Table Of ContentBiologically Active Peptides
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Biologically Active Peptides
From Basic Science to Applications
for Human Health
Edited by
Fidel Toldra´
Department of Food Science, Institute of Agrochemistry and
Food Technology (CSIC), Valencia, Spain
Jianping Wu
Faculty of Agricultural Life and Environmental Sciences,
Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, AB, Canada
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Contents
List of contributors.............................................................................................xxiii
Preface...............................................................................................................xxix
Chapter 1:Bioactive peptides in health and disease: an overview.............................. 1
JuDong Yeo and Fereidoon Shahidi
1.1 Introduction...........................................................................................................1
1.2 Preparation of bioactive peptides...........................................................................2
1.3 Absorption of peptides in the small intestine.........................................................3
1.3.1 Paracellular transport..................................................................................3
1.3.2 Transcellular transport................................................................................4
1.3.3 Absorption of peptides in the large intestine (colon)...................................5
1.3.4 Approaches for enhancing the absorption of peptides.................................6
1.3.5 Structure-activity relationship of bioactive peptides....................................8
1.4 Bioactivities of food-derived bioactive peptides focusing on
inhibiting chronic diseases.....................................................................................9
1.4.1 Anticancer activity......................................................................................9
1.4.2 Anti-inflammatory effect...........................................................................11
1.4.3 Antimicrobial activity...............................................................................12
1.4.4 Antihypertensive effect.............................................................................13
1.4.5 Immunomodulatory peptides.....................................................................15
1.4.6 Antidiabetic effect.....................................................................................16
1.5 Conclusion...........................................................................................................18
References...................................................................................................................18
Chapter 2:Enzymatic mechanisms for the generation of bioactive peptides.............. 27
Fidel Toldra´and Leticia Mora
2.1 Introduction.........................................................................................................27
2.1.1 Enzymatic mechanisms in the hydrolysis of food proteins........................27
v
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2.1.2 Bioactive peptides generated during food processing................................30
2.1.3 Bioactive peptides generated through the hydrolysis of
proteins with commercial peptidases.........................................................30
2.2 Degree of hydrolysis............................................................................................37
2.2.1 Definition..................................................................................................37
2.2.2 Precursor techniques and alternative methods/procedures.........................38
2.3 Assay of endopeptidase activity...........................................................................38
2.3.1 Definition..................................................................................................38
2.4 Assay of exopeptidase activity.............................................................................40
2.4.1 Definition..................................................................................................40
2.4.2 Materials, equipment, and reagents...........................................................40
2.4.3 Protocol....................................................................................................41
2.4.4 Pros and cons............................................................................................41
2.4.5 Summary...................................................................................................42
References...................................................................................................................42
Chapter 3:Novel technologies in bioactive peptides production and stability............ 47
Aı´da Jimena Velarde-Salcedo, Gabriela Va´zquez-Rodrı´guez,
Antonio De Leo´n-Rodrı´guez and Ana Paulina Barba de la Rosa
3.1 Introduction.........................................................................................................47
3.2 Expression of recombinant peptides.....................................................................48
3.2.1 Escherichia coli expression vectors and strains for recombinant
protein production.....................................................................................50
3.3 Stability of proteins and peptides.........................................................................52
3.4 Definition: production of recombinant bioactive peptides in
Escherichia coli...................................................................................................57
3.4.1 Antihypertensive peptides.........................................................................57
3.4.2 Antiangiogenic peptides............................................................................58
3.5 Protocol...............................................................................................................59
3.5.1 Antihypertensive cassette design...............................................................59
3.5.2 Amplification of the encrypted vasoinhibin peptide..................................60
3.5.3 DNA cloning into a suitable vector...........................................................61
3.5.4 Transformation of the host cells................................................................63
3.5.5 Induction of the expression of the desired protein under
controlled conditions.................................................................................66
3.5.6 Recovery and purification of the recombinant product..............................68
3.5.7 Preparation and encapsulation of recombinant peptides............................69
3.6 Summary.............................................................................................................70
References...................................................................................................................71
Contents vii
Chapter 4:Methodologies for extraction and separation of short-chain
bioactive peptides................................................................................ 75
Andrea Cerrato, Sara Elsa Aita, Carmela Maria Montone,
Anna Laura Capriotti, Susy Piovesana and Aldo Lagana`
4.1 Introduction.......................................................................................................75
4.2 Definition: Short-chain peptide enrichment........................................................77
4.3 Materials, equipment and reagents.....................................................................77
4.4 Protocols............................................................................................................78
4.5 Pros and cons.....................................................................................................78
4.6 Alternative methods/procedures.........................................................................79
4.7 Troubleshooting & Optimization........................................................................79
4.8 Materials, equipment and reagents.....................................................................80
4.9 Protocols............................................................................................................81
4.10 Pros and cons.....................................................................................................82
4.11 Alternative methods/procedures.........................................................................82
4.12 Troubleshooting & Optimization........................................................................83
4.13 Summary...........................................................................................................83
References...................................................................................................................84
Chapter 5:Methodologies for peptidomics: Identification and quantification............ 87
Leticia Mora and Fidel Toldra´
5.1 Introduction.........................................................................................................87
5.2 Identification of naturally generated peptides......................................................88
5.3 Materials, equipment, and reagents......................................................................89
5.3.1 Protocol....................................................................................................89
5.3.2 Analysis and statistics...............................................................................90
5.3.3 Pros and cons............................................................................................90
5.3.4 Alternative methods/procedures................................................................90
5.3.5 Troubleshooting and optimization.............................................................91
5.4 Label-free relative quantitation of naturally generated peptides...........................91
5.4.1 Materials, equipment, and reagents...........................................................92
5.4.2 Protocols...................................................................................................92
5.4.3 Analysis and statistics...............................................................................93
5.4.4 Pros and cons............................................................................................95
5.4.5 Alternative methods/procedures................................................................96
5.4.6 Troubleshooting and optimization.............................................................96
5.5 Absolute quantitation of naturally generated peptides..........................................96
5.5.1 Materials, equipment, and reagents...........................................................97
5.5.2 Protocols...................................................................................................97
viii Contents
5.5.3 Analysis and statistics...............................................................................97
5.5.4 Pros and cons............................................................................................99
5.5.5 Alternative methods/procedures................................................................99
5.5.6 Troubleshooting and optimization.............................................................99
5.6 Summary...........................................................................................................100
References.................................................................................................................101
Chapter 6:Methodologies for bioactivity assay: biochemical study.........................103
Miryam Amigo-Benavent, Mohammadreza Khalesi, Ganesh Thapa
and Richard J. FitzGerald
6.1 Introduction.......................................................................................................103
6.2 Antioxidant activity assays................................................................................104
6.2.1 Ferric-reducing antioxidant power assay.................................................105
6.2.2 Oxygen radical absorbance capacity (ORAC) assay................................108
6.2.3 Trolox-equivalent antioxidant capacity assay..........................................110
6.2.4 Other antioxidant activity assays.............................................................113
6.3 Enzyme inhibitory assays...................................................................................114
6.3.1 Assay of angiotensin-I-converting enzyme inhibition............................114
6.3.2 Assay of renin inhibition.......................................................................117
6.3.3 Assay of dipeptidyl peptidase IV inhibitory activity.............................119
6.3.4 Assay of α-amylase inhibitory activity.................................................121
6.3.5 Assay of α-glucosidase inhibitory activity............................................124
6.3.6 Assay of lipase inhibitory activity.........................................................126
6.3.7 Assay of tyrosinase inhibitory activity..................................................129
6.3.8 Assay of trypsin inhibitory activity.......................................................131
6.3.9 Assay of chymotrypsin inhibitory activity.............................................133
6.3.10 Assay of acetylcholinesterase inhibitory activity...................................135
6.3.11 Pros and cons........................................................................................137
6.3.12 Troubleshooting and optimization.........................................................138
6.4 Summary...........................................................................................................139
Acknowledgments......................................................................................................140
References.................................................................................................................141
Chapter 7: Methodologies for bioactivity assay: cell study.....................................155
Nan Shang, Khushwant S. Bhullar and Jianping Wu
7.1 Introduction.......................................................................................................155
7.2 Cell culture basics..............................................................................................156
7.2.1 Basic equipment for cell culture.............................................................156
7.2.2 Safety aspects of cell culture...................................................................156
Contents ix
7.2.3 Aseptic technique and contamination control..........................................159
7.2.4 Cell types and sourcing of cell lines.......................................................160
7.2.5 Cell culture conditions............................................................................163
7.3 Basic cell culture protocols................................................................................165
7.3.1 Protocol 1. Subculturing adherent cultures..............................................165
7.3.2 Protocol 2. Subculturing suspension cultures..........................................167
7.3.3 Protocol 3. Quantification of total cell number and cell viability............167
7.3.4 Protocol 4. Freezing cells........................................................................167
7.3.5 Protocol 5. Thawing cryopreserved cells.................................................168
7.4 Study bone health-promoting peptide.................................................................169
7.4.1 Bone formation cells...............................................................................169
7.4.2 Bone resorption cells..............................................................................172
7.5 Biochemical and molecular analysis of cell study..............................................177
7.5.1 Protocol 12. Western blotting..................................................................177
7.5.2 Protocol 13. Quantitative reverse transcription polymerase
chain reaction..........................................................................................182
7.6 Summary...........................................................................................................186
References.................................................................................................................186
Chapter 8:Methodologies for bioactivity assay: animal study................................191
Feiran Xu and Elvira Gonzalez de Mejia
Abbreviations.............................................................................................................191
8.1 Introduction.......................................................................................................191
8.2 Administration of food peptides and animal safety............................................193
8.2.1 Safety and toxicological evaluation of peptides......................................193
8.2.2 Meal feeding information........................................................................194
8.2.3 Distribution of gender and age................................................................195
8.2.4 Development of oral and injectable peptides derived from food.............199
8.3 Animal models to evaluate hypertension............................................................199
8.3.1 Classical animal models to evaluate hypertension...................................200
8.3.2 Newfangled animal models to evaluate hypertension and
cardiovascular disease.............................................................................203
8.4 Animal models to evaluate metabolic dysfunction.............................................204
8.4.1 Animal models to evaluate metabolic dysfunction..................................206
8.4.2 Knockout mice models to evaluate metabolic dysfunction......................208
8.5 Analysis and statistics........................................................................................209
8.5.1 Sample size: power analysis....................................................................209
8.5.2 Handling of normal and nonnormal distributed data...............................209
8.5.3 Multivariate analysis of animal studies...................................................209
