Agents of Change: Enzymes in Milk and Dairy Products

Preface
Contents
Chapter 1: Enzymology of Milk and Dairy Products: Overview
	1.1 Introduction
	1.2 Indigenous Enzymes of Bovine Milk
	1.3 Endogenous Enzymes in Milk
	1.4 Exogenous Enzymes
		1.4.1 Rennets
		1.4.2 β-Galactosidase (Lactase)
		1.4.3 Lipases
		1.4.4 Lysozyme
		1.4.5 Catalase
		1.4.6 Glucose Oxidase
	1.5 Exogenous Enzymes in Food Analysis
	1.6 Conclusion
	References
		Further Reading
Chapter 2: The Plasmin System in Milk and Dairy Products
	2.1 Introduction
	2.2 Plasmin and Plasminogen
		2.2.1 Proteolytic Specificity of Plasmin on Milk Proteins
			2.2.1.1 β-casein
			2.2.1.2 αs2-casein
			2.2.1.3 αs1-casein
			2.2.1.4 κ-casein
			2.2.1.5 Whey Proteins
	2.3 The Plasmin System in Bovine Milk
		2.3.1 Activators of the Plasmin System
		2.3.2 Inhibitors of the Plasmin System
	2.4 Distribution of Plasmin System Components in Milk
		2.4.1 Factors Affecting the Distribution of Plasmin System Components Within Milk
	2.5 Factors Affecting Plasmin Activity and Consequent Proteolysis
		2.5.1 Mastitis
		2.5.2 Stage of Lactation
		2.5.3 Age of Cow
		2.5.4 Breed of Cow
		2.5.5 pH of Milk
		2.5.6 Heat Treatment
		2.5.7 Presence of β-Lactoglobulin
		2.5.8 Storage Temperature of Milk
			2.5.8.1 Cold Storage
			2.5.8.2 Warm (Room Temperature) Storage
		2.5.9 Microbial Proteases
		2.5.10 Membrane Filtration
		2.5.11 High Pressure
	2.6 Characterisation of Plasmin and Plasminogen
		2.6.1 Assay Methods for Plasmin and Plasminogen
		2.6.2 Assay Methods for Activators and Inhibitors of the Plasmin System
		2.6.3 Monitoring of Plasmin-Mediated Proteolysis
			2.6.3.1 Soluble Protein in Supernatant
			2.6.3.2 Analysis of Free Amino Groups
			2.6.3.3 Urea-Polyacrylamide Gel Electrophoresis
			2.6.3.4 Reversed Phase-High Performance Liquid Chromatography
			2.6.3.5 Mass Spectrometry
	2.7 Applications of Plasmin and Its Significance in Various Dairy Products
		2.7.1 Cheese
			2.7.1.1 Cheese-making Properties
			2.7.1.2 Cheese Ripening
		2.7.2 Milk-Based UHT Products
		2.7.3 High Protein Milk Products
			2.7.3.1 Whey Protein-Containing Ingredients
			2.7.3.2 Casein-Containing Ingredients
	2.8 Conclusions and Areas for Additional Research
	References
Chapter 3: Lysosomal and Other Indigenous Non-plasmin Proteases in Bovine Milk
	3.1 Introduction
	3.2 Origin of Non-plasmin Proteolytic Enzymes in Bovine Milk
	3.3 The Lysosomal and Other Non-plasmin Proteases in Bovine Milk
		3.3.1 Cathepsin B
		3.3.2 Cathepsin C: Dipeptidyl Peptidase 1
		3.3.3 Cathepsin D
		3.3.4 Cathepsin E
		3.3.5 Other Lysosomal Cysteine Proteases
		3.3.6 Cathepsin G
		3.3.7 PMN Elastase
		3.3.8 Kallikrein
		3.3.9 Amino-and Carboxypeptidases
	3.4 Measurement of Lysosomal and Other Non-plasmin Proteases in Milk
	3.5 Thermal Inactivation
	3.6 Impact on Dairy Products
	3.7 Studies of Indigenous Non-plasmin Milk Proteases in Cases of Induced Mastitis
	3.8 Biological Significance
	3.9 Future Aspects and Missing Links
	References
Chapter 4: Phosphatases in Milk
	4.1 Introduction
	4.2 Alkaline Phosphatase
	4.3 Origin, Isolation and Characterization of ALP in Milk
	4.4 Assay Methods for ALP Activity
	4.5 Significance of ALP in Dairy Products
	4.6 Reactivation of ALP
	4.7 Acid Phosphatase
	4.8 Distribution, Isolation and Characterization of ACP
	4.9 Significance of ACP
	4.10 Conclusions
	References
Chapter 5: Antimicrobial Enzymes in Milk, and Their Role in Human Milk
	5.1 Introduction
	5.2 Antimicrobial Enzymes in Human Milk
		5.2.1 Lysozyme
			5.2.1.1 General Characteristics of Lysozyme
			5.2.1.2 Mechanism of Antibacterial Activity of Lysozyme
			5.2.1.3 Factors Affecting Antimicrobial Activity of Lysozyme
			5.2.1.4 Assay Methods for Lysozyme Activity
		5.2.2 Lactoperoxidase
			5.2.2.1 General Characteristics of Lactoperoxidase
			5.2.2.2 Mechanism of Antibacterial Activity of Lactoperoxidase
			5.2.2.3 Factors Affecting Antimicrobial Activity of Lactoperoxidase
			5.2.2.4 Assay Methods for Lactoperoxidase Activity
		5.2.3 Xanthine Oxidase
			5.2.3.1 General Characteristics of Xanthine Oxidase
			5.2.3.2 Mechanism of Antibacterial Activity of Xanthine Oxidase
			5.2.3.3 Factors Affecting Antimicrobial Activity of Xanthine Oxidase
			5.2.3.4 Assay Methods for Xanthine Oxidase Activity
		5.2.4 The XO-LPO “Oxidative Enzyme System’ in Human Milk and Its Activation by Infant Saliva
		5.2.5 Polyamine Oxidases
			5.2.5.1 General Characteristics of Polyamine Oxidase
			5.2.5.2 Mechanism of Antibacterial Activity of Polyamine Oxidase
			5.2.5.3 Factors Affecting Activity of Polyamine Oxidase
			5.2.5.4 Assay Methods for Polyamine Oxidase
		5.2.6 Lipases
			5.2.6.1 General Characteristics of Lipases
			5.2.6.2 Mechanism of Antibacterial Activity of Lipases
			5.2.6.3 Factors Affecting Activity of Lipases
			5.2.6.4 Assay Methods for Lipases
		5.2.7 N-Acetyl-β-D-Glucosaminidase (NAGase)
			5.2.7.1 General Characteristics of NAGase and Its Activity
			5.2.7.2 Assay Method for Activity of NAGase
		5.2.8 Platelet-Activating Factor (PAF) Acetylhydrolase
			5.2.8.1 General Characteristics, Mechanism and Activity of PAF Acetylhydrolase
			5.2.8.2 Assay Method for Activity of PAF Acetylhydrolase
	5.3 Summary
	References
Chapter 6: Enzymes Associated with Milk Phospholipid Membrane Structures: Milk Fat Globule Membranes and Extracellular Vesicles
	6.1 Introduction
	6.2 Sulfhydryl Oxidase
		6.2.1 Structure of Flavin-Dependent Sulfhydryl Oxidase
		6.2.2 Biological Role of Flavin-Dependent Sulfhydryl Oxidase and Significance in Milk
	6.3 Catalase
		6.3.1 Structure of Catalase
		6.3.2 Biological Role of Catalase and Significance in Milk
	6.4 Lactoperoxidase
		6.4.1 Structure of Lactoperoxidase
		6.4.2 Biological Role of Lactoperoxidase and Significance in Milk
	6.5 Xanthine Oxidoreductase
		6.5.1 Structure of Xanthine Oxidoreductase
		6.5.2 Biological Role of Xanthine Oxidoreductase and Significance in Milk
	6.6 γ-Glutamyltransferase
		6.6.1 Structure of γ-Glutamyltransferase
		6.6.2 Biological Role and Significance of Glutamyltransferase in Milk
	6.7 5’-Nucleotidase
		6.7.1 Structure of 5’-Nucleotidase
		6.7.2 Biological Role and Significance of 5’-Nucleotidase in Milk
	6.8 Conclusion
	References
Chapter 7: Milk and Other Glycosidases
	7.1 Introduction
	7.2 Origin and Significance of Glycosidases in Milk
	7.3 Cleavage Specificity and Biological Relevance in Relation to Substrates
	7.4 Glycosidases as Indicators of Mastitis
	7.5 Industrial Relevance and Effect of Milk Glycosidases during Processing
		7.5.1 Effect of Processing on Milk Glycosidases
		7.5.2 Applications of Lactases in Processing of Dairy Products
	7.6 Detection and Methods for Analysis of Glycosidases
	7.7 Concluding Remarks
	References
Chapter 8: The Enzymology of Non-bovine Milk
	8.1 Introduction
	8.2 Enzymology of Ovine and Caprine Milk
		8.2.1 Proteolytic Enzyme Activities
		8.2.2 Activities of Other Enzymes
		8.2.3 Effect of Enzyme Activity on Milk and Dairy Product Quality
	8.3 Enzymology of Buffalo Milk
	8.4 Enzymology of Camel Milk
	8.5 Enzymology of Non-ruminant Milk
	8.6 Conclusion
	References
Chapter 9: The Enzymology of Human Milk
	9.1 Introduction
	9.2 Proteases
		9.2.1 Protease Activators
		9.2.2 Antiproteases
		9.2.3 Protease Activity in the Mammary Gland
		9.2.4 Protein Digestion in Human Neonates
		9.2.5 Proteolytic Systems and Peptides
		9.2.6 Specific Protease Systems
			9.2.6.1 Plasmin System
			9.2.6.2 Cathepsin System
			9.2.6.3 Elastase System
			9.2.6.4 Trypsin System
			9.2.6.5 Chymotrypsin
			9.2.6.6 Thrombin
			9.2.6.7 Kallikrein
			9.2.6.8 Amino- and Carboxypeptidase Systems
			9.2.6.9 Matrix Metalloproteinase System
	9.3 Lipases
		9.3.1 Lipoprotein Lipase
		9.3.2 Bile Salt-Stimulated Lipase
	9.4 Glycosidases
		9.4.1 α-L-Fucosidase
		9.4.2 α-1,3/4-Fucosyltransferase
		9.4.3 N-Acetyl-β-D-hexosaminidase
		9.4.4 Neuraminidase
		9.4.5 α-Amylase
		9.4.6 Pro- and Anti-Glycosidases
	9.5 Other Enzymes in Human Milk
		9.5.1 Phosphatases
		9.5.2 Xanthine Oxidase
		9.5.3 Antioxidant Enzymes
		9.5.4 Additional Enzymes
	9.6 Conclusion
	References
Chapter 10: Lipases from Milk and Other Sources
	10.1 Introduction
	10.2 The Milk Enzymes
		10.2.1 Lipoprotein Lipase
		10.2.2 Somatic Cell Lipase
		10.2.3 Colostral Lipase
		10.2.4 Milk Esterases
	10.3 Lipolysis Due to Milk Lipase in Milk
		10.3.1 Spontaneous Lipolysis
		10.3.2 Induced Lipolysis
	10.4 Lipase-Catalysed Acyl-Transfer Reactions on Milk Fat
		10.4.1 Interesterification
		10.4.2 Acidolysis
		10.4.3 Alcoholysis
	10.5 Conclusion
	References
Chapter 11: Heat-Stable Microbial Peptidases Associated with the Microbiota of Raw Milk
	11.1 The Microbiota of Raw Milk
		11.1.1 The Genus Pseudomonas
	11.2 Microbial Peptidase from Raw Milk Isolates
		11.2.1 Peptidases Secreted by Pseudomonas Species
		11.2.2 Interactions of Plasmin and Microbial Peptidases in Milk
	11.3 Destabilization of Milk by Peptidases
	11.4 Determination of Peptidase Activity in Milk
	11.5 Thermal Inactivation of Peptidases
	11.6 Conclusion and Outlook
	References
Chapter 12: The Heat Stability of Indigenous and Bacterial Enzymes in Milk
	12.1 Enzyme Sources and Problems Encountered in Milk Products
	12.2 Inactivation Methods
	12.3 Basics of Heat Inactivation Kinetics
	12.4 Heat Stability of Enzymes
		12.4.1 Heat Stability of Indigenous Enzymes
	12.5 Heat Stability of Bacterial Enzymes
	12.6 Conclusion
	References
Chapter 13: The Role of Proteases in the Stability of UHT-Treated Milk
	13.1 Introduction
	13.2 Principles of UHT Processing
		13.2.1 Definition of UHT Treatment
	13.3 Chemical Changes Occurring in Milk During UHT Processing
	13.4 Physico-Chemical Changes in Ultra-High-Temperature (UHT) Treated Milk During Storage
		13.4.1 Gelation of UHT Milk
	13.5 Strategies to Control Destabilisation of UHT Treated Milk During Storage
		13.5.1 Raw Milk Quality
		13.5.2 Accelerated Shelf-Life Testing of UHT Milk
	13.6 Conclusions
	References
Chapter 14: Milk-Clotting Enzymes
	14.1 Historical Background
	14.2 Different Types of Rennets and Coagulants
		14.2.1 Bovine Rennets
		14.2.2 Ovine and Caprine Rennets
		14.2.3 Porcine and Chicken Pepsin
		14.2.4 Microbial Coagulants
		14.2.5 Plant Coagulants
		14.2.6 Fermentation Produced Chymosin (FPC)
		14.2.7 General Considerations Around Rennets and Coagulants
	14.3 Production of Rennets and Coagulants
	14.4 Market Shares of Different Rennets and Coagulants
	14.5 Molecular and Catalytic Properties of Milk-Clotting Enzymes
	14.6 Analysis of Rennets and Coagulants
	14.7 Interpretation of the Strength of Rennets
	References
Chapter 15: Enzymes in Cheese Ripening
	15.1 Introduction
	15.2 Lactose Metabolism
		15.2.1 Starter Cultures
		15.2.2 Glycolysis and Regeneration of NADH
		15.2.3 Lactic Acid Metabolism
	15.3 Citrate Catabolism
	15.4 Milk Fat Catabolism
		15.4.1 Milk Fat Hydrolysis
		15.4.2 Fatty Acid Metabolism
		15.4.3 Methods to Accelerate Lipolysis
	15.5 Proteolysis
		15.5.1 Proteolytic Activities in Cheese
		15.5.2 Indigenous Milk Proteases
		15.5.3 Coagulants
		15.5.4 Lactic Acid Bacterial Protease: Lactocepin
		15.5.5 Bacterial Cell Membrane Transport Systems
		15.5.6 Peptidases
		15.5.7 Phosphatases
		15.5.8 Synthesis of Specific Glu-Peptides
		15.5.9 Acceleration of Proteolysis in Cheese
	15.6 Amino Acid Catabolism
		15.6.1 Lyases
		15.6.2 Dehydratases
		15.6.3 Aminotransferases
		15.6.4 Deaminases
		15.6.5 Decarboxylases
	15.7 Breakdown of Microbial Cell Membranes and Cell Lysis
	15.8 Nucleic Acid Catabolism
	15.9 Catabolism of Vitamins and Other Compounds
	15.10 Conclusions
	References
Chapter 16: Enzyme Modified Cheese
	16.1 Introduction
	16.2 General Principles
	16.3 The Application and Advantages of EMC as a Cheese Flavor Ingredient
	16.4 Production Methods of EMC
		16.4.1 Emulsification of the Substrate
		16.4.2 Enzyme Addition and Incubation
		16.4.3 Termination of Enzyme Activity/Standardization of the Final Product
		16.4.4 Blue Cheese Flavor
	16.5 Enzymes in EMC Production
		16.5.1 Proteolytic Enzymes
		16.5.2 Lipolytic Enzymes
		16.5.3 Use of Starter Cultures in EMC Production
	16.6 Health Effects and Functional Properties of EMCs
	16.7 Conclusion and Future Trends
	References
Chapter 17: Enzymatic Protein Cross-Linking in Dairy Science and Technology
	17.1 Introduction
	17.2 Chemical Aspects and Production of Enzymes for Protein Cross-Linking
		17.2.1 Transglutaminase
		17.2.2 Oxidoreductases
	17.3 Analysis of Cross-Linking Reactions
		17.3.1 Quantification of Enzymatically Induced Covalent Cross-Links
			17.3.1.1 N-ε-(γ-Glutamyl)-Lysine Isopeptide Content
			17.3.1.2 Cross-Links from Other Enzymatic Reactions
		17.3.2 Static and Dynamic Light Scattering
		17.3.3 Size Separation of Polymerised Milk Proteins
			17.3.3.1 Gel Electrophoresis
			17.3.3.2 Size-Exclusion Chromatography
			17.3.3.3 Field Flow Fractionation
	17.4 Application of Cross-Linking Enzymes in Dairy Science and Technology
		17.4.1 Overview of Review Articles and Book Chapters
		17.4.2 Limitations of Transglutaminase Application for Acid-Induced Milk Gels
		17.4.3 Application of Laccase in Stirred Yoghurt Manufacture
		17.4.4 Enzymatic Protein Cross-Linking in Cheese Making
		17.4.5 Creation of Well-Defined Micro- and Nanostructures
			17.4.5.1 Preparation of Microgel Particles by Cross-Linking of Casein Micelles
			17.4.5.2 Cross-Linking of Casein Nanoparticles: Molecular Aspects and Gelation Properties
			17.4.5.3 Formation and Physical Properties of Nanoparticles from Cross-Linked α-Lactalbumin
	17.5 Conclusions and Outlook
	References
Chapter 18: The Production of Bioactive Peptides from Milk Proteins
	18.1 Introduction: Milk Proteins and Bioactive Peptides
	18.2 Production of Milk Protein-Derived BAPs
		18.2.1 In Vitro Enzymatic Hydrolysis
			18.2.1.1 Enzymatic Hydrolysis for the Generation of Anti-Diabetic Peptides
			18.2.1.2 Enzymatic Hydrolysis for the Generation of Antihypertensive Peptides
			18.2.1.3 Enzymatic Hydrolysis for the Generation of Antioxidant Peptides
			18.2.1.4 Enzymatic Hydrolysis for the Generation of Immunomodulatory Peptides
			18.2.1.5 Enzymatic Hydrolysis for the Generation of Anticancer Peptides
			18.2.1.6 Enzymatic Hydrolysis for the Generation of Antimicrobial Peptides
			18.2.1.7 Enzymatic Hydrolysis for the Generation of Opioid-Like Peptides
			18.2.1.8 Enzymatic Hydrolysis for the Generation of Other Bioactive Peptides
		18.2.2 Microbial Fermentation
			18.2.2.1 Bacteria
			18.2.2.2 Yeasts and Moulds
		18.2.3 Production of BAPs During Gastrointestinal Digestion
		18.2.4 In silico Approaches for Peptide Generation
	18.3 Fractionation and Enrichment of BAPs
		18.3.1 Membrane Filtration
		18.3.2 Chromatography
	18.4 Conclusion
	References
Chapter 19: Reducing Allergenicity by Proteolysis
	19.1 Cow’s Milk Allergy
		19.1.1 Bovine Milk Proteins
		19.1.2 Bovine Milk Epitopes
		19.1.3 Bovine Milk Proteolysis
	19.2 Digestion
		19.2.1 Digestion Process
		19.2.2 Digestibility of Bovine Milk Proteins
		19.2.3 Digestion Parameters Affecting the Digestibility of Bovine Milk Proteins
		19.2.4 Evaluation of Digestibility
		19.2.5 Evaluation of Residual Allergenicity of Digestion Products
	19.3 Infant Formulas
		19.3.1 Hypoallergenic Infant Formulas
		19.3.2 Production Process
		19.3.3 Clinical Use of eHF and pHF
		19.3.4 Allergenicity Evaluation
	19.4 Conclusions
	References
Chapter 20: Future Opportunities and Challenges in Dairy Enzymology
	20.1 Introduction
	20.2 Future Challenges in Indigenous and Endogenous Milk Enzymology
	20.3 Processing and Milk Enzymes
	20.4 Emerging Enzymes
		20.4.1 Protein Glutaminase
		20.4.2 Glutaminase
		20.4.3 Lactose Oxidase
		20.4.4 Phospholipase
	20.5 Application of Molecular Methods: Omics
	20.6 Conclusion: Remaining Gaps in Our Knowledge and Future Outlook
	References
Index