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

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