adjacent to the large intestine. Lactulose is prebiotic that can help lower the risk of colon carcinogenesis by

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Table 16.2 Studies related to the health benefits of lactulose. Health benefits

Experimental conditions

References

Prebiotic effect

8 healthy adults; 4 g/day 10 postmenopausal women; 10 g/day 32 patients with stages 3 and 4 of chronic kidney disease; 30 mm lactulose syrup, 3 times/day; 8 weeks 52 healthy females; 2 g/day, 2 weeks

Mizota et al. (2002) Venema et al. (2003) Tayebi-Khosroshahi et al. (2016) Sakai, Seki, Hamano, Ochi, Abe, Masuda, et al. (2019) Sakai, Seki, Hamano, Ochi, Abe, Shimizu, et al. (2019) Seki et al. (2007)

26 healthy females; 1-3 g/day, 2 weeks Enhancing mineral absorption

Purgative effect Hepatic encephalopathy (HE) treatment

24 healthy men; 2 g/day 12 41 12 20 30 35

postmenopausal women; 10 g/day postmenopausal women with osteopenia; 10 g/day, months healthy females; 14 g/day constipated elderlies; 20 g/day patients with minimal HE; 20-40 g/day

Data derived from various studies; 30-110 g/day 60 adult patients; 30-60 mL/day; 2 weeks 61 patients with minimal HE; 30-60 mL/day, 3 months

Van Den Heuvel et al. (1999) Blanch et al. (2013) Oku & Okazaki (1998) Lederle et al. (1990) Sharma et al. (2008)

Avery et al. (1972) Shavakhi et al. (2014) Prasad et al. (2007)

facilitating the growth of Bifidobacterium, which suppressed the precursor of colonic adenoma and the formation of tumors (Aït-Aissa & Aïder, 2014; Panesar & Kumari, 2011; Schuster-Wolff-Bühring et al., 2010).

16.5

Conclusion

Whey is a major waste generated by the dairy industry. Lactose makes up around 70% of whey powder. Applications of lactose in the food industry are limited due to various factors, that is, lactose intolerance. One alternative to whey valorization is lactulose synthesis from whey lactose. Lactulose synthesis can be done by isomerization or transgalactosylation. The methods of lactulose synthesis can be chemical, enzymatic, or EA. The chemical method uses alkaline catalysts and is performed at a high temperature. The EA method uses an electric field in place of catalysts and can be done at a milder temperature. The enzymatic method can be done by isomerization (using the enzyme cellobiose 2-epimerase) or transgalactosylation (using β-galactosidase or β-glycosylase). From the perspective of reaction yield and ease of separation, lactulose synthesis through cellobiose 2-epimerase-catalyzed isomerization in the EMR

References

is a promising approach. Following this strategy, such industrial preparative chromatography is the only main unit operation to separate lactose and lactulose. Alongside its applications in the food industry, lactulose also has various health benefits and studies have been done to demonstrate them.

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Seo, Y. H., Park, G. W., & Han, J. I. (2015). Efficient lactulose production from cheese whey using sodium carbonate. Food Chemistry, 173, 1167 1171. Available from https://doi.org/ 10.1016/j.foodchem.2014.10.109. Seo, Y. H., Sung, M., & Han, J. I. (2016). Lactulose production from cheese whey using recyclable catalyst ammonium carbonate. Food Chemistry, 197, 664 669. Available from https://doi. org/10.1016/j.foodchem.2015.10.078. Sharma, P., Sharma, B. C., Puri, V., & Sarin, S. K. (2008). An open-label randomized controlled trial of lactulose and probiotics in the treatment of minimal hepatic encephalopathy. European Journal of Gastroenterology and Hepatology, 20(6), 506 511. Available from https:// doi.org/10.1097/MEG.0b013e3282f3e6f5. Shavakhi, A., Hashemi, H., Tabesh, E., Derakhshan, Z., Farzamnia, S., Meshkinfar, S., Shavakhi, S., Minakari, M., & Gholamrezaei, A. (2014). Multistrain probiotic and lactulose in the treatment of minimal hepatic encephalopathy. Journal of Research in Medical Sciences, 19(8), 703 708. Available from http://jrms.mui.ac.ir/index.php/jrms/article/download/10015/4696. Shen, Q., Zhang, Y., Yang, R., Pan, S., Dong, J., Fan, Y., & Han, L. (2016). Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Food Chemistry, 207, 60 67. Available from https://doi.org/10.1016/j.foodchem.2016.02.067. Sitanggang, A. B., Drews, A., & Kraume, M. (2016). Development of a continuous membrane reactor process for enzyme-catalyzed lactulose synthesis. Biochemical Engineering Journal, 109, 65 80. Available from https://doi.org/10.1016/j.bej.2016.01.006. Sitanggang, A. B., Drews, A., & Kraume, M. (2015). Influences of operating conditions on continuous lactulose synthesis in an enzymatic membrane reactor system: A basis prior to long-term operation. Journal of Biotechnology, 203, 89 96. Available from https://doi.org/10.1016/j.jbiotec.2015.03.016. Sitanggang, A. B., Drews, A., & Kraume, M. (2014). Rapid transgalactosylation towards lactulose synthesis in a small-scale enzymatic membrane reactor (EMR). Chemical Engineering Transactions, 38, 19 24. Available from https://doi.org/10.3303/CET1438004. Sitanggang, A. B., Drews, A., & Kraume, M. (2016). Recent advances on prebiotic lactulose production. World Journal of Microbiology and Biotechnology, 32(9). Available from https://doi.org/ 10.1007/s11274-016-2103-7. Song, Y. S., Lee, H. U., Park, C., & Kim, S. W. (2013). Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase. Food Chemistry, 136(2), 689 694. Available from https://doi.org/10.1016/j.foodchem.2012.08.074. Tayebi-Khosroshahi, H., Habibzadeh, A., Niknafs, B., Ghotaslou, R., Yeganeh Sefidan, F., Ghojazadeh, M., Moghaddaszadeh, M., & Parkhide, S. (2016). The effect of lactulose supplementation on fecal microflora of patients with chronic kidney disease; a randomized clinical trial. Journal of Renal Injury Prevention, 5(3), 162 167. Available from https://doi.org/10.15171/jrip.2016.34. Van Den Heuvel, EGHM, Muijs, T., Van Dokkum, W., & Schaafsma, G. (1999). Lactulose stimulates calcium absorption in postmenopausal women. Journal of Bone and Mineral Research, 14 (7), 1211 1216. Available from https://doi.org/10.1359/jbmr.1999.14.7.1211. Venema, K., Van Nuenen, MHMC, Van Den Heuvel, E. G., Pool, W., & Van Der Vossen, JMBM (2003). The effect of lactulose on the composition of the intestinal microbiota and shortchain fatty acid production in human volunteers and a computer-controlled model of the proximal large intestine. Microbial Ecology in Health and Disease, 15(2-3), 94 105. Available from https://doi.org/10.1080/08910600310019895. Wang, H., Yang, R., Hua, X., Zhao, W., & Zhang, W. (2013). Enzymatic production of lactulose and 1-lactulose: Current state and perspectives. Applied Microbiology and Biotechnology, 97(14), 6167 6180. Available from https://doi.org/10.1007/s00253-013-4998-3. Wang, M., Yang, R., Hua, X., Shen, Q., Zhang, W., & Zhao, W. (2015). Lactulose production from lactose by recombinant cellobiose 2-epimerase in permeabilised Escherichia coli cells.

References

International Journal of Food Science and Technology, 50(7), 1625 1631. Available from https:// doi.org/10.1111/ijfs.12776. Wu, L., Xu, C., Li, S., Liang, J., Xu, H., & Xu, Z. (2017). Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor. Bioresource Technology, 233, 305 312. Available from https://doi.org/10.1016/j.biortech.2017.02.089. Zimmer, F. C., Souza, A. H. P., Silveira, A. F. C., Santos, M. R., Matsushita, M., Souza, N. E., & Rodrigues, A. C. (2017). Application of factorial design for optimization of the synthesis of lactulose obtained from whey permeate. Journal of the Brazilian Chemical Society, 28(12), 2326 2333. Available from https://doi.org/10.21577/0103-5053.20170083.

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Determination of lactose in milk and milkderived ingredients using biosensor-based techniques Caleb Wagner1,2, Richa Singh3 and Jayendra K. Amamcharla1 1

Department of Animal Sciences and Industry, Food Science Institute, Kansas State University, Manhattan, KS, United States, 2School of Food Science, Washington State University, Pullman, WA, United States, 3Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

17.1

Introduction

Lactose is the principal carbohydrate found in mammalian milk, composing 4.5% 4.8% of cow or caprine milk (Huber & BeMiller, 2017). The amount of lactose in milk is directly proportional to the fat, casein, and ash content of the milk regardless of animal species (Fox, 2009). The factors that impact the relative proportions of these components in milk include the animal’s environment, lactation cycle timing (Fox, 2009), and overall health (Booth et al., 2017). The disaccharide lactose consists of one D -glucose and one D -galactose unit each in a 6-carbon cyclic hemiacetal ring conformation connected by a β-1,4 glycoside bond as shown in Fig. 17.1(Durham, 2009). While lactose is found primarily in free disaccharide form, small amounts naturally polymerized into higher oligosaccharides (Huber & BeMiller, 2017).

FIGURE 17.1 Chemical structure of ß-lactose. Source: From wiki commons (public domain).

427 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00017-5 © 2023 Elsevier Inc. All rights reserved.

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17.2

Importance of lactose in milk and dairy ingredients

Lactose is especially significant because of its economic importance as well as its contributions to the sensory and functional qualities of dairy products (Lynch et al., 2007). In low-moisture foods, lactose crystallization causes grittiness. The crystallization to α-lactose monohydrate sequesters moisture, causing casein micelles to become unstable. Lactose determination is crucial in cheese whey fermentation process (Abboud et al., 2010; Jeon et al., 1984) as well as in membrane separation of lactose from cheese whey (Cuartas-Uribe et al., 2009). Milk products are diverse in terms of their intended applications, sources, compositions, and manufacturing styles. The presence of lactose can have a significant impact on the overall quality, value, and utility of dairy ingredients. While dairy ingredients can be used in their liquid form when convenient and economical, they are more often used in powdered form since that allows more convenient storage and improves shelf life qualities (Schuck, 2009). Some of the most common ingredients include skim milk powder, sweet whey, whey protein concentrate or isolate, milk protein concentrate (MPC) or isolate, caseinates, and whey or milk permeate. Lactose is present in most dairy ingredients, comprising ,2% of dry protein isolates to 100% of pure lactose powders (Huppertz & Gazi, 2016). The amount of lactose in an ingredient is generally inversely proportional to the ingredient’s protein content. Fig. 17.2 conveys how dairy ingredients can be compositionally diverse. As a result, the availability of a rapid and low-cost method for determining lactose has implications for the dairy business (Iwasaki et al., 2008). The routine analysis of lactose in dairy ingredients could lead to improved utilization of dairy ingredients, as well as help to better understand lactose’s role in ingredient quality. For example, measuring the lactose content of permeate and retentate streams can be a process optimization benchmark during the membrane concentration of dairy proteins (Olabi et al., 2015; Sluková et al., 2016). Also, incorporation of the correct amount of starting lactose is important for whey solids fermentation optimization (Sansonetti et al., 2010). Lastly, quality defects such as stickiness, caking, or excessive browning in dairy powders are largely dependent on the amount and form of lactose present in a dried ingredient (Gulzar & Jacquier, 2018; Olabi et al., 2015).

17.3

Lactose quantification methods

Many tests exist to measure lactose in dairy products. Table 17.1 provides a nonexhaustive list of some documented lactose quantification methods and the objective shortcomings of each.

17.3 Lactose quantification methods

FIGURE 17.2 Composition of some common dairy ingredients. Source: Prepared from Wisconsin Center for Dairy Research, Dried Dairy Ingredients, 1st Ed. May 15, 2008.

Currently available methods for determination of lactose have limitations, including long analysis time requirements, the need for expensive instrumentation, and skilled technicians. Consequently, the dairy industry is always in need of a rapid and inexpensive method that can be performed with minimal training of the analyst. Sharma et al. (2002) developed a quick, simple, and economical biostripbased method that has shown potential for estimating lactose in milk and milk products. The biostrips are prepared by immobilizing β-galactosidase, galactose oxidase, and horseradish peroxidase on Whatman filter paper. Estimated concentration of lactose is correlated with different color changes observed on the strips: Blue (20 g/L), yellowish-brown (40 g/L), light brown (60 g/L), brown (80 g/L), and dark brown (100 g/L). The strip did not show any color change when the concentration of lactose was less than 20 g/L. While this is promising, the quantification of lactose by visual color change of a strip is likely to be inconsistent, and accuracy of the quantification will always questionable. Another rapid method was developed by CDR Lab for determination of lactose content in lactose-free dairy products within 10 min using CDR FoodLab

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Table 17.1 Lactose quantification methods for dairy foods. Reference(s)

Method identification

Objective shortcomings

AOAC 896.01

Polarimetry

Cheng and Christian (1977) Cheng and Christian (1977)

Idda et al. (2016)

Redox titration Phenol-sulfuric acid colorimetric assay Gravimetric precipitation of copper ions Gas chromatography (GC)

Interference with other optically active components, nonspecific, empirical calculation Cumbersome, error-prone Nonspecific detection and reaction chemistry

Gabbanini et al. (2010); Upreti et al. (2006), AOAC 984.22 Lynch et al. (2007)

High-performance liquid chromatography (HPLC) Calculation by difference

Lynch et al. (2007), AOAC 972.16 Lynch et al. (2007), AOAC 2006.06

Infrared spectroscopy

Jhonson (1987)

Enzymatic spectrophotometric absorbance Raman spectroscopy

Silveira et al. (2016) Hu et al. (2007) Jager et al. (2007)

Nuclear magnetic resonance (NMR) Electrophoresis

Luzzana et al. (2003), Gille et al. (2018)

Differential pH (Microlab EFA)

Tedious sample preparation, interference from all reducing carbohydrates Expensive equipment, sample derivatization necessary, trained personnel needed Expensive equipment, tedious sample preparation, trained personnel needed Mistakes other compounds for lactose, relies on the accuracy of multiple other tests Expensive equipment, nonspecific, tedious and inflexible calibration required Expensive equipment, tedious sample preparation, and trained personnel are needed Expensive equipment, nonspecific, tedious and inflexible calibration required Expensive equipment and trained personnel are needed Expensive equipment and trained personnel are needed Requires diluted system to have a very specific buffering capacity

analyzer. This analyzer is based on the conversion of lactose into glucose and galactose. Through an enzymatic reaction with peroxidase, glucose combines with a phenolic molecule to generate a pink-colored complex, the absorbance of which is measured at 505 nm. The absorbance value is proportional to the amount of lactose in the sample. This principle is similar to commercial enzyme kits for lactose determination, which are very time-consuming and require analysts with trained laboratory skills; The CDR FoodLab analyzer cuvettes are prefilled with required reagents, and therefore the method is much simpler to perform with less chances for error in measurements to be made. Biosensors are another rapid measurement option, and arguably the most promising. Biosensor-based detection methods are very specific, simple, relatively low-cost, and possibly portable (Mello & Kubota, 2002; Sharma & Leblanc, 2017), making them an attractive alternative to currently available

17.4 Biosensors

methods. The working principles of biosensors and some examples relevant to the dairy industry are reviewed in the next section.

17.4

Biosensors

Biosensors principally consist of a biological recognition element coupled to a transducer. In the presence of a target analyte, the biological recognition element creates a response and the transducer “translates” that response into an analytical signal that can be measured conveniently. Biologically produced signals and the transducer type that characterizes the name of common biosensors are summarized in Table 17.2 (Mello & Kubota, 2002).

17.4.1

Biosensors used in dairy foods

Biosensors are available for measuring many analytes of interest in milk, including hormones, antibiotics, pathogens, contaminants, and lactose. However, while there are many examples of these biosensors in the literature, there are disproportionately few commercial examples. This has been attributed to slow transfer of information from academia to industry, inability to reproducibly prepare biosensor surfaces, and matrix interferences of milk. The milk matrix itself seems to be the most major issue hindering the development of commercial biosensors for dairy products, as most commercially viable sensors would be based around electrochemical analyte quantification principles that are prone to interference from the many electroactive species in milk (Booth et al., 2017).

17.4.2

Biosensors used for lactose quantification

The most promising sensors for commercial lactose quantification have been based on enzymes in conjunction with amperometric transducers, reflecting general biosensor trends. Several examples of sensors with enzymatic recognition elements based around β-galactosidase in conjunction with other Table 17.2 Common biological signals and corresponding transducer types found in biosensors. Biologically produced signal

Typical transducer required

Electron tunneling, ion mobility, creation of a current, diffusion of electroactive or charged particles Temperature change Absorption or emission of electromagnetic radiation Mass and/or microviscosity alterations of wave propagation

Electrochemical (amperometric, potentiometric, etc.) Thermal Optical Piezoelectric

Adapted with permission from Mello, L. D., & Kubota, L. T. (2002). Review of the use of biosensors as analytical tools in the food and drink industries. Food Chemistry, 77(2), 237 256. https://doi.org/10.1016/S0308-8146(02)00104-8.

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enzymes have been used to quantify lactose in milk, blood, and wastewater. However, as of 2017, it was found to be difficult to commercially immobilize the enzyme cocktails in these types of sensors to the transducer (Sharma & Leblanc, 2017). Another promising group of sensors are those based around a cellobiose dehydrogenase (CDH) enzymatic recognition element, which have successfully been applied to measure lactose in milk, yogurt, and cheese plant-waste water streams (Glithero et al., 2013; Lopez et al., 2017; Safina et al., 2010; Yakovleva et al., 2012). However, the CDH enzyme is not commercialized yet and CDH with the greatest commercial viability has shown some activity toward glucose and other sugars when complex sensor manufacturing strategies are not employed (Lopez et al., 2017). Despite setbacks reported in the literature, there are some proprietary commercial biosensors available to quantify lactose, including the Lactosens (DirectSens GMBH, marketed by Chr. Hansen in the United States), BioMilk 300 (Biolan, marketed by DSM), and YSI biochemistry analyzer (Xylem inc.). Both the BioMilk 300 and Lactosens are claiming as being third party validated against a reference method. The BioMilk 300 was documented as being an excellent tool for measuring low (,0.3%) lactose levels in lactose-free milks. Accuracy was found to be at par with HPLC-PAD (pulsed amperometric detector) (Churakova et al., 2019). However, the BioMilk 300 was unable to reliably quantify milk samples with high lactose concentration (4.65%, w/w). The BioMilk 300 mechanism of action is unknown, although it is likely to involve a sequence of successive redox processes in the presence of lactose that results in the release of electrons at a rate proportional to the lactose concentration. The inability of BioMilk 300 to quantify high lactose concentration is most likely due to the change in the sample matrix upon dilution. The BioMilk 300 is developed to determine the low lactose concentration (,1%) in dairy matrices and three different variants are available for measuring ranges of 0.005% 0.02 %, 0.02% 0.2 %, or 0.05% 0.6%. The reliability of measuring lactose even in low-lactose samples using the BioMilk 300 is questionable as high RSD (24%) was observed and more replicate measurements are required for arriving at accurate values. The YSI biochemistry analyzer (Xylem Inc.) is designed for determining lactose concentration in complex matrices like cheese after calibration with known lactose standard solutions. It is claimed that the equipment is able to accurately quantify lactose concentrations ranging between 1% and 10%. In the YSI biosensor, the lactose from samples diffuse into the galactose oxidase containing membrane, where it is converted to hydrogen peroxide and a galactose dialdehyde derivative. The hydrogen peroxide is detected on a platinum electrode surface and an amperometric signal is generated. The amount

17.5 Blood glucose meter biosensors as an option for determination of lactose

of current flowing through the electrode is proportional to the amount of hydrogen peroxide present, and hence to the amount of lactose present. (Churakova et al., 2019) also evaluated the YSI biosensor 2700 for measurement of lactose in milk and low lactose milk. The Biosensor was able to detect normal concentration of lactose (B4.80%, w/w) in milk with good precision (RSD 0.9%). However, the biosensor was not found suitable for quantifying low-lactose levels in milk, presumably because any side reaction that produces even a trace amount of hydrogen peroxide will result in a false positive signal for lactose at low analyte concentrations Lactosens was validated with HPLC-PAD method (Halbmayr-Jech et al., 2020) as per Standard Method Performance Requirements (SMPRs) for lactose in lowlactose or lactose-free milk, milk products, and products containing dairy ingredients (AOAC SMPR 2018.009). The method’s performance was within acceptable limitations as defined in SMPR 2018.009, allowing it to be adopted as a First Action Official Method with only a few restrictions. The Lactosens determines lactose in lactose-free or low-lactose milk, dairy, and infant formula products produced with yeast-neutral lactases. In the Lactosens, lactose in a given test product is oxidized by an enzyme (proprietary) immobilized on a disposable test strip, and the resultant electrons are amperometrically detected by the Lactosens Reader. According to the factory-set calibration function, proprietary software translates the electrical signal into a lactose concentration. For quality assurance, each test strip is labeled with a QR code for sample tracking and batch-specific information, along with a ready-to-use positive control standard. CertusBio biosensor for lactose estimation uses lactose-specific enzyme (proprietary) which is capable of direct electron transfer. The biosensor was validated over a range of lactose concentrations (0.5% 8%) against an HPLC method and found highly accurate (R2 5 0.9998). The biosensor is highly precise in measuring lactose content in whey and milk products as RSD was ,10%.

17.5 Blood glucose meter biosensors as an option for determination of lactose Besides using a purpose-built biosensor for estimating lactose in milk and milk product, the existing technology of a blood glucose meter (BGM) biosensor can be used. The primary advantage of a BGM method over the aforementioned proprietary options would be lower costs, with meters typically demanding a one-time investment of B$20 USD and individual test strips often costing ,$0.50 USD each. It has been shown that BGM biosensors can be adopted for measuring lactose in milk (Amamcharla & Metzger, 2011), in model lactose solutions (Booth et al., 2017; Heinzerling et al., 2012) and dairy ingredients (Wagner et al., 2020).

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BGMs were developed after decades of extensive research and clinical trials. They are used worldwide to monitor blood glucose concentration. The technology of glucose meters and their clinical and technological evaluations can be found in various publications (Bergenstal et al., 2000; Briggs & Cornell, 2004; Hönes et al., 2008; Kimberly et al., 2006). BGM biosensors are very adaptable to uses outside of their original design, having been shown to quantify not only lactose but also enzymes such as β-galactosidase or pathogens such as influenza virus and Escherichia coli when appropriate sample preparation is performed (Das et al., 2018). BGMs are extremely userfriendly and require a very small sample size. The first step in utilizing a BGM for the measurement of lactose in dairy products is to hydrolyze lactose’s β-1,4 glycoside bond using β-galactosidase enzyme (commonly referred to as lactase) to separate D-glucose and D-galactose constituents (Fig. 17.3). The amount of glucose formed during the hydrolysis of lactose is proportional to the amount of lactose present in the sample. Subsequently, the concentration of glucose is determined using a BGM, and the output from the BGM is then used to determine the lactose concentration in the sample.

FIGURE 17.3 Hydrolysis of lactose by beta-galactosidase (lactase) enzyme. Source: Adapted with permission from Halpin, G., McEntee, S., Dwyer, C., Lawless, F., & Dempsey, E. (2022). Lactose biosensor development and deployment in dairy product analysis. Journal of the Electrochemical Society, 169(3), 03752.

17.5 Blood glucose meter biosensors as an option for determination of lactose

Enzyme kinetics of lactose hydrolysis was successfully measured by using a BGM (Heinzerling et al., 2012). The KM for lactose hydrolysis was calculated for both in buffer and unbuffered milk system and with reported values of 70 6 2 and 73 6 2 mmol/L, respectively. These values were slightly lower than published values of 85 mmol/L. They have also found the optimum pH (pH 4.5) and temperature (45 C) for lactose hydrolysis by monitoring with a BGM. Therefore the BGM can be applied for quantifying lactose in milk and milk products. However, for quantifying lactose in unknown milk and milk product samples, a calibration curve is required to be developed between known lactose concentrations and BGM readings. The lactose estimation in different dairy products using a BGM is slightly difficult due to the presence of measurement interferents such as solution pH and different types or amounts of nonlactose solids that can impact BGM output. Dairy product manufactures could readily adopt the BGM for routine lactose monitoring activities given their commercial availability, simple use, small size, and very low cost. However, an understanding of BGM operating principles and theory is necessary to select an appropriate BGM for lactose analysis. A better understanding of how BGM biosensors work will also help to clarify, what sample matrix characteristics are important to account in a generalized method for lactose analysis in dairy products beyond milk.

17.5.1

Blood glucose meter operating principles

BGM biosensors are key tools for patient and health-care provider management of diabetes mellitus (Lan et al., 2011; Link et al., 2015; Sode et al., 2017). They are available over the counter at most drugstores, are fast and simple to operate, and require very small sample sizes (Vanavanan et al., 2010). Proper operation of the meters takes little training, with an operator needing only to insert a new test strip into the corresponding BGM, and then dripping sample onto the capillary site of the test strip; glucose quantification results are usually achieved in ,5 s. Most commercial BGM biosensors use an enzymatic biological recognition element. Common enzymes used include glucose oxidase (GOx) or glucose dehydrogenase (GDH) coupled with nicotinamide adenine dinucleotide or flavin adenine dinucleotide (referred to as GDH-NAD or GDH-FAD, respectively) as cofactors (Sode et al., 2017). Most commercial meters available are first- or second-generation amperometric biosensors in principle; glucose is selectively oxidized by the enzyme and mediators transfer the created electrochemical current to an electrode that is under an applied voltage. The resulting current is proportional to glucose concentration. An example of this scheme in a GOx-based amperometric biosensor is shown in Fig. 17.4.

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FIGURE 17.4 Glucose measurement scheme of a second-generation amperometric glucose oxidase based blood glucose meter. Source: Adapted with permission from Wang, J. (2008). Electrochemical glucose biosensors. Chemical Reviews, 108(2), 814 825.

17.5.2 Potential issues with a blood glucose meter based lactose assay One key issue associated with a BGM-based lactose assay is the lack of standardized tools. There is no certification or assurance that a BGM will perform adequately in any application outside its intended use for blood. Instead, validation of results falls upon the user and associated third parties if desired. A certain level of manufacturing quality for commercial BGM sensors is assured via compliance to International Organization for Standardization (ISO) standard ISO 15197 that is updated periodically, most recently ISO 15197-2013 (Klonoff, 2014; Link et al., 2015; Pftzner et al., 2012). However, these compliances are largely meaningless outside of a meter’s intended use, so more attention should be paid to the actual design and operation principles of the BGM to choose the correct tools. When selecting a BGM for use in a lactose assay, selection of the correct meter begins with assessing what technologies are available and what can act as an interferent. According to manufacture instructions and Link et al. (2015), most BGM brands are rated for measuring glucose concentrations between 20 and 600 mg/ dL, temperatures between 10 C and 40 C, relative humidity between 10% and 85 %, and hematocrit levels between 10% and 60 %. These parameters are relevant for blood, but they offer guidance when establishing quantification procedures for other matrices such as milk and dairy ingredients. The next step is to consider known interferences since BGM performance can be impacted by a variety of environmental conditions and nonglucose compounds. Table 17.3 lists common enzyme recognition elements used in BGM biosensors and known measurement interferents for each of those technologies. Universal interferents, whether they be environmental or compound specific, must be considered regardless of what BGM is used. Universal environmental interferents such as pH, temperature, humidity, and hematocrit all have an impact on the enzymatic recognition element: glucose measurements in

17.5 Blood glucose meter biosensors as an option for determination of lactose

Table 17.3 Measurement environment and nonglucose compound interferents known to bias blood-glucose meter readings.

Reference(s)

Enzyme recognition element

de Mol et al. (2010); Dungan et al. (2007); Ramljak et al. (2013)

Glucose oxidase (GOx)

Dungan et al. (2007); Ramljak et al. (2013)

Glucose dehydrogenaseflavin adenine dinucleotide (GDH-FAD) Glucose dehydrogenasenicontinamide adenine dinucleotide (GDH-NAD) Glucose dehydrogenasepyrroloquinoline quinone (GDH-PQQ)

Dungan et al. (2007); Perera et al. (2011); Ramljak et al., (2013) Dungan et al. (2007); Perera et al. (2011); Ramljak et al. (2013); Schleis (2007)

Measurement environment interferents

Nonglucose compound interferents

Hematocrit, pH, temperature, humidity, atmospheric oxygen levels Hematocrit, pH, temperature, humidity

Ascorbic acid, acetaminophen, mannitol

Hematocrit, pH, temperature, humidity

Ascorbic acid, acetaminophen, dopamine, maltose, maltotriose, maltotetrose, xylose Ascorbic acid, acetaminophen, dopamine, maltose, maltotriose, maltotetrose, galactose, xylose

Hematocrit, pH, temperature, humidity

Ascorbic acid, acetaminophen, dopamine

blood outside the pH range of 6.8 7.8 are inaccurate due to changes in the sensing enzyme’s activity (Dungan et al., 2007). Temperature, and to a lesser extent humidity, impacts enzymatic activity (Erbach et al., 2016). High hematocrit levels (volume of red blood cells relative to plasma volume in blood) has been shown to artificially lower the BGM output for glucose measurements in blood due to physical hinderance of the test strip’s enzyme site, and some BGM brands employ algorithms to correct for the effect (Ramljak et al., 2013). Compounds that universally interfere with amperometric BGM measurements include ascorbic acid and acetaminophen due to the ease at which these compounds are nonspecifically oxidized by the BGM electrode’s reference voltage . While these compounds are usually not of concern in milk and dairy ingredients, the principle that any easily oxidized substance can bias BGM output must be considered. Interferents can also be specific to a given BGM technology. For example, GOx-based sensor output can be artificially lowered in areas where oxygen is sparse (i.e., high-altitude locations) due to these meters getting their molecular oxygen mediator from the environment (de Mol et al., 2010). Also, galactose and certain small glucose polymers are interfering compounds for glucose dehydrogenase-nicontinamide adenine dinucleotide (GDH-NAD) and glucose dehydrogenase-pyrroloquinoline quinone (GDH-PQQ) based BGM biosensors. In general, attention must be paid to the BGM technology

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being used in order to select a BGM that will be optimal for the intended application.

17.5.3 Practical applications reported in literature for use of blood glucose meter in measurement of lactose 17.5.3.1 Measurement of lactose in milk using a blood glucose meter BGM was successfully applied for rapid determination of lactose in milk (Amamcharla & Metzger, 2011). They have determined temperature and time required for the near-complete hydrolysis of lactose and reported 2% enzyme (β-galactosidase) addition with incubation at 40 C for a minimum of 10 min is required for complete hydrolysis. Subsequently, the influence of glucose meters and their test strip lots were evaluated. The authors concluded that for test strips, between-lot differences were significant and found as much as a 1.3 mmol/L (23.4 mg/dL) difference in control solutions. In further experiment, the proposed method was validated using different concentrations of lactose solutions (1.9% 6.5%) and compared with an HPLCbased reference method. The average CV, calculated by considering the 5 lots as replications, was found to be 3.8%. The relatively high CV and lot-to-lot variation in the mean difference between the glucose meter method and HPLC-based method suggested that development of lot-specific calibration equations may be required. They suggested to prepare calibration samples that contain 3.5% protein and varying concentrations of lactose (2% 6%) using MPCconcentrate 85, deproteinized whey powder, and distilled water. Then dilute 0.25 mL of sample with 5 mL of distilled water in a test tube and mix thoroughly. Add 0.1 mL of lactase enzyme (approximately 270 yeast lactase units) and incubate at 40 C for 10 min. Using a suitable BGM (ReliOn Ultima or any other compatible BGM) and corresponding test strips, a calibration model is to be prepared. For unknown sample also, take 0.25mL sample and follow the same procedure. From slope and intercept of linear calibration model and dilution factor (0.0467), the lactose concentration in the sample can be calculated.

17.5.3.2 Measurement of lactose in dairy ingredients using a blood glucose meter BGM was also evaluated for lactose analysis in whey- and skim milk-derived (WD and SMD, respectively) ingredients (Wagner et al., 2020). Measurement interferents such as solution pH and different types or amounts of nonlactose solids present in the measurement background were investigated for their impact on BGM output bias; it was found that measurement solution pH must be between 6.7 and 7.0, and that calibration procedures must be designed to correct for large differences in nonlactose solids that may be

17.6 Conclusion

present when measuring different materials. Therefore a standardized calibration procedure is required to be developed for measuring the lactose content of WD and SMD ingredients. The composition of the material used in the calibration standards should loosely resemble the ingredients ultimately being measured (i.e., SMD vs WD ingredients). It is necessary that solids of commercial samples to be diluted for 0.5%, 3.0%, and 27% that will represent 31% 100%, 3.5% 18%, and ,2.0% dry-basis lactose concentration, respectively. Pure lactose can be used to adjust the lactose concentration of the calibration standards anywhere from 0.08% 0.6% as-is lactose. With careful calibration, lactose was accurately measured in 15 dried commercial dairy ingredients of both WD and SMD type over a broad range of lactose contents (0.01% 81.9% lactose as-is) using the BGM method and an enzymatic absorbance (EZA) as a reference method. Precision of the BGM method in terms of agreement between the BGM results and EZA results was found to depend on the meter used (Nova Max Plus % CV ,2.83, FreeStyle Precision Neo meter % CV ,3.54). In the experiments, one outlier was noted when measuring lactose in micellar casein (% bias was .10%). This consistently high error in terms of average absolute percentage bias difference (AD) for micellar casein was observed. It may be presumed that random BGM measurement error is inflated on a percent basis at lower lactose concentrations (about 0.14%). Thus accurately measuring low amounts of lactose for a given calibration series may be a limitation of the BGM method. An alternative hypothesis for the high AD associated with this particular micellar casein ingredient could be due to glycosylation of the κ-casein macropeptide portion of the protein. Glycosylated dairy proteins can be covalently linked to glucose, galactose (Recio et al., 2009), or lactose (Lillard et al., 2009), and the extent of glycosylation in κ-casein macropeptide in particular can be difficult to predict since it depends largely on a cow’s lactation cycle (O’Riordan et al., 2014). While it is unlikely that these protein-attached carbohydrates could cause measurement inaccuracies with the proposed BGM lactose analysis, it is a point worth mentioning for further exploration if applicable.

17.6

Conclusion

Lactose is a sugar with unique functional properties and is of great consequence to the nutritional value and marketability of dairy products. Lactose is a ubiquitous component of milk and dairy ingredients and plays a principal role in the quality and shelf life of dried dairy ingredients containing great amounts of the substance. Many processes involving dairy ingredient utilization and manufacture could benefit from routine application of an accurate, rapid, and low-cost way to quantify lactose. BGM biosensors have

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been identified as a potential tool to fit in this need. However, there are several key items that need to be investigated to create a generalized BGM-based lactose assay for milk and dairy ingredients. Firstly, since BGM sensors can only quantify a certain range of glucose concentrations, different dilution schemes tailored to accommodate the wide range of lactose contents available in milk and dairy ingredients in particular will be necessary. This dictates that relevant measurement environment factors such as pH or nonlactose solid amount and composition must be studied to create a robust lactose assay. Secondly, great care must be put into selecting BGM brands that are appropriate for measuring lactose. For example, GDH-PQQ-based meters are inappropriate since galactose is an interfering analyte for these meters, and galactose will be generated during the mandatory lactose hydrolysis step of the proposed BGM analysis. Also, some meters are known to algorithmically correct for different hematocrit levels, and this may be useful for lactose analysis in dairy ingredients assuming that nonlactose solids such as protein would have an analogous effect as red blood cells on BGM output.

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CHAPTER 18

Enzyme-based analytical methods pertinent to dairy industry C.G. Harshitha1, Rajan Sharma1 and Y.S. Rajput2 1 2

Dairy Chemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India

18.1

Introduction

Milk contains numerous biomolecules differing in size and quantity. Although the level of these molecules depends on species and their breed, physiological state, lactation stage, and health, the level remains within a certain range. Lactose, fat, and protein are major contributors to the total solids (TSs) present in milk. On a weight basis, the contribution of minerals, vitamins, growth factors, and amino acids is noticeable but remains low in comparison to lactose, fat, and protein. TS in milk refers to the weight in gram of solids that remain after evaporation of water from milk and fall in the range of 12%
15%. The term “solid non-fat (SNF)” is also very popular for defining milk quality and is calculated by subtracting fat levels from TSs. The consumers are provided information on nutrients such as protein, fat, lactose, minerals, vitamins, saturated fat, TS, and SNF available on packaged milk packets. The level of these nutrients is regulated by each country so that consumers are not deprived of nutrient as well as milk marketing companies do not dilute milk with water. Milk diluted with water or the addition of exogenous solids to milk is called milk adulteration and is prohibited across the countries. Lactose is a disaccharide secreted in milk and its level is about 5%. Certain individuals are deficient in enzyme hydrolyzing lactose and are unable to utilize this sugar. For such individuals (lactose intolerant), lowlactose milk and milk products are available on market. Milk is a good medium for microorganisms. Lactic acid bacteria grow in milk and convert lactose into lactic acid. Thus the level of lactic acid in milk is related to microbiological load and is a marker for hygiene maintained during milk production, processing, and storage. Antibiotics, pesticides, and other environmental contaminants also find an entry in milk and are of concern for human health. Although urea is naturally present in milk, this is a common adulterant for maintaining TS in milk diluted with water. For the same Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00018-7 © 2023 Elsevier Inc. All rights reserved.

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purpose, milk is also adulterated with sucrose, glucose, maltose, maltodextrin, and starch. Milk is a perishable food and for enhancing shelf life, milk is adulterated with hydrogen peroxide, formaldehyde, and antibiotics. Over the years, several analytical methods are developed for measuring fat, protein, lactose, urea, lactic acid, cholesterol, sucrose, glucose, maltose, maltodextrin, antibiotics, pesticides, hydrogen peroxide, minerals, melamine, and many more. Several of them can be estimated with the involvement of enzymes. Needless to emphasize that involvement of enzymes provides accuracy to analytical methods. This chapter has dealt with enzyme-based analytical methods that are applicable to the dairy industry or useful for further perusing research in dairy science. Analytical methods for estimation of urea, lactate, lactose (in low-lactose milk), cholesterol, and ascorbic acid in milk have been discussed. Also, principles and methods of strip tests for quick detection of adulterants such as urea, hydrogen peroxide, glucose, sucrose, maltodextrin, and starch in milk are explained.

18.2

Urea estimation in milk

Urea is a nitrogen-containing compound present in a soluble fraction of milk and contributes about 55% to the nonprotein nitrogen content of milk. Urea level in milk is linked to an unbalanced diet of dairy animals, particularly with respect to energy intake and protein content. Urea levels in blood and milk are correlated (Bendelja et al., 2011). Milk urea level is associated with nitrogen use efficiency as well as the reproductive performance of the dairy herd (Kananub et al., 2020). Urea level in most milk samples lies between 20 and 40 mg/100 mL. The heat stability of milk is also dependent on urea level (Rajput et al., 1984, 1982). Urea has been recognized as one of the major adulterants in milk and the Food Safety Standards Authority of India (FSSAI) has prescribed the upper limit of urea in liquid milk as 70 mg/100 mL beyond which milk is treated as adulterated one. Over the years, researchers have made efforts to develop quantitative analytical methods for estimating urea content in milk. Both enzymatic and nonenzymatic methods for detection as well as estimation of urea in milk are developed. In enzymatic methods, milk components do not interfere and thus these methods are preferred over nonenzymatic methods. In most of the enzymatic assays reported in the literature, urea is hydrolyzed by urease (EC 3.5.1.5), resulting in the formation of ammonium ion and carbamate which is further decomposed to another molecule of ammonium ion and bicarbonate (Mazzei et al., 2016) as shown in the following equation: Urease

H3 O1

1 2 1 CH4 N2 O 1 H2 O - CH2 NO2 2 1 NH4 - HCO3 1 2NH4

18.2 Urea estimation in milk

Ammonium ion under alkaline conditions converts to ammonia gas. Urease reaction on urea results in a change in pH, ammonium ion concentration, and gas pressure which are dependent on urea concentration. Urease reaction can be carried out in biological fluids such as milk, and blood as well as on electrode surfaces. A typical urea sensor requires immobilization of urease on the electrode surface. Also, urease reaction can be used as a prestep before employing sensors responsive to changes in hydrogen ion concentration, ammonium ion concentration, ammonia gas pressure, and carbon dioxide gas pressure. Even, ammonium ions can be measured spectrophotometrically with the involvement of other enzymes which enable specificity to assay. The principles and a brief description of the urea estimation method based on these concepts are described below.

18.2.1

Monitoring pH change

Change-in-pH of milk on the action of urease can be monitored by either indicator dye or pH electrode. Indicator dye provides results almost instantaneously but these are qualitative in nature. In a differential pH-based method for estimation of urea, change-in-pH is monitored upon hydrolysis of milk urea by urease (Luzzana & Giardino, 1999). Milk pH is increased by the action of urease on urea and the change in pH is directly proportional to the amount of urea in milk. The method requires differential pH apparatus consisting of two electrodes. In 2004 International Dairy Federation (IDF) has adopted this method (IDF, 2004). The method requires the preparation of a standard curve in a buffer solution (pH 6.7, phosphate buffer), and the method can be used in milk samples preserved with bronopol and sodium azide. Change-in-pH of milk on its treatment with urease has been exploited for developing strip tests impregnated with urease and cresol red dye. The paper strip changes color from yellow to red on dipping said strip in milk samples having extraneously added urea (Sharma, Gautam, Rajput, & Mann, 2022a).

18.2.2

Monitoring change in pressure

The action of urease on urea results liberation of carbon dioxide which results in a change in pressure (ΔP) (Jenkins et al., 1999, 2000). The change in pressure is correlated linearly with the urea concentrations as per the following equation:  ΔP 5

 kP RTVf ½Urea kP Vg 1 RT½H2 OVf

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FIGURE 18.1 Hardware setup for pressure assay using urease for estimation of urea in milk. Source: From Jenkins, D. M., Delwiche, M. J., Depeters, E. J., & Bondurant, R. H. (2000). Journal of Dairy Science, 83(9), 2042
2048. https://doi.org/10.3168/jds.S0022-0302(00)75085-5.

where kp is Henry’s law constant (which increases with temperature), R is the universal gas constant, T is absolute temperature, and Vf and Vg are the volumes of liquid and gas in the enclosure, respectively. The assay conditions require lowering of pH after urease reaction by employing citric acid to completely convert the carbonate ions produced during the reaction to carbon dioxide. The assay also requires a reaction cell with a pressure transducer (Fig. 18.1). Varying levels of milk constituents do not affect this assay. Piezoelectric pressure sensors measure the voltage across a piezoelectric element generated by the applied pressure. Renny et al. (2005) developed a piezoelectric sensor for the estimation of urea using reaction cell containing milk and urease.

18.2.3

Potentiometric approach

One of the products of the urease reaction is ammonium ions (NH41 Þ which can be measured using an ion-selective electrode (ISE). In this approach, urease is either added to the reaction mixture or immobilized in proximity to the NH41 -sensitive ISE. Change-in-ammonium ion leads to change-inelectrode potential across the membrane which is related to ammonium ion concentration as per the Nernst equation E 5 E0 1

RT lna nF

ð18:1Þ

where E is the measured electrode potential; E0 is the reference electrode potential (constant); RT/nF is the slope of the electrode (mv per decade), and a is the concentration of ammonium ions in solution. Using this concept, Verma and Singh (2003) reported a microbial biosensor for the estimation of urea in milk.

18.2 Urea estimation in milk

In an alternate approach, ammonium ions formed following urease reaction are converted to ammonia which is then measured using ammoniasensitive electrode. Ammonium ions under alkaline conditions are converted to ammonia gas. Sharma et al. (2008) applied this approach for the estimation of urea in milk wherein a high pH-ionic strength adjustor (pH in the range of 11
14) containing sodium hydroxide, methanol, and an indicator was used in the reaction to completely convert ammonium ions to ammonia.

18.2.4 Spectrophotometric measurement of ammonium ion concentration Ammonium ions produced by the action of urease on urea can be measured spectrophotometrically. In a commercial urea estimation kit marketed by Megazyme (https://www.megazyme.com/), the ammonium ion reacts with 2-oxoglutarate in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and the reaction is catalyzed by glutamate dehydrogenase (GDH). GDH

1 2 2 Oxoglutarate 1 NADPH 1 NH1 4 - L 2 glutamic acid 1 NADP 1 H2 O

In the above reaction, the decrease in NADPH content is spectrophotometrically monitored at 340 nm which is related to NH1 4 and ultimately with urea content in the sample. The method requires deproteinization of the milk sample by trichloroacetic acid.

18.2.5

Urea biosensor

A typical urea biosensor comprises of immobilized urease, transducer, and data processor. Transducer senses reaction products of urease reaction on urea. Urease reaction is already elaborated in this chapter and reaction products can be sensed by various devices, including amperometric, potentiometric, conductometric, thermal, optical, manometric, and piezoelectric. In this regard, readers are advised to refer to recent reviews published in this area (Botewad et al., 2021; Pundir et al., 2019). Immobilization of urease on some support is central to all the approaches. One of the reasons for a large number of articles on urea biosensors is perhaps enhanced stability of urease on immobilization which enables its repeated use (Jamwal et al., 2020; Kutlu et al., 2020; Mangaldas et al., 2010). In a recent trend, urease immobilization on polymer matrices, both conductive (e.g., polypyrrole films, polyaniline films, polythiophene films) and nonconductive polymers (activated polyvinyl alcohol, polyvinyl chloride, chitosan, alginate, etc.), has been attempted. Other approaches include immobilization of urease on nanoparticles and deposition of such

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nanoparticles in the vicinity of transducers, for example, ammonia selective electrode (Önde¸s et al., 2021) or using an amperometric sensor (Korkut Uru et al., 2021). In spite of all these advancements, commercially available urea biosensor is still elusive.

18.3

Lactose estimation

Lactose is one of the major constituents of milk and its content in cow milk is around 4.9%. The contribution of lactose in SNF in human, cow, goat, buffalo, and sheep milk is around 83%, 54%, 52%, 48%, and 42% respectively (Fox et al., 2015; Patel & Mistry, 1997). Lactose is a key ingredient being metabolized during the production of fermented dairy products. It also affects the texture of some frozen and concentrated dairy products (Fox et al., 2015). Lactose intolerance among the population has led to the development of lactose-free or low-lactose milk and milk products (Rao et al., 2021). It is critical to quantify lactose in these foods in order to verify the reported lactose concentration in lactose-free milk and cheeses. Various methods based on polarimetry, oxidation
reduction, colorimetry, chromatography, and enzymology have been developed (Fox et al., 2015). The traditional approach for the estimation of lactose is based on the fact that lactose is a reducing sugar that has the ability to reduce cupric sulfate to cuprous oxide (water-insoluble) and measure the latter gravimetrically (Teles et al., 1978). Colorimetrically lactose can be quantified by boiling it with anthrone reagent (under strongly acidic conditions) and spectrophotometrically measuring the colored product at 625 nm (Fox et al., 2015). Also, lactose can be separated on the amide column and then detected using a single quadrupole MS (Yang et al., 2021). Among all mentioned methods, enzymatic methods are preferred because of their specificity and rapidity (Portnoy & Barbano, 2021). Enzymatically lactose can be measured by using two different approaches: (1) spectrophotometric method and (2) differential pH method.

18.3.1

Spectrophotometric method

Milk is turbid and therefore milk samples are clarified before the analysis using Carrez solution or trichloroacetic acid (Portnoy & Barbano, 2021). During estimation, lactose is first hydrolyzed into glucose and galactose by the β-galactosidase enzyme. The lactose can be quantified either by measuring glucose or galactose. Glucose can be estimated by employing either of the following enzymatic reactions.

18.3 Lactose estimation

1. Action of glucose oxidase (GOD) on glucose resulting in the formation of hydrogen peroxide which is measured by employing reduced o-anisidine acted and peroxidase. Glucose oxidase

Glucose 1 H2 O 1 O2








- H2 O2 1 D 2 gluconic acid Peroxidase

H2 O2 1 Reduced o 2 anisidine



- Oxidized o 2 anisidine

(2) Action of hexokinase on glucose resulting in the formation of glucose-6phosphate. Then, glucose-6-phosphate dehydrogenase (G-6-P-DH) acts on glucose-6-phosphate and simultaneously converts NADP1 into NADPH. The amount of NADPH is stoichiometrically related to the amount of lactose present in the product and can be measured by recording the absorbance at 340 nm. Hexokinase

D 2 glucose 1 ATP



- Glucose 2 6 2 phosphate 1 ADP G 2 6 2 P 2 DH; NADP1

Glucose 2 6 2 phosphate














- Gluconate 2 6 2 phosphate 1 NADPH 1 H1

Galactose can be measured by employing galactose dehydrogenase (GalDH) which converts galactose into galactonic acid and NADH. The amount of NADH is stoichiometrically related to the amount of lactose present in the product and can be measured by recording the absorbance at 340 nm. GalDH

D 2 galactose 1 NAD1 - Galactonic acid 1 NADH 1 H1

Similar enzymatic methods have been adopted by IDF for measuring lactose in dried milk, dried ice mixes, and processed cheese involving the measurement of either glucose (ISO 5765-1:2002/ IDF 79-1:2002) or galactose (ISO 5765-2:2002/ IDF79-2:2002) (IDF, 2002a, 2002b). Colorimetric estimation of lactose involving enzymes has the limitation of interferences from monosaccharides such as glucose or galactose which can inhibit enzymes. However, the use of enzymes makes estimation rapid and specific and involves simpler sample preparation steps. Gille et al. (2018) have described an enzymatic method wherein free glucose is first eliminated by use of GOD and catalase and then lactose is estimated by the application of β-galactosidase and hexokinase enzymes in the presence of ATP. Glucose-6-phosphate-dehydrogenase acts on glucose-6-phosphate and simultaneously converts NADP into NADPH stoichiometrically. NADPH can be assayed at 340 nm. Measurement of generated glucose and galactose concentration is the basis of lactose estimation in kits marketed by Megazyme (https://www.megazyme.com/), R-Biopharm AG (https://r-biopharm.com/), and Sigma-Aldrich (https://www.sigmaaldrich.com). In these kits, lactose is first hydrolyzed into glucose and galactose by the β-galactosidase enzyme. Glucose formed in the reaction can be

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estimated directly using the combination of enzymes such as hexokinase, G-6-PDH and 6-phosphogluconate dehydrogenase (6-PGDH) in the presence of NADP1. The amount of NADPH formed in the reaction is measured spectrophotometrically which is then correlated with lactose concentration. Galactose formed in the reaction catalyzed by β-galactosidase can also be used for the estimation of lactose. However, in this case, α-D-galactose formed is required to be converted to β-D-galactose using galactose mutarotase. The β-D-galactose is then further oxidized at pH 8.6 by NAD1 by β-galactose dehydrogenase leading to the formation of D-galactonic acid and NADH. The amount of NADP formed is measured spectrophotometrically and is correlated with lactose concentration.

18.3.2

By measuring the change in pH

The first step is similar to that of the spectrophotometric method where lactose is hydrolyzed into glucose and galactose using β-galactosidase. The produced glucose reacts with ATP in the presence of hexokinase to yield glucose6-phosphate, ADP, and H1 ions. The production of H1 ions causes a change in pH. The pH difference between the electrodes was recorded to quantify the lactose content (Luzzana et al., 2003). β 2 galactosidase

Lactose







- Glucose 1 Galactose Hexokinase

Glucose 1 ATP





- Glucose 2 6 2 P 1 ADP 1 H1

The method can be used even in turbid samples such as milk.

18.4 Estimation of lactate or lactic acid in dairy products In fresh raw milk, lactic acid or lactate is less than 20 mg per liter. Once the microbial activity initiates on exposure of milk to environmental conditions, lactic acid in milk starts rising. Lactic acid is produced by the aerobic breakdown of lactose by lactic acid bacteria and its level in milk provides microbial as well as hygienic status of milk (Kumar et al., 2016). The lactic acid level in milk can be easily assessed by titration with a standard alkali using phenolphthalein as an indicator. However, when milk is neutralized by the addition of neutralizers such as NaOH, Na2CO3, or NaHCO3; lactic acid gets converted to lactate which is now not titratable with alkali and thus estimation of titratable acidity in such milk provides false (lower acidity) values of milk. Under these conditions, it is best to measure the lactate content of milk by a suitable method to know the hygienic status of milk. The lactate content of nonfermented milk products can also provide an idea about the hygienic status of milk used for the preparation of products. As per the Bureau of Indian Standards, lactate content in extra grade skimmed milk powder should not be more than 1.5 mg per g (BIS, 2014).

18.4 Estimation of lactate or lactic acid in dairy products

Lactate measurements in fermented milk products such as cheese are also of interest to know the status of ripening as lactate is utilized as a precursor during ripening (McSweeney, 2004). Further, the type of lactate isomer produced during ripening provides the idea of the type of predominant microorganisms in ripened cheeses. During fermentation and ripening, mainly L-lactate is produced by the starter cultures while the formation of D-lactate indicates the predominance of nonstarter lactic acid bacteria (McSweeney et al., 2017). Although chemical methods for estimation of lactate in milk/milk products are available (AOAC, 2000; BIS, 1984), these methods are cumbersome. Now, enzymatic methods are preferred (IDF, 2005). The enzymatic method for estimation of lactate content in dairy products usually involves the conversion of lactate to pyruvate by L-lactate dehydrogenase (L-LDH) and/or D-lactate dehydrogenase (D-LDH) in the presence of NAD1. In this reaction, NAD1 is reduced to NADH which can be measured spectrophotometrically at 340 nm. In this measurement, glutamate pyruvate transaminase is used to remove pyruvate formed in the reaction into L-alanine in the presence of L-glutamate to tilt the reaction equilibrium (IDF, 2005). D 2 LDH

D 2 lactate 1 NAD1



- Pyruvate 1 NADH 1 H1 L 2 LDH

L 2 lactate 1 NAD1



- Pyruvate 1 NADH 1 H1 GPT

Pyruvate 1 L 2 glutamate

- L 2 alanine 1 2 2 oxoglutarate

Commercial lactate or lactic acid estimation kits using these reactions are marketed by Megazyme (https://www.megazyme.com/) and R-Biopharma AG (https://r-biopharm.com/). In another approach, Shapiro and Silanikove (2010) developed an enzymebased fluorometric coupled reaction for determining lactate content. D- and L-lactate are transformed into D- and L-pyruvate, respectively, by D-LDH and 1 1 L-LDH, simultaneously reducing NAD to NADH 1 H . Further, the diaphorase enzyme converts nonfluorescent resazurin in the presence of NADH 1 H1 into fluorescent resorufin which can be measured using a fluorometer at Ex/Em 5 540/590 (Shapiro & Silanikove, 2010). D 2 LDHðorL 2 LDHÞ

D 2 lactateðor L 2 lactateÞ 1 NAD1












- Dðor LÞ 2 pyruvate 1 NADH 1 H1 Resazurin diaphorase

Resazurin 1 NADH 1 H1












- NAD1 1 Resoruf in

In yet another approach, Kumar et al. (2016) developed a rapid method for qualitative and quantitative estimation of lactate in dairy products using lactate oxidase. The approach relies on lactate oxidase (LOD) oxidizing lactate while simultaneously producing H2O2, which then oxidizes 2, 4, 6-tribromo 3-hydroxy benzoic acid (TBHBA) in the presence of peroxidase. Oxidized TBHBA then complexes with 4-amino antipyrine to produce a magenta color

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(λmax 5 523 nm) as shown in Figure 18.2. In the qualitative assay, raw milk samples spiked with 300 mg lactate/L show a color change. For cow and buffalo milk, the LOD values for the established quantitative lactate assay were found to be 43.88 and 48.22 mg/L, respectively, while LOQ values were 146.25 and 171.11 mg/L, respectively. Some researchers developed enzyme-based amperometric biosensors for the detection of lactate in dairy products using LOD and LDH enzymes. In such cases, the surface of a carbon screen-printed electrode is altered with a ternary composite, electrochemically reduced graphene oxide, and poly (allylamine) hydrochloride, and the LDH enzyme is then immobilized by cross-linking

FIGURE 18.2 Action of LDH and peroxidase enzymes and involvement of 2, 4, 6-tribromo 3-hydroxy benzoic acid and 4-amino antipyrine for estimation of lactate. Source: From Kumar, B., Sharma, R., Thakur, R., Barui, A. K., Rajput, Y. S., & Mann, B. (2016). Rapid lactate oxidase-based assay for lactate content in milk to ascertain its hygienic quality. International Journal of Dairy Technology, 69(3), 460
467. https://doi.org/10.1111/1471-0307.12285.

18.5 Estimation of cholesterol in dairy products

using glutaraldehyde. The biosensor has a specific sensitivity of 1.08 μA/ mM  cm2 and a detection limit of 1 μM in a range of up to 3 mM L-lactic acid (Istrate et al., 2021). Ozoglu et al. (2021) immobilized LOD enzyme on the platinum electrode to estimate lactate using cyclic voltammetry and chronoamperometry. The developed biosensor has a detection limit of 31 μM of lactate (Ozoglu et al., 2021).

18.5

Estimation of cholesterol in dairy products

Cholesterol is a waxy substance found in all human cells and is an essential component of cell membranes, hormones, and other body components. Cholesterol is a part of the unsaponifiable matter of milk fat and its content in milk fat is around 300 mg/100 g milk fat. Many studies have demonstrated that dietary cholesterol, serum cholesterol, and the development of coronary heart disease are related. Ghee and other fat-rich dairy products contribute to cholesterol consumption. Also, some cholesterol oxidation products are cytotoxic, atherogenic, mutagenic, and carcinogenic and therefore these are of concern for human health. Foods with a reduced level of cholesterol are commercially available. Blending milk fat with vegetable oils, extraction with organic solvents, adsorption with activated charcoal and saponin, vacuum distillation, molecular distillation, cholesterol degradation by the enzyme [cholesterol oxidase (CO)], and cholesterol removal by supercritical carbon dioxide are some of the methods used. β-cyclodextrin (a starch hydrolyzed product) is used for removing cholesterol from milk, cream, cheese, fat, and egg yolk. Beta cyclodextrin is nontoxic, nonhygroscopic, chemically stable, and edible (Shingla & Mehta, 2018). Cholesterol levels in dairy products can be monitored using a variety of methods. These techniques can be classified into three groups: (1) Liebermann
Burchard (L
B) reaction for color development, (2) fluorometric and colorimetric enzymatic assays typically used in assay kits and automated plate readers, and (3) analytical instrumental approaches such as gas (Fletouris et al., 1998) and liquid chromatography (Jeffrey Hurst et al., 1983) or mass spectrometry. The colorimetric methods such as the Abell method (Abel et al., 1952) are multiple-step assays and take a long time and necessitate the use of corrosive chemicals, whereas enzymatic approaches, on the other hand, are adaptable and have essentially superseded the more time-consuming methods. Cholesterol esterase (CE) hydrolyzes cholesteryl esters into cholesterol, which is subsequently oxidized by CO to generate ketone and hydrogen peroxide. Generated hydrogen peroxide reflects the concentration of cholesterol and its

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measurement relies on horseradish peroxidase (HRP)-coupled oxidation of H2O2 (Li et al., 2019). Probes can be either chromogenic (4-aminoantipyrine and phenol) or fluorogenic (homovanillic acid) (Figure 18.3). Amundson and Zhou (1999) described method for estimation of cholesterol using CE, CO, and peroxidase enzymes but different fluorescent probes. Peroxidase enzyme converts nonfluorescent amplex red [10-Acetyl-3,7dihydroxyphenoxazine (ADHP)] into fluorescent resorufin that has excitation and emission maximum at 563 and 587 nm, respectively. HRP

H2 O2 1 Amplex Red ðnon 2 f luorescentÞ - Resoruf in ðHighly f luorescentÞ

In milk, cholesterol exists both in free and esterified forms. Larsen developed a novel enzyme-based fluorometric method to quantify both free cholesterol and total cholesterol in bovine milk (Larsen, 2012). Mild detergents and enzymes such as phospholipase C, lipoprotein lipase, and lipase are used to quantify milk cholesterol, primarily inside the milk fat globule membrane (MFGM). CE is included in total cholesterol analysis but not in free cholesterol analysis. Furthermore, cholesterol is then oxidized by CO to produce cholest-

FIGURE 18.3 Schematic representation of enzyme coupled reaction of fluorometric enzymatic assay. Source: From Li, L. H., Dutkiewicz, E. P., Huang, Y. C., Zhou, H. B., & Hsu, C. C. (2019). Journal of Food and Drug Analysis, 27(2), 375
386. https://doi.org/10.1016/j.jfda.2018.09.001.

18.6 Ascorbic acid estimation in dairy products

4-en-3-one and hydrogen peroxide, which combines stoichiometrically with the nonfluorescent “ADHP” under the action of peroxidase to produce an equal amount of the fluorescent “resorufin”. The author has used lipases and CE in the initial step as they are critical for hydrolyzing cholesterol from its lipid matrix in the MFGM. About 6%
7% of the cholesterol in milk is esterified (Larsen, 2012). Despite the use of heat treatment, incubation without lipase and lipoprotein lipase, the method detects only 82%
83% of total cholesterol and incubation without lipases and CE it detects only 56% of the possible cholesterol quantity. This condition emphasizes the importance of heat treatment as well as hydrolytic enzyme preincubation. Cholesterol's structural relationships with other membrane components are complex. The bulk of cholesterol may be structurally trapped in the membrane matrix and maintained there by van der Waals forces in the hydrophobic environment, aside from esterification with triacylglycerols (TAG), phospholipids, and may be other chemicals. The hydrolytic enzymes used in this work have an affinity for these nearby molecules, allowing free cholesterol to be released (Larsen, 2012). Milk sample is processed prior to use of CE and CO and involves following steps and cholesterol is analyzed by using fluorescent probe “ADHP”. 1. Prior to analysis, milk samples are diluted, emulsified, and blended to obtain a uniform mixture. 2. Phospholipids and TAG are degraded by enzymes. Lipoprotein lipase, lipase, phospholipase C, and CE (for determining total cholesterol) are incubated with emulsified milk samples. Phospholipase C hydrolyzes phospholipids on the glycerol side of the phospholipid, resulting in diacylglycerols and free “P-side chains” in the MFGM and milk serum. The lipoprotein lipase and lipase enzymes hydrolyze TAG and diacylglycerols to yield fatty acids and ultimately break down the MFGM's main structure. Free glycerol and free fatty acids are the end products. Cholesterol that can be esterified into TAG and phospholipids is hydrolyzed by CE. Parmar et al. (2016) have described a method for estimation of cholesterol in dairy products such as butter, kalakand, khoa, and paneer wherein fat is saponified using methanolic KOH. Cholesterol is estimated in unsaponifiable materials (Parmar et al., 2016). Cholesterol is extracted from unsaponifiable materials by hexane and after hexane evaporation, cholesterol is estimated using CE, CO, and peroxidase enzymes.

18.6

Ascorbic acid estimation in dairy products

Ascorbic acid is a sugar acid that can be synthesized from D-glucose or D-galactose by most species. In the presence of transition metal ions, heat, light, or weakly

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alkaline circumstances, ascorbate can be oxidized reversibly to dehydroascorbate without losing its vitamin function. With the loss of activity, dehydroascorbate can be converted irreversibly to 2,3-diketogulonic acid. In the end, 2,3-diketogulonic acid decomposes into oxalic and L-threonic acids and brown pigments (Fox et al., 2015). Ascorbic acid is an important antioxidant in many biological systems because it is a potent reducing agent (Lindmark-Månsson & Åkesson, 2000). It is also required for the functioning of the hydroxylase, which catalyzes the conversion of proline to hydroxyproline and lysine to hydroxylysine after translation. Collagen, the main protein in connective tissue, requires this post-translational hydroxylation. Ascorbate helps in maintaining iron in the proper oxidation state (ferrous) and aids in absorption. Vitamin C is also involved in the metabolism of amino acids, iron absorption, and infection resistance (Khan et al., 2019). As compared to fruits and vegetables, milk is a poor source of ascorbic acid. Cow milk contains roughly 1 mg of ascorbate for every 100 g of milk although stated levels range from 0.85 to 2.75 mg per 100 g. In milk, the ratio of ascorbate to dehydroascorbate is 4: 1. Ascorbate levels in human milk and colostrum are approximately 4 and 7 mg per 100 g, respectively. Ascorbic acid in caprine milk is similar to cow’s milk. However, sheep's milk contains higher ascorbate (5 mg per 100 g) than bovine milk. At the pH of milk, ascorbate is easily oxidized. Temperature, light, oxygen content, and the presence of catalytic trace elements all have an impact on the rate of oxidation. Ascorbic acid is essential for the establishment and maintenance of redox equilibria in milk, as well as the protection of folate and the avoidance of oxidized flavor development. The oxidation of ascorbate is catalyzed by the photochemical degradation of riboflavin (Aurand et al., 1966; Birch & Bonwick, 2019). Ascorbic acid can be detected by several methods such as the electrochemical method (Mazzara et al., 2021), hydrophilic interaction HPLC method (Zuo et al., 2015), a combination of liquid chromatography and amperometric detection (Pachla & Kissinger, 1979), and by an enzymatic method (Badrakhan et al., 2004; Bensinger et al., 1978). The enzymatic method involves the measurement of dehydroascorbic acid (DHAsA) as a result of ascorbic acid oxidation by oxygen or hydrogen peroxide in the presence of ascorbate oxidase and peroxidase (Shekhovtsova et al., 2006). Beutler and Beinstingl (1980) have described ascorbic acid estimation method in foodstuffs and the method follows three key steps: 1. In the presence of the electron carrier 5-methylphenazinium methosulfate (PMS) at pH 3.5, L-ascorbic acid (L-ascorbate) and a few other reducing chemicals reduce the tetrazolium salt MTT [3-(4,5dimethyl thiazolyl-2)-2,5- diphenyltetrazolium bromide] to a formazan. The sum of the reducing substances is measured.

18.7 Detection of common adulterants

2. The sample is also separately treated with ascorbate oxidase (AAO) for preparing sample blank. MTT/PMS does not react with the produced dehydroascorbate. 3. The amount of L-ascorbate in the sample is equal to the absorbance difference between the sample and sample blank. The measuring parameter is the MTT-formazan, which is measured at 578 nm. PMS

L 2 ascorbate ðX 2 H2 Þ 1 MMT - Dehydroascorbate ðX Þ 1 MMT 2 f ormazon 1 H1 1 AAO L 2 ascorbate ðX 2 H2 Þ 1 O2 - Dehydroascorbate ðXÞ 1 H2 O 2

The enzyme-based test kit commercially marketed under the name Enzytec Generic L-Ascorbic acid (R-Biopharma) and L-ascorbic acid (L-ascorbate) assay procedure (Megazyme) for detection of ascorbic acid in foods follows the same principle as mentioned above with limit of detection of 0.3 and 0.175 mg/L, respectively (https://www.megazyme.com/). An alternate method for ascorbic acid estimation in fruit juices, milk, and sour milk products for infants is described by Shekhovtsova et al. (2006). Ascorbic acid is oxidized to dehydroascorbic acid in the presence of hydrogen peroxide. A part of the excess amount of hydrogen peroxide added to the sample is used for oxidation of ascorbic acid while unused hydrogen peroxide is measured by employing peroxidase, 3,3’,5,5’-tetramethylbenzidine (TMB) or TMB.

18.7

Detection of common adulterants

Milk is one of the food items which is adulterated extensively across the world (Poonia et al., 2017; Windarsih et al., 2021) with the situation being particularly bad in impoverished and underdeveloped countries due to a lack of sufficient monitoring and control systems. Adulteration of milk not only raises ethical and financial concerns but also poses a health risk to milk consumers. Most of the time, adulteration is done with the purpose to increase profits; however, the other factors which led to adulteration of milk include demand and supply gaps, the perishable nature of milk, poor customer purchasing power, etc. The lack of effective and rapid detection tests which may act as a deterrent for such adulteration is also one of the possible causes. Chemical substances such as sucrose, salt, starch, urea, and ammonium sulfate are added to milk for fraudulently increasing SNF content. Formalin and hydrogen peroxide as preservatives are added for reducing costs during milk transportation. Neutralizers such as NaOH, Na2CO3, and NaHCO3 are employed to offset the increase in the acidity of milk during storage of milk at nonrefrigeration temperature (Kamal & Karoui, 2015). Rapid and simple tests are needed for the screening of samples for adulteration.

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18.7.1 milk

Detection and estimation of hydrogen peroxide in

Hydrogen peroxide (H2O2) acts as a preservative and thus extends the shelf life of products. The presence of H2O2 not only efficiently limits microbial growth that causes milk spoiling but also activates the lactoperoxidase enzyme system, significantly improving the quality of dairy products to which it has been added (Li et al., 2017). Although hydrogen peroxide is a preservative, it is not permitted in milk in India (Food Safety and Standards (Food Products Standards and Food Additives) Regulation, 2011). The residue of H2O2 in dairy products is a public health danger because of its ability to increase oxidative stress, which can lead to health issues such as gastritis, bowel inflammation, and bloody diarrhea (Handford et al., 2016). Almost all enzymatic assays for the detection of hydrogen peroxide in milk or other matrices use HRP (HRP, EC 1.11.1.7). HRP catalyzes the following reaction: Horseradish peroxidase

Substrate ðReducedÞ 1 H2 O2












- Substrate ðOxidizedÞ 1 2H2 O

Often the substrate in its reduced form is colorless and oxidized substrate is either fluorescent or colored which can be either perceived visually or measured through a spectrophotometer/fluorimeter. In a commercial hydrogen peroxide estimation kit marketed by Megazyme (https://www.megazyme.com/), HRP-mediated oxidation of Megaplex Red is utilized. The principle of assay involves the conversion of the colorless Megaplex red probe into a colored resorufin (Megaplex red probe) in a 1:1 stoichiometry in the presence of HRP and H2O2. Resorufin is measured either fluorometrically using a fluorometer for excitation maxima at 571 nm and emission maxima at 585 nm or colorimetrically using a spectrophotometer tuned at 570 nm. HRP

H2 O2 1 Megaplex red - Resoruf in 1 H2 O

Recently bioactive paper-based methods have been developed for rapid detection of hydrogen peroxide in milk in which peroxidase-catalyzed reaction between hydrogen peroxide and guaiacol to produce a tetraguaiacol (red), which is then quantified via digital imaging (Lima et al., 2020). Recently, Sharma and coworkers have developed paper strips for the detection of hydrogen peroxide in milk. The said strip is prepared by impregnation of peroxidase and potassium iodide. With pure milk, the strip will remain white, while the strip color changes to yellow or brown on dipping in milk adulterated with hydrogen peroxide (Sharma, Rajput, Mann, & Gautam, 2022e). The developed paper strip can detect the presence of 0.001% level of hydrogen peroxide in milk.

18.7 Detection of common adulterants

18.7.2

Detection of glucose in milk

The normal glucose content in milk is around 50 mg per liter (Fox et al., 2015). Extraneous glucose is normally added to milk either to increase SNF content or to mask the added water in milk. In solution, glucose can exist as one of two anomers α, β, or as the open-chain glucose aldehyde. At pH 7 and 25 C, roughly 63% of the glucose will adopt the β-glucopyranose conformation, 37% as the α-glucopyranose, and less than 1% existing as either the aldehyde or glucofuranose. A number of enzymes accept glucose as a substrate and produce a product in a detectable form. GOD and hexokinase activities, in particular, have been used in the creation of spectrophotometric and colorimetric glucose assays. A commercialized kit marketed by Sigma Aldrich (https://www.sigmaaldrich. com) utilizes hexokinase to catalyze the phosphorylation of glucose in presence of ATP. Glucose-6-phosphate is then oxidized to 6-phospho-gluconate by glucose-6-phosphate dehydrogenase (G6PDH). An equimolar amount of NAD is reduced to NADH during this oxidation. NADH can be monitored by measuring absorbance at 340 nm. Hexokinase

Glucose 1 ATP




- Glucose 2 6 2 phosphate 1 ADP G 2 6 2 P 2 DH; NAD1

Glucose 2 6 2 phosphate












- 6 2 phosphogluconate 1 NADH 1 H1

Glucose assay kit marketed by Megazyme (https://www.megazyme.com/) utilizes GOD and peroxidase enzyme. GOD generates H2O2, whereas peroxidase acts on H2O2 and simultaneously oxidizes 4-aminoantipyrine into quinoneimine dye which can be measured at 510 nm. Glucose oxidase

D 2 glucose 1 H2 O 1 O2








- H2 O2 1 D 2 gluconic acid Peroxidase

H2 O2 1 p 2 hydrobenzoicacid 1 4 2 aminoantipyrine



- Quinoneimine

Luther et al., (2017) also used GOD and peroxidase enzymes for glucose assay. These workers employed o-dianisidine as substrate for peroxidase. Glucose oxidase

Glucose 1 H2 O 1 O2







- H2 O2 1 D 2 gluconic acid Peroxidase

H2 O2 1 Reduced o 2 dianisidine



- Oxidized o 2 dianisidine

Sharma and coworkers have developed paper strips for the detection of glucose in milk. The said strip is prepared by impregnation of GOD, peroxidase, 2,4,6- tribromo-3-hydroxybenzoic acid, and 4-aminoantipyrine. With pure milk, the strip will remain white, while strip color changes to pink with milk adulterated with glucose (Sharma, Rajput, Mann, & Bhaveshkumar, 2022c). The paper strip has sensitivity to detect 0.04% level of glucose in milk.

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18.7.3 Detection and estimation of sucrose in milk and milk products Sucrose is absent in milk and sucrose detected in milk points toward adulteration of milk. Sucrose is added to milk for fraudulently increasing SNF content of milk. However, sucrose is also used as an additive in many dairy products, and sometimes its addition is controlled by regulations. In such cases, correct estimation of sucrose is required. Apart from the traditional Lane
Eynon titration procedure, enzymatic assays can be used. The enzymatic methods for the detection/estimation of sucrose in milk usually rely on the action of invertase which converts sucrose to glucose and fructose. Commercial sucrose estimation kit marketed by Sigma Aldrich (https://www. sigmaaldrich.com) utilizes invertase to break down sucrose into glucose and fructose. Hexokinase catalyzes the phosphorylation of glucose and fructose in the presence of ATP. In the presence of NAD, glucose-6-phosphate (G6P) is oxidized to 6-phosphogluconate by glucose-6-phosphate dehydrogenase (G6PDH). An equimolar amount of NAD is reduced to NADH during this oxidation. The increase in absorbance at 340 is proportional to the content of sucrose. Hexokinase

D 2 glucose 1 ATP



- Glucose 2 6 2 phosphate 1 ADP G 2 6 2 P 2 DH; NADP1

Glucose 2 6 2 phosphate












- Gluconate 2 6 2 phosphate 1 NADPH 1 H1

A commercial sucrose assay kit marketed by Megazyme (https://www.megazyme.com/) works on a similar principle where the concentration of D-glucose is measured utilizing hexokinase and G6PDH before and after hydrolysis of sucrose by β-fructosidase (invertase). The difference in D-glucose concentrations before and after hydrolysis by β-fructosidase is used to calculate the sucrose content. Also, D-fructose on action of hexokinase can be converted into D-fructose-6-P which on further action of phosphoglucose isomerase enzyme is isomerized to D-glucose-6-P. Luther et al., (2017) developed a paper test card for the determination of sucrose in milk. Sucrose is first hydrolyzed into glucose and fructose by invertase and then glucose is determined by GOD-peroxidase-o-dianisidine system. Invertase

Sucrose 1 H2 O



- Glucose 1 Fructose Glucose oxidase

Glucose 1 H2 O 1 O2







- H2 O2 1 D 2 gluconic acid Peroxidase

H2 O2 1 Reduced o 2 anisidine



- Oxidized o 2 anisidine

Paper strips for the detection of sucrose in milk can also be prepared by depositing invertase, GOD, HRP, 2,4,6- tribromo-3-hydroxybenzoic acid, and

18.7 Detection of common adulterants

4-amino antipyrine on paper (Sharma, Gautam, Rajput, & Mann, 2022b). The strip remains white on dipping in pure milk, while the paper turns pink with milk adulterated with sucrose. The strip has the sensitivity to detect a 0.1% level of sucrose in milk.

18.7.4

Detection of maltodextrin in milk

Maltodextrin is a carbohydrate derived from corn starch and consists of glucose monomers joined together. Maltodextrin is a starch hydrolysate having a dextrose equivalent (DE) of less than 20. In contrast to natural starches, maltodextrin is water-soluble. Maltodextrin is used in the production of a variety of dairy products, including yogurt, ice cream, milk powders, and cheeses owing to its functional properties such as bulking, gelling, binding, and prevention of crystallization, control of freezing, and fat replacement (Chronakis, 1998). Maltodextrin has been added to milk as an adulterant, mostly to raise the SNF and the yield of dairy products. Maltodextrin in milk can be detected using a variety of techniques. Enzymatically maltodextrin in milk can be detected using a combination of three enzymes: amyloglucosidase (AMG, EC 3.2.1.3), GOD, and HRP. Amyloglucosidase converts maltodextrin into glucose which can be assayed by using the following enzymatic reactions. AMG

Maltodextrins 1 H2 O - D 2 glucose Glucose oxidase

- H2 O2 1 D 2 gluconate D 2 glucose 1 H2 O 1 O2








2H2 O2 1 p 2 hydroxybenzoic acid Peroxidase

1 4 2 aminoantipyrine



- Quinoneimine dye 1 4H2 O

Similar to other paper strips, strips for maltodextrin can be prepared by employing amyloglucosidase, GOD, HRP, and potassium iodide. Strip turns yellow on dipping in milk adulterated with maltodextrin (Sharma, Rajput, Mann, & Bhaveshkumar, 2022d).

18.7.5

Paper strip for urea detection

Action of urease on urea present or added in milk results in increase-in-pH. Immobilization of urease along with indicator dye on paper constitutes paper strips for detection of added urea in milk. Increase-in-pH results in change-incolor of indicator dye which can be visually observed (Kumar et al., 2000). The developed paper is yellow in color and changes to peach, reddish-brown, pink, and magenta in the presence of increasing urea content in milk. Recently, the author's laboratory has developed improved strips for the detection of urea in milk (Sharma, Gautam, Rajput, and Mann, 2022a). The method involves the impregnation of paper with urease and cresol red dye. Urea detection in milk involves dipping the strip in the milk sample for 1
2 s followed by visualization

463

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CHAPTER 18:

Enzyme-based analytical methods pertinent to dairy industry

of the color change of the dye. With pure milk, the strip remains yellow, while paper turns red on dipping in adulterated milk.

18.7.6 Detection and estimation of starch in milk and milk products Thickening additives such as starch and flour are added to milk to counteract the dilution resulting from added water (Kamal & Karoui, 2015). Starch is also a normal additive in certain dairy products. Traditional methods for starch estimation in milk-based matrices are based on the conversion of starch into glucose using acid hydrolysis followed by estimation of glucose either using Lane
Eynon’s procedure or through a polarimeter (Charles et al., 2009). In such assays, lactose present in the milk interferes. Enzymatic assay of starch requires hydrolysis of α-D-(l-4), α-D-(l-6), and α-D-(l-3) links to release D-glucose by amyloglucosidase. The rate of hydrolysis is dependent on the linkage; with α-D-(l-4) glucosidic linkages being faster than α-D-(l-6) and α-D-(l-3) glucosidic links (Luo et al., 2021). The use of amyloglucosidase allows for the quantitative conversion of starch to D-glucose, which is subsequently measured using the GOD/POD combination. Megazyme Ltd. (https://www.megazyme.com/) commercialized the enzyme kit for the detection of total starch in food products based on the ability of α-amylase to catalyze the hydrolysis of starch into soluble, branched, and unbranched maltodextrins. Predissolving resistant starch in the sample using cold 1.7 M NaOH, neutralization with sodium acetate buffer, and hydrolysis with α-amylase are performed when necessary. Dissolution in DMSO at 100 C is also a viable option. AMG hydrolyzes maltodextrins to D-glucose in a quantitative manner. D-glucose is converted to D-gluconate with the release of equimolar levels of H2O2. Then peroxidase acts on H2O2 in the presence of phydroxybenzoic acid and 4-aminoantipyrine and quinoneimine is formed. α 2 amylase

Starch 1 H2 O





- Maltodextrins AMG

Maltodextrins 1 H2 O - D 2 glucose Glucose oxidase

D 2 glucose 1 H2 O 1 O2







- H2 O2 1 D 2 gluconate Peroxidase

2H2 O2 1 p 2 hydroxybenzoic acid 1 4 2 aminoantipyrine




- Quninoneimine dye 1 4H2 O

18.8

Conclusion

There are several enzymes that are used in the detection and estimation of analytes relevant to the dairy industry. Milk is turbid fluid and comprises numerous biomolecules. These molecules invariably interfere with

References

nonenzymatic methods. Enzyme-based analytical methods provide specificity in assay and this has enabled the availability of commercial kits for estimation of cholesterol, ascorbic acid, lactate, lactose, sucrose, glucose, and hydrogen peroxide. Several International organizations such as IDF, ISO, and AOAC have adopted these methods for the estimation of various components in food and dairy products. Even, enzyme reactions can be carried out on paper and this enabled the preparation of paper strip tests for several adulterants. These strips are simple in use and results are available quickly. There will be increasing use of enzyme-based methods for ensuring milk quality and composition of milk and milk products.

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CHAPTER 19

Lactate biosensor for assessing milk microbiological load Gurdeep Rattu1 and P. Murali Krishna2 1

Department of Biotechnology, School of Applied Sciences, Reva University, Bengaluru, Karnataka, India, 2Basic and Applied Science, National Institute of Food Technology Entrepreneurship and Management (NIFTEM) Kundli, Haryana, India

19.1

Introduction

Milk is an important food for much of the world's population, and it is also an excellent growth medium for a variety of microorganisms (Rahman et al., 2016; Rattu & Krishna, 2021). In the case of milk spoiling, numerous microbial metabolites (lactate) are produced, resulting in changes to the milk's odor, taste, texture, and nutritive property. The shelf life of milk is influenced by its initial microbial load, the type and distribution of microorganisms, and the ability of these microbes to grow under storage conditions (Ziyaina et al., 2018). Lactic acid bacteria (LAB) are the primary cause of milk deterioration due to the production of lactic acid. LAB are indigenous microorganisms in raw milk and can withstand the pasteurization process. Bacterial growth leads to fermentation, which produces lactic acid that raises the acidity, thereby curdling of milk. 2-hydroxypropanoic acid, or lactic acid, is an organic acid (C3H6O3 or CH3CHOHCOOH). It can dissociate into its nonvolatile component, lactate, which is an odorless, colorless, and crystalline substance (Hosoya et al., 2001). It is the prevalent acidic constituent of fermented milk and dairy products, sports medicine, bioprocess engineering, and food manufacturing (Nikolaus & Strehlitz, 2008; Rattu et al., 2021). In the fermentation process of dairy products, lactate is utilized as an indicator of quality, freshness, flavor, stability, storage longevity, and bacterial load (Rattu et al., 2021; Rawoof et al., 2021). The detection of spoiled milk relies heavily on the measurement of lactic acid or lactate produced by LAB. In food microbiology, microbial growth on selective media, screening, and serological confirmation is used to detect microbial deterioration (Ziyaina et al., 2020). Conventional methods of lactate qualitative and quantitative detection include; High-performance Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00019-9 © 2023 Elsevier Inc. All rights reserved.

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liquid chromatography (HPLC) (Milagres et al., 2012) and liquid chromatography-mass spectrometry (LC-MS/MS) (Jackson et al., 2017). Although estimation is very accurate and precise with low detection limits but these methods are expensive, time consuming, require confirmation to verify results by the expertise of trained technicians (Rattu et al., 2021). For this reason, it is imperative to investigate new and rapid methods for real-time detection of milk spoiling that are reliable, rapid, and inexpensive. The metabolites (lactate) produced by bacteria in dairy products can be consistently and quickly identified using modern techniques (Fig. 19.1). This chapter examines the sensor technology for detecting milk spoiling, as well as the requirements for their industrial use. To help the dairy business, this chapter provides guidance on the creation of modern and integrated analytical systems.

FIGURE 19.1 Overview of lactate detection methods (conventional and sensors) for milk analysis.

19.2 Lactic acid for assessing milk microbial load

19.2

Lactic acid for assessing milk microbial load

In the process of milk fermentation, microorganisms produce lactate, which has a variety of sensory, texture, flavor, and organoleptic effects on the finished product (Quigley et al., 2013). Typically, cows’ milk contains a significant LAB population that includes Lactococcus (8.2 3 101
1.4 3 104 cfu/mL), Streptococcus (1.41 3 101
1.5 3 104 cfu/mL), Lactobacillus (1.0 3 102
3.2 3 104 cfu/mL), Leuconostoc (9.8 3 101
2.5 3 103 cfu/mL), and Enterococcus spp. (2.57 3 101
1.58 3 103 cfu/mL). A number of other microbes (psychrotrophic) can be present in significant proportions such as Aeromonas, Pseudomonas, and Acinetobacter spp. (Quigley et al., 2013; Raats et al., 2011). Hammer and Hix (1916) found a general relationship between the rise in acidity and the number of organisms present in sterile milk inoculated with LAB. Similar results were found by Gould and Jensen (1944) in random samples of milk which were taken as received at a creamery and allowed to stand at 22 C
24 C until noticeable off-flavors developed (Gould & Jensen, 1944). Velasco and Moats (1959) used four recently developed methods for lactic acid determination to analyze samples of nonfat dry milk of known direct microscopic counts for apparent lactic acid. About 50 spray-dried samples were analyzed in duplicate by the Golden State, Davidson, and Ling procedures. Also, single analyses were run on 25 samples by the ion-exchange method. The reproducibility of each of the four methods was determined by statistical analysis of the data and an estimate was made of their accuracy. Correlation coefficients between the direct microscopic clump counts and the apparent lactic acid present also were calculated for each method. The approximate time required to run a set of samples of each method was recorded and the probable usefulness of lactic acid content in estimating the direct microscopic count was evaluated statistically (Table 19.1). Among all the

Table 19.1 Statistical data for lactic acid analyses by four methods.

Number of samples analyzed Coefficient of variation Standard deviation between duplicates Coefficient of correlation—lactic acid Regression line Time required

Ion-exchange

Ling

Davidson

Golden state

25 3.86% 122

57 11.7% 87

48 18.7% 68.4

50 15.5%

0.828

0.838

0.810

0.844

y 5 0.895x 1 181 8 h for 4 samples

y 5 1.337x 1 774 4 h for 10 samples

y 5 1.229x 1 233 4 h for 8 samples

y 5 1.091x 1 230 6.5 h for 8 samples

Adapted from Velasco, W., & Moats, A. (1959). Relation between lactic acid and direct microscopic counts for bacteria in nonfat dry milk. Journal of Dairy Science. 42 (11),1785
1791.

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methods tested, the ion-exchange method was found to be the most accurate and also the slowest. The Ling method was definitely the fastest. The value of lactic acid content as an estimate of bacteria count was calculated. Thus it is possible to estimate the microbial load in the milk sample by lactic acid concentration in the sample medium (Velasco & Moats, 1959). Mistry and Kosikowski (1985) estimated a correlation between lactic acid production and bacterial count in skim milk. In this research, the objective was to produce highly concentrated ultrafiltered retentates and observe lactic acid production, numbers of viable bacteria, and pH when such retentates were fermented by direct set, frozen concentrated lactic starters. Doubling times for starter bacteria and for lactic acid production in the exponential phase were calculated using the following equation:   1=Td 5 ðlogN 2 logNo Þ= 0:301 3 ðt 2 to Þ

ð19:1Þ

where Td is the doubling time, N is the colony-forming units or lactic acid concentration at time t, No represents the colony-forming units or lactic acid concentration at time to. Specific lactic acid production rates (µmol lactic acid/cfu/min) were calculated using the following equation: Spla 5 ðCt =Nt Þ 3 ðIn2=Td Þ

ð19:2Þ

where Spla is the specific lactic acid production rate, Ct is the lactic acid concentration at time t, Nt is the bacterial population at time t, and Td is the doubling time. Lactic acid fermentation of skim milk and skim milk retentates has been studied at 32 C. In milk retentates, the lactic acid production continued for 11 h, but pH slowed considerably after the first 5 h. The effect of protein concentration on the pH and lactic acid relationship is presented in a three-dimensional plot using values from lactic acid cultures. With rise in total protein more lactic acid was needed to change pH (Fig. 19.2), particularly below pH 5.5. Fast starters had longer growth doubling times than slow starters. This time period increased slightly with higher protein concentration. Maximum lactic acid production averaged 1.2 3 10-6 µmol lactic acid/cfu/min and did not differ between retentates or starters (Mistry & Kosikowski, 1985).

19.3 19.3.1

Methods of detection Analytical conventional techniques

19.3.1.1 High-performance liquid chromatography HPLC is an analytical liquid chromatographic technique used for the purification, identification, and quantification of the different components in a mixture.

19.3 Methods of detection

FIGURE 19.2 Effect of protein concentration in ultrafiltration skim milk retentates on pH change during starter culture growth. Source: Adapted from Mistry, V. V., & Kosikowski, F. V. (1985). Growth of lactic acid bacteria in highly concentrated ultrafiltered skim milk potentates. Journal of Dairy Science, 68(10), 2536
2543. Available from https://doi.org/10.3168/jds.S0022-0302(85)81134-6.

Lactic acid in milk samples can be quantified and validated using ion exclusion
HPLC with a UV detector (Milagres et al., 2012). In both the samples, NaOH neutralized fermented milk and the fermented milk, the lactic acid concentration was measured after the 9-h fermentation period. An analysis of variance (ANOVA) combined with the Tukey test was used to demonstrate that the method used was insensitive to the variations between neutralized and fermented milk. As the peak height rises, the concentration of lactic acid also rises. This method showed good accuracy (97.6%
99.6%) and precision (variation coefficient 7.0%). Limit of detection and quantification for lactic acid were 0.5 and 1.0 mmol/L, tested within the linear range from 0.5 to 25.0 mmol/L (R2 . 0.996) (Milagres et al., 2012). Therefore it has been reported that the proposed method is suitable as a quality control unit in laboratories to monitor the acidity of milk. Biagi et al. (2012) reported conventional reversed-phase procedures (RP-HPLC) for monitoring L-lactate levels. The fast chromatographic method developed with C18 as reversed-phase column and UV
vis detector set at 220 nm for lactate. The sample was eluted at 6.54 min and the detection limit for L-lactate reported as 0.03 mM.

19.3.1.2 Liquid chromatography
mass spectrometry HPLC and a mass spectrometry detection system are used together in the conventional analytical approach known as LC/MS. An extensive range of

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samples can be used for identification, production, and then separation of charged species through LC-MS (Al Kadhi et al., 2017; Hird et al., 2014; Zhang et al., 2017). Lactic acid was separated from the sample using a ZICHILIC guard column under isocratic conditions by 100 mmol/L ammonium acetate/acetonitrile (80:20, v/v) mobile phase (Jackson et al., 2017). A flow rate of 0.4 mL/min was used to maintain a pH of 4.7. Analyses were carried out in negative ion mode of electrospray ionization (ESI) source and by extracting the target mass (m/z): 89.1 for lactic acid. Linear range of calibration was 2.5
2500 g/mL with a detection limit of 0.75 µM. Moldoveanu et al. (2018) reported the LC-MS method for simultaneously measuring lactic acid and other organic acids such as malic, oxalic, fumaric, pyruvic, and trihydroxy butanoic based on mass difference. Separation was performed using a mobile phase composed of 5% methanol and 95% water and a Synergy Hydro-RP column under isocratic conditions with a flow rate of 0.06 mL/ min and the column's temperature was kept at 20 C. The calibration range of lactic acid was 0.3
50 µg/mL. The method was successfully applied to measure a variety of organic acids (Moldoveanu et al., 2018).

19.3.2

Lactate biosensors

The most common sensors used in milk analysis are electrochemical, optical, and mass-sensitive. One sort of biosensor, which converts a biological response into an electrical signal, can be used to assess the concentration of organic compounds, either directly or indirectly. The oxidation and reduction of ATP, NAD/NADH, and the generation of Krebs cycle compounds are some of the most common applications. In order to capture target analytes, amplify signals, and fabricate sensors, biosensors take advantage of breakthroughs in micro- and nanotechnology. These sensors have a number of advantages including low cost, ease of use, facile, and rapid analysis, depending on the sensor transducer type (Mortari & Lorenzelli, 2014; Nikolaus & Strehlitz, 2008; Rattu et al., 2021; Ziyaina et al., 2020).

19.3.2.1 Electrochemical methods Microbial metabolism leads to the breakdown of macromolecules (protein, carbohydrates, lipids) in a food matrix such as milk. During the breakdown, hydrolytic reactions frequently produce charged molecules, which might alter the milk's electrical conductivity. In order to monitor and assess the load of bacteria in the media, these changes can be observed and quantified (McMeekin, 2003). For estimating the shelf life of milk by monitoring changes in the growth medium's impedance and conductivity, Ziyaina electrochemical method is used in dairy microbiology. A loss in quality of 1.0 3 106 cfu/mL has been determined using these methods in cheese and raw and pasteurized milk (Ziyaina, Rasco, Coffey, Mattinson, et al., 2019a).

19.3 Methods of detection

In cow milk, lactate acts as a biomarker for any number of microbial contamination levels. An amperometric biosensor was reported using conducting polymer, poly-5,20 -50 ,200 -terthiophene-30 -carboxylic acid (pTTCA), and multiwall carbon nanotube (MWNT) composite fabricated on a gold electrode (Rahman et al., 2009). The pTTCA/MWNT composite film was then used to immobilize lactate dehydrogenase (LDH) and the oxidized form of nicotinamide adenine dinucleotide (NAD1). The detection signal was amplified by the assembly of pTTCA/MWNT onto which LDH was immobilized by the covalent bond between the enzyme's amino groups and carboxylic acid groups of the composite film, which stabilized the enzyme. The calibration plot was linear from 5 to 90 µM (R2 5 0.995) and sensitivity was 0.0106 µA/µM at a detection limit of 1 µM. L-lactate concentration in commercial milk samples was accurately determined using the developed sensor (Rahman et al., 2009). A novel nickel-metal organic framework (Ni-MOF)-modified platinum electrode was developed for the detection of lactate in cow milk samples (Manivel et al., 2018). The Ni-MOF with a mean length of 12 nanometers and an average width of 0.7 nanometers was synthesized using a mild solution approach. In order to design a potentiostatic three-electrode configuration device, Ni-MOF-modified platinum, platinum wire, and Ag/AgCl electrodes were used as working, counter, and reference electrodes, respectively. Scan rates of 0.01 V/s were used to record cyclic voltammograms (CVs) of the bare Pt and Pt/Ni-MOF electrodes in the absence and presence of 0.7 mM lactate as shown in Fig. 19.3 and Fig. 19.4. In the absence of lactate, no detectable redox reaction was observed at bare Pt electrode, suggesting that bare Pt electrode cannot oxidize lactate to pyruvate at applied potential range. The CV of the bare Pt electrode in the presence of 0.7 mM lactate provides additional evidence of lactate oxidation inefficiency because no significant redox peak is observed in the applied potential range. The

FIGURE 19.3 A novel electrochemical method towards the detection of lactate in milk samples based on the fabrication of Pt/Ni-MOF electrode. Source: Adapted from Manivel, P., Suryanarayanan, V., Nesakumar, N., Velayutham, D., Madasamy, K., Kathiresan, M., Kulandaisamy, A. J., & Rayappan, J. B. B. (2018). A novel electrochemical sensor based on a nickel-metal organic framework for efficient electrocatalytic oxidation and rapid detection of lactate. New Journal of Chemistry, 42(14), 11839
11846. Available from https://doi.org/10.1039/C8NJ02118J.

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(iv)

(iii)

200

(ii)

150

60 40

Current (PA)

Current (P PA)

478

(i)

20 0

100 50 0.09 Vs-1

0

-20 -0.6

0.01 Vs-1

-50

-40 -0.4

-0.2

0.0

Potential (V) vs. Ag/AgCl

0.2

-0.8

-0.6

-0.4

-0.2

0.0

Potential (V) vs. Ag/AgCl

FIGURE 19.4 Cyclic voltammograms reported for (i) bare Pt without 0.7 mM of lactate, (ii) bare Pt with 0.7 mM of lactate, (iii) Pt/Ni-MOF without 0.7 mM of lactate, (iv) Pt/Ni-MOF with 0.7 mM of lactate, and Pt/Ni-MOF electrode at different scan rates (0.01
0.09 Vs) in the presence of 0.7 mM of lactate. Source: Adapted from Manivel, P., Suryanarayanan, V., Nesakumar, N., Velayutham, D., Madasamy, K., Kathiresan, M., Kulandaisamy, A. J., & Rayappan, J. B. B. (2018). A novel electrochemical sensor based on a nickel-metal organic framework for efficient electrocatalytic oxidation and rapid detection of lactate. New Journal of Chemistry, 42(14), 11839
11846. Available from https://doi.org/10.1039/C8NJ02118J.

developed Ni-MOF-modified platinum electrodes have good lactate sensitivities (106.61 and 29.533 µA/mM) over wide linear ranges of 0.01
0.9 and 1
4 mM, with correlation coefficient of R2 5 0.99, a detection limit of 5 µM, repeatability of 0.57% RSD, and stability of 95.3% over 20 days. There are no potential interfering species in the proposed electrode, so lactate in cow milk samples can be easily quantified (Manivel et al., 2018). Recently, Carrillo-Gómez and coworkers reported detection of bacterial contamination (Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica) in the pasteurized milk samples using multisensory methods (electronic nose, electronic tongue) as shown in Fig. 19.5. Each of the bacterial suspensions was tested by E-nose and E-tongue and measurements were repeated 10 times. It was observed that both the system obtained stable and adequate results with 94.7%
98.7% success rates. These results concluded that the developed electrochemical device can be used for the real-time monitoring of bacterial load in commercial sample testing (Carrillo-Gómez et al., 2021). A limitation associated with impedance and conductivity measurements is that rather high levels of bacterial growth (6.0
7.0 log10 cfu/mL) are needed to stimulate electrical signal in order to assess milk microbial load or spoilage. To identify specific microbes in milk, the Bactometer is one instrument that employs these concepts to detect specific microbe in milk (Kowalik & Ziajka, 2005), milk powder (Neaves et al., 1988), and yogurt (Schaller et al., 1998) and to monitor the quality of raw and pasteurized milk.

19.3 Methods of detection

FIGURE 19.5 Schematic illustration of monitoring of bacterial load in milk samples by electrochemical multisensory methods. Source: Adapted from Carrillo-Gómez, J. K., Durán Acevedo, C. M., & García-Rico, R. O. (2021). Detection of the bacteria concentration level in pasteurized milk by using two different artificial multisensory methods. Sensing and Bio-Sensing Research, 33, 100428. Available from https://doi.org/10.1016/j.sbsr.2021.100428.

19.3.2.2 Optical spectroscopic methods Optical sensing methods show many improvements over electrochemical transduction methods. Optical transducers provide rapid, facile, and low-cost sensing for selective and sensitive detection (Rattu et al., 2021). Colorimetry, UV
visible near-infrared (NIR) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy are among the most popular spectroscopic techniques (Borshchevskaya et al., 2016; P˘aucean et al., 2017; Rahman et al., 2016; Rattu et al., 2021; Ziyaina, Rasco, Coffey, Mattinson, et al., 2019b). Optical transduction methods for assessing milk microbial load are less established as compared to electrochemical techniques, presenting at the same time new possibilities to investigate potential methods to develop for various applications. In the next section, an overview of the existing reports in optical sensors is presented.

19.3.2.2.1 UV
vis spectroscopy An analysis approach that relies on spectrophotometers is extremely selective and sensitive. Because of their ease of use and accessibility, they are commonly

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employed in laboratories (Borshchevskaya et al., 2016). Additionally, UV
vis spectroscopy can be used to identify specific cell wall lipid or protein characteristics, or ketone metabolites. Borshchevskaya et al. (2016) developed an easy-to-use spectrometric technique for the detection of lactic acid in food and biological liquids. Catalysis of L-lactate with iron (III) chloride is used to detect spectrometric response at 390 nm due to the formation of ferric lactate ((CH3CHCOO)3Fe) as given by the following equatio: FeCl3 1 3CH3 CHOHCOOH 5 ðCH3 CHOHCOOÞ3 1 3HCl

ð19:3Þ

The calibration curve of lactic acid reported at 0.3
10 g/L having a correlation coefficient of 0.9999 (Borshchevskaya et al., 2016).

19.3.2.2.2 Colorimetric The colorimetric sensors have been established as efficient methods due to its high sensitivity, easy fabrication, rapid detection, and naked-eye or visual color change reaction phenomenon. Nanoparticles are widely used in colorimetric detection as an efficient signal transducer element because of their unique optical properties and the ease of surface modification (Ziyaina, Rasco, Coffey, Mattinson, et al., 2019a). The most common application of this technique in food analysis is to detect temperature changes during food storage. Temperature sensors provide the basic link for the development of colorimetric sensors in smart packaging, such as those that detect the formation of microbial metabolites indicative of flavor development or food spoilage. Staphylococcus aureus and E. coli can be detected in culture media and apple juice using a colorimetric biosensor based on polydiacetylene vesicles trapped in cellulose strips (Pires et al., 2011). This colorimetric sensor was built using 10,12-pentacosadyinoic acid (PCDA) and N-[(2-tetradecanamide) ethyl]-ribonamide (TDER) vesicles. The colorimetric change of DER/PCDA vesicles occurred when they were exposed to bacterial supinate (Pires et al., 2011). Cavallo et al. (2014) developed a colorimetric sensor for milk deterioration using modified polypropylene film with methylene blue (MB) dye. The color of the dye is reduced to colorless as a result of the growth of microorganisms, as shown in Fig. 19.6. Because of its short response time (300 cells/hour) and low detection limit (104
107 cfu/mL), this particular MB reaction is well suited for use in the detection of spoilage in liquid milk at various stages of decomposition. The developed MB-based sensors can also be used in smart packaging (Cavallo et al., 2014).

19.3.2.2.3 Fourier transform infrared spectroscopy FTIR spectroscopy is a facile and noninvasive method for testing the quality of food. FTIR can be used to track chemical changes during hydrolysis of food

19.3 Methods of detection

FIGURE 19.6 Oxidized and reduced methylene blue species present in redox reactions for spoilage detection in milk samples. Source: Adapted from Cavallo, J. A., Strumia, M. C., & Gomez, C. G. (2014). Preparation of a milk spoilage indicator adsorbed to a modified polypropylene film as an attempt to build a smart packaging. Journal of Food Engineering, 136, 48
55. Available from https://doi.org/10.1016/j.jfoodeng.2014.03.021.

components, metabolite production from microbes. This provides an estimate of the microbial load, either qualitatively or quantitatively (Lu et al., 2011). Al-Qadiri and coworkers reported FTIR combined with visible- and shortwavelength NIR spectroscopy that can be used to detect deterioration in pasteurized skim milk. At three different storage temperatures, they monitored the deterioration of pasteurized skim milk using visible and short wavelengths NIR. In order to determine the shelf life of milk, they compared spectral properties to the total number of aerobic plates and pH. Clustering and segregation of milk due to the formation of microbial metabolites, the use of glucose and protein, may all be monitored using principal component analysis, which can be used to measure these changes in milk stored for 30 min (Al-Qadiri et al., 2008). P˘aucean and co-workers reported the use of FT-IR spectroscopy to monitor lactic acid levels in various systems (culture media and food). Analysis of lactic acid produced by Lactobacillus plantarum ATCC 8014 and Lactobacillus casei ATCC 393 by FTIR coupled with multivariate statistical analysis was the focus of this study. Lactic acid spectra after four hours of fermentation were depicted in Fig. 19.7. Common absorption bands appeared in all of the spectra. In the C~O stretching band at 1730 cm
1 carboxylic acid groups are identified. The OsH stretching of the acid component is responsible for the peaks between 2500
3000 cm
1 and the bands in the region 1200
950 cm
1 observed due to the CsC and CsO functional groups (P˘aucean et al., 2017). The coefficients of determination (R2) between the predicted and the reference values were 0.986 and 0.965. Results confirmed the detection efficiency of FTIR spectroscopy as a facile rapid for monitoring of lactic acid (P˘aucean et al., 2017).

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FIGURE 19.7 (A) FTIR spectrum for standard lactic acid and (B) FTIR spectra for fermenting media. FTIR, Fourier transform infrared. Source: Adapted ˘ from Paucean, A., Vodnar, D. C., Muresan, ¸ V., Fetea, F., Ranga, F., Man, S. M., Muste, S., & Socaciu, C. (2017). Monitoring lactic acid concentrations by infrared spectroscopy: A new developed method for Lactobacillus fermenting media with potential food applications. Acta Alimentaria, 46(4), 420
427. Available from https://doi.org/10.1556/066.2017.0003.

19.3 Methods of detection

19.3.3

Nanotechnology applications in sensors

With the recent advancements in nanotechnology, the use of nanomaterials in analytical chemistry has received a lot of attention (Bollella et al., 2019; Huang et al., 2020; Naresh & Lee, 2021). At the nanoscale, with the reduction in size, surface energy and surface area of materials increase tremendously offers excellent optical, electrical, catalytic properties for sensor development (Refer Fig. 19.8). Lactate sensors have been investigated using a variety of nanocomposites, including metal-oxide, polymeric, and composite materials. This is due to the absorption of reactants onto nanoparticle surfaces, as well as changes in the underlying chemistry of the nanoparticle state for milk sample analysis (Lakade et al., 2017; Rattu & Krishna, 2021; Yata, 2019). Colorimetric nanosensor based on cysteine modified silver nanoparticles (AgNPs) was reported to detect milk spoilage (Lakade et al., 2017). The

FIGURE 19.8 Overview of the functional properties of nanoparticles as compared to bulk.

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absorbance of cysteine biofunctionalized nanoparticles at 394 nm steadily dropped as lactic acid concentration increased, but the absorbance at 600 nm increased. As milk deterioration progresses, the color of the milk changes from yellow to dark crimson, as shown in Fig. 19.9. Ziyaina and coworkers designed colorimetric sensor using silicon dioxide (SiO2) coated with Schiff's reagent to detect volatile organic compounds (VOCs) generated by the growth of spoilage bacteria in pasteurized whole milk maintained at different storage temperatures. Pasteurized whole milk contains microorganisms that produce VOCs, which interact with Schiff's base adsorbed to silica and change the milk's color from pink to purple (Ziyaina, Rasco, Coffey, Ünlü, et al., 2019b). Boronic acids are employed as receptor molecules for various sensing applications (Pizer & Selzer, 1984; Zaryanov et al., 2017). L-lactate sensors rely on 3-aminophenylboronic acid (3-APBA) receptor molecules, which have been proven to be particularly effective at adhering to hydroxy acids when immobilized on a metal surface (Sartain et al., 2008). Recently, a label-free optical sensor based on gold nanoparticles (AuNPs) functionalized with boronic acid was reported for lactate quantification in commercial milk samples for assaying microbiological load (Rattu & Krishna, 2021). L-lactate binds to the trigonal form of 3-APBA, resulting in the formation of a tetrahedral boronate complex stabilized by the AuNPs that causes the aggregationinduced blue shift in the UV
vis absorption wavelength (300 to 288 nm) at pH 7 (Fig. 19.10). Indian milk powder Nestle-Lactogen was used in the lactate sensing standardization experiment. Increased UV
vis absorption peak intensity was seen when milk samples were spiked with up to 20 mM of L-Lactate. The sensor showed the LOD of 1.1 mM (Rattu & Krishna, 2021). Furthermore, this sensor approach work can be used in conjunction with a smartphone to construct an on-site food and pharma device application.

FIGURE 19.9 Different colors produced in cysteine modified silver nanoparticles with increasing lactic acid concentration in milk spoilage. Source: Adapted from Lakade, A. J., Sundar, K., & Shetty, P. H. (2017). Nanomaterial-based sensor for the detection of milk spoilage. LWT - Food Science and Technology, 75, 702
709. Available from https://doi.org/10.1016/j.lwt.2016.10.031.

19.3 Methods of detection

FIGURE 19.10 Milk samples analysis for L-lactate content by AuNPs functionalized with boronic acid using UV
vis spectroscopy. Source: Adapted from Rattu, G., Khansili, N., Maurya, V. K., & Krishna, P. M. (2021). Lactate detection sensors for food, clinical and biological applications: A review. Environmental Chemistry Letters, 19(2), 1135
1152. Available from https://doi.org/10.1007/s10311-020-01106-6.

Table 19.2 Lactate biosensor for assessing milk microbiological load and metabolites. Method of detection

Sensing material

Electrochemical Nanomaterial based optical Optical Electrochemical Optical Solid-phase microextraction Optical

Nanomaterialbased optical

Detection principle

Linear range (mM)

LOD

Application

Reference(s)

pTTCA/MWNT composite film Cys-Ag NPs

Amperometric

0.005
0.09

1 µM

Milk samples

Colorimetric

5
100

Milk spoilage

Iron(III)chloride @lactate Pt/Ni-MOF

UV
vis spectroscopy

0.3
10 g/L

5 mM -

Voltametric

0.01
4

5 µM

Milk samples

SPR-based optical fiber SiO2 NPs

UV
vis spectroscopy

0
10

-

Gas chromatography

-

-

FT-NIR@ lactate

Near infrared spectroscopy, FTNIR UV
vis spectrometric

4.5
13.8

0.5 mM

Dairy, baby food, tomato juices, purees Milk, dairy products and food products Food and clinical samples

Rahman et al. (2009) Lakade et al. (2017) Borshchevskaya et al. (2016) Manivel et al. (2018) Kassal et al. (2018) Ziyaina et al. (2019a) Baishya et al. (2021)

1
20

1.1 mM

Milk, milk powder, Milk pasteurized

Rattu et al. (2021)

3-APBA@AuNPs

Dairy food samples

3-APBA@AuNPs, 3-aminophenyl boronic acid functionalized with gold nanoparticles; Cys-AgNPs, cysteine-modified silver nanoparticles; FTNIR; Fourier transform near-infrared spectroscopy; LDH, lactate dehydrogenase; LOx, lactate oxidase; pTTCA/MWNT, 5,20 -50 ,20 0 -terthiophene-30 -carboxylic and multiwall carbon nanotube; Pt/Ni-MOF, nickel-metal organic framework modified platinum electrode; SiO2 NPs, silicon dioxide nanoparticles; SPR, surface plasmon resonance.

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Sensors based on nanotechnology have the potential to identify spoilage compounds quickly and accurately for assessing microbial load. The development of detection systems for shelf life expiration dates (best-before dates) using these colorimetric sensors could pave the way for significant enhancements in quality assurance. Table 19.2 summarized the list of biosensing technologies for identifying bacteria or metabolites in milk samples. The use of nanotechnology in sensors for food packaging still has some intrinsic limitations, such as low resolution or sensitivity in response to changes in bacterial load at sufficiently low numbers, poor responsiveness of sensors when refrigerated, among other things.

19.4

Conclusion and future prospective

Microbial contamination has been detected using a variety of new methods in recent years. These methods are easier to use and faster than older ways, but it is also more expensive and time-consuming. Assessing microbial load particularly those associated with milk spoilage was the focus of this work. These innovations have the potential to improve the quality and safety of milk samples. For public health, they offer better food processing, storage, handling, and marketing. As a first step in the creation of future quality control systems for the dairy industry, these rapid approaches provide numerous advantages. These lactate detection methods can be paired with a smartphone app (colorassist/Color Picker/Colormeter) that has been obtained online. Both spectrophotometry and colorimetry can be used on smartphones with good built-in cameras that have high pixel values. As a result, these methods can be applied to the development of tiny mobile sensors and gateway devices for the dairy food industry.

Acknowledgment This research article was supported by The National Institute of Food Technology Entrepreneurship and Management (NIFTEM), set up by the Ministry of Food Processing Industries (MOFPI), Government of India, Kundli, Sonipat district, Haryana, under Delhi NCR.

Conflicts of interest The authors declare no conflict of interest, financial or otherwise.

Ethical approval Ethical approval was not required for this work.

References

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CHAPTER 20

Enzymes for cleaning-in-place in the dairy industry Angela Boyce and Gary Walsh Department of Chemical Sciences and Bernal Institute, University of Limerick, Limerick, Ireland

20.1 Introduction: fouling and cleaning-in-place in the dairy industry Fouling, the accumulation of deposits of milk solids on surfaces, occurs under normal processing conditions in the dairy industry. While milk will adhere to unheated surfaces such as pipelines and tanks, problematic and difficult to remove fouling occurs where milk is in contact with heated surfaces during thermal processing, for example, in heat exchangers used for pasteurization. Under these conditions (70 C 110 C), a voluminous fouling deposit (commonly referred to as Type A) composed of 50% 70% protein, 30% 50% mineral, and 4% 8% fat forms due to heat-induced protein denaturation, the formation of whey protein aggregates, and reduced solubility of milk salts leading to precipitation of calcium phosphate (Changani et al., 1997; Fryer et al., 2006; Huppertz & Nieuwenhuijse, 2022). The fouling deposit may also contain trace amounts of lactose which may be conjugated to the protein due to Maillard reaction (Huppertz & Nieuwenhuijse, 2022). Fouling deposits obstruct product flow, increase pressure drop, and decrease heat transfer efficiency, limiting processing run times and potentially leading to a deterioration in product quality and safety (Bansal & Chen, 2006; Prakash et al., 2005). These fouling deposits, as well as milk and dairy products in general, provide favorable conditions for microbial growth (Bansal & Chen, 2006; Gopal et al., 2015). The formation of biofilms (aggregations of bacterial cells attached to a surface) is a major concern in dairy processing as biofilms are potential sources of product contamination resulting in product spoilage and food safety issues and are more challenging to eliminate than planktonic cells (Kumari & Sarkar, 2016, 2018; Teh et al., 2014). Residual fouling not removed during cleaning can protect bacteria during sanitization and reduce the effectiveness of sanitization (Reinemann, 2003). 491 Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00020-5 © 2023 Elsevier Inc. All rights reserved.

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The removal of fouling by regular and effective cleaning is therefore essential to avoid operational problems, maintain production efficiency, ensure food quality, safety, and shelf life, and avoid economic loss. This is undertaken by automated cleaning-in-place (CIP) procedures during which cleaning solutions are circulated through the plant or sprayed on surfaces without dismantling the equipment with removal of fouling achieved mainly via chemical and also thermal and mechanical actions (Chisti, 2000; Reinemann, 2003). While the exact conditions vary, CIP is typically undertaken as outlined in Fig. 20.1 by circulation of sodium hydroxide solution or formulated caustic detergent at 70 C 80 C to remove organic deposits followed by acid circulation for removal of mineral deposits (Bylund, 1995). Conventional chemical-based CIP, involving high temperatures, biologically nondegradable cleaning agents, and significant water consumption, is a source of environmental concern. Cleaning represents 30% of energy use in dairy processing, makes a significant contribution to wastewater volume, and shows the highest proportion of chemicals used, some of which have a negative effect on the environment and have health and safety

FIGURE 20.1 Overview of a typical cleaning-in-place procedure undertaken in the dairy industry. The exact conditions and cleaning regime will vary from plant to plant. Source: Details from Bylund, 1995; Reinemann, 2003; Fryer et al., 2006; Fryer and Asteriadou, 2009; and Chisti, 2000.

20.2 Industrial enzymes and their use for cleaning-in-place in the dairy industry

implications (Boyce et al., 2010; Danalewich et al., 1998; Eide et al., 2003; Fryer et al., 2006; Grasshoff, 2005; Kushwaha et al., 2011; Wildbrett, 2003). Alkali cleaning solutions result in high pH wastewater, necessitating neutralization prior to treatment which contributes to high salt concentrations (Grasshoff, 2005; Stasinakis et al., 2022). Several recent studies examining the environmental impact of the dairy processing industry have reported energy use and water consumption as areas requiring attention (Finnegan, Clifford, et al., 2018; Finnegan, Goggins, et al., 2018; Üçtu˘g, 2019) with many highlighting the contribution and negative environmental effect of CIP (Finnegan, Clifford, et al., 2018; Stasinakis et al., 2022; Zouaghi et al., 2019). Moreover, it is generally considered that current CIP procedures using caustic and acid vary in terms of their efficiency in relation to biofilm removal, necessitating the assessment of alternative means to achieve more satisfactory biofilm removal (Gopal et al., 2015; Kumar et al., 2021; Kumari & Sarkar, 2016; Meireles et al., 2016).

20.2 Industrial enzymes and their use for cleaning-inplace in the dairy industry The use of enzymes for cleaning is well established in other industries especially in laundry and dishwasher detergents and also for cleaning membranes, contact lenses, and surgical instruments (Boyce et al., 2010). Within the dairy industry, enzyme-based cleaning of membranes to maintain membrane permeability and selectivity is commonly undertaken and is considered advantageous in terms of reduced rinsing and wastewater volumes and decreased energy costs and chemical usage (D’Souza & Mawson, 2005). In comparison to chemical-based approaches, enzymatic cleaning is also considered milder and less aggressive which is favorable to extend membrane lifetime, particularly for the more sensitive membranes used for some applications (Argüello et al., 2005; Grasshoff, 2005). Apart from membrane cleaning and a proteolytic enzyme product (P3-Paradigm) suitable for cleaning nonheated surfaces at low temperatures (50 C 55 C) (Pottchoff & Serve, 1997), little attention was traditionally given to the use of enzymes on a wider scale for CIP in dairy processing. This has changed in recent years mainly due to concerns around the environmental implications and energy consumption of conventional chemical-based CIP (Fryer et al., 2006; Reinemann, 2003). As a result, the use of enzymes to remove the more problematic fouling associated with heated surfaces has gained increasing attention, driven by the potential to overcome some of the disadvantages of chemical cleaning outlined above. Enzymes are biodegradable and work at low temperatures relative to chemicalbased CIP, resulting in less energy use and associated costs (Olsen & Falholt,

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1998). The wastewater generated has a lower pH and is more compatible with subsequent treatment processes reducing the requirement for pH neutralization (Graßhoff, 2002). In addition to reducing chemical usage, the use of enzymes is favorable from a health and safety standpoint as it eliminates the storage and handling of corrosive and hazardous chemicals and the equipment being cleaned is exposed to less thermal and chemical stress (Pottchoff & Serve, 1997). Enzyme cleaning has also been associated with less rinsing and hence decreased water consumption and wastewater volumes (D’Souza & Mawson, 2005). The enzymes of interest for CIP are ubiquitous in nature being produced by eukaryotes and prokaryotes and are already used in various industrial applications, including the food and detergent industries. Industrial enzymes are predominantly of microbial origin (B60% from fungi, 24% from bacteria, and 4% from yeast) due to the ease and speed of production and downstream processing, high yields, lower investment requirements, favorable enzyme stability, and ease of optimization and genetic manuipulation (Fasim et al., 2021; Matkawala et al., 2021; Solanki et al., 2021). Such enzymes are generally produced in large quantities via submerged or solid-state fermentation using the native producing microorganism or in many cases by recombinant means to achieve higher enzyme yields. Microbial strains used for the production of industrial enzymes are usually classified as GRAS (generally recognized as safe) (Paul et al., 2021). After fermentation, cell separation, enzyme concentration, and possibly some purification are undertaken. This is followed by enzyme formulation (in liquid or solid form) with emphasis on long-term enzyme stability and compatibility with the intended application (Arbige et al., 2019). The global market for industrial enzymes is expected to reach US$7.0 billion by 2023 (Fasim et al., 2021) and the main producers include Novozymes, DuPont, BASF, and DSM (Arbige et al., 2019). Several studies, outlined in Section 20.3, have examined the potential of various enzyme cleaning solutions to remove both the Type A fouling deposits and biofilms found in the dairy industry. The composition of Type A fouling was outlined in Section 20.1. In biofilms, the microorganisms (which may be viable or nonviable, single species or multispecies) are embedded in EPS (extracellular polymeric substances) composed of polysaccharides, proteins, lipids, phospholipids, and nucleic acids. The exact composition of the (selfproduced) EPS varies depending on the bacterial species and growth conditions (Anand et al., 2014; Flint et al., 2020) with proteins and polysaccharides reported to account for 75% 89% of the EPS (Simões et al., 2010). The EPS is known to play a role in the adhesion of microorganisms, including attachment to surfaces, and behaves like a protective shield enabling the bacteria to resist environmental stresses, including cleaning and disinfection (Anand et al., 2014; Boels, 2011). Biofilm bacterial cells are

20.2 Industrial enzymes and their use for cleaning-in-place in the dairy industry

100 1000 times more resistant to cleaning and sanitizing agents than planktonic cells (Fysun et al., 2019) which may not be able to penetrate into the biofilm matrix affecting only microbes on the outer surface (Anand et al., 2014; Meireles et al., 2016). Ideally, cleaning should disrupt the EPS to facilitate access of disinfectants, otherwise any residual microorganisms will be able to continue their growth or redeposit elsewhere producing a biofilm (Boels, 2011; Simões et al., 2010). The enzymes studied to date in relation to cleaning in the dairy industry are predominantly proteases and to a lesser extent lipases and amylases. Proteases (EC 3.4.-.-), also referred to as proteinases or peptidases, are proteolytic enzymes that catalyze the hydrolytic cleavage of peptide bonds in protein chains. Among the classification systems reported, proteases may be divided into two major groups, endopeptidases and exopeptidases. Endopeptidases, which act on peptide bonds found internally in the protein chain generating shorter peptides are considered more suitable for cleaning applications than exopeptidases which act on peptide bonds at the chain termini. Such endopeptidases are further classified based on their mechanism of action and of the classes of serine proteases (EC 3.4.21) and in particular, the subtilisin subclass are widely used for cleaning in the detergent industry due to their high stability, broad substrate specificity, and high level secretion by many microorganisms (Contesini et al., 2018; Matkawala et al., 2021; Solanki et al., 2021). Serine proteases have an essential nucleophilic serine residue in their active site, which together with aspartic acid and histidine residues makes up the catalytic triad (Gupta et al., 2002; Vojcic et al., 2015). Alkaline serine proteases are generally optimally active at pH 7 11 and 30 C 80 C, are reasonably stable over a wide pH and temperature range, and display activity and stability in the presence of various detergents, surfactants, organic solvents, and alcohols as well as broad substrate specificity on proteinaceous substrates. While several microbial species are capable of producing alkaline serine proteases, to date only a few are established as commercial producers, in particular various Bacillus species, with industrial applications in detergent, food, leather, baking, pharmaceutical, and animal feed industries. In 2019 the global protease market was US$ 2.76 billion with a compound annual growth rate of almost 6.1% forecasted for 2019 24 (Matkawala et al., 2021). Proteases account for 60% of global enzyme sales with detergent proteases representing 20% of global enzyme sales and 30%-40% of global enzyme revenues (Matkawala et al., 2021; Solanki et al., 2021). Lipases (EC 3.1.1.3), also referred to as triacylglycerol acyl hydrolases, catalyze the hydrolysis of triglycerides (in an oil water interface) producing more water-soluble mono- and diglycerides, fatty acids, and glycerol. On the basis of positional specificity, lipases may be classified as nonspecific, 1,3-specific, or fatty acid specific. Lipases have a conserved pentapeptide

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sequence Gly-X-Ser-X-Gly containing the active site serine residue and display a catalytic triad somewhat similar to serine hydrolases. Common microbial sources of lipase include bacteria (Bacillus sp., Pseudomonas sp., Staphylococcus sp., Burkholderia sp.), fungi (Thermomyces lanuginosus, Rhizopus oryzae, and Aspergillus niger), and yeast (Candida antarctica and Candida rugosa). Recently studied lipases reportedly displayed optimal activity from 15 C 70 C (generally 37 C 60 C) and pH 5.0 10.8 (generally pH 8 9). One of the major industrial applications of lipase is in the laundry and dishwash detergent industry (32% of total lipase sales), where lipases with low substrate specificity are desirable to hydrolyze lipid-based stains of various composition. Additional applications include the food, pharmaceutical, leather, cosmetics, pulp and paper, biodiesel, and chemical industries. Lipases represent the third most used enzyme class next to proteases and amylases (Javed et al., 2018; Yao et al., 2021). Amylases or amylolytic enzymes are starch hydrolyzing enzymes. α-amylase (EC 3.2.1.1), also known as α-1,4-glucan-4-glucanohydrolase, is the most commonly used and is an endo-acting enzyme that cleaves internal α-1,4-Dglucosidic bonds linking the glucosyl residues in starch and other α-glucans in a random manner producing oligosaccharides of various length. The products produced (glucose, short oligosaccharides, limit dextrin, maltotriose, maltose) have increased solubility. In the hydrolysis of starch (composed of unbranched amylose and branched amylopectin), α-amylases are active only on α-1,4-glucosidic linkages and cannot hydrolyze the α-1,6-glucosidic linkages found at the branch points of amylopectin. α-Amylases are metalloenzymes, requiring metals such as calcium for stabilization. The α-amylases used industrially are from microbial sources, particularly Bacillus and Aspergillus species and have application ranges of 15 C 100 C and pH 6 11 with some more suitable in the lower pH range of 4 8. Bacterial α-amylases are usually resistant to high temperatures. α-Amylases are used widely in the detergent industry (90% of all liquid detergents) to facilitate the removal of starch-containing stains and to prevent swollen starch from adhering to surfaces. Additional industrial applications of lipase include food, textile, paper, biofuel, and pharmaceuticals with α-amylase accounting for about 25% 30% of the global enzyme market (Farias et al., 2021; Lahiri et al., 2021; Olsen & Falholt, 1998; Paul et al., 2021).

20.3 Reported studies on the effectiveness of enzymes for cleaning-in-place in the dairy industry 20.3.1

Removal of Type A fouling deposits

Several studies have been reported (Table 20.1) examining the potential of various enzyme cleaning solutions for CIP in dairy processing. These studies

Table 20.1 Published studies assessing the use of enzymes for removal of fouling deposits (Type A) in the dairy industry. Enzymes assessed from (1) Novozymes and (2) Genencor. Enzyme(s)

Foulant

Cleaning conditions

Analysis

Main findings

References

8 commercial enzymes Proteases: Alcalase1, Savinase1, Esperase1, Purafect L2, Properase L2, Flavourzyme1, Neutrase1 Protease/lipase: CIPzyme1

Milk fouling deposit on a lab heat exchanger Field test: milk pasteurizer

Visual examination

Lab scale: Experimentally generated milk fouling deposit on stainless steel Preliminary process scale: pasteurizer after operation at 80 C for 6.75 8.5 h

Lab scale: deposit-free surfaces observed with Esperase, Properase L, and Savinase. Optimum conditions: Esperase (0.05%; pH 12; 60 C), Properase L (0.05%; pH 10; 50 C) Savinase (0.025%, pH 9.5; 55 C) Field test: deposit-free surfaces observed upon opening of plate heat exchanger; no indication of microbial contamination. Lab scale: Initial assessment of proteases: 6 of the 8 proteases visibly clean surfaces after cleaning at 40 C, 50 C, or 60 C and 0% 0.5% residual organic matter and 0 1.12 mg protein detected. Performance of several proteases at 40 C comparable to 1% (w/v) NaOH at 60 C. Further assessments undertaken on 3 most suitable proteases including confirmation of satisfactory cleaning by LSCM. Incorporation of lipase activity in the protease cleaning solution did not significantly improve cleaning Preliminary process scale: No issues maintaining milk temperature at 80 C during subsequent production run indicating satisfactory cleaning.

Graßhoff (2002)

10 commercial enzymes Proteases: Liquanase 2.5L1, Esperase 8.0L1, Alcalase 3.0T1, Savinase 6.0T1, Protex 6L2, Protex 30L2, Protex 40L2, Protex 89L2 Lipases: Lipolase 100T1 Lipex 100L1

Lab scale: acid treatment (15 min; 0.5% nitric acid; 60 C) followed by enzyme (including NaOH/KOH for pH adjustment and a surfactant) 45 min; initial assessment 25 C 75 C, pH 7 12 Field test: 15 min acid wash at 55 C followed by enzyme solution (Savinase, surface active agents, NaOH) pH 9 9.5, 55 C 60 C, 45 min. Initial assessment of proteases: pH 10 11.3, 40 C 60 C, 0.05 units/mL; 60 min compared to 1% (w/w) NaOH at 40 C 60 C Further assessment of 3 most suitable proteases: pH 10 10.5; 40 C; 0.025 0.05 units/mL; 60 min Lipases: 1% (w/v) alone or in combination with proteases; 40 C, pH 10 10.5 Preliminary process scale: Single protease product; 45 C 50 C; 60 min.

Lab scale: quantification of residual organic matter and protein after cleaning, laser scanning confocal microscopy (LSCM)

Boyce et al. (2010)

Continued

Table 20.1 Published studies assessing the use of enzymes for removal of fouling deposits (Type A) in the dairy industry. Enzymes assessed from (1) Novozymes and (2) Genencor. Continued Enzyme(s)

Foulant

Cleaning conditions

Analysis

Main findings

References

Crude protease produced by Schizophyllum commune

Experimentally generated milk fouling deposit on stainless steel

Quantification of residual organic matter and protein after cleaning

Satisfactory cleaning observed using B25 units/ mL enzyme, Triton X-100 and propylene glycol, Satisfactory cleaning observed using enzyme only at a higher concentration of B46 49 units/mL.

Boyce and Walsh (2012)

Crude keratinolytic protease produced by Bacillus tequilensis hsTKB2

Milk fouling deposit on stainless steel panels

Quantification of residual organic matter and protein

Heat-denatured whey protein on stainless steel surface Laboratory milk fouling model on stainless steel

Crude enzyme: no organic matter or protein detected after 1-h cleaning period. Surface immobilized enzyme: no organic matter or protein detected after 2-h cleaning period. Detergency value observed for enzyme similar to 0.5% (w/w) NaOH. Fouling removal effectiveness: commercial enzyme cleaner (78%), commercial alkaline cleaner (73%). Enzymatic solutions containing protease, amylase, and surfactant at three different pH values (75.35% 80.43%), protease and surfactant at pH 9.5 (72.89%), surfactant only at pH 9.5 (69.5%). Cleaning efficiency: enzyme solution (87.1%), alkaline cleaner (86.9%). Enzymatic cleaning of the fouling components (sugar, fat and protein) more homogeneous than chemical cleaning.

Paul et al. (2014)

Protease (Everlase1)

Sodium carbonate (50 mM) prerinse (40 C; 30 min; pH 11.5) followed by enzyme cleaning solution (25 units/mL crude enzyme, 0.05% w/v Triton X-100, 0.05% w/v propylene glycol) 40 C; 60 min; pH 5.8. Crude enzyme (40 U/mL): 70 C, pH 10.5; 1 3 h; 0.1% (w/v) Triton X-100 and propylene glycol Surface immobilized enzyme alginate beads (40 U/mL); 70 C; 1 3 h. Enzyme (1g/L) 30 C, pH 8.1.

Commercial enzymatic cleaner (protease, amylase, and lipase); additional enzymatic solutions containing nonionic surfactant and protease (Savinase1) with or without amylase (Termamyl Ultra1)

Enzyme cleaning solution containing 0.12% protease (Savinase1), 0.1% v/v amylase (Termamyl Ultra1), and a nonfoam surfactant (Tensio CIP)

Laboratory milk fouling model on stainless steel

Enzymes: 50 C; 30 min; pH 8.5, 9.2, or 9.5; commercial alkaline cleaner: 70 C, 45 min; pH 10 12.

Enzyme solution: 55 C; pH 8.5; 30 min Alkaline cleaner 6% v/v; 70 C, pH 10 12; 45 min.

Detergency expressed as amount of initial deposited soil which was removed. Weight after cleaning and turbidity of cleaning solution

Weight after cleaning; microscopic analysis

JuradoAlameda et al. (2014) GuerreroNavarro et al. (2019)

GuerreroNavarro et al. (2020)

Protease (Savinase1) and amylase (Termamyl Ultra 300L1)

1

Novozymes Genenor

2

Dairy fouling on pilot plant indirect plate heat exchanger and spray dryer

Plate heat exchanger: enzyme: 1% v/v (3% v/v Savinase and 3.2% v/v Termamyl Ultra 300L), diluted in 3% (v/v) buffer solution [nonylphenoxy poly (ethyleneoxy) ethanol 15EO (10.0% v/v)]; 50 C, 20 min chemical: alkaline step: 3% Brio Complex (NaOH and phosphonates); 80 C; 20 min Acid step: 1% Acimix CIP (nitric acid); 50 C; 20 min spray dryer: enzyme solution as above with 1% v/ v alkaline foaming detergent; 50 C; 20 min chemical: alkaline step: 3% jet foam (H2O2 and surfactants); 80 C; 20 min acid step: 1% Acid Jet (ortophosphoric acid, surfactants); 80 C; 20 min.

Amount of fouling removed (g/L) based on weight of dried residues in cleaning solution, microbiological analysis, fluorescence marker detection, identification of residual fouling components

Efficacy of enzyme cleaning solution to remove fouling comparable to alkaline-acid cleaning. Amount of dairy fouling removed (plate heat exchanger): enzyme cleaning 33.21 g/L; chemical cleaning 23.95 g/L (20.61 g/L alkaline step and 3.3 g/L acid step). Spray dryer: enzyme cleaning 5.30 g/L; chemical treatment 5.10 g/L. Microbiological analysis: effective microbiological cleaning observed in both cases.

GuerreroNavarro et al. (2022)

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were undertaken mainly at laboratory scale using experimentally generated milk fouling deposits representative of the Type A fouling deposit formed during heat treatment of milk as described above. For some of the enzyme cleaning studies shown in Table 20.1, assessment at pilot or process scale was also undertaken. The enzymes used were generally commercial enzymes, developed and optimized for use in the detergent industry with emphasis mainly on protease activity. Cleaning performance was assessed by various techniques as shown in Table 20.1. For illustration, Fig. 20.2 shows a typical

FIGURE 20.2 Stainless steel panels, with an experimentally generated milk fouling deposit, before and after cleaning with water, buffer, sodium hydroxide, or two different protease enzymes (A and B). The images of the stainless steel panels after cleaning show different cleaning performances for proteases A and B, with complete removal of the fouling deposit observed after cleaning with Protease B compared to significant fouling observed after cleaning with Protease A. The images of the stainless steel panels after cleaning highlight the satisfactory cleaning performance achievable using Protease B at 40 C compared to water, buffer or NaOH at 40 C, with the enzymatically cleaned panel similar to that cleaned with NaOH at 60 C. Generation of milk fouling deposits and enzyme cleaning was undertaken as described by Boyce et al. (2010).

20.3 Reported studies on the effectiveness of enzymes

laboratory-generated milk fouling deposit before and after cleaning with water, buffer, sodium hydroxide, or buffered protease solution, highlighting the satisfactory cleaning performance achievable using protease activity at 40 C. Several commercial protease products and a single protease/lipase product were assessed by Grasshoff (2005) and Graßhoff (2002) with the overall objective of minimizing the requirement for pH neutralization of the cleaning effluent and reducing its (nonbiodegradable) chemical loading. Following enzyme-based cleaning at lab-scale after an initial acid pretreatment (conditions shown in Table 20.1), deposit-free surfaces were observed for three of the protease products assessed. One of these proteases (Savinase, produced by a genetically modified Bacillus strain) was subsequently used to clean a milk pasteurizer with satisfactory cleaning observed. Reported environmental benefits of the enzyme-based cleaning approach included a lower pH effluent (pH B9 compared to pH 12 13) more suitable for release without neutralization and a lower cleaning temperature (55 C) with associated energy savings. Boyce et al. (2010) assessed additional commercial enzyme products (proteases and lipases) at lab scale with quantitative evaluation of cleaning undertaken. Pretreatment with acid was not carried out prior to cleaning as in the Grasshoff study and enzyme cleaning was assessed at temperatures as low as 40 C with the objective of decreasing energy consumption. Initial cleaning studies at the enzyme optimum pH showed that satisfactory removal of a milk fouling deposit could be achieved using 6 of the 8 proteases assessed at a cleaning temperature of 40 C, 50 C, or 60 C. At 40 C, the cleaning performance of 7 of the 8 proteases assessed was better than that of 1% (w/w) NaOH, with ,0.4% residual organic matter and ,0.1 mg protein detected on the enzyme cleaned panels compared to B1.5% residual organic matter and 1 mg protein on the NaOH cleaned panel. The removal of the milk fouling deposit by the buffered protease solutions was further underlined by the significant residual fouling observed after cleaning with buffer only at pH 10 11.3 (B43% 59% residual organic matter and B8 20 mg protein) or water only (95% residual organic matter and 37 mg protein). For the three most favorable proteases identified, cleaning was further optimized in terms of enzyme concentration and cleaning pH. A preliminary process-scale study was undertaken by circulation of one of these protease products through an area of a commercial milk processing plant (including a pasteurizer) at 45 C 50 C, pH B10 for 60 min, for 4 consecutive days after routine production, where the pasteurizer was run at 80 C for 6.75 8.5 h each day. Satisfactory cleaning, judged by no issues maintaining milk temperature at 80 C during subsequent processing after the enzyme cleaning step, was observed. The results of the study show the potential of

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proteases to achieve satisfactory cleaning at low temperatures with associated benefits in terms of energy consumption. Preliminary cost analysis undertaken indicates that the cost of the enzyme cleaning solutions (h0.015/L) is comparable to that of 1.5% NaOH (h0.012 0.016/L) and caustic formulated detergent (h0.011 0.019/L). The suitability of proteases for cleaning at low temperature was also reported in a separate study based on the ability of the commercial protease product Everlase (produced by an engineered Bacillus) to remove heat-denatured whey protein from stainless steel at 30 C (Jurado-Alameda et al., 2014). Guerrero-Navarro et al. (2019) assessed the efficacy of various enzymatic cleaning solutions to remove a laboratory milk fouling model from stainless steel. A commercial enzymatic product (containing protease, amylase, and lipase) used at 50 C, pH 9.5 for 30 min compared favorably with a commercial alkaline cleaner used at 70 C, pH 10 12 for 45 min (77.99% effectiveness compared to 73.31%). Other enzyme-based cleaning solutions containing protease with or without amylase were also assessed at 50 C (Table 20.1) with a nonionic surfactant included to increase wettability and solubility. The authors reported no statistical difference between the cleaning solutions with reduced wastewater ( 2 33.3%), temperature ( 2 28.5%), and cleaning time ( 2 33.3%) observed for the enzymatic alternative. The economic cost of the enzymatic cleaning (h0.045/L) was similar to that of the alkaline chemical cleaning (h0.047/L) with enzymatic cleaning cost dependent on the enzymes used and their concentration. In a later study, Guerrero-Navarro et al. (2020) again reported no difference between the efficacy of enzymatic cleaning using protease, amylase, and surfactant (55 C; 30 min; pH 8.5) compared to an alkaline cleaner (70 C; 45 min; pH 10 12) (Table 20.1). Guerrero-Navarro et al. (2022) examined the effectiveness of enzymatic cleaning at pilot plant scale including a plate heat exchanger and spray dryer equipment. Details of the cleaning solutions and conditions used and results observed are summarized in Table 20.1. The enzyme components of the cleaning solution (3.0% protease and 3.2% α-amylase) were similar to that reported by Rodriguez Jerez et al. (2020), in which improved reduction of fouling was observed upon inclusion of the α-amylase with the protease at pH 8 9 (87.51% compared to 68.95%) but less so at pH 9.5 (73.99% compared to 72.25%). At pilot plant scale, enzymatic cleaning using both the protease and amylase at 50 C was comparable to alkaline-acid cleaning at 80 C. In terms of cost, the alkalineacid cleaning cost h12.20/200L compared to h18 for the enzyme cleaning solution. However, half the cost of the enzyme cleaning solution is due to the buffer component, the concentration of which could be reduced, resulting in an enzyme cleaning cost of h9.90 (Guerrero-Navarro et al., 2022). In addition to commercial protease products, crude native proteases isolated from microorganisms have also been assessed for CIP in the dairy industry.

20.3 Reported studies on the effectiveness of enzymes

For example, screening of 14 fungi for their ability to produce proteases suitable for this application resulted in a lab-scale CIP procedure based on the protease produced by Schizophyllum commune (Boyce & Walsh, 2012). This procedure involving a sodium carbonate prerinse followed by enzyme cleaning at 40 C and pH 5.8 resulted in satisfactory cleaning with a reduced concentration of enzyme required upon inclusion of Triton X-100 and propylene glycol (Table 20.1). Improved cleaning in the presence of Triton X-100 may be attributed to improved enzyme accessibility, weakened protein lipid interactions, and decreased protein readsorption after cleaning (Allie et al., 2003). Propylene glycol is considered an enzyme stabilizer although improved protease stability at 40 C was not observed with addition of up to 1.0% (w/v) propylene glycol in this study. Similarly, satisfactory cleaning of a milk fouling deposit using the crude protease produced by Bacillus tequilensis hsTKB2 at 70 C and pH 10.5 in the presence of Triton X-100 and propylene glycol (Table 20.1) has also been reported (Paul et al., 2014). The CIPzyme product (containing both protease and lipase activity) assessed by Graßhoff (2002) removed only 1% 20% of the test milk deposit which was significantly less than 5 of the proteases tested. Under the lab-scale conditions used in the study by Boyce et al. (2010), inclusion of lipase activity did not significantly improve cleaning despite the lipases retaining relatively high activity in the cleaning solution. Further studies indicated little or no fat breakdown when the lipases were used alone with increased fat breakdown reported when used with protease activity, which was attributed to improved lipase access to the fat component upon degradation of the protein component of the fouling. Rodriguez Jerez et al. (2020) observed that for a cleaning solution containing protease, amylase, and lipase, the lipase component was not necessary when cleaning was undertaken at the optimum pH of the protease and amylase components. While lipases did not significantly improve cleaning in these cases, it is noteworthy that lipase activity may be beneficial for cleaning fouling deposits of higher fat content (e.g., those associated with processing of recombined milk, pasteurization of homogeneized milk, or formed at lower pH) (Bansal & Chen, 2006; Jeurnink et al., 1996) and that lipase-induced fat breakdown may be beneficial for subsequent wastewater treatment (Boyce et al., 2010). From the studies reported in the literature to date, proteases appear to be the most promising enzymes for removal of Type A milk fouling deposits, which is not surprising considering that protein is the main component of the deposit. Furthermore, proteins in the fouling deposit are known to interact with proteins on the MFGM (milk fat globule membrane) which surround milk fat globules and with minerals and protein-stabilized mineral precipitates leading to the incorporation of fat globules and minerals into the deposit (Huppertz & Nieuwenhuijse, 2022; Jeurnink et al., 1996). On this

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basis, it is possible that the action of proteases in hydrolyzing the protein component of the fouling layer would also contribute towards the removal of associated non-protein components. While a limited number of enzyme cleaning studies have included α-amylase activity with protease activity (Table 20.1), the specific role of α-amylase activity in removing fouling is not clear considering the reported composition of Type A fouling deposits and satisfactory cleaning has been reported using proteases only. Further assessment is required to establish if inclusion of α-amylase activity is beneficial for the removal of Type A fouling deposits.

20.3.2

Removal of biofilms

In recent years there has been increased interest in bacterial fouling in dairy processing, in particular, the control and removal of biofilms with the aim of reducing their negative effect on product quality and safety and equipment impairment (Flint et al., 2020; Gopal et al., 2015). Bacteria found in dairy processing facilities include those of the genera Enterobacter, Lactobacillus, Listeria, Micrococcus, Streptococcus, Bacillus, Pseudomonas, Chryseobacterium, Lactococcus, Klebsiella, and Staphylococcus (Anand et al., 2014; Flint et al., 2020). Several studies have reported the efficiency of enzyme-based cleaning solutions to remove biofilms when used alone or in combination with chemical or physical treatments (Araújo et al., 2017; Meireles et al., 2016; Simões et al., 2010). This has been attributed to their ability to target and degrade the components of the EPS matrix, cutting the links between cells and thus weakening the biofilm structure rendering it easier to remove (Molobela et al., 2010). The action of enzymes reduces the protective effect of the biofilm on the embedded microorganisms and improves the ability of disinfectants to kill the cells (Coughlan et al., 2016; Meireles et al., 2016). Owing to the heterogeneity of the EPS, a mixture of enzymes with different specificities may be required. Several studies have assessed the effectiveness of various enzymes in removing single- and mixed-species bacterial biofilms, including those relevant to the dairy industry, from solid surfaces, some of which are summarized in Table 20.2. Enzyme selection is generally based on the composition of the targeted EPS and beneficial results have been reported using readily available commercial enzymes such as serine proteases and α-amylase with some studies also including additional components such as (bio)surfactants. For example, Lequette et al. (2010) reported the use of enzymes to remove biofilms of bacterial species isolated in the food industry, including some from dairy plants and products (Table 20.2). Of the enzymes assessed, which included serine proteases, papains, α-amylase, and polysaccharidase mixes containing cellulase, hemicellulase, and β-glucanase, the two serine proteases and α-amylase were found

Table 20.2 Selected published studies assessing the use of enzymes for removal of biofilms. Enzymes from Enzyme(s)

Biofilm

Conditions

Main findings

References

2 serine proteases , 2 papains2,3, α-amylase1, polysaccharidase Mix A1 (cellulase, hemicellulase), polysaccharidase Mix B (Mix A, α-amylase1, β-glucanase1)

Biofilms of various bacterial species on microtiter plates; biofilms of Bacillus mycoides, Bacillus cereus, and Pseudomonas fluorescens on stainless steel.

Enzyme solutions in PBS at pH 4, 7 or 10 (pH 10 most efficient for biofilm removal) or in a formulated alkaline buffer (pH 10) with anionic surfactants, dispersing and chelating agents 45 C; 30 min

Lequette et al. (2010)

Proteases1: (Savinase, Everlase, and Polarzyme) Amylases1: (Bacterial α-amylase, amyloglucosidase)

Pseudomonas fluorescens biofilm on glass wool.

26 C; Proteases (pH 8.3); Amylases (pH 7.0 or pH 5)

Reflux E20014 (protease and lipase) Reflux E10004 (protease) QuatroZyme4 (lipase, protease, cellulase, amylase)

Klebsiella oxytoca biofilms on dairy industry ultrafiltration membranes

Enzymes used at 0.2%(v/v), 48 C, pH 7 10; 45 min except for QuatroZyme (30 min) Compared with standard CIP (alkali & sodium hypochlorite at 50 C for 30 min).

α-Amylases5 from Aspergillus oryzae, Bacillus subtilis and human saliva and β-amylase5 from sweet potato

Staphylococcus aureus biofilm in polystyrene microtiter dishes or polyurethane tubing.

37 C

Microtiter plate assay: 2 serine proteases most efficient; α-amylase most efficient polysaccharidase Biofilms on stainless steel: Serine proteases more efficient than polysaccharidases for removal of Bacillus biofilm cells but polysaccharidases more efficient for P. fluorescens biofilms Improved efficiency observed in buffer containing surfactants, dispersing and chelating agents. Comparison with NaOH: cells were similarly removed however enzyme treatments removed EPS more efficiently. Everlase and Savinase most effective in biofilm removal and EPS degradation; Polarzyme not effective; Amyloglucosidase partially effective in degrading EPS carbohydrate; α-amylase less effective. No significant difference observed in the effectiveness of the four different cleaners. QuatroZyme was slightly better than the others. Concluded that enzyme cleaner followed by a sanitizer effective. Inhibition of biofilm formation: Reduction in biofilm buildup observed with α-amylases but less so with β-amylase Biofilm reduction: α-amylases from A. oryzae and B. subtilis reduced existing biofilms for all isolates and surfaces tested; α-amylase from human saliva less effective and no reduction with β-amylase A. oryzae α-amylase reduced/dissociated cell aggregates in liquid.

1

Molobela, Cloete, and Beukes (2010)

Tang et al. (2010)

Craigen, Dashiff, and Kadouri (2011)

Continued

Table 20.2 Selected published studies assessing the use of enzymes for removal of biofilms. Enzymes from Continued Enzyme(s)

Biofilm

Conditions

Main findings

References

β-glucanase (Ultraflo ), α-amylase (Fungamyl1), Lipase (Lecitase1), Protease (Alcalase1)

Pseudomonas fluorescens biofilm.

Enzymes alone or with cetyltrimethylammonium bromide (CTAB) 1 h

Araújo et al. (2017)

Pronase6, cellulase5, Dnase I5

Listeria monocytogenes biofilm and mixed biofilms (L. monocytogenes, Escherichia coli and L. monocytogenesP. fluorescens) on stainless steel.

Enzymes alone or with benzalkonium chloride (BAC) Pronase (pH 7.5; 37 C; 30 min), cellulase (pH 6.0; 32 C; 30 min); DNaseI (pH 7.5; 32 C; 30 min) BAC treatment after 30 min with Pronase or DNaseI

Biofilm mass removal: β-glucanase, protease, and α-amylase-moderate biofilm mass removal (B25%); lipase-no removal Increased biofilm removal upon combination of CTAB with β-glucanase or α-amylase; CTAB and protease no removal Biofilm CFU/cm2 reduction: Protease best (1.59) followed by lipase, β-glucanase and α-amylase (1.34, 1.25, and 1.05, respectively) Significant increase upon combination with CTAB (2.07 and 1.93 for β-glucanase and protease, respectively) Protease long-term effect on biofilm mass reduction; β-glucanase- significant biofilm regrowth after treatment Complete biomass removal/CFU reduction not achieved and biofilm integrity and viability not compromised. L. monocytogenes and E. coli: greatest effect (B2 log reduction) using 400 μg/ml DNaseI; greatest removal using DNaseI followed by Pronase and cellulase L. monocytogenes more sensitive than E.coli to the enzymes with exception of DNaseI at high concentration. Pronase/DNaseI in combination with BAC: speciesdependent effect: L. monocytogenes BAC better after DNaseI compared to Pronase; E. coli- BAC better after Pronase Dual-species biofilm: DNaseI-BAC treatment reduced number of viable attached cells when assessed on biofilms of both L. monocytogenes and E. coli and L. monocytogenes and P. fluorescens.

1

RodríguezLópez, CarballoJusto, Draper, and Cabo (2017)

Serine protease5

24-h old biofilms of various B. cereus strains on stainless steel coupons.

9 commercial enzymes: pronase6, cellulase5, pectinase5, DNaseI5, lysozyme5, phospholipase5, peroxidase5, β-glucanase5 and chitinase5

Mono- and dual-species biofilms of Listeria monocytogenes with bacteria from dairy, meat and seafood processing (P. fluorescens used to represent the dairy industry).

Enzy-CIP7 (1 , 2.5% subtilisin; 1 , 2.5% α-amylase & a non-foaming detergent)

Pseudomonas fragi; UHT milk Streptococcus spp., Bacillus spp., Micrococcus spp.; Pasteurized milk On PTFE-hose liner in dairyfilling. hose Macrococcus caseolyticus biofilm on stainless steel.

Protease (Alcalase1) Lipase (pancreatic lipase5) DNase I8, 1,4(1,3-1,4) β-D-glucan4glucanohydrolase (CMCase5) α-amylase (Fungamyl1)

RSM-optimized enzyme CIP: 1.0 U/ml enzyme; pH 8.5; 60 C; 20 min followed by water rinse and 0.158 M HNO3; 65 C; 10 min compared to optimized CIP (0.375 M NaOH; 65 C; 30 min) followed by HNO3 as for enzyme cleaning 1 h at room temperature

45 C; 1.5 h; pH 8 (except CMCase pH 5 and amylase pH 7.2) Cocktail 1: protease, lipase, amylase and CMCase Cocktail 2: as for Cocktail 1 but at higher concentrations and DNase included Cocktail 1 and 2 prepared in Tris HCl buffer containing surfactin CTC: alkaline detergent (BASO CTC) (1% v/v); 60 C; 1.5 h

Optimized enzyme CIP resulted in complete removal of biofilm cells (compared to a reduction of B4.92 log biofilm cells/cm2 for optimized alkali CIP) and amount of biofilm matrix removed also significantly higher. B1 log CFU/cm2L. monocytogenes reduction (B90% of population) For dairy biofilm (L. monocytogenes and P. fluorescens), Pronase, Pectinase, and DNaseI reduced biofilm thickness by 0%, 17%, and 29%, reduced covered area by 37%, 36%, and 90% and reduced biovolume by 40%, 70%, and 99%, respectively. Sufficient cleaning observed.

Kumari and Sarkar (2018)

Individual enzymes: protease, lipase, DNase significant biofilm elimination; CMCase and amylase not significant Cocktail 2 most efficient, followed by Cocktail 1 and CTC (cfu log reduction values of 4.2, 3.1, and 2.95, respectively). Concluded that enzymatic cocktail resulted in more efficient biofilm removal than the chemical detergent BASO CTC.

Mnif, Jardak, Yaich, and Aifa (2020)

Puga et al. (2018)

Fysun et al. (2019)

Continued

Table 20.2 Selected published studies assessing the use of enzymes for removal of biofilms. Enzymes from Continued Enzyme(s) 9

Deterzyme 520/180 (alkaline protease and α-amylase)

1

Novozymes, Enzybel, Blue Star Chemicals, 4 Orica, 5 Sigma, 5 Roche, 7 iTram Hygiene, 8 Promega, 9 ENMEX. 2 3

Biofilm

Conditions

Main findings

References

E. coli, Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa, L. monocytogenes, and B. cereus. Mixed-species biofilms developed in whole milk on stainless steel and polypropylene B.

30 min; 25 C

Enzymes followed by paracetic acid as a disinfectant effective in removing the biofilms developed on stainless steel EPS removal after enzyme use evident upon microscopic analysis

IñiguezMoreno, GutiérrezLomelí, & Avila-Novoa (2021)

20.4 Considerations for the development of optimal enzyme-based cleaning solutions

to be the most efficient. Enhanced biofilm removal was observed when the enzymes were used with surfactants and dispersing and chelating agents. Upon comparison with NaOH treatment, both at 45 C, the removal of cells was similar but EPS was removed more efficiently by the enzyme treatment. Kumari and Sarkar (2018) optimized a serine protease-based approach for removal of Bacillus cereus biofilm, a bacteria of concern in dairy processing due to its ability to produce spores and survive pasteurization temperature and its association with food poisoning (Gopal et al., 2015; Kumari & Sarkar, 2016). The optimized approach resulted in complete removal of biofilm cells and removal of biofilm matrix was also greater than observed for akalibased CIP. The use of enzymes on membrane-associated biofilms has also been reported (Anand & Singh, 2013; Tang et al., 2010). As evident from Table 20.2, the effectiveness of specific enzymes has been observed to vary depending on the bacteria being targeted (Lequette et al., 2010; RodríguezLópez, Puga, et al., 2017) which can be attributed to differential EPS composition. In addition, multispecies biofilms are generally a more challenging target for enzymes due to their increased complexity. As shown in Table 20.2 some studies (Mnif et al., 2020; Puga et al., 2018) have also assessed the use of DNaseI, which targets the extracellular DNA in the biofilm (Craigen et al., 2011), and Pronase, from Streptomyces griseus, which contains several nonspecific endo- and exoproteases and is used for total degradation of proteins to single amino acids. At present, the widespread use of these two enzymes for CIP in the dairy industry is likely to be limited by cost and lack of bulk availability as these enzymes are not currently produced at truly industrial scale. It is noteworthy that while enzymes may disrupt and disperse the biofilm structure, they do not necessarily have bactericidal activity and therefore their use in combination with antimicrobials is necessary to achieve both biofilm removal and disinfection and limit dissemination of live cells which could redeposit and form a biofilm elsewhere (Boels, 2011; Iñiguez-Moreno et al., 2021; RodríguezLópez, Carballo-Justo, et al., 2017). The action of enzymes in terms of biofilm disruption can facilitate easier mechanical removal and improve access to the cells potentially reducing the amount of disinfectant required (Boels, 2011; Meireles et al., 2016; Rodríguez-López, Puga, et al., 2017).

20.4 Considerations for the development of optimal enzyme-based cleaning solutions Enzymes exhibit substrate specificity and therefore to achieve effective enzyme-based cleaning, enzymes should be selected based on the composition of the fouling deposit being targeted. This is relatively straightforward in

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the case of the Type A fouling which occurs during heat treatment of milk at 70 C 110 C, and predominantly consists of proteins which can be removed using protease activity (Huppertz & Nieuwenhuijse, 2022). The selection of enzymes for removal of microbial fouling or biofilms is more challenging due to species-dependent variations in EPS composition, which may be further complicated by the formation of multispecies biofilms, the presence of milk fouling components, poor substrate accessibility, and the presence of complex polysaccharides and numerous types of linkages requiring the activity of specific enzymes for hydrolysis. Knowledge of the composition of the fouling deposit is therefore an essential prerequisite for the development of an effective enzyme-based cleaning solution. Furthermore it is essential that initial lab-scale cleaning studies are undertaken using reproducible fouling deposits as representative as possible as that which will be encountered at industrial scale, including the surface on which the fouling deposit is formed. While such studies facilitate initial assessment and optimization of the enzyme cleaning procedure, subsequent industrial-scale cleaning studies under realistic processing conditions are essential to confirm enzyme cleaning performance and achieve more widespread acceptance of enzyme-based CIP in dairy processing. To achieve the best cleaning performance, enzymes should be used under conditions at which they are optimally active. With growing interest on reducing energy consumption, enzymes which are optimally active at low temperatures and which can achieve satisfactory cleaning at temperatures lower than those currently used for chemical-based CIP are preferable. In terms of pH, slightly alkaline conditions are preferable to promote swelling of milk proteins and EPS, with the latter reported to be insoluble at low pH and therefore more difficult to remove (Fryer & Asteriadou, 2009; Lequette et al., 2010). Such pH conditions (up to BpH 10) are more compatible with wastewater treatment processes than highly alkaline cleaners, minimizing the need for neutralization as outlined above. Enzymes exhibiting high activity at a combination of low temperature and slightly alkaline pH are therefore desirable. The effect of pH on the cleaning performance of a selected protease is shown in Fig. 20.3. Where two or more enzymes are used, enzymes with compatible temperature and pH optima should be chosen where possible, otherwise a compromise in terms of cleaning pH and temperature will have to used which may result in reduced enzyme activity and stability. An appropriate buffer, the components of which do not negatively affect the performance of the cleaning enzyme(s) and which are capable of maintaining the cleaning solution at the required pH throughout the cleaning period should be identified. In addition to buffering components, several studies reported beneficial cleaning effects upon inclusion of other additives in the

20.4 Considerations for the development of optimal enzyme-based cleaning solutions

FIGURE 20.3 Enzyme cleaned panels showing the effect of pH on cleaning. Cleaning was undertaken using a commercial protease product (40 C for 60 min) using half the optimum enzyme concentration required for satisfactory cleaning. Generation of milk fouling deposits and enzyme cleaning was undertaken as described by Boyce et al. (2010).

enzyme cleaning solution, such as surfactants (Table 20.1). The inclusion of such additives may have possible cost and environmental implications and where this is the case their use should be limited. The enzymes used should exhibit good stability during storage and for the duration of the cleaning period and the possible effect of any additives in the cleaning solution, as well as any potential inhibitors on enzyme activity and stability should be considered. Moreover, enzymes are heat sensitive and care must be taken to avoid any possible overheating. Determination of the optimum concentration of enzyme is also important as too low an enzyme concentration may result in insufficient cleaning or necessitate long cleaning times, while excessively high enzyme concentrations would unnecessarily increase the cost. The effect of enzyme concentration on cleaning performance is shown in Fig. 20.4. A statistical design of experiments approach such as response surface methodology (RSM) can be used to systematically assess the effect of significant factors such as cleaning temperature, pH, duration, and enzyme concentration as well as their interactions on cleaning and to develop an optimized CIP method, as reported by Kumari and Sarkar (2018). To prevent any deleterious/adverse effects on dairy products processed after enzyme-based cleaning, it is essential that all traces of the cleaning enzyme(s) are removed or inactivated after cleaning (Grasshoff, 2005; Kumar et al., 2021; Pottchoff & Serve, 1997). Studies have shown that under normal cleaning conditions, this is not an issue as any residual enzyme activity will almost certainly be removed during the water rinse step undertaken after cleaning, inactivated by circulation of acid often undertaken to remove mineral

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FIGURE 20.4 Enzyme cleaned panels showing the effect of enzyme concentration on cleaning. Cleaning was undertaken using a commercial enzyme product containing protease and lipase activity (40 C, pH 10 for 60 min). Generation of milk fouling deposits and enzyme cleaning was undertaken as described by Boyce et al. (2010).

deposits or by the sanitation step if undertaken (Fig. 20.1). In the case of three protease enzymes, Boyce et al. (2010) reported complete removal of residual enzyme activity after rinsing enzymatically cleaned stainless steel panels with water with no protein detected on the panels by laser scanning confocal microscopy after rinsing and no protease activity detected in the third 20-mL rinse solution collected. In the same study, total enzyme inactivation was observed upon incubation of the proteases with 0.5% or 1.0% nitric acid at room temperature (1 min) or upon incubation of the enzymes at 80 C (3 min), 90 C (1 min), or with hypochlorite (1 min). This strongly indicates that these enzymes would be inactivated under the conditions to which they would be exposed during the acid circulation step or during heat or chemical-based sanitation undertaken after cleaning. The commercial enzyme product CIPzyme, containing protease and lipase activities, is also reported to be inactivated by acid and high temperature and removal of the enzyme cleaning product, P3-Paradigm, by the final water rinse and inactivation by the disinfection cycle has been reported (Pottchoff & Serve, 1997). Moreover, simulated worst case scenario testing where cleaning enzyme was intentionally mixed with dairy product to determine possible effects on dairy starter cultures concluded that the tested enzyme did not pose an additional risk to cheese or yogurt manufacture (Grasshoff, 2005; Pottchoff & Serve, 1997). The complete removal/inactivation of any enzymebased cleaning product should be validated under industrial conditions.

References

20.5

Conclusions and future outlook

Numerous studies have highlighted the potential contribution that enzymes can make toward achieving physical, chemical, and microbiological cleanliness in the dairy industry. While these (mainly lab-based) studies have contributed toward addressing initial concerns in relation to low enzyme cleaning efficiency, further assessment of enzyme-based CIP under realistic processing conditions at pilot and industrial scale would be beneficial in terms of achieving more widespread acceptance and implementation of enzyme-based CIP in the dairy industry. An additional issue traditionally associated with enzyme-based cleaning was the perceived high cost of enzymes in comparison to chemical cleaning agents. The cost of enzymes has decreased several fold in recent years due to the development of more efficient production strains and fermentation processes with the cost of producing industrial enzymes now approaching that of producing high-purity nutritional proteins (Arbige et al., 2019). As outlined above in Section 20.3, recent studies have reported comparable costs for both enzyme and chemical cleaning solutions with additional savings likely in the case of enzyme-based CIP due to potential reductions in energy consumption and water use and decreased costs in relation to storage and handling of corrosive chemicals. While successful cleaning can be achieved using commercially available industrial enzymes, developed mainly for the detergent industry, even greater cleaning efficiency would almost certainly be possible using enzymes developed specifically for CIP in the dairy industry. The development of such enzymes should ideally focus on those specific to the fouling deposit being targeted and which exhibit high activity and stability during cleaning. The discovery of new enzymes and improvement of existing enzymes has become much more feasible due to advances in bioprospecting, recombinant DNA technology, and protein engineering (which can be used to improve the properties of the enzyme, e.g., its specificity, activity, and stability) (Arbige et al., 2019; Fasim et al., 2021; Solanki et al., 2021). As a result, enzymes from unculturable microorganisms can be assessed and the conditions under which an enzyme can be used in industry are no longer limited by the properties of the native enzyme. Exploiting such technological advances will contribute toward improving the commercial applicability of enzymebased CIP in the dairy industry, maximizing the associated environmental and sustainability benefits.

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CHAPTER 21

Regulatory policies on use of food enzymes Smita Sahu1, Shubhangi Agrawal2 and Ashwani Sahu3 1

21.1

Amity IPR Cell, Amity University, Noida, Uttar Pradesh, India, 2Novartis, Hyderabad, 3 Telangana, India, Bharat Heavy Electricals Limited (BHEL), New Delhi, India

Introduction

Most enzymes are proteins that act as catalysts in all living organisms (microorganisms, plants, animals, and humans) increasing the rate of chemical reactions in biological systems. Enzymes can increase the rate of reactions up to 10 million times with a very small quantity. Enzymes operate within a narrow set of conditions, such as temperature and pH (acidity). A survey on global revenue of enzymes ascribes 31% and 6% for food and feed enzymes, respectively, while the remaining accounts for technical enzymes (Chandran, 2019). Classification of enzymes is based on the type of reaction and the substrate they act upon. It is customary to attach the suffix "-ase" to the name of the principal substrate upon which the enzyme acts. Enzymes extracted from edible plants and the tissues of food animals, as well as those produced by microorganisms (bacteria, yeasts, and fungi), have been used for centuries in food manufacturing. Rennet is an example of a natural enzyme mixture from the stomach of calves or other domestic animals that has been used in cheese making. It contains a protease enzyme that coagulates milk, causing it to separate into solids (curds) and liquids (whey). Also, for centuries enzymes produced by yeast have been used to ferment grape juice to make wine. In the 20th century, increased isolation of enzymes from living cells led to a large-scale commercial production and wider application in the food industry. Today, microorganisms are the most important source of commercial enzymes. Although microorganisms do not contain the same enzymes as plants or animals, a microorganism that produces a related enzyme to catalyze the desired reaction can be found. Enzyme manufacturers have optimized microorganisms for the production of enzymes through screening and selection techniques. Enzymes Beyond Traditional Applications in Dairy Science and Technology. DOI: https://doi.org/10.1016/B978-0-323-96010-6.00021-7 © 2023 Elsevier Inc. All rights reserved.

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Genetic modification encompasses the most precise methods for optimizing microorganisms for the production of enzymes. These methods are used to obtain high-yielding production organisms. Biotechnology also provides the tools to have a genetic sequence from a plant, animal, or a microorganism, from which commercial-scale enzyme production is not adequate, to be transferred to a microorganism that has a safe history of enzyme production for food use (Government of Canada, 2014). Enzymes produced through biotechnology are identical to those found in nature. Additionally, enzymes produced by microorganisms are extracted and purified before they are used in food manufacturing. Enzymes have been important to food technology because of their ability to transform raw material into an improved food product. Food technologist selects those enzymes which can improve one particular unit operation of food production. Substituting fish protein hydrolysates for milk in calf feed is an example of improvement for saving energy and money in production process and modifying the functional properties of proteins. Production of dairy foods make use of diverse enzymes including proteases to reduce the allergenicity of bovine milk products and lipases to improvise the flavor of cheese. The scope of minor enzymes in milk food applications needed a better production of dairy-related products and for the better scope of dairy technology. The worldwide dairy product market is impressively increasing demand of microbial enzymes; however, there are a limited number of enzymes producing industries in contrary. The production of proteinase, microbial rennet, lactase, and lipase is increasing in the laboratory- and small-scale industries. In the near future, the need for such enzymes would be increasing, essentially due to the increasing demand of significant nutritional dairy products to overcome the prevalence of malnutrition and obesity that necessitates a shift toward low-fat and nutritional foods. Enzymes derived from microorganisms and used in food technology are listed in Table 21.1. Table 21.1 Enzymes derived from microorganisms and used in food technology. Enzyme

Source

Action

Application

α-Amylase

Aspergillus spp., Bacillus spp., Microbacterium imperiale

Wheat starch hydrolysis

Lipase and Esterase

Aspergillus spp., Candida spp., Rhizomucor miehei, Penicillium roqueforti, Rhizopus spp., Bacillus subtilis Aspergillus spp., Penicillium funiculosum

Hydrolyzes triglycerides to fatty acids and glycerol; hydrolyzes alkyl esters to fatty acids and alcohol Hydrolyzes pectin

Dough softening, increased bread volume, aid production of sugars for yeast fermentation Flavor enhancement in cheese products; fat function modification by interesterification; synthesis of flavor esters Clarification of fruit juices by depectinization

Pectinase (polygalacturonase)

21.1 Introduction

In the United States, enzymes are classified as food additives or as Generally Recognized as Safe (GRAS), while certain enzymes are regulated as secondary direct food additives. The act permits an individual or company to decide whether an ingredient is GRAS for an intended use and further market the ingredient without any prior contact with the agency, based on the appropriate data. In the EU, food enzymes and novel foods follow a different regulatory pathway. In 2008, the EU adopted Regulation EC 1331/2008 (a formal approval procedure for food additives, enzymes, and flavorings) and EC 1332/2008 (a harmonized regulatory framework and safety approval process for food enzymes). The legislation became fully applicable in 2010, whereas before the introduction of this legislation, food enzymes were not regulated at the EU level or were regulated inconsistently by member countries. According to this regulation, the European Commission is responsible for approval of all currently marketed and new food enzymes in the EU with prior evaluation by the European Food Safety Authority (EFSA). The ultimate aim is to establish a list of approved enzymes for the EU. Australia and New Zealand enzyme regulation is contained within horizontal standards of processing aids. This standard regulates the use in food manufacturing and thus stopping them from being used in food unless there is a specific permission as per standard. Food safety institutions and regulations for Ethiopia and India have been established more recently. It is likely that the regulatory approaches by high-income countries will pave the way in informing other countries. The Codex Alimentarius is a joint commission of the Food and Agriculture Organization of the United Nations and the World Health Organization Food Standards Programme established in 1962. It aims for the protection of consumer health and promotion of fair practices in food trade food guidelines, standards, and codes. Some of the enzymes generally employed in food industry are listed below: 1. Alpha-amylase: It is used to solubilize the carbohydrates found in barley and other cereals used in brewing. 2. Beta-glucanase: Breakdown of glucans in malt and other materials. 3. Lipase: Used to shorten the time for cheese ripening. It is employed in the production of enzyme-modified cheese/butter from cheese curd or butterfat. 4. Papain: It is widely used as a meat tenderizer. 5. Chymosin: Helps in the curdling of milk by breaking down kappacaseins in cheese making. 6. Microbial proteases: Used in the production of fish meals, meat extracts, texturized proteins, etc. 7. Pectinase: Treatment of fruit pulp to facilitate juice extraction. It also helps in the clarification and filtration of fruit juice. 8. Lactase: Additive for dairy products for individuals lacking lactase.

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9. Glucoseoxidase: Conversion of glucose to gluconic acid to prevent Maillard reaction (reaction that gives browned food a particular flavor) in products caused by high heat used in dehydration. 10. Cellulase: Conversion of cellulose waste to fermentable feedstock for ethanol or single-cell protein production.

21.2

Regulatory framework regarding food enzymes

Food enzyme business worldwide worths nearly US$ 5.5 billion and its number and annual turnover have been steadily increasing for many years along with the kind of applications (Agarwal & Sahu, 2014; Sutay Kocaba¸s & Grumet, 2019). As of 2015 the major industry association, AMFEP (Association of Manufacturers and Formulators of Enzyme Products) lists about 70 commercially available enzymes (Sutay Kocaba¸s & Grumet, 2019). Considering that a known enzyme can be reproduced from multiple microorganisms, more than 200 commercial enzyme products are currently available in the market. GM microorganisms produced enzymes account nearly 50% of the total industrial enzyme market. Food and feed applications together dominate the global enzyme market by 55% 60% of the market share. Enzymes are used to replace steps in food processing still being in line with both sustainable industrial production and careful processing of food in order to maintain nutritionally important ingredients. Enzymes started becoming a focal issue for regulators, consumer group, or general public when consumer and environmental groups were alarmed by the increasing use of genetically modified microorganisms (GMMs) for enzyme production. This eventually led to reviews of safety concerns and regulatory aspects, especially in Germany, Switzerland, and Austria. The fundamental aspects whether a premarket approval is needed or some particular information from manufacturers is required during the course of safety evaluation remain same. In legislation on food enzymes, there has been a distinction on the basis of food additives and processing aids. Food additives are the substances not normally consumed as food in itself or as a characteristic ingredient with or without nutritive value instead used for technological purpose or to obtain some reasonable results whereas processing aids do not have any technological effect on finished food product. Moreover, no health risks are related. Unlike food additives, its use does not affect the intrinsic characteristics of the food. The use of substance in food processing determines how it should be classified. A substance that is regulated as a food additive in one situation might be considered as a processing aid in a different situation. In Canada and United States, for instance, all food enzymes are regulated as food additives. In Australia food enzymes are considered as processing aids.

21.3 Specific aspects of intellectual property right protection on enzymes

FDA requires most detailed information on enzymes including technical data, structural modifications of GMMs and also the manufacturing and purification process and takes long-term experience with certain enzymes; a GRAS status may be assigned to such enzymes.

21.3 Specific aspects of intellectual property right protection on enzymes Enzymes can strongly reduce or even remove the need for inorganic content in food/animal feed and replace artificial coloring, flavors, and preservatives. Enzyme technology is considered as a “green technology” and contributes to saving of resources and increasing the utilization of alternative natural resources. It can be foreseen that a better valorization of plant biomass by adequate new enzymes will increase in the years to come. The development of industrial microbial enzymes implies to selection, genetic engineering, expression, upscale production, formulation, registration, and promotion. Screening a new enzyme and its practical development is quite expensive so the Intellectual Property (IP) generated must be protected against unauthorized usage by competitors. This is usually done by patenting the enzyme or its production method or the process in which it is to be used. IP is related to creations for which a monopoly is assigned to designated owners by law. The biggest enzyme developers and producers have a generally aggressive strategy of patenting (Sutay Kocaba¸s & Grumet, 2019). There still seems to be a huge technical gap between in vitro production of enzymes and the final commercial product obtained. The world’s principal enzymes producers (AB Vista, BASF, Novozymes, DSM, VTR Bio-Tech, DuPont, Youtell Biochemical, Smistyle, SunHY, and Adisseo) have a global objective to select and develop the microbial catalysts which are efficacious for a number of different applications such as detergents, starch, agrofuels, ethanol, textiles, human food, and animal feed. In recent years, the global industrial enzyme market size increased year by year, registering an annual growth of 5%. The oligopoly global enzyme market accounted for a 44% market share by Novozyme while share for Dupont and DSM was 20% and 6%, respectively, in 2014. Europe and North America bring the highest demand for industrial enzymes of nearly 80% in sharp contrast to 9.4% in China (2018). According to Section 3(c) of the Indian Patent Act 1970, the mere discovery of a scientific principle or the formulation of an abstract theory or discovery of any living thing or nonliving substance occurring in nature is not a patentable invention. Products such as microorganisms, nucleic acid sequences, proteins, enzymes, and compounds, which are directly isolated from nature, are not patentable subject matter. However, processes of

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isolation of these products can be considered subject to requirements of Section 2(1)(j) of the Act (Guidelines for Examination of Biotechnology Applications, n.d.). As per Indian Patent Act 1970, patent can be granted for the product, process, or system that fulfills basic patentability criteria including novelty, nonobviousness (involves inventive step), and industrial applications and it does not fall under excluded subject matter. For determining inventive step, the technology must not be an obvious development relative to the earlier public disclosures. It means technology would not be obvious to a person “skilled in the art". Examples of obviousness include obvious modifications (e.g., for production, administration, stability, and other process parameters), homologues of known enzymes, and usage of related enzymes for the same application/use. Grant of patent (1) prohibits others to work commercially in claimed subject matter/scope, (2) provides monopoly right for the claimed invention for next 20 years from the date of filing, and (3) presents opportunity to license or sell the invention. Patent rights can only be enforced if granted. Patent for enzymes can be granted for product which includes (1) specific novel/synergistic composition containing the enzyme; (2) specific DNA encoding the particular enzyme; (3) a vector containing the DNA; (4) host cells containing the vector; and (5) particular antibody which recognizes the enzyme, a process which includes (1) method of synthesizing the enzyme, (2) method of functional using the enzyme, (3) a method of screening for the presence of the enzyme, enzyme applications which include (1) usage of the enzyme for a specific purpose and enzyme for a medical application. Examples for nonpatentable inventions involving enzymes include (1) homologue of a known enzyme for a known application/use (e.g., from a different species), (2) enzyme with similar properties to a known enzyme for a known functionality (particularly if the sequence is similar), (3) enzyme optimized relative to known enzyme for a known functionality, using known techniques, and yielding no more than expected improvements (Dr E. Jones, n.d.).

21.4

Government policies toward food in biotechnology

International government bodies have embraced biotechnology as an emerging technology similar in potential to microelectronics and informatics technology. Just as the development of very large-scale integrated computers, biotechnology holds the promise of curing for almost every disease and also manufactures high-value products at low cost. The Cartagena Protocol is an international protocol adopted in 2000. It focuses on living modified organism’s safe handling, transportation, and its use. The protocol aims to prevent risks and adverse effects to both human health and

21.4 Government policies toward food in biotechnology

biological diversity. Cartagena Protocol defines a living modified organism as “any living organism that possesses a novel combination of genetic material obtained through the use of modern biotechnology.” The EU, Ethiopia, and India are among the 171 countries to have signed and ratified the protocol, unlike the United State. Modern biotechnology method to culture milk and egg proteins are not impacted by the protocol since the end product does not include any living modified organism (Sutay Kocaba¸s & Grumet, 2019).

21.4.1

Enzyme regulation in Canada

In Canada, food processing enzymes are regulated as food additives. An enzyme meets the definition of a food additive, as set out in Section B.01.001 of the Food and Drug Regulations, when it affects the characteristics of the food or its by-products become part of the food. Instead of enzyme activity, the physical enzyme residues are considered in determining if enzyme residues remain in or on a food. Health Canada is responsible for conducting the premarket safety assessment of enzymes and approving their use in foods along with other food additives. Enzymes that are permitted for use in foods sold in Canada, along with the permitted sources of each enzyme, are listed in the List of Permitted Food Enzymes which is published on Health Canada's website (Government of Canada, 2014). The safety of the source organism is the primary consideration in assessing an enzyme product. Food animals and edible plants have a history of safe use as sources of enzymes for the food industry. A microorganism used for food enzyme production must be well characterized and not produce any pathogens, toxins, or antibiotics. These are usually soil microorganisms to which humans are commonly exposed to through their environment and diet, and which have a history of safe use in food enzyme manufacture. Health Canada's safety assessment considers toxicity tests on the enzyme product and the process used to commercially produce the enzyme. The safety assessment of an enzyme produced by a GMM expands on the approach above. In this case, Health Canada also reviews the technique used to transfer the genetic material along with the safety of the genetic material that has been introduced and expressed in the production of microorganism. The genome of the production microorganism must be fully characterized for a safety review to be completed.

21.4.2

Enzyme regulation in Australia and New Zealand

Food processing enzymes are regulated as food additives. In proposal P276 of the Food Standards Australia New Zealand (FSANZ), an enzyme is defined

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as a food additive and the regulation of enzymes as processing aids in Clause 15, 16, and 17 of Standards 1.3.3 processing aids. A separate proposal, P277 Review of Processing Aids (other than enzymes), was finalized and gazetted on February 15, 2007. The act is now undergoing its first major review in almost 30 years (the review), which presents an exciting opportunity for modernization (Australia and New Zealand, 2019). Proposal P276 focuses on the review of enzymes, apart from the other processing aids. FSANZ considers the assessment of safety and management of risk and technological issues which are different for enzymes and processing aids. The review includes: G

G G G

G

The safety assessment of recently approved enzymes and the guidelines for the same. By-products of enzymatic reactions. The nomenclature of the enzymes and source organism. Listing the enzymes which are not being used in Australia and New Zealand currently. Other issues raised by submitters.

Over the time FSANZ has reviewed clauses and proposed a number of variations thus bringing changes to maintain health and safety and update nomenclature of enzymes and their sources, limiting errors, removing duplication and anomalies, enhancing the consistency, and thus improving the function of standard (Food standards Australia New Zealand & Ta Mana, 2007).

21.4.2.1 Proposed amendments G

G

G

G

G

Ensure protection of public health and safety since no safety concerns were identified during the safety assessment. Ensure consistency within the code and improvements with other international food standards. Include submissions on issues received, as well as advice from an expert advisory group, made up of experts external to FSANZ. Would not lead to any expected added costs to food manufacturers, consumers, or regulatory agencies. Turned the regulations more cost effective.

21.4.3

European Union regulation

Regulation (EC) No 1332/2008 - The so-called “Framework regulation” harmonized the rules on food enzymes for the first time in the EU and fixed a deadline of 2 years for the submission of applications for authorization. According to that regulation, all food enzymes have to be subject to safety evaluation by the

21.4 Government policies toward food in biotechnology

EFSA and subsequently approved by the European Commission by means of their inclusion into the Union list of food enzymes. The commission could understand and conclude that the initial deadline for submitting applications was insufficient as it was limiting the stakeholders and in particular small- and medium-size enterprises to produce all necessary data within that period. Therefore the 24 month period was extended to 42 months by Commission Regulation (EU) No 1056/2012, amending Regulation (EC) No 1332/2008. Industry now has 3 or 5 years to make the information available for the risk assessment of food enzymes and submit applications on existing and new enzymes, starting from September 11, 2011 (Regulation (EC) No 234/2011). These food enzymes are in the process of being evaluated for safety by EFSA and will be approved by “comitology” procedure (establishing the EU list). A food enzyme will be included in the EU list only if: G G G

It does not pose a health concern to the consumer. There is a technological need. Its use does not mislead the consumer.

The process to draw up the Union list requires the submission of applications for approval of food enzymes. Numerous applications were received and have undergone the safety evaluation by the EFSA. There is currently no Union list of authorized food enzymes, it will be established once the evaluations are finalized. Until then, the placing on the market and the use of food enzymes and of food produced with food enzymes are subject to the EU Member States' legislation. Other pieces of EU legislation relevant to food enzymes are the following: Regulation (EC) No 178/2002 lays down the general principles and requirements of food law. Regulation (EC) No 1331/2008 establishes the common authorization procedure for food additives, food enzymes, and food flavorings. Both Regulation (EC) No 178/2002S and Regulation (EC) No 1331/2008 were amended by Regulation (EU) 2019/1381 on the transparency and sustainability of the EU risk assessment in the food chain amended with effect from March 27, 2021. Regulation (EU) No 234/2011 implements the common authorization procedure and applies from September 11, 2011. That regulation has been amended by Commission Implementing Regulation (EU) No 562/2012 which lays down derogation from submitting toxicological data in some specific cases and the possibility of grouping food enzymes under one

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application under certain conditions. It has also been adjusted by Commission Implementing Regulation (EU) 2020/1823 to accommodate the changes linked to Regulation (EU) 2019/1381 on the transparency and sustainability of the EU risk assessment in the food chain. These new provisions are applicable to applications submitted from March 27, 2021 (EU Rules, n.d.).

21.4.4

Scope of enzyme regulation

The food enzymes added for some technological purpose in the manufacturing, processing, preparing, treating, packaging, transporting, or storing of food, including enzymes used as processing aids.

21.4.5 G

G

Limitations

Enzymes intended for nutritional or digestive purposes of human consumption; Food enzymes used in the production of food additives under Regulation EC 1333/2008 and in the production of processing aids.

Microbial cultures traditionally used in the production of food (cheese, wine), which may incidentally produce enzymes but are not specifically used to produce them, are not considered food enzymes (Fig. 21.1).

21.4.6

US regulations

Enzyme preparations used in food processing contain an active enzyme to cause the intended technical effect in food. The enzyme is derived from a variety of biological sources and preparation may contain a blend of two or more active enzymes. Diluents, preservatives, stabilizers, or other substances (suitable for use in food) are added to formulate the enzymes. Their preparations may also contain constituents derived from the source of enzyme or manufacturing process. Food processing involves very low levels of enzyme preparations which are either inactivated or removed from the end food product. Enzyme preparations can be regulated as secondary direct food additives under Title 21 of the Code of Federal Regulations, Part 173 (21 CFR 173). Petition process establishes the regulatory status of food additives, including secondary food additives. G

Section 409(b) (1) of the Federal Food, Drug, and Cosmetic Act (the Act) [21 U.S.C. 348(b) (1)]: Anyone can file the issuance of a regulation.

21.4 Government policies toward food in biotechnology

FIGURE 21.1 Food enzyme and novel food regulatory pathways in the EU.

G

G

Section 409(b) (2) of the Act [21 U.S.C. 348(b) (2)]: Prescribes the statutory requirements for food additive petitions. Section 201(s) of the Act [21 U.S.C. 321(s)]: Generally recognized as safe (GRAS) substance is exempted from use of a food additive.

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A substance can be determined to be GRAS by qualified experts under the intended conditions of use. An evidence of its safety and a basis to conclude is accepted. Title 21 of the CFR contains regulations for food ingredients that are either listed as GRAS or affirmed as GRAS through GRAS affirmation petitions. In the past, GRAS affirmation petitions were reviewed by FDA for enzyme preparations. A successful review of a GRAS affirmation petition resulted in a regulation in 21 CFR, Part 184, now a voluntary notification program under the agency’s proposed regulation [Proposed 21 CFR 170.36 (62 FR 18938); April 17 1997; Substances Generally Recognized as Safe (GRAS)] (U.S. Department of Health and Human Services Food and Drug Administration, 2010; Bernadene et al., 2013).

21.4.6.1 Petitions for enzyme preparations Section 409(b) (2) of the act describes the statutory requirements for food additive petitions, which focuses on five the information: G G G G G

Identification of additive. The proposed use of the additive. The intended technical effect of the additive. Analytical method for the additive in food. Intensive report on safety investigations of the additive.

21 CFR 171.1(c) details the data and administrative information required in food additive petitions, pertinent to the above five basic areas. 21 CFR 171.1(h) mentions the public disclosure of certain data and information contained in food additive petitions, while other data and information is restricted.

21.4.6.2 Generally Recognized as Safe notices for enzyme preparations GRAS may be based either on scientific procedures (21 CFR 170.30(b)) or on experience based on common use in food (21 CFR 170.30(c)), for substances used in food prior to January 1, 1958. GRAS can be determined by qualified experts outside of government through scientific procedures which require common knowledge about the substance and its safety under the intended conditions. A person may notify FDA of its determination that a substance is GRAS [Proposed 21 CFR 170.36 (62 FR 18938); April 17, 1997; Substances Generally Recognized as Safe (GRAS)] through a voluntary notification program. If the data and information provided in the GRAS notice do not raise safety concerns about the use of the substance at the time of the review, FDA issues a letter to the notifier stating that the agency has no questions

21.4 Government policies toward food in biotechnology

FIGURE 21.2 GRAS framework timeline.

regarding the notifier’s conclusion that the substance is GRAS under the intended conditions of use. GRAS framework timeline is shown in Fig. 21.2.

21.4.7

FAO/WHO

Joint FAO/WHO Expert Committee on Food Additives (JECFA) serves as an independent scientific expert committee, which performs risk assessments and provides advice to FAO, WHO, and the member countries of both organizations, as well as to the Codex Alimentarius Commission (CAC) (Food safety and quality, 2021; Sutay Kocaba¸s & Grumet, 2019). JECFA members are prominent scientists from across the world, of recognized scientific excellence with competencies spreading across disciplines within JECFA remit: G

G G

G

Risk assessments / safety evaluations of food additives, processing aids, residues of veterinary drugs in animal products, contaminants and natural toxins. Exposure assessments to chemicals. Specifications and analytical methods, residue definition, Maximum Residue Limits proposals on veterinary drugs. Guidelines for the safety assessment of chemicals in foods consistent with current thinking on risk assessment in toxicology and other relevant sciences.

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Table 21.2 Safety assessment of certain enzyme processing aids by Joint FAO/WHO Expert Committee on Food Additives. Safety assessment conclusions

Enzyme processing aid α-Acetolactate decarboxylase (EC 4.1.1.5) from Bacillus brevis α-Amylase (EC 3.2.1.1) from Bacillus licheniformis Hexose oxidase (EC 1.1.3.5) from Chondrus crispus Invertase (EC 3.2.1.26) from Saccharomyces cerevisiae Maltogenic amylase (EC 3.2.1.133) from Bacillus stearothermophilus Xylanases (EC 3.2.1.8) from Bacillus subtilis Mixed β-glucanase (EC 3.2.1.6) and xylanase (EC 3.2.1.8) enzyme preparation, produced by a strain of Humicola insolens

No No No No No No No

toxicological toxicological toxicological toxicological toxicological toxicological toxicological

concerns concerns concerns concerns concerns concerns concerns

Safety assessment of certain enzyme processing aids by JECFA is given in Table 21.2.

21.5 Policy and regulatory framework: lower middle income countries Ethiopia: The Food jurisdiction is regulated by multiple government agencies and ministries [Ethiopian Standards Agency (ESA), the Ethiopian Food and Drug Authority (EFDA), the Ministry of Agriculture and Livestock Resources (MOA&L), and the Ministry of Trade and Industry (MoTI)]. The ESA established a National Codex Committee in 2003 to provide guidance to the government on national implementation of the Codex and is also a participating member of the Codex Alimentarius Commission. In addition, the EFDA works along with the MOA&L to regulate the import of products of plants and livestock. Finally, the MoTI is responsible for the quality control of food imports and exports. At present, information specific to microbial food enzyme regulation in Ethiopia has not been identified (Sutay Kocaba¸s & Grumet, 2019; food safety and quality, n.d.). India: FSSAI published the Food Safety and Standards (Food Products Standards and Food Additives) Regulations in 2011. This set of regulations details standards for Indian market manufacturing, storage, distribution, sale, and import of commonly used food products and food enzymes. For example, nonanimal rennet (a microbial enzyme in cheese and other dairy products widely used in India) is approved for use under this regulation. Amendment is a continuous process, updating the subregulations on specific food products was announced by the FSSAI in the 16th amendment (2017).

21.6 Future prospect

As per Food Safety and Standards (Food Products Standards and Food Additives) Regulations, 2011 of FSSAI, a list of 48 enzymes (All enzymes are from nongenetically modified sources) for treatment or processing of raw materials, foods, or ingredients has been approved. Although, regulations generally indicate GMP usage for enzymes, however, at some places, usage level is defined. For example, pectinase enzyme can be added up to a level of 0.2% during manufacture of tea as processing aid. Comparison of the international regulatory systems on food enzymes in the United States, EU, Turkey, Argentina, and China are provided in Table 21.3 (Magnuson et al., 2013).

21.6

Future prospect

To ensure sustainable production and consumption of nutritional and safe food products is the core mandate and objective of the prevalent food regulations. Currently, an increasing worldwide preference of recombinant DNA technologies for food products, the individual countries and international bodies have enlisted specific legal regulations and political approaches on food products and food additives. The well-recognized systems prevalent in the United States and EU, along with the CODEX, usually serve as a benchmark for the adoption and formulation of regulatory approaches for food and food products in individual countries. Several countries have adopted new concepts and new procedures over the time to assess functional food risks. The provision of providing safe, nutritious, high-quality, and affordable food to consumers is the central objective of the policies, which should cover all stages of the food supply chain, "from farm to fork". The standards and requirements aim to ensure a high level of food safety and nutrition within an efficient, competitive, sustainable, and innovative global market. There are several challenges in developing countries for both further development of food enzyme regulation and for safety evaluation that can be identified. These challenges can be attributed to differences in both existing legislation and in requirements for safety evaluation. Functional foods need to be safe according to all criteria defined in the current regulations. In the near future, the need for such enzymes would be increasing, essentially due to the increasing demand of significant nutritional dairy products in a country to overcome the prevalence of malnutrition and obesity that necessitates a shift toward low-fat and nutritional foods. The regulation is set to improve the functioning of the internal market by removing disparities and bringing more legal certainty to the market. Only authorized food enzymes shall be allowed to be commercialized and /or

533

Table 21.3 Comparison of the international regulatory systems on food enzymes in the United States, EU, and Turkey, Argentina, and China. Countries

United States

EU

Turkey

Argentina

China

Regulatory Body

USDA (FDA)

EFSA

Ministry of Agriculture and Forestry

Ministry of Health (MOH)

Regulation

Code of Federal Regulations (CFR) Title 21 (Part 170)

Regulation (EC) No 1332/2008

Turkish Food Codex Food Enzymes Regulation

Assumption safety on the basis of general use/reasonable evidence

GRAS

QPS

Simplified Procedure

Ministry of Health (Ministerio de Salud) & The National Administration of Drugs, Foods, and Medical Technology (Administración Nacional de Medicamentos, Alimentos y Tecnología Médica ANMAT) CAA, Chapter XVI, Articles 1261, 1262 and 1263. Enzymes regulations are not harmonized in Mercosur Codigo Alimentario Argentino CAA GMC 26/03

Definition of food enzyme

“Food additives which are used to improvise food processing and the quality of the finished food.’’

“A product obtained from plants, animals or microorganisms or products there of including a product obtained by a fermentation process using microorganisms:(i) containing one or more enzymes capable of catalyzing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.’’

“A product obtained from plants, animals or microorganisms or a product containing one or more enzymes capable of catalyzing a specific biochemical reaction and obtained by a fermentation process using microorganisms; or, a product obtained by fermentation using various microorganisms and added to food for a technological purpose at any stage of the production, processing, preparation, treatment, packaging, transport or storage of foods.’’

“Enzymes or enzyme preparations are defined as substances of animal, plant or microbial origin that act by promoting the desirable chemical reactions’’

Hygienic Standard for Enzyme Preparations Used in Food Processing (Notified to the WTO as G/SPS/N/CHN/112 on January 5, 2009) Rules on Administrative Licensing of FoodRelated New Product Varieties (Notified to the WTO as G/SPS/N/CHN/ 120 on August 13, 2009) “Biological products directly extracted from edible or non-edible parts of a plant or animal or fermented and extracted from traditional or genetically modified microorganisms (including but not limited to bacteria, actinomycetes, and fungi) that are used in food processing and have a special catalytic function.” A list of allowable food enzyme preparations is available (Annex C, People's Republic of China, 2010)’’

References

used in foods sold irrespective whether they are used as processing aids or ingredients.

References Agarwal, S., & Sahu, S. (2014). Safety and regulatory aspects of food enzymes: An industrial perspective. IJIMS, 1, 253 267. Australia and New Zealand Ministerial Forum on Food Regulation, Review of the Food Standards Australia New Zealand Act 1991 - draft Regulatory Impact Statement (2019). Retrieved December 14, 2021 from https://consultations.health.gov.au/chronic-disease-andfood-policy-branch/fsanz-act-review-draft-ris/. Chandran, R. P. (2019). Enzymes in food industry. Retrieved December 14, 2021, https://amrita. edu/news/enzymes-food-industry/. Dr. Jones, E. Patenting an enzyme NorZymeD. (n.d.). Retrieved December 14, 2021, from https://norzymed.nmbu.no/sites/default/files/pdfattachments/elizabeth_jones.pdf. EU Rules. (n.d.). efsa, Food enzymes, Retrieved December 26, 2021 from EU Rules (efsa.europa.eu) https://www.efsa.europa.eu/en/topics/topic/food-enzymes#:B:text 5 A%20food%20enzyme% 20will%20be,and%20Regulation%20EC%201332%2F2008. Food safety and quality. (n.d.). Joint FAO/WHO Expert Committee on Food Additives (JECFA), Retrieved December 21, 2021 from https://www.fao.org/food-safety/scientific-advice/jecfa/en/. Food standards Australia New Zealand, Ta Mana kounga kal (2007). Food Standards Australia and New Zealand: Final Assessment Report Proposal P276 Review of processing Aids (enzymes), 1-6. Retrieved December 12, 2021 from https://www.foodstandards.gov.au/code/ proposals/documents/FAR_Final_P276_Review_of_Enzymes.pdf. Government of Canada, Health Canada. (2014). Enzymes used in food processing. Retrieved December 22, 2021. , https://www.canada.ca/en/health-canada/services/food-nutrition/ food-safety/food-additives/enzymes-used-processing.html . . Kumar, V., & Nunes, C. S., (2018). Enzymes in human and animal nutrition: Principles and perspectives (Vikas Kumar & Carlos Simões Nunes, Eds.; 1st edition (17 March 2018), Vol. 1). Academic Press. Magnuson, B., Munro, I., Abbot, P., Baldwin, N., Lopez-Garcia, R., Ly, K., McGirr, L., Roberts, A., & Socolovsky, S. (2013). Review of the regulation and safety assessment of food substances in various countries and jurisdictions. Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment, 30(7), 1147 1220. Available from https://doi. org/10.1080/19440049.2013.795293. Sutay Kocaba¸s, D., & Grumet, R. (2019). Evolving regulatory policies regarding food enzymes produced by recombinant microorganisms. GM Crops and Food, 10(4), 191 207. Available from https://doi.org/10.1080/21645698.2019.1649531. The Office of the Controller General of Patents, Designs & Trade Marks (CGPDTM), DPIIT, Ministry of Commerce & Industry, Government of India, Guidelines for Examination of Biotechnology Applications for Patent (2013), Retrieved December 8, 2021, http://ipindia. gov.in/writereaddata/Portal/IPOGuidelinesManuals/1_38_1_4-biotech-guidelines.pdf. U.S. Department of Health and Human Services Food and Drug Administration Center for Food Safety and Applied Nutrition, Guidance for Industry: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices for Enzyme Preparations (2010). Retrieved December 26, 2021, from https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/guidance-industry-recommendations-submission-chemical-and-technological-data-food-additive-petitions.

535

Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A

AAO. See Ascorbate oxidase (AAO) AAs. See Amino acids (AAs) Abstract theory, 523
524 ABTS assay. See 2,2'-Azinobis-3ethylbenzothiazoline-6-sulfonic assay (ABTS assay) ACE. See Angiotensin-converting enzyme (ACE) Acetate, 475
476 Acetonitrile, 475
476 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP), 456 Acid phosphatase (ACP), 16
18, 37
39, 69
70, 87
88 Acid whey, 403
404 Acid β-galactosidases, 368 Acidic enzymes, 365
366 ACP. See Acid phosphatase (ACP) Acremonium sp. L1
4B, 165
166 Acrylamide-co-acrylic acid, 356 Activities of β-galactosidases, 352f Adenine, 21 Adiabatic heating, 59
60 Administración Nacional de Medicamentos, Alimentosy Tecnología Médica (ANMAT), 534t Adulterants, 459
464 detection and estimation of hydrogen peroxide in milk, 460 starch in milk and milk products, 464 sucrose in milk and milk products, 462
463 detection of glucose in milk, 461 detection of maltodextrin in milk, 463 paper strip for urea detection, 463
464

Adulteration, 459 AG. See α-glucosidase (AG) AGC. See Aspiration of gut content (AGC) Aged cheeses, 334 Agitation, 14 Agro-industrial residues, 384
385 AIEX. See Anion-exchange chromatography (AIEX) Airways, 19
20 Alanine aminotransferase (ALT), 45
46, 272
273 Albutensin A, 217
218 Alcalase, 164
165, 190, 213
214, 241 Alginate, 356 Alkali cleaning solutions, 492
493 Alkaline phosphatase (ALP), 16
17, 37
39, 45
46, 58, 66, 78
87 activity in nonbovine milk, 85 effect of high-pressure processing on, 67 effect of mastitis, 85 methods for estimation of alkaline phosphatase activity, 80
85 analytical methods for ALP activity determination in dairy products, 81t AOAC method for cheese, 84
85 colorimetric methods, 80
83 fluorimetric methods, 83
84 immunochemical methods, 84 reaction catalyzed by alkaline phosphatase, 16f reactivation of, 85
87 significance of, 79
80 Alkaline-acid cleaning, 502 ALP. See Alkaline phosphatase (ALP)

Alpha-amylase, 521 α whey protein concentrate (αWPC), 324 α-1,4-glucan-4-glucanohydrolase, 496 α-acetolactate decarboxylase, 532t α-amylase (AM), 263, 520t inhibitory peptides from camel milk proteins, 270
271 α-glucosidase (AG), 263 inhibitory peptides from camel milk proteins, 271
273 α-lactalbumin (α-la), 26, 143, 189, 196t, 313
314, 403
404 α-lactorphin, 315t αs1-casein, 143, 168t, 171t, 175t, 313
314 αs2-casein, 143, 168t, 171t, 175t, 313
314 ALT. See Alanine aminotransferase (ALT) AM. See α-amylase (AM) American Public Health Association (APHA), 84
85 AMFEP. See Association of Manufacturers and Formulators of Enzyme Products (AMFEP) AMG. See Amyloglucosidase (AMG) Amino acids (AAs), 119, 126
127, 137, 313, 445
446 multiple alignment, 144f sequence of milk proteins, 138
143, 139t Aminopeptidase P (PepP), 199 3-Aminophenylboronic acid (3APBA), 484 Aminotransferases, 45
46, 49
50 Ammonium ions, 446
447, 449

537

538

Index

Amorphous phosphated titanium oxide (APTO), 323
324 Amperometric biosensor, 476 Amperometric sensor, 449
450 Amperometric transducers, 431
432 AMPs. See Antimicrobial peptides (AMPs) Amyloglucosidase (AMG), 463 Analysis of variance (ANOVA), 474
475 Analytical conventional techniques, 474
476 high-performance liquid chromatography, 474
475 liquid chromatography
mass spectrometry, 474
475 Analytical methods, 445
446, 530 for identification of bioactive peptides, 201
206 application of MS, 204t enrichment and fractionation of bioactive peptides, 201
203 peptide characterization, 203
206 Angiotensin-converting enzyme (ACE), 127
128, 166
167, 190, 316 ACE-I, 145, 290
291 inhibitory peptides, 163
164 Animal species, 289, 427 Anion-exchange chromatography (AIEX), 324 Anion-exchange membranes, 411
412 Anorexigenic hormones, 217 ANOVA. See Analysis of variance (ANOVA) Antibacterial enzymes, 43
44 Antibiotics, 445
446 Anticancer peptides, 212
213 Antidiabetic, 290
291 activity, 170
173 agent, 417
418 peptides, 206
207 from bovine casein, 171t derived from camel milk proteins, 263
274, 266t Antiendotoxin, 417
418 Antihypertensive activity, 167
170 Antihypertensive peptides, 207
208 from bovine casein, 168t

Antihypertensive properties of camel milk protein hydrolysates, 245
250, 246t, 251t Antiinflammatory peptides, 214
216 Antimicrobial Peptide Database (APD), 199
200 Antimicrobial peptides (AMPs), 208
210, 257 from camel milk, 250
263, 258t, 260t Antiobesity peptides from camel milk proteins and structural activity relationship, 274
276 Antioxidant activity, 174
177 effects of protein hydrolysates, 236
245, 236t structural activity relationship of camel milk
derived antioxidant peptides, 243
245 enzymes, 41
43 catalase, 42 glutathione peroxidase, 42 SOD, 42 xanthine oxidase, 43 peptides, 210
212 identified from camel milk via enzyme hydrolysis or fermentation, 238t AOAC method for cheese, 84
85 AOAC phosphatase method, 84
85 APD. See Antimicrobial Peptide Database (APD) APHA. See American Public Health Association (APHA) APTO. See Amorphous phosphated titanium oxide (APTO) Arrhenius-type volume model, 64 Arthrobacter sp., 334, 353
354 Arylesterases, 41 Aschaffenburg and Mullen test, 80
83 Ascorbate oxidase (AAO), 459 Ascorbic acid estimation in dairy products, 457
459 Aseptic process, 370 Aspartate aminotransferase (AST), 37
39, 45
46, 272
273 Aspergillus, 496 A. aculeatus, 375t, 394t A. niger, 356, 364, 385

A. oryzae, 356, 375t, 385, 394t, 409t Aspergillus oryzae. See Yeast cells (Aspergillus oryzae) Aspiration of gut content (AGC), 123 Association of Manufacturers and Formulators of Enzyme Products (AMFEP), 522 AST. See Aspartate aminotransferase (AST) Automated milking systems, 48
49 AYFYPEL peptides, 163
164 2,2'-Azinobis-3ethylbenzothiazoline-6-sulfonic assay (ABTS assay), 128
129

B

Bacillus, 496 B. circulans, 356, 375t, 385, 394t B. clausii, 198 B. metalloendopeptidase, 174 B. tequilensis, 502
503 Bacteria, 363t, 491, 519 Bacterial enzymes, 352 Bacterial load in milk samples by electrochemical multisensory methods, 479f Bacterial β-galactosidases, 366t Bactometer, 478 BAPs. See Bioactive peptides (BAPs) Basification reaction, 411
412 Batch process, 369 Beta cyclodextrin, 455 Beta-glucanase, 521 β-casein, 143, 168t, 171t, 175t, 313
314 β-casokinin-10, 177
178 β-casomorphin-7, 177
178 β-D-Galactosidase, 357 β-galactosidase, 351, 361, 363, 383
384, 450 activities of, 352f characteristics of, 363
372 future scope, 371
372 optimal reaction conditions for, 365
367 production and purification of, 367 reactions catalyzed by, 364
365 technologies for producing lowlactose milk, 369
371 classification of, 353
354, 354f

Index

immobilization of, 355
357 immobilized β-galactosidase for production of galactooligosaccharides, 393
395 for production of galactooligosaccharides, 385
393 future scope, 392
393 optimal reaction conditions for production of galactooligosaccharides, 387 production and purification of galactooligosaccharides, 388
391 reactions catalyzed by β-galactosidases for production of galactooligosaccharides, 386 sources of industrial β-galactosidase and galactooligosaccharides, 391
392 sources of β-galactosidases, 385
386 reactions of, 354
355, 354f sources of, 352, 353f, 363
364 sources of industrial, 368
369 structure of, 353 β-galactosidase, 431
432 β-glucanase, 504
509 β-hydroxy β-methylglutaryl-CoA reductase (HMG-CoA reductase), 155 β-lactoglobulin (β-lg), 143, 189, 196t, 313
314, 403
404 β-lactorphin, 315t β-lactotensin, 217
218 β/κ-casein, 171t β2-microglobulin, 39
40 BGM biosensor. See Blood glucose meter biosensor (BGM biosensor) Bifidobacteria, 338, 404
405 Bifidobacterium, 363
364, 384, 417 B. bifidum, 356
357, 375t B. lactis, 314
315 B. longum, 338 KACC91563, 174 Bile salt
stimulated lipase (BSSL), 3
4, 15 Bioactive compounds, 331
340

Bioactive peptides (BAPs), 119, 189, 313, 315, 321
322, 335. See also Antimicrobial peptides (AMPs) bioactive functional compounds, 314f bioactive peptides derived from cows' milk proteins, 315t bioactive peptides from dietary milk proteins, 316f from camel milk proteins, 235
278 downstream processing of, 319
325 estimation of bioactive peptide content in food items using insilico methods, 157
160 from fermented bovine milk products, 291
295 from fermented camel milk products, 298
299 from fermented goat milk products, 296
297 from fermented mare milk products, 300
302 from fermented sheep milk products, 302
304 function assessment methods, 127
130 importance of quantifying, 130
131 methods of isolation and identification/characterization of food-derived peptides, 126
127 predicted concentration of BPs in milk and milk proteins from different species, 158t production mechanisms of bioactive peptides, 315
319 enzymatic hydrolysis, 316 enzymes derived from proteolytic microorganisms, 317
319 fermentation, 316
317 production of bioactive peptides from bovine casein, 164
167 protein digestion methods, 120
126 released by the action of exogenous or milk-origin enzymes, 336t released by the action of microbial origin enzymes, 335t Biocatalyst, 406

Biochemistry, 368 Bioengineering tools, 364 Biofilms bacterial cells, 494
495 removal of, 504
509 Biological fluids, 447 Biological oxygen demand, 403
404 Biological properties of camel milk
derived hydrolysates, 276
278 Biological sources, 528 BioMilk 300, 432 BIOPEP-UWM database, 137
138, 145, 199
200 Bioprocess engineering, 471 Bioreactor systems, 371 Biosensors, 430
433, 431t Biotechnology, 384
385, 520, 524
532 Bisimidoesters, 355
356 Blood glucose meter biosensor (BGM biosensor), 433 in measurement of lactose, 438
439 in dairy ingredients blood glucose meter, 438
439 in milk blood glucose meter, 438 as option for determination of lactose, 433
439 Blue Star Chemicals, 505t Bombyx mori, 22
23 Boric acid, 416 Boronic acids, 484 Bos taurus. See Cow (Bos taurus) Bovine casein peptides, 167
178 antidiabetic activity, 170
173 antidiabetic peptides from bovine casein, 171t antihypertensive activity, 167
170 antihypertensive peptides from bovine casein, 168t antioxidant activity, 174
177 antioxidant peptides from bovine casein, 175t bioactivities, 177
178 production of bioactive peptides from, 164
167 degradation by digestive enzymes, 166

539

540

Index

Bovine casein (Continued) hydrolysis by enzymes from plant or microorganism, 164
166 proteolysis during fermentation, 166
167 Bovine liver catalase, 20
21 Bovine mastitis, 37 Bovine milk, 19, 291 Bovine plasminogen, 90
91 Bovine serum albumin (BSA), 189, 196t, 313
314 Bovine whey protein, 189 Box-Behnken Design, 410
411 BPs. See Bioactive peptides (BAPs) Brevibacterium linens group, 340 Brie, 334 Bromelain, 241 BSA. See Bovine serum albumin (BSA) BSSL. See Bile salt
stimulated lipase (BSSL) Bubalus bubalis. See Buffalo (Bubalus bubalis) Buffalo (Bubalus bubalis), 139t, 146t, 151t

C

CAC. See Codex Alimentarius Commission (CAC) Calcium, 366 Calcium phosphate, 491 Caldicellulosiruptor bescii (CbCEP), 413
414 Caldicellulosiruptor saccharolyticus, 413
414 Calibration curve, 435, 480 California Mastitis Test (CMT), 37 Calpis sour milk, 163
164 Camel (Camelus dromedarius), 77, 139t, 146t, 151t Camel milk, 85, 298 Camel milk proteins, 233
234 bioactive peptides from, 235
278 antidiabetic peptides derived from camel milk proteins, 263
274, 266t antihypertensive, 245
250 antimicrobial peptides from camel milk, 250
263, 258t, 260t

antiobesity peptides from camel milk proteins and structural activity relationship, 274
276 antioxidant, 236
245 biological properties of camel milk
derived hydrolysates, 276
278 in vitro studies, 264t future perceptions, 278 Camelus dromedarius. See Camel (Camelus dromedarius) Camembert cheese, 334 CAMP. See Collection of Antimicrobial Peptides (CAMP) Candida antarctica, 495
496 Candida kefir, 291 Capra hircus. See Goat (Capra hircus) Caprine milk, 15, 19 Carbamate, 446
447 Carbohydrate-Active enZYmes (CAZy), 353 Carbohydrates, 57
58, 392
393, 427 carbohydrate-based functional food ingredient, 383
384 digestive enzyme, 263 6-Carbon cyclic hemiacetal ring, 427 Carbon dioxide gas, 417, 447
448 Carboxyl ester hydrolases, 15
16 Carboxylesterases, 15
16, 41 Carboxylic chains, 364
365 Cardosin A and B, 192 Carotenoids, 340 Carrez solution, 450 Cartagena Protocol, 524 Casecidin, 178, 315t Casein, 163, 313
314, 332 beneficial effects, 163
164 bioactivities of bovine casein peptides, 167
178 production of bioactive peptides from bovine casein, 164
167 Casein glycomacropeptide (CGMP), 197
198 Casein glycomacropeptide hydrolysates, 173 Caseinate hydrolysates, 170
172 Caseino phosphor peptides (CPP), 316 Caseinomacropeptide (CMP), 321
322 Caseinophosphopeptides (CPPs), 178

Casokinins, 315t Casomorphins, 315t Casoplatelins, 315t Casoxins, 315t Caspase-3, 40
41 Caspases, 40
41 CAT. See Catalase (CAT) Catalase (CAT), 20
21, 42, 94
96, 174
177, 211
212, 272
273 activity in milk, 95 measurement of catalase activity, 95
96 physicochemical properties, 94
95 significance in dairy industry, 96 Cataloging of milk enzymes, 3 Catalysis, 479
480 Catalytic mechanism, 364 of β-galactosidase for hydrolysis and transgalactosylation reactions, 365f Cathepsin, 40 Cathepsin D, 10
14, 40 Cathode solution, 411 Cation-exchange membranes, 411
412 Cation-exchange resins, 390 CAZy. See Carbohydrate-Active enZYmes (CAZy) CbCEP. See Caldicellulosiruptor bescii (CbCEP) CCK. See Cholecystokinin (CCK) CDH. See Cellobiose dehydrogenase (CDH) CDR Food Lab analyzer, 429
430 CE. See Cholesterol esterase (CE); Cholesteryl esterase (CE) CEH. See Cholesterol ester hydrolase (CEH) Cell counters, 48
49 Cell envelope proteinases (CEPs), 194
195 Cell membrane, 367 Cellobiose 2-epimerase, 413 Cellobiose dehydrogenase (CDH), 431
432 Cellulase, 522 CEPs. See Cell envelope proteinases (CEPs) CertusBio biosensor, 433 CGMP. See Casein glycomacropeptide (CGMP)

Index

Cheddar, 18, 333
334 Cheese, 93, 316
317, 338, 362 AOAC method for, 84
85 making process, 338
339 plant-waste water streams, 431
432 Cheese ripening, 331 bioactive compounds, 332
340 carotenoids, 340 CLA, 337
338 gama aminobutyric acid and Lornithine, 338
340 peptides, 332
336 Cheese whey health benefits of lactulose, 417
418 lactulose production, 405
416 isomerization-based lactulose synthesis, 406
414 transgalactosylation-based lactulose synthesis, 414
416 separation of lactulose, 416
417 Chemical agents, 371 Chemical isomerization-based lactulose synthesis, 416 Chemical methods, 383, 406
411, 452
453 Chemical oxygen demand, 403
404 Chemical reactions, 406 Chemical-based CIP, 493
494 Chitosan, 356 Cholecystokinin (CCK), 172
173 Cholesterol, 455 enzyme coupled reaction of fluorometric enzymatic assay, 456f estimation of cholesterol in dairy products, 455
457 Cholesterol ester hydrolase (CEH), 15 Cholesterol esterase (CE), 455
456 Cholesterol oxidase (CO), 455 Cholesteryl esterase (CE), 274
275 Cholinesterases, 15
16, 41 Chromatographic methods, 202
203, 322
325 ion-exchange chromatography, 323
324 reversed-phase liquid chromatography, 324
325 size-exclusion chromatography, 322
323 Chromatography, 450

Chronoamperometry, 454
455 Chymosin, 521 Chymotrypsin, 190, 316 CIP. See Cleaning-in-place (CIP) Cis
urocanic acid, 45 Citric acid, 448 CLA. See Conjugated linoleic acid (CLA) Classical breeding techniques, 519 Cleaning-in-place (CIP), 492
493 considerations for development of optimal enzyme-based cleaning solutions, 509
512 fouling and, 491
493 industrial enzymes for, 493
496 reported studies on effectiveness of enzymes for, 496
509 removal of biofilms, 504
509 removal of Type A fouling deposits, 496
504 Clostridium tyrobutyricum, 22 “Clustal omega” tool, 143 Clustering, 481 CMP. See Caseinomacropeptide (CMP) CMT. See California Mastitis Test (CMT) CO. See Cholesterol oxidase (CO) Coagulants, 331 Codex Alimentarius, 521 Codex Alimentarius Commission (CAC), 531
532 Cold active enzymes, 363
364 Collagenase, 40 Collection of Antimicrobial Peptides (CAMP), 199
200 Colloidal calcium phosphate, 403
404 Colorimetric enzymatic assays, 455 Colorimetric estimation, 451 Colorimetric methods, 80
85 Colorimetric nanosensor, 483
484 Colorimetric sensors, 480 oxidized and reduced methylene blue species present in redox reactions, 481f Colorimetrically lactose, 450 Colostrum, 26 Column reactors with immobilized β-galactosidases, 376
377 Comitology, 527 Commercial β-galactosidases, 368t Commission Regulation (EU), 527

Compounds, 331 Compression of energy and heat, 59
60 con A. See Concanavalin A (con A) ConA-β-galactosidase, 355
356 Concanavalin A (con A), 215 Condensed milk, 362 Conjugated linoleic acid (CLA), 337
338 biological activity, 338 Conventional thermal death timetype model, 64 Conversion process, 412
413 Cooling/warming, 14 Corolase 2TS, 192 Correlation coefficients, 473
474 Coulter concept, 48
49 Covalent bond method, 372
373 Cow (Bos taurus), 139t, 146t, 151t Coxiella burnetii, 79
80 CPP. See Caseino phosphor peptides (CPP) CPPs. See Caseinophosphopeptides (CPPs) CVs. See Cyclic voltammograms (CVs) Cyclic voltammograms (CVs), 477
478, 478f Cytosolic Cu/Zn-SOD, 23

D

D-lactate

dehydrogenase (D-LDH), 453 D-LDH. See d-lactate dehydrogenase (D-LDH) Dahi, 297 Dairy animals, efforts in diagnosing mastitis in, 46
50 Dairy foods, 520 biosensors used in, 431 Dairy ingredients composition of common dairy ingredients, 429f importance of lactose in milk and, 428 measurement of lactose in dairy ingredients blood glucose meter, 438
439 Dairy products, 57, 63
64, 316
317, 435, 471, 520 DE. See Dextrose equivalent (DE) Debaryomyces hansenii, 166
167, 334

541

542

Index

Degradation by digestive enzymes, 166 Degree of hydrolysis (DH), 165 Dehydroascorbic acid (DHAsA), 458 Dephosphorylation, 18 Detection analytical conventional techniques, 474
476 high-performance liquid chromatography, 474
475 of common adulterants, 459
464 detection and estimation of starch in milk and milk products, 464 and estimation of hydrogen peroxide in milk, 460 and estimation of sucrose in milk and milk products, 462
463 of glucose in milk, 461 of maltodextrin in milk, 463 paper strip for urea detection, 463
464 lactate biosensors, 476
482 methods of, 474
486 Detergent industry, 495 Dextrose equivalent (DE), 463 DH. See Degree of hydrolysis (DH) DHAsA. See Dehydroascorbic acid (DHAsA) Diagnosis of mastitis, 39
40 Digestive enzymes, degradation by, 166 Dimethyl adipimidate (DMA), 355
356 Dimethyl suberimidate (DMS), 355
356 Dipeptidyl peptidase IV (DPP-IV), 127
128, 145, 163
164, 192, 263 inhibitory peptides from camel milk proteins, 263
270 2,2 Diphenyl-1 picrylhydrazyl (DPPH), 128
129 Directed evolution method, 385
386 Disaccharide lactose, 427 DMA. See Dimethyl adipimidate (DMA) DMS. See Dimethyl suberimidate (DMS) DNA shuffling, 385
386

Domestic animals, 519 Donkey (Equus asinus), 10, 139t, 146t, 151t Downstream processing of bioactive peptides, 319
325 bioactive peptides released from milk proteins, 319t chromatographic methods, 322
325 fractionation methods, 320 membrane separation techniques, 320
322 DPP-IV. See Dipeptidyl peptidase IV (DPP-IV)

E

EA. See Electroactivation (EA) EC. See Enzyme Commission (EC) Economical biostrip-based method, 429 EFDA. See Ethiopian Food and Drug Authority (EFDA) EFSA. See European Food Safety Authority (EFSA) Elastase, 40 Electric current density, 412 Electric field, 411 Electrical signal, 476 Electro-activation-based isomerisation, 411
413 Electroactivation (EA), 406 Electrochemical analyte, 431 Electrochemical methods, 476
478. See also Optical spectroscopic methods bacterial load in milk samples by electrochemical multisensory methods, 479f cyclic voltammograms, 478f novel electrochemical method towards detection of lactate, 477f Electrode potential, 448 Electrolysis reaction, 411 Electronic nose, 478 Electronic tongue, 478 Electrospray ionization (ESI), 203
206, 391, 475
476 Electrostriction, 59
60 Emmental, 333 EMR. See Enzymatic membrane reactor (EMR)

Endopeptidases, 495 Engineered enzymes, 389t ENMEX, 505t Enrichment and fractionation of bioactive peptides, 201
203 Ensure protection, 526 Enterococcus spp, 338, 473 E. faecalis, 318t Environment-friendly method, 383 Environmental contamination, 374 Enzybel, 505t Enzymatic absorbance (EZA), 438
439 Enzymatic digestion, 119
120 Enzymatic hydrolysis, 190
192, 316, 361, 363, 403
404 bioactive peptides released from milk proteins by enzymes, 317t Enzymatic isomerisation, 413 Enzymatic membrane reactor (EMR), 415
416 Enzymatic methods for production of GOS, 389t for protein digestion, 120
122 for urea detection, 446
447 Enzymatic recognition element, 431
432 Enzyme Commission (EC), 5 Enzyme photo-activated systems (EPAS), 83
84 Enzyme regulation in Australia and New Zealand, 525
526 amendments, 526 in Canada, 525 scope of, 528 Enzyme-based analytical methods pertinent to dairy industry ascorbic acid estimation in dairy products, 457
459 detection of common adulterants, 459
464 detection and estimation of hydrogen peroxide in milk, 460 detection and estimation of starch in milk and milk products, 464 detection and estimation of sucrose in milk and milk products, 462
463 detection of glucose in milk, 461

Index

detection of maltodextrin in milk, 463 paper strip for urea detection, 463
464 estimation of cholesterol in dairy products, 455
457 estimation of lactate or lactic acid in dairy products, 452
455 lactose estimation, 450
452 measuring change in pH, 452 spectrophotometric method, 450
452 urea estimation in milk, 446
450 monitoring change in pressure, 447
448 monitoring pH change, 447 potentiometric approach, 448
449 spectrophotometric measurement of ammonium ion concentration, 449 urea biosensor, 449
450 Enzyme:substrate ratio (E:S), 165, 190 Enzymes, 3
5, 57
58, 62
63, 69
70, 351, 493, 519, 524 cleaning, 493
494 coupled reaction of fluorometric enzymatic assay, 456f in dairy science and technology acid phosphatase, 87
88 alkaline phosphatase, 78
87 catalase, 94
96 GGT, 100
101 lactoperoxidase, 96
98 milk lipoprotein lipase, 88
90 plasmin, 90
94 xanthine oxidoreductase, 98
99 derived from microorganisms and used in food technology, 520t derived from proteolytic microorganisms, 317
319 enzyme-based isomerisation, 413
414 enzyme
substrate interaction, 62
63 immobilization, 392
393 kinetics, 435 levels in cow, buffalo, goat, sheep, camel, and human milk, 11t in mastitis milk, 39
46 antibacterial enzymes, 43
44 antioxidant enzymes, 41
43

esterases, 41 LDH, 45 NAGase, 44
45 phosphatases and aminotransferases, 45
46 proteases, 39
41 in milk, 77 molecules, 415
416 preparations, 528
530 for removal of fouling deposits type A in dairy industry, 497t reported studies on effectiveness of enzymes for cleaning-in-place in dairy industry, 496
509 removal of biofilms, 504
509 removal of Type A fouling deposits, 496
504 stability, 387 thermal stability, 374
375 Enzymology, 450 Eosinophil peroxidase (EPO), 19
20 EPAS. See Enzyme photo-activated systems (EPAS) EPO. See Eosinophil peroxidase (EPO) Equus asinus. See Donkey (Equus asinus) Equus caballus. See Horse (Equus caballus) ERK1/2. See Extracellular signalregulated kinases 1 and 2 (ERK1/ 2) ESA. See Ethiopian Standards Agency (ESA) Escherichia coli, 17, 352, 363
364, 434, 478 GGTP, 24
25 ESI. See Electrospray ionization (ESI) Esterases, 3
4, 14
16, 41, 520t Ethiopia, 532 Ethiopian Food and Drug Authority (EFDA), 532 Ethiopian Standards Agency (ESA), 532 EU. See Commission Regulation (EU) European Food Safety Authority (EFSA), 69
70, 521 European Union regulation, 526
528 Exocrine secretions, 19
20 Exopeptidases, 495 Exposure time, 61
62

Externally crusted ripened cheeses, 334 Extracellular DNA, 509 Extracellular EC-SOD, 23 Extracellular signal-regulated kinases 1 and 2 (ERK1/2), 265 EZA. See Enzymatic absorbance (EZA)

F

Factory-set calibration function, 433 FAD. See Flavin adenine dinucleotides (FAD) FAO. See Food and Agriculture Organization (FAO) Fast protein liquid chromatography, 322 Fatty acids, 495
496 Federal Food, Drug, and Cosmetic Act, 528 FeptideDB, 155
156 Fermentation, 119
120, 194
199, 289, 316
317, 452
453, 471 bioactive peptides released from milk proteins, 318t process, 428 Fermented dairy products, 365 Fermented milk products, 289, 405
406 animal species, 290f bioactive peptides from fermented bovine milk products, 291
295, 295f from fermented camel milk products, 298
299 from fermented goat milk products, 296
297 from fermented mare milk products, 300
302 from fermented sheep milk products, 302
304 Kefir, 294
295 Koumiss, 293
294 Soful, 292 yogurt, 293 Fermented products, 289 Ferric reducing antioxidant power (FRAP), 241 First Action Official Method, 433 First-order kinetic model, 64
65 Flavin adenine dinucleotides (FAD), 22

543

544

Index

Flavin-dependent sulfhydryl oxidase, 24 Flavourzyme, 164, 190 Fluidized bed reactors, 394
395 Fluorescent resorufin, 453 Fluorimetric methods, 83
84 Fluoro-yellow, 83
84 Fluorophos, 83
84 test system method, 83
84 Foaming, 14 Food and Agriculture Organization (FAO), 521, 531
532 Food chain, 527 Food enzymes government policies toward food in biotechnology, 524
532 enzyme regulation in Australia and New Zealand, 525
526 enzyme regulation in Canada, 525 European Union regulation, 526
528 FAO/WHO, 531
532 limitations, 528 scope of enzyme regulation, 528 US regulations, 528
531 policy and regulatory framework, 532
533 regulatory framework regarding food enzymes, 522
523 specific aspects of intellectual property right protection on enzymes, 523
524 Food guidelines, 521 Food labeling information, 61 Food microbiology, 471
472 Food processing concept, 59 enzymes, 525
526 Food Safety Standards Authority of India (FSSAI), 446, 532
533 Food Standards Australia New Zealand (FSANZ), 525
526 Food technology, 520, 520t Food-derived peptides, 119 methods of isolation and identification/characterization of, 126
127 Formaldehyde, 445
446 FOS. See Fructooligosaccharides (FOS) Fouling, 491
493

CIP procedure undertaken in dairy industry, 492f Fourier transform infrared spectroscopy (FTIR spectroscopy), 479
482 Fractionation methods, 320 Framework regulation, 526
527 FRAP. See Ferric reducing antioxidant power (FRAP) Free enzymes, 371, 389t Free fatty acids, 331 Freezing, 14 Frozen milk, 362 Fructooligosaccharides (FOS), 404
405 Fructose, 383, 410
411 FSANZ. See Food Standards Australia New Zealand (FSANZ) FSSAI. See Food Safety Standards Authority of India (FSSAI) Function assessment methods, 127
130 in vitro methods, 127
129 in vivo methods, 129
130 Functional enzyme aggregates, 355
356 Functional peptides, 57
58 Fungal β-galactosidases, 365
366, 366t Fungi, 363
364, 495
496, 519

G

G-6-P-DH. See Glucose-6-phosphate dehydrogenase (G-6-P-DH) GABA. See Gama aminobutyric acid (GABA) GAD. See Glutamic acid decarboxylase (GAD) Galactooligosaccharides (GOS), 352, 362, 383
384, 389t, 404
405 β-galactosidase for production of, 385
393 immobilized β-galactosidase for production of, 393
395 industrial β-galactosidase and, sources of, 391
392 optimal reaction conditions for production of, 387 production and purification of, 388
391

reactions catalyzed by β-galactosidases for production of, 386 Galactose, 353
355, 361, 366
367, 383, 410
411, 451 Galactose dehydrogenase (GalDH), 451 Galactose monosaccharides, 362 Galactosyl moiety, 414
415 Galactosyl-enzyme complex, 364
365 Galactosyltransferase, 39
40 Galactotransylation reactions, 375
376 GalaxyPEPDOCK, 155
156 GalDH. See Galactose dehydrogenase (GalDH) Gama aminobutyric acid (GABA), 331, 338
340 synthesis of GABA from L-glutamic acid, 339f γ-glutamyl transferase (GGT), 24, 58, 66, 69
70, 100
101 effect of high
pressure processing on, 68
69 physicochemical properties, 100 significance in dairy industry, 100
101 γ-glutamyl transpeptidase (GGTP), 3
4, 24
25, 100 Gas chromatography (GC), 430t Gas chromatography-MS (GC
MS), 391 Gastrointestinal digestion, hydrolysis during, 192
194 Gastrointestinal tract (GIT), 119
120, 383 Gaussian software, 155 GC. See Gas chromatography (GC) GC
MS. See Gas chromatographyMS (GC
MS) GDH. See Glucose dehydrogenase (GDH); Glutamate dehydrogenase (GDH) GDH-FAD. See Glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD) GDH-NAD. See Glucose dehydrogenase nicontinamide adenine dinucleotide (GDH-NAD) GDH-PQQ. See Glucose dehydrogenase pyrroloquinoline quinine (GDH-PQQ)

Index

Gel beads, 356 Gel filtration chromatography, 323 Gel permeation chromatography, 367 Genencor, 497t Generally Recognized as Safe (GRAS), 363
364, 494, 521, 529
531 framework timeline, 531f notices for enzyme preparations, 530
531 Genetic material, 525 Genetic modification (GM), 369, 520 Genetic sequence, 520 Genetically modified microorganisms (GMMs), 522 Geotrichum, 334 GGT. See γ-glutamyl transferase (GGT) GGTP. See γ-glutamyl transpeptidase (GGTP) GH. See Glycosyl hydrolase (GH) Ghee, 455 Ghrelin, 172
173 GIP. See Glucose inhibitory polypeptide (GIP); Glucosedependent insulinotropic peptide (GIP) GIT. See Gastrointestinal tract (GIT) GLP-1. See Glucagon-like peptide 1 (GLP-1) Glucagon-like peptide 1 (GLP-1), 172
173, 206, 263 Gluconic acid, 522 Glucose, 353
355, 383, 391
392, 431
432, 450, 522 detection of glucose in milk, 461 measurement of second-generation amperometric glucose oxidase, 436f meter method, 438 Glucose dehydrogenase (GDH), 435 Glucose dehydrogenase flavin adenine dinucleotide (GDH-FAD), 437t Glucose dehydrogenase nicontinamide adenine dinucleotide (GDH-NAD), 437
438, 437t Glucose dehydrogenase pyrroloquinoline quinine (GDHPQQ), 437
438, 437t

Glucose inhibitory polypeptide (GIP), 206 Glucose oxidase (GOx), 435, 437t, 451 Glucose-6-phosphate dehydrogenase (G-6-P-DH), 451, 461 Glucose-dependent insulinotropic peptide (GIP), 172
173, 263 Glucoseoxidase, 522 Glutamate dehydrogenase (GDH), 449 Glutamic acid decarboxylase (GAD), 338
339 Glutaraldehyde, 355
356, 454
455 Glutathione (GSH), 272
273 Glutathione peroxidase (GSH-Px), 42, 174
177, 211
212 Glycolysis, 331 Glycomacropeptids, 315t fraction, 403
404 Glycosidic bonds, 383
384 Glycosyl hydrolase (GH), 353 Glycosyn, 391
392 GM. See Genetic modification (GM) GMMs. See Genetically modified microorganisms (GMMs) Goat (Capra hircus), 77, 139t, 146t, 151t milk, 87 GOS. See Galactooligosaccharides (GOS) Government policies toward food in biotechnology, 524
532 enzyme regulation in Australia and New Zealand, 525
526 enzyme regulation in Canada, 525 European Union regulation, 526
528 FAO/WHO, 531
532 limitations, 528 scope of enzyme regulation, 528 US regulations, 528
531 food enzyme and novel food regulatory pathways in EU, 529f GOx. See Glucose oxidase (GOx) Gram-positive bacteria, 25
26, 367 Grana Padano, 18 GRAS. See Generally Recognized as Safe (GRAS) Green technology, 523 GROMACS software, 155 Growth factors, 57
58

GSH. See Glutathione (GSH) Guanidine hydrochloride (GuHCL), 126
127 GuHCL. See Guanidine hydrochloride (GuHCL) Gut microflora, 404
405 Gut microorganisms, 404

H

HCC. See Hepatocellular carcinoma (HCC) HE. See Hepatic encephalopathy (HE) Health benefits of lactulose, 417
418, 418t Healthy mammalian species, 313 Heat exchangers, 491 Hematocrit levels, 436
437 Hemicellulase, 504
509 Henry’s law, 448 Hepatic encephalopathy (HE), 417
418, 418t Hepatocellular carcinoma (HCC), 125
126 Heterogeneity, 504 Heterogeneous catalysts, 408
410 Heterologous expression, 363
364 Heterologous secretion, 367 Hexokinase, 451 Hexose oxidase, 532t HHP. See High hydrostatic processing (HHP) High hydrostatic processing (HHP), 59
61 High pH anion-exchange chromatography with pulsed amperometric detection (HPAECPAD), 391 “High Pressure Certified” seal, 61 High-performance liquid chromatography (HPLC), 322, 430t, 471
472, 474
475 protein concentration in ultrafiltration skim milk retentates, 475f High-pressure processing technology (HPP technology), 57, 59
61 effect of high pressure on activity and structure of milk enzymes, 61
63 effect of high-pressure processing on

545

546

Index

High-pressure processing technology (HPP technology) (Continued) alkaline phosphatase, 67 lipoprotein lipase, γ-glutamyltransferase, and lactoperoxidase, 68
69 milk enzyme system, 65
69 plasmin, 68 kinetics of high pressure on milk enzyme inactivation, 63
65 milk enzymes as high
pressure processing indicator, 69
70 need for alternate processing of milk, 58
59 schematic representation of highpressure processing of milk, 60f significance of milk enzymes, 57
58 High-temperature cooked cheeses, 333 High-temperature short-time pasteurization (HTST pasteurization), 10
14 hIR. See Human insulin receptor (hIR) HLPLP peptides, 163
164 HMG-CoA reductase. See β-hydroxy β-methylglutaryl-CoA reductase (HMG-CoA reductase) Homo sapiens. See Human (Homo sapiens) Homogeneous catalysts, 408
410 Homogenization, 14 Hormones, 57
58 Horse (Equus caballus), 139t, 146t, 151t Horseradish peroxidase (HRP), 455
456 Horses, 10 HPAEC-PAD. See High pH anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD) HPLC. See High-performance liquid chromatography (HPLC) HPLC-PAD method, 433 HPP technology. See High-pressure processing technology (HPP technology) HRP. See Horseradish peroxidase (HRP) HTST pasteurization. See Hightemperature short-time

pasteurization (HTST pasteurization) Human (Homo sapiens), 139t, 146t, 151t BSSL, 15 cancer cell lines, 212
213 digestive system, 417 health, 383 Human insulin receptor (hIR), 265 Human milk, 15, 85 lysozyme, 26 Hydraulic fluid, 60
61 Hydrogen ion concentration, 447 Hydrogen peroxide (H2O2), 19
20, 43, 96, 99, 432
433, 445
446, 455
456, 460 detection and estimation in milk, 460 Hydrolases, 5 Hydrolysis, 447 by enzymes from plant or microorganism, 164
166 during gastrointestinal digestion, 192
194 of lactose by beta-galactosidase enzyme, 434f reaction, 362, 387 Hydrolytic cleavage, 495 Hydrolytic enzymes, 383 Hydrophilic interaction chromatography, 322 Hydroxyl ions, 411
412 2-Hydroxypropanoic acid, 471 Hypercholesterolemia, 417
418 Hypertension, 167
170, 207 Hypothiocyanite ion, 20 Hypothiocyanous acid, 20

I

Ice cream, 362 IDF. See International Dairy Federation (IDF) IEC. See Ion exchange chromatography (IEC) IFN-γ. See Interferon-γ (IFN-γ) IL-8. See Interleukin-8 (IL-8) Ile-Pro-Pro (IPP), 167
170 Ile-Pro-Pro-Leu (IPPL), 167
170 IMCU. See International Milk Clotting Units (IMCU) Immobilization, 372, 449
450 of β-galactosidases, 355
357

chitosan, 356 functional enzyme aggregates, 355
356 gel beads and lattices, 356 metal affinity columns, 357 methacrylate and variants, 357 nanoparticles, 356
357 process, 362 Immobilized enzyme bioreactors, 362 Immobilized β-galactosidases, 372
376 column reactors with, 376
377 for production of galactooligosaccharides, 393
395 Immune system, 384 Immunochemical methods, 84 Immunoglobulins, 57
58, 189, 313
314 Immunomodulatory cytokine, 384 Immunomodulatory peptides, 213
214 Immunopeptides, 315t In-silico aided enzymatic release of bioactive peptides, 199
201 In-silico methods for milk-derived bioactive peptide prediction, 137
138 A, AE, and W scores of milk proteins, 151t amino acid sequence of milk proteins, 138
143, 139t estimation of bioactive peptide content in food items using insilico methods, 157
160 fragments, 146t molecular docking simulation, 155
156 in-silico digestion of milk proteins, 143
155 in vitro confirmatory experiments after in-silico prediction, 156
157 In vitro confirmatory experiments after in-silico prediction, 156
157 In vitro methods, 119
122 enzymatic method, 120
122 In vivo methods, 119
120, 122
126 aspiration of gut content, 123 characteristics of proteolysis methods, 124t

Index

identification/characterization in blood, 125
126 measurement in blood, 123
125 Inactive apoenzyme, 338
339 Indian Patent Act 1970, 524 Indicator dye, 447 Indigenous enzymes in milk, 77 Indigenous microorganisms, 471 Indigenous milk enzymes, 4 principal indigenous milk enzymes, source, substrates, kinetic parameters, and reaction catalyzed, 9t reaction catalyzed, and major location in milk, 6t significance of, 5f Industrial enzymes, 383
384 for cleaning-in place in dairy industry, 493
496 Industrial β-galactosidase, sources of, 368
369 Inflammatory bowel diseases, 417
418 Influenza virus, 434 Intellectual Property (IP), 523 specific aspects of intellectual property right protection on enzymes, 523
524 Interferents, 437
438 Interferon-γ (IFN-γ), 213
214 Interleukin-8 (IL-8), 213
214 International Dairy Federation (IDF), 58
59, 447 International Milk Clotting Units (IMCU), 172
173 International Organization for Standardization (ISO), 436 International regulatory systems on food enzymes, 534t International Union of Biochemistry and Molecular Biology (IUBMB), 5 Interventions, 278 Intestinal bacterial microbiota, 384 Ion exchange chromatography (IEC), 322
324 Ion-exchange membrane chromatography, 367 Ion-exchange method, 473
474 Ion-selective electrode (ISE), 448 IP. See Intellectual Property (IP) IPI peptides, 163
164 IPIQY peptides, 163
164 IPP. See Ile-Pro-Pro (IPP)

IPP peptides, 163
164 IPPL. See Ile-Pro-Pro-Leu (IPPL) ISE. See Ion-selective electrode (ISE) ISO. See International Organization for Standardization (ISO) Isolation of bioactive peptides, 319
320 Isomerases, 5 Isomerisation process, 406, 409t Isomerization-based lactulose synthesis, 406
414 chemical method, 406
411 electro-activation-based isomerisation, 411
413 enzyme-based isomerisation, 413
414 Isooctane reverse micelles, 387 Isostatic rule, 59
60 Isracidin, 178, 315t iTram Hygiene, 505t IUBMB. See International Union of Biochemistry and Molecular Biology (IUBMB)

J

JAK. See Janus kinase (JAK) Janus kinase (JAK), 214
215 JECFA. See Joint FAO/WHO Expert Committee on Food Additives (JECFA) Joint FAO/WHO Expert Committee on Food Additives (JECFA), 531
532 Joule heating effect, 412
413

K

K-E linkages, 334 K-I linkages, 334 K-V linkages, 334 κ-casein, 143, 168t, 175t, 313
314 Kay and Graham method, 83 Kefir, 294
297 Kinetics of high pressure on milk enzyme inactivation, 63
65 parameters, 393 Klebsiella pneumoniae, 478 Kluyveromyces fragilis, 291, 356, 385 Kluyveromyces lactis, 166
167, 352
353, 364, 375t, 385, 394t, 414
415

Kluyveromyces marxianus, 166
167 Koopeh, 303
304 Koumiss, 293
294, 301
302 KVLPVPEK, 174
177

L

L-lactate dehydrogenase (L-LDH), 453 L-LDH. See L-lactate dehydrogenase (L-LDH) L-ornithine, 338
340 LA rearrangement. See Lobry de Bruyn-Alberda van Ekenstein rearrangement (LA rearrangement) LAB. See Lactic acid bacteria (LAB) Laboratory milk fouling model, 502 Lactase, 351, 355, 521 Lactase deficiency (LD), 361 Lactase non-persistence (LNP), 361 Lactate biosensors, 476
482 electrochemical methods, 476
478 cyclic voltammograms, 478f novel electrochemical method towards detection of lactate, 477f lactic acid for assessing milk microbial load, 473
474 lactate detection methods for milk analysis, 472f statistical data for lactic acid analyses by four methods, 473t and metabolites, 485f methods of detection, 474
486 analytical conventional techniques, 474
476 lactate biosensors, 476
482 nanotechnology applications in sensors, 483
486 nanotechnology applications in sensors, 483
486 optical spectroscopic methods, 479
482 colorimetric, 480 Fourier transform infrared spectroscopy, 480
482 UV
vis spectroscopy, 479
480 Lactate dehydrogenase (LDH), 37
39, 45, 49
50, 477 and peroxidase enzymes and involvement, 454f

547

548

Index

Lactate detection methods for milk analysis, 472f Lactate in dairy products, estimation of, 452
455 Lactate oxidase (LOD), 453
454 Lactate sensors, 483 Lactic acid for assessing milk microbial load, 473
474 estimation of lactic acid in dairy products, 452
455 production, 474 Lactic acid bacteria (LAB), 166
167, 194
195, 289, 316
317, 333
334, 338, 363
364, 445
446, 471 Lactobacillus, 333
334, 384, 473 GG, 317
319 L. acidophilus, 314
315 ATCC 4356, 177
178 La-5, 195 L. bulgaricus, 242, 291 DSM 20081, 356 L. casei, 166
167, 314
315 L. delbrueckii WS4, 174 L. helveticus, 122, 314
315, 390
391 LH-2, 195 L. kefiri, 174 L. paracasei, 339 L. plantarum, 356
357 L. reuteri L103, 356 L. rhamnosus, 317
319, 318t, 339 Lactobionic acid, 404
405 Lactococcus, 333
334, 473 L. lactis, 291 L. lactis subsp. lactis GR5, 177
178 Lactoferampin, 208
209 Lactoferricin (Lfcin), 208
209, 315t Lactoferrin (LF), 195, 196t, 208
209, 313
314, 323
324 Lactoferroxin, 315t Lactoperoxidase (LP/LPO), 19
20, 43
44, 58, 65
66, 96
98 concentration in milk and colostrum, 97 effect of high
pressure processing on, 68
69 physicochemical properties, 97 reaction catalyzed by, 19f significance of lactoperoxidase enzyme, 97
98

system in milk, 98 Lactoperoxidase system (LPS), 20, 98 Lactose, 58, 404f, 427, 445
446 biosensors, 431
433 used for lactose quantification, 431
433 used in dairy foods, 431 biotransformation, 384
385 blood glucose meter biosensors as option for determination of, 433
439 blood glucose meter in measurement of lactose, 438
439 blood glucose meter operating principles, 435 potential issues with a blood glucose meter
based lactose assay, 436
438 blood glucose meter in measurement of, 438
439 blood glucose meter in measurement of lactose, 438
439 in dairy ingredients blood glucose meter, 438
439 in milk blood glucose meter, 438 blood glucose meter operating principles, 435 chemical structure of ß-lactose, 427f crystallization, 362, 428 derivatives of, 405f estimation, 450
452 measuring change in pH, 452 spectrophotometric method, 450
452 importance of lactose in milk and dairy ingredients, 428 lactose quantification methods, 428
431 for dairy foods, 430t lactose-free dairy products, 362 lactose-free product market, 362 potential issues with blood glucose meter
based lactose assay, 436
438 quantification methods, 428
431 biosensors used for, 431
433 for dairy foods, 430t transgalactosylation with β-galactosidase as catalyst, 414f

Lactose intolerance (LI), 361 Lactosens, 431
433 Lactulose, 404f, 417 health benefits of, 417
418 isomerization-based lactulose synthesis, 406
414 chemical method, 406
411 electro-activation-based isomerisation, 411
413 enzyme-based isomerisation, 413
414 production, 405
416, 407f separation of, 416
417 synthesis process, 416 transgalactosylation-based lactulose synthesis, 414
416 Lactulosylamine, 408
410 LAP. See Leucine aminopeptidase (LAP) Laser scanning confocal microscopy, 509
510 Lattices, 356 L
B reaction. See Liebermann
Burchard reaction (L
B reaction) LBU. See Lovibond Blue Units (LBU) LC. See Liquid chromatography (LC) LC-MS method. See Liquid chromatography-mass spectrometry method (LC-MS method) LC
MS/MS. See Liquid chromatography followed by tandem mass spectrometry (LC
MS/MS) LD. See Lactase deficiency (LD) LDH. See Lactate dehydrogenase (LDH) Le Chatelier’s principle, 59
60 Leucine aminopeptidase (LAP), 167
170 Leuconostoc, 473 L. lactis PTCC1899, 243 LF. See Lactoferrin (LF) Lfcin. See Lactoferricin (Lfcin) Liebermann
Burchard reaction (L
B reaction), 455 Ligases, 5 Linear calibration model, 438 Linkages, 334 Lipases, 3
4, 14, 77, 88, 495
496, 520t, 521 reaction catalyzed by, 14f

Index

Lipids, 57
58 Lipolysis, 88
89, 331 Lipopolysaccharide (LPS), 215 Lipoprotein lipase (LPL), 3
4, 15, 88, 457 effect of high
pressure processing on, 68
69 Liquid cheese whey, 198 Liquid chromatography (LC), 201, 322, 455, 474
475 Liquid chromatography followed by tandem mass spectrometry (LC
MS/MS), 324 Liquid chromatography-mass spectrometry method (LC-MS method), 471
472, 474
476 LLM. See Low-lactose milk (LLM) LMW peptides. See Low-molecularweight peptides (LMW peptides) LNP. See Lactase non-persistence (LNP) Lobry de Bruyn-Alberda van Ekenstein rearrangement (LA rearrangement), 406
408, 407f LOD. See Lactate oxidase (LOD) Lovibond Blue Units (LBU), 80
83 Low-lactose milk (LLM), 362 characteristics of β-galactosidases, 363
372 future scope, 371
372 optimal reaction conditions for β-galactosidases, 365
367 production and purification of β-galactosidases, 367 reactions catalyzed by β-galactosidases, 364
365 sources of industrial β-galactosidase, 368
369 sources of β-galactosidases, 363
364 technologies for producing LLM, 369
371 column reactors with immobilized β-galactosidases, 376
377 immobilized β-galactosidases, 372
376 Low-molecular-weight peptides (LMW peptides), 120
122 LPL. See Lipoprotein lipase (LPL) LPQNIPPL, 170
172 LPS. See Lactoperoxidase system (LPS); Lipopolysaccharide (LPS) LPYPY peptides, 163
164

Lyases, 5 Lysozyme, 3
4, 25
26 Lytic enzymes, 367

M

Macrophages, 37
39 Maillard reaction, 491, 522 MALDI. See Matrix-assisted laser desorption ionization (MALDI) MALDI coupled to time-of-flight MS (MALDI-TOF MS), 391 MALDI-time-of-flight (MALDI-TOF), 324 MALDI-TOF. See MALDI-time-offlight (MALDI-TOF) MALDI-TOF MS. See MALDI coupled to time-of-flight MS (MALDI-TOF MS) Malnutrition, 520 Malondialdehyde (MDA), 174
177 Maltodextrin, 463 in milk, detection of, 463 Maltogenic amylase, 532t Mammalian digestive system, 405
406 Mammalian GGTP, 24
25 Mammalian heme-containing peroxidase (XPO), 19
20 Mammary gland, 37
39 of dairy cows, 43 Mammary infection, 37
39 Manufacturing process, 403
404 MAPK. See Mitogen-activated protein kinases (MAPK) Mass spectrometry (MS), 119
120, 324, 391, 455 Mastitis diagnosis, 37
39, 49
50 Mastitis milk effect of, 85 efforts in diagnosing mastitis in dairy animals, 46
50 enzymes in, 39
46 gross structure of mammary gland different enzymes coming in milk at onset of infection, 38f Matrix-assisted laser desorption ionization (MALDI), 324, 391 Matrix-assisted laser desorption/ ionization-quadrupole-time-offlight, 203 MBC. See Minimum bactericidal concentration (MBC)

MBPDB. See Milk Bioactive Peptides DataBase (MBPDB) MDA. See Malondialdehyde (MDA) Megazyme, 451 Membrane fouling, 415
416 Membrane process, 321
322, 403
404 Membrane proteins, 39
40 Membrane separation techniques, 320
322, 367 pressure-driving membrane separation mode, 321f Mesenteric lymph nodes (MLN), 214 Metabolite production, 480
481 Metal affinity columns, 357 Metallic ions, 86
87 Methacrylate and variants, 357 Methanol, 475
476 Methanolic procedure, 410
411 Methicillin-resistant Sta. aureus (MRSA), 261 5-Methylphenazinium methosulfate (PMS), 458 MFGM. See Milk fat globule membrane (MFGM) MIC. See Minimum inhibitory concentration (MIC) Micellar casein, 439 Micelles, 332 Micro organisms, 494
495, 519 Microbial catalysts, 523 Microbial cultures, 528 Microbial deterioration, 471
472 Microbial fermentation, 390
391, 404
405 Microbial genera assessed as potential sources of β-galactosidases, 363t Microbial inhibitory activity, 128 Microbial membranes, 20 Microbial method, protein digestion, 122 Microbial proteases, 521 Microbial β-Galactosidases, 393
394, 394t Micrococcus lysodeikticus, 25
26 Microelectronics, 524 Microflora, 417
418 Microorganisms, 317
319, 338, 363, 445
446, 471, 494
495, 523
524 Microsomes, 17

549

550

Index

Microwave heating method, 408
410 Milk, 3, 57
58, 77, 289, 313, 331
332, 362, 445
446, 471 adulteration, 445
446 ALP, 78 deterioration, 4 enzymes from psychrotrophs origin in, 26
27 fermentation, 472 fouling deposit, 501
502 lipoprotein lipase, 88
90 concentration in bovine milk, 89 lipolysis, 89 physicochemical characteristics, 88
89 significance in dairy industry, 89
90 matrix, 431 measurement of lactose in milk blood glucose meter, 438 need for alternate processing of, 57
59 PMNs, 37
39 powder, 452
453 products, 93
94 processing plant, 501
502 products, 428 detection and estimation of starch in milk and, 464 detection and estimation of sucrose in milk and, 462
463 sodium, 39
40 somatic cells, 37
39 spoiling, 471 Milk Bioactive Peptides DataBase (MBPDB), 199
200 Milk enzymes enzymes from psychrotrophs origin in milk, 26
27 effect of high pressure on activity and structure of, 61
63 effect of high-pressure processing on, 65
69 γ-Glutamyl transpeptidase, 24
25 as high
pressure processing indicator, 69
70 kinetics of high pressure on milk enzyme inactivation, 63
65 lipases and esterases, 14
16 bile salt
stimulated lipase, 15 lipoprotein lipase, 15

lysozyme, 25
26 N-Acetyl-β-D-glucosaminidase, 25 oxidases, 19
24 phosphohydrolases, 16
19 proteinases, 10
14 significance, nomenclature, reaction catalyzed, and activity levels, 4
10 indigenous milk enzymes, reaction catalyzed, and major location in milk, 6t levels of enzymes in cow, buffalo, goat, sheep, camel, and human milk, 11t principal indigenous milk enzymes, source, substrates, kinetic parameters, and reaction catalyzed, 9t significance of indigenous milk enzymes, 5f significance of, 57
58 Milk fat globule membrane (MFGM), 3, 77, 456
457, 503
504 Milk protein concentrate (MPC), 428 Milk protein isolate (MPI), 165 Milk proteins, 233, 313
314 amino acid sequence of, 138
143, 139t components, 313 products, 93 Milk-derived bioactive peptides, 314
315 Minerals, 57
58, 491 absorption, 404, 417 Minimum bactericidal concentration (MBC), 197
198 Minimum inhibitory concentration (MIC), 197
198 Ministry of Agriculture and Livestock Resources (MOA&L), 532 Ministry of Trade and Industry (MoTI), 532 Mitochondrial Mn-SOD, 23 Mitogen-activated protein kinases (MAPK), 214
215 MLN. See Mesenteric lymph nodes (MLN) MOE. See Molecular Operating Environment (MOE) Molds, 334 Molecular docking simulation, 155
156

Molecular Operating Environment (MOE), 155 Molecular weight (MW), 316 Monopoly, 523 Monosaccharides, 451 polymers, 383 MoTI. See Ministry of Trade and Industry (MoTI) Moving bed chromatography, 390 MPC. See Milk protein concentrate (MPC) MPI. See Milk protein isolate (MPI) MPO. See Myeloperoxidase (MPO) MRSA. See Methicillin-resistant Sta. aureus (MRSA) MS. See Mass spectrometry (MS) Multicyclic process, 63 Multisensory methods, 478 Multivalent peptides, 235 Multiwall carbon nanotube (MWNT), 477 Mutagenesis, 385 MW. See Molecular weight (MW) MWNT. See Multiwall carbon nanotube (MWNT) Mycobacterium tuberculosis, 67, 79
80 Myeloperoxidase (MPO), 19
20, 43
44

N

N-((2-tetradecanamide) ethyl)ribonamide (TDER), 480 N-acetyl-D-glucosaminidase (NAGase), 37
39, 44
45 N-acetyl-β
D-glucosaminidase (NAGase), 3
4, 25, 49
50, 58 N-acetylglucosamine (NAG), 25
26 N-acetylmuramic acid (NAM), 25
26 NADPH. See Nicotinamide-adenine dinucleotide phosphate (NADPH) NAG. See N-acetylglucosamine (NAG) NAGase. See N-acetyl-Dglucosaminidase (NAGase); Nacetyl-β
D-glucosaminidase (NAGase) NAM. See N-acetylmuramic acid (NAM) Nanofiltration (NF), 321
322 system, 388 Nanomaterials, 483

Index

Nanoparticles, 356
357, 480 Nanotechnology applications in sensors, 483
486 different colors produced in cysteine modified silver nanoparticles, 484f lactate biosensor for assessing milk microbiological load and metabolites, 485f National Center for Biotechnology Information (NCBI), 137, 143 National Codex Committee, 532 Natural enzyme, 519 NCBI. See National Center for Biotechnology Information (NCBI) Near-infrared spectroscopy (NIR spectroscopy), 479 Nernst equation, 448 Nestle, 391
392 Neutral protease I (NP I), 167
170 Neutral β-galactosidases, 368 Neutralization, 492
493 Neutralizers, 459 Neutrase, 164, 190, 192 Neutrophils, 40, 45
46 NF. See Nanofiltration (NF) NF-κB. See Nuclear factor kappa B (NF-κB) NFDM. See Non-fat dry milk (NFDM) Ni-MOF-modified platinum electrodes, 477
478 Ni21-nitrilotriacetic acid (NTA), 357 Nickel-metal organic framework (NiMOF), 477
478 Nicotinamide adenine dinucleotide (NAD 1 ), 477 Nicotinamide-adenine dinucleotide phosphate (NADPH), 449 NIR spectroscopy. See Near-infrared spectroscopy (NIR spectroscopy) Nitrate (NO3
), 22 Nitric oxide (NO), 215 Nitrite (NO22), 22 NMR. See Nuclear magnetic resonance (NMR) NO. See Nitric oxide (NO) Nola Fit enzymes, 369 Non-fat dry milk (NFDM), 92
94 Nonbovine milk, alkaline phosphatase activity in, 85 Nonbovine species, 77

Nondegradable cleaning agents, 492
493 Nondigestible prebiotics, 384 Nonenzymatic methods, for urea detection, 446
447 Nonphotosynthetic bacteria, 340 Novozymes, 494, 497t, 505t NP I. See Neutral protease I (NP I) Nrf2. See Nuclear factor erythroid-2related factor 2 (Nrf2) NTA. See Ni21-nitrilotriacetic acid (NTA) Nuclear factor erythroid-2-related factor 2 (Nrf2), 174
177 Nuclear factor kappa B (NF-κB), 40
41, 214
215 Nuclear magnetic resonance (NMR), 391, 430t Nucleic acid sequences, 523
524 Nucleophilic acceptor, 364
365 Nutrients, 57
58, 313

Orange pigmented Brevibacterium linens, 340 Organic acid, 471, 475
476 Orica, 505t Ornithine, 331, 339
340 Ortho-nitrophenyl β-Dgalactopyranoside (ONPG), 374 Ovis aries. See Sheep (Ovis aries) Oxidases, 3
4, 19
24 catalase, 20
21 LPO, 19
20 reduction, 450 SOD, 22
23 sulfhydryl oxidase, 23
24 XO, 21
22 Oxidoreductases, 5 2-Oxoglutarate, 449 Oxygen radical absorbance capacity (ORAC), 210
211 Oxygen-dependent antimicrobial system, 43
44

O

P

Obesity, 520 Occurrence frequency, 143
144 Oligopoly global enzyme market, 523 Oligosaccharides, 57
58, 383, 427 ONPG. See Ortho-nitrophenyl β-Dgalactopyranoside (ONPG) Operational principles, 59
60 Opioid-like peptides, 216
217 Optical sensing methods, 479 Optical spectroscopic methods, 479
482. See also Electrochemical methods colorimetric, 479
480 Fourier transform infrared spectroscopy, 480 UV
Vis spectroscopy, 479
482 Optical transduction methods, 479 Optimal enzyme-based cleaning solutions, considerations for development of, 509
512 Optimal reaction conditions for β-galactosidases, 365
367 for production of galactooligosaccharides, 387 Optimization, 428 ORAC. See Oxygen radical absorbance capacity (ORAC)

Packaging, 528 Pancreatic lipase (PL), 274
275 PAO1. See Pseudomonas aeruginosa (PAO1) Papain, 164, 192, 241, 521 Paper strip for urea detection, 463
464 Parameter, 412
413 Parmigiano Reggiano, 18 Pasteurization process, 471 Pasteurizer, 501
502 Pathogen trap receptors, 384 Pathogenic bacteria, 404 Pathogens, 40, 79
80 Pectinase, 520t, 521 Pediococcus acidilactici, 291 Penicillium spp., 334 P. camemberti, 334 P. roqueforti, 334 10,12-Pentacosadyinoic acid (PCDA), 480 Pepbank, 199
200 PepP. See Aminopeptidase P (PepP) Pepsin, 213
214, 316, 333 hydrolysis, 212
213 Peptidases, 332 Peptide characterization, 203
206 Peptide YY (PYY), 172
173 PeptideDB, 199
200

551

552

Index

PeptideLocator, 199
200 PeptideRanker tool, 150
155, 199
200 Peptides, 332
336 bioactive peptides released by the action of exogenous or milkorigin enzymes, 336t bioactive peptides released by the action of microbial origin enzymes, 335t Peptidoglycan, 25
26 Peroxidase (POD), 96 Peroxides, 96 Pesticides, 445
446 Petitions for enzyme preparations, 530 Phenolic molecule, 429
430 Phosphatases, 45
46, 49
50 level of different enzymes present in healthy, subclinical, and clinical mastitis milk of dairy animals, 47t 6-Phosphogluconate dehydrogenase (6-PGDH), 451
452 Phosphohexoseisomerase, 66 Phosphohydrolases, 3
4, 16
19 acid phosphatase, 17
18 alkaline phosphatase, 16
17 ribonuclease, 18
19 Phospholipids, 331, 456
457 Phosphopeptides, 315t Phosphotungstic acid (PPTA), 320 Physical methods, 383 Pichia pastoris, 364 Piezoelectric pressure sensors, 448 PL. See Pancreatic lipase (PL) Planktonic cells, 491 Plasmin, 10, 39
40, 77, 90
94 cheese, 93 effect of high-pressure processing on, 68 inactivation, 91
92 milk powder products, 93
94 milk protein products, 93 significance of plasmin in milk, 92 ultrahigh temperature milk, 92
93 Plasminogen, 39
40, 68 activators, 91 PLP-dependent enzyme. See Pyridoxal 50 -phosphatedependent enzyme (PLPdependent enzyme)

PMN cells. See Polymorphonuclear cells (PMN cells) PMP. See Pyridoxamine 50 -phosphate (PMP) PMS. See 5-methylphenazinium methosulfate (PMS) POD. See Peroxidase (POD) Policy framework, 532
533 Poly-5,20 -50 ,200 -terthiophene-30 carboxylic acid (pTTCA), 477 Polygalacturonase, 520t Polymer matrices, 449
450 nanoparticles, 356
357 Polymeric materials, 372
373 Polymerization, 386 Polymorphonuclear cells (PMN cells), 37
39 Polypropylene (PP), 357 film, 480 Posthydrolysis processes, 369 Potential issues with blood glucose meter
based lactose assay, 436
438 Potentiometric approach, 448
449 PP. See Polypropylene (PP) PP-8. See Proteose peptone-8 (PP-8) PPTA. See Phosphotungstic acid (PPTA) Prebiotics, 384, 404 effect, 418t properties, 365 Prehydrolysis processes, 369 Premarket safety assessment, 525 Pressure (p), 60
62 assisted thermal sterilization, 59
60 monitoring change in, 447
448 hardware setup for pressure assay, 448f pressure-assisted thermal processing, 59
60 Pro-Hyp, 129
130 Problematic fouling, 493 Producing low-lactose milk, technologies for, 369
371 Product isolation, 377 Production process, 403
404 and purification of galactooligosaccharides, 388
391 and purification of β-galactosidases, 367

Promega, 505t Propionic acid bacteria, 338 Propylene glycol, 502
503 Protamex, 164, 190, 192 Proteases, 39
41 caspases, 40
41 elastase, collagenase, and cathepsin, 40 plasminogen and plasmin, 39
40 products, 501 protease S, 192 Protein(s), 57
58, 61
62, 313, 503
504 breakdown, 331 concentration in ultrafiltration skim milk retentates, 475f denaturation, 62
63 digestion methods, 120
126 process flow diagram for identification of food-derived BPs, 121f in vitro methods, 120
122 in vivo methods, 122
126 engineering, 385
386 Proteinases, 3
4, 10
14, 332 cathepsin D, 10
14 plasmin, 10 Proteolysis, 119
120, 331 during fermentation, 166
167 Proteolytic enzymes, 332 Proteolytic hydrolysis process, 332 Proteolytic microorganisms, enzymes derived from, 317
319 Proteome analysis, 49 Proteose peptone-8 (PP-8), 15 Pseudomonas aeruginosa (PAO1), 261 Psychrotrophs origin in milk, enzymes from, 26
27 Purgative effect, 418t Purification of xanthine dehydrogenase, 21 Purification process, 408
410, 523 Pyridoxal 50 -phosphate-dependent enzyme (PLP-dependent enzyme), 338
339 Pyridoxamine 50 -phosphate (PMP), 338
339 PYY. See Peptide YY (PYY)

Index

Q

QSAM. See Quantitative structure
activity modeling (QSAM) QSAR. See Quantitative structure
activity relationship (QSAR) QSOX. See Quiescin-sulfhydryl oxidase (QSOX) QTMS. See Quantum topological molecular similarity (QTMS) Qualitative data, 471
472 Quantification of biomarkers, 49 Quantitative data, 471
472 Quantitative structure
activity modeling (QSAM), 200
201 Quantitative structure
activity relationship (QSAR), 137
138, 200
201, 249 Quantum topological molecular similarity (QTMS), 200
201 Quiescin-sulfhydryl oxidase (QSOX), 24

R

Rapid method, 429
430, 453
454 Rational method, 385
386 RCSB-PDB. See Research Collaboratory for Structural Bioinformatics Protein Database (RCSB-PDB) Reaction mixture, 416 Reactions catalyzed by β-galactosidases, 364
365 for production of galactooligosaccharides, 386 Reactive nitrogen species (RNS), 99 Reactive oxygen species (ROS), 37
39, 43, 99, 174
177 Realistic processing, 509
510 Recombinant DNA technology, 363
364 Recombinant expression systems, 372 Recombinant microorganisms, 368t Redox processes, 432 Reduction
oxidation reactions, 411
412 Reference sequences (RefSeq), 143 Refining process, 333 RefSeq. See Reference sequences (RefSeq)

Regulatory framework, 532
533 regarding food enzymes, 522
523 Reindeers, 10 Relative release frequency, 145 Release frequency, 145 Rennet, 519 Research Collaboratory for Structural Bioinformatics Protein Database (RCSB-PDB), 155 Residual organic matter, 501
502 Response surface methodology (RSM), 510
511 Reverse phase (RP), 201 Reverse-phase liquid chromatography-mass spectrometry (RPLC-MS), 190 Reversed-phase high-performance liquid chromatography (RPHPLC), 322, 474
475 Reversed-phase liquid chromatography, 324
325 Reviewed/unreviewed filter, 138
143 RfCEP. See Roseburia faecis (RfCEP) Ribonuclease (RNase), 16, 18
19 Ripened cheeses, 67 Ripening process, 199, 333 Risk assessments, 531 RNase. See Ribonuclease (RNase) RNase A, 18
19 RNS. See Reactive nitrogen species (RNS) Roche, 505t Roquefort cheese, 334 ROS. See Reactive oxygen species (ROS) Roseburia faecis (RfCEP), 413
414 RP. See Reverse phase (RP) RPLC-MS. See Reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) RSM. See Response surface methodology (RSM) RYLGY peptides, 163
164

S

Saccharomyces cerevisiae, 364, 390
391 Safety approval process, 521 Safety assessment, 526 of certain enzyme processing aids by JECFA, 532t

Safety evaluation, 522 Saheli-SGID hydrolysates, 245
247 Saliva, 19
20 Salmonella carriers, 417
418 S. enteric, 478 Sanitization, 491 Saphera enzymes, 369 Satiety hormone-inducing peptides, 217
218 SCC. See Somatic cell count (SCC) SCFAs. See Short-chain fatty acids (SCFAs) Scharer modified method for spectrophotometric quantification, 84
85 Scharer’s rapid phosphatase test, 83 Schizophyllum commune, 502
503 SDS-PAGE. See Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SEC. See Size exclusion chromatography (SEC) Secondary hydrolysis, 415
416 Semirational method, 385
386 Sensor technology, 472 Separation, 475
476 Sephadex G-10, 390 Sephadex G-25-based fraction, 241
242 Sequence homology of lysozyme, 26 Serine proteases, 495 SGID. See Simulated gastrointestinal digestion (SGID) Sheep (Ovis aries), 77, 139t, 146t, 151t Short-chain fatty acids (SCFAs), 384 SHR. See Spontaneously hypertensive rats (SHR) Sigma, 505t Silicon dioxide (SiO2), 484 Silver nanoparticles (AgNPs), 483
484 Simulated gastrointestinal digestion (SGID), 241 Size exclusion chromatography (SEC), 320, 322
323, 390 Size-exclusion membrane reactor, 415
416 Skim milk, 474 SMPRs. See Standard Method Performance Requirements (SMPRs)

553

554

Index

SOD. See Superoxide dismutase (SOD) SOD1, 23 Sodium caseinate, 164 Sodium dicotyl-sulfosuccinate, 387 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 120
122 Sodium hydroxide solution, 492
493 Soful, 292 Solid non-fat (SNF), 445
446 Solid-state fermentation, 494 Soluble enzyme, 371 Somatic cell count (SCC), 37 Sour milk, 316
317 Spectrophotometers, 479
480 Spectrophotometric measurement of ammonium ion concentration, 449 Spectrophotometric method, 450
452 Spoilage, 478, 483
484 Spontaneous lipolysis, 14 Spontaneously hypertensive rats (SHR), 129
130, 207
208 SSA. See Succinic semialdehyde (SSA) Stainless steel panels, with experimentally generated milk fouling deposit, 500f Standard Method Performance Requirements (SMPRs), 433 Staphylococcus aureus, 44
45 Starch in milk and milk products, detection and estimation of, 464 Starter microorganisms, 331, 338
339 Statistical data for lactic acid analyses by four methods, 473t Sterile milk, 473
474 Sterile β-galactosidase, 371 Sterilization process, 406 Storing of food, 528 Streptococcus, 333
334, 473 S. thermophilus, 166
167, 291, 339 Streptomyces griseus, 509 Structural activity relationship (SAR) camel milk
derived antidiabetic peptides, 273
274 camel milk
derived antihypertensive peptides, 249
250

camel milk
derived antimicrobial peptides, 261
263 camel milk
derived antioxidant peptides, 243
245 Subsequent processing, 501
502 Subsequent treatment processes, 493
494 Subtilisin A. See Alcalase Succinic semialdehyde (SSA), 338
339 Sucrose, 462 Sugar acid, 457
458 Sulfhydryl groups (-SH groups), 17, 62, 85
86 Sulfhydryl oxidase, 23
24 Sulfosalicylic acid, 320 Supercritical fluid extraction, 390 Superoxide, 99 radicals, 43 Superoxide dismutase (SOD), 21
23, 42, 174
177, 211
212, 272
273 Surfactants, 510
511 Sustainability, 527
528 Sweet whey, 403
404 Sweetening properties, 383 Syrup, 405
406

T

T2DM. See Type 2 diabetes mellitus (T2DM) TAA. See Thioacetamide (TAA) TAG. See Triacylglycerols (TAG) TBHBA. See 2,4,6-tribromo 3hydroxy benzoic acid (TBHBA) TCA. See Trichloroacetic acid (TCA) TDER. See N-((2-tetradecanamide) ethyl)-ribonamide (TDER) TEAC. See Trolox equivalent antioxidant capacity (TEAC) Tears, 19
20 Technologies for producing lowlactose milk, 369
371 Temperature (T), 61
62 3,30 ,5,50 -Tetramethylbenzidine (TMB), 459 TFA. See Trifluoroacetic acid (TFA) Th1 cell. See Type 1 T helper cell (Th1 cell) Thermal processing, 491 Thermal stability, 364 Thermomyces lanuginosus, 495
496

Thermotolerant enzymes, 392
393 Thioacetamide (TAA), 278 Thiocyanate, 19
20 Thiocyanate ion (SCN2), 19
20, 98 Three-dimensional plot, 474 Thyroid peroxidase (TPO), 19
20 Tissue plasminogen activator (tPA), 91 Title 21 of Code of Federal Regulations, Part 173 (21 CFR 173), 528
530 Total solids (TSs), 445
446 tPA. See Tissue plasminogen activator (tPA) TPO. See Thyroid peroxidase (TPO) Transcriptome analysis, 49 Transducer, 449 Transferases, 5 Transgalactosylation activity, 383
384 Transgalactosylation reaction, 362, 409t, 413
414 for synthesis of GOS, 386f Transgalactosylation-based lactulose synthesis, 414
416 Transglycosylation, 406 Triacylglycerol acyl hydrolases, 495
496 Triacylglycerols (TAG), 456
457 2,4,6-Tribromo 3-hydroxy benzoic acid (TBHBA), 453
454 Trichloroacetic acid (TCA), 320, 450 Trifluoroacetic acid (TFA), 126
127 Trolox equivalent antioxidant capacity (TEAC), 210
211 Trypsin, 192
194, 213
214, 316 Tryptic casein hydrolysate, 178 TSs. See Total solids (TSs) Two column liquid chromatography, 416 Two iron-sulfur (2Fe-2S), 22 Two-dimensional gel electrophoresis, 120
122 Two-phase systems, 387 Type 1 T helper cell (Th1 cell), 213
214 Type 2 diabetes mellitus (T2DM), 206 Type A fouling deposits, removal of, 496
504, 497t

Index

U

UF. See Ultrafiltration (UF) UHHP. See Ultra-HHP (UHHP) UHT. See Ultra high temperature (UHT); Ultrahigh-temperature (UHT) UHT-treated milk. See Ultrahigh temperature-treated milk (UHTtreated milk) UK. See United Kingdom (UK) Ultra high temperature (UHT), 58 Ultra-HHP (UHHP), 59 Ultrafiltration (UF), 320 Ultrafiltration membrane reactors, 388 Ultrahigh temperature-treated milk (UHT-treated milk), 85
86, 92
93 Ultrahigh-performance liquid chromatography
MS/MS (UPLC
MS/MS), 203 Ultrahigh-temperature (UHT), 362 UniProtKB. See Universal Protein Resource Knowledgebase (UniProtKB) United Kingdom (UK), 391
392 United States (US), 79
80 Universal Protein Resource Knowledgebase (UniProtKB), 137
143 uPA. See Urokinase plasminogen activator (uPA) UPLC
MS/MS. See Ultrahighperformance liquid chromatography
MS/MS (UPLC
MS/MS) Urea biosensor, 449
450 Urea detection, paper strip for, 463
464 Urea estimation in milk, 446
450 monitoring change in pressure, 447
448 hardware setup for pressure assay monitoring pH change, 447 potentiometric approach, 448
449 spectrophotometric measurement of ammonium ion concentration, 449 urea biosensor, 449
450 Urea levels, 446 Urease reaction, 449

Urokinase plasminogen activator (uPA), 91 US. See United States (US) US regulations, 528
531 generally recognized as safe notices for enzyme preparations, 530
531 GRAS framework timeline, 531f petitions for enzyme preparations, 530 UV
vis absorption, 484 UV
Vis spectroscopy, 479
480

V

Val-Pro-Pro (VPP), 167
170 peptides, 163
164 Val-Pro-Pro-Phe (VPPF), 167
170 Vegetable oils, 455 Viili, 295 Vitamin C, 457
458 Vitamins, 57
58 VOCs. See Volatile organic compounds (VOCs) Volatile organic compounds (VOCs), 484 VPP. See Val-Pro-Pro (VPP) VPPF. See Val-Pro-Pro-Phe (VPPF) VPYPQ peptides, 163
164

W

Wastewater, 492
493 treatment processes, 509
510 Water, 353
354 consumption, 493
494 molecule, 386 water-soluble extracts, 333 Whey protein, 189 analytical techniques for identification of bioactive peptides, 201
206 biological effects of whey-derived bioactive peptides, 206
218 anticancer peptides, 212
213 antidiabetic peptides, 206
207 antihypertensive peptides, 207
208 antiinflammatory peptides, 214
216 antimicrobial peptides, 208
210

antioxidant peptides, 210
212 immunomodulatory peptides, 213
214 opioid-like peptides, 216
217 satiety hormone-inducing peptides, 217
218 generation of whey protein
derived bioactive peptides, 190
201, 191t enzymatic hydrolysis, 190
192 fermentation, 194
199 fermented dairy products, 196t hydrolysis during gastrointestinal digestion, 192
194 in-silico aided enzymatic release of bioactive peptides, 199
201 Whey protein concentrate (WPC), 190
192, 403
404 Whey protein hydrolysates (WPHs), 190, 241
242 Whey protein isolate (WPI), 194
195, 403
404 Whey utilization, 404
405 World Health Organization, 521, 531
532 WPC. See Whey protein concentrate (WPC) WPHs. See Whey protein hydrolysates (WPHs) WPI. See Whey protein isolate (WPI)

X

X-PDAP. See X-prolyl-dipeptidyl aminopeptidase (X-PDAP) X-prolyl-dipeptidyl aminopeptidase (X-PDAP), 199 Xanthine dehydrogenase (XDH), 21, 43, 98
99 Xanthine oxidase (XO), 21
22, 43, 69
70, 98
99 Xanthine oxidoreductase (XOR), 98
99 XDH. See Xanthine dehydrogenase (XDH) XO. See Xanthine oxidase (XO) XOR. See Xanthine oxidoreductase (XOR) Xylose, 383

555

556

Index

Y

Yaks, 10 Yarrowia lipolytica, 334 Yeast cells (Aspergillus oryzae), 314
315

Yeasts, 289, 334, 363
364, 495
496, 519 β-galactosidases, 366t enzymes, 352, 366
367 Yogurt, 293, 296, 304, 316
317, 362

YSI biochemistry analyzer, 432
433

Z

Ziyaina electrochemical method, 476