isolation and characterisation of bioactive peptides derived ......isolation and characterisation of...
TRANSCRIPT
Isolation and characterisation of bioactive
peptides derived from milk and cheese
By
Stephanie Rae Pritchard
A Thesis submitted in fulfilment of the requirements for
the degree of Doctorate of Philosophy
University of Western Sydney, Australia
2012
i
STATEMENT OF AUTHENTICATION
The work presented in this thesis is to the best of my knowledge and belief, original
except as acknowledged in the text. I, hereby declare that I have not submitted this
material, either in full or in part, for a degree at this or any other institution.
Stephanie Rae Pritchard
ii
ACKNOWLEDGEMENTS
I firstly would like to acknowledge my supervisors Kaila and Michael for their
support, encouragement, guidance and especially, patience, during my PhD. I would
like to thank Kaila for his expertise particularly at the start of my candidature and in
relation to publishing- without your input in regards to publishing my work, it may
not have happened. I would like to thank Michael for his enthusiasm and guidance
especially at the end of my candidature.
I would like to thank Mark Jones, all the technical staff and assistants especially
Karen Stephenson, Julie Svenberg, Mahnez Shahnaseri, Linda Westmoreland,
Rosalie Liang and Gillian Wilkins for their encouragement and support. Also,
Rosalie Durham for her advice on the proximate composition analysis and constant
support throughout my PhD.
I would like to thank Russell Pickford for his help with Mass Spectrometry analysis
at UWS Campbelltown as well as Narsimha Reddy and Allan Torres for their help
regarding the NMR analysis at UWS Campbelltown.
I would also like to acknowledge my colleagues Sarah Moore, Junus Salampessy,
Mariam Farhad for their great advice, support and friendship over the past years.
Finally, I would like to acknowledge my family, particularly Mum, Dad and
Grandma, and friends for their continuing love, encouragement and support
throughout my PhD.
iii
DEDICATION
I dedicate this work to my daughter Isabel Evelyn.
“Learn from yesterday, live for today, hope for tomorrow.
The important thing is not to stop questioning”- Albert Einstein.
iv
LIST OF PUBLICATIONS
Journal Publications
1. Pritchard S.R., Phillips M., Kailasapathy K., 2010. Identification of bioactive peptides in commercial Cheddar cheese. Food Research International. 43, 1545-1548.[Short Communication]
2. Pritchard S.R., Phillips M., Kailasapathy K., 2010. Identification of bioactive peptides in commercial Australian organic cheddar cheeses. Australian Journal of Dairy Technology. 65, 170-173. [Short Presentation]
Book Chapters
1. Pritchard S.R. and Kailasapathy K., 2011. Chemical, Physical and Functional Characteristics of Milk and Dairy Ingredients. IN CHANDAN, R. C. & KILARA, A. (Eds.) Dairy Ingredients for Food Processing. Wiley Blackwell.
Conference Presentations
1. Pritchard S.R., Phillips M., Kailasapathy K., 2010. Identification of bioactive peptides derived from fermentation of organic milk. Graduate Dairy Foods Poster Competition. ADSA-PSA-AMPA-CSAS-ASAS Joint Annual Meeting in Denver, Colorado, USA. (Poster Presentation).
2. Pritchard S.R., Phillips M., Kailasapathy K., 2010. Identification of bioactive peptides in commercial Australian organic cheddar cheeses. DIAA cheese science conference. International Dairy Federation (IDF) World Dairy Summit, Auckland, NZ. (Oral Presentation).
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TABLE OF CONTENTS
STATEMENT OF AUTHENTICATION .............................................................................. i
ACKNOWLEDGEMENTS .................................................................................................... ii
DEDICATION ........................................................................................................................ iii
LIST OF PUBLICATIONS ................................................................................................... iv
TABLE OF CONTENTS ......................................................................................................... v
LIST OF FIGURES ............................................................................................................... ix
LIST OF TABLES ................................................................................................................ xii
LIST OF ABBREVIATIONS ............................................................................................. xiv
LIST OF APPENDICES ..................................................................................................... xvi
LIST OF AMINO ACIDS: STRUCTURES AND CODES ............................................. xvii
ABSTRACT .............................................................................................................................. 1
Chapter 1 Introduction and Literature Review .................................................................... 4
1.1 Background ...................................................................................................................... 4
1.2 Aim ................................................................................................................................... 6
1.3 Objectives ......................................................................................................................... 6
1.4 Significance and justification ........................................................................................... 6
1.5 Milk constituents .............................................................................................................. 8
1.5.1 Types of bovine milk ................................................................................................ 8
1.5.2 Types of milk protein .............................................................................................. 12
1.5.2.1 Proteins: Separation and analysis ......................................................................... 15
1.6 Functional Foods and Nutraceuticals ............................................................................. 19
1.7 Probiotic bacteria ........................................................................................................... 20
1.7.1 Lactobacillus genus ................................................................................................. 23
1.7.1.1 Lactobacillus acidophilus .................................................................................... 24
1.7.1.2 Lactobacillus casei ............................................................................................... 24
1.7.1.3 Lactobacillus helveticus ....................................................................................... 25
1.7.1.4 Lactobacillus rhamnosus (also known as L. casei subsp. rhamnosus) ................ 25
1.8 Milk production and organic farming ............................................................................ 26
1.9 Fermentation and proteolysis by lactic acid bacteria ..................................................... 29
1.9.1 Cheddar cheese fermentation and processing ......................................................... 32
1.10 Enzymes and enzymatic hydrolysis of food proteins ................................................... 33
1.10.1 Flavourzyme (Protease from Aspergillus oryzae) ................................................. 34
1.10.2 Bromelain from pineapple stem (E.C 3.4.22.32) .................................................. 34
1.10.3 Papain from papaya latex (E.C 3.4.22.2) .............................................................. 35
1.10.4 Fromase 750 XLG ................................................................................................. 35
1.10.5 Rennin from calf stomach (E.C 3.4.23.4) ............................................................. 35
1.11 Bioactive peptides ........................................................................................................ 36
1.11.1 Techniques used to isolate and characterise bioactive peptides ........................... 41
1.11.2 The gastrointestinal tract, peptide stability and absorption ................................... 42
1.11.3 Antimicrobial peptides .......................................................................................... 45
1.11.4 Antitumour peptides .............................................................................................. 50
1.11.5 Antioxidant peptides ............................................................................................. 52
1.11.6 Antihypertensive / ACE-inhibitory Peptides ........................................................ 55
1.11.6.1 Role of ACE and hypertension .......................................................................... 58
1.11.7 Other bioactive peptides ........................................................................................ 60
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1.11.8 Commercial bioactive peptide applications and production problems ................. 70
1.12 Pathogenic bacteria ...................................................................................................... 71
1.12.1 Escherichia coli ATCC 8739 ................................................................................ 71
1.12.2 Bacillus cereus ATCC 11778 ............................................................................... 72
1.12.2.1 Bacillus cereus and food poisoning ................................................................... 72
1.12.3 Staphylococcus aureus ATCC 6538 ..................................................................... 73
1.12.4 Streptococcus mutans ............................................................................................ 73
1.12.4.1 Streptococcus mutans and dental caries ............................................................. 74
1.13 Scope of this of study ................................................................................................... 74
Chapter 2 General Methods .................................................................................................. 75
2.1 Bacterial cultures and growth......................................................................................... 75
2.2 Chemicals, media, stock solutions, buffers and reagents ............................................... 76
2.2.1 Chemicals ................................................................................................................ 76
2.2.1.1 RP-HPLC solvents ............................................................................................... 76
2.2.2 Bacterial Media ....................................................................................................... 77
2.2.3 Stock solutions ........................................................................................................ 79
2.2.4 Buffers ..................................................................................................................... 80
2.2.5 Reagents .................................................................................................................. 82
2.3 Analytical Instruments ................................................................................................... 82
2.3.1 Shimadzu Reverse Phase High Performance Liquid Chromatography .................. 82
2.3.2 Bio-Rad Benchmark Plus Microplate Spectrophotometer ...................................... 83
2.3.3 Bio-Rad Gel Electrophoresis Unit .......................................................................... 83
2.3.4 Freeze-dryer ............................................................................................................ 83
2.3.5 Autoclaving and sterilisation .................................................................................. 83
2.3.6 Quadruple Time-of-Flight Liquid Chromatography-Electronspray Ionisation- Tandem Mass Spectrometer (QToF-LC-ESI-MS/MS) ................................. 83
2.3.7 Nuclear Magnetic Resonance spectrometer (NMR) ............................................... 84
2.4 Bioactivity analysis general overview ........................................................................... 84
2.5 Bioactive Screening Assays ........................................................................................... 85
2.5.1 Antimicrobial assay ................................................................................................. 85
2.5.2 ACE-inhibitory assay .............................................................................................. 85
2.5.2.1 ACE-inhibitory peptide: stability assay ............................................................... 87
2.5.2.2 ACE-inhibitory peptides: gastrointestinal stability assay .................................... 88
2.5.3 Antioxidant assay .................................................................................................... 89
2.6 Fractionation and purification of selected bioactive peptides ........................................ 89
2.7 SDS-PAGE reagents, preparation and casting ............................................................... 90
2.7.1 Gel imaging ............................................................................................................. 92
2.8 Bradford protein assay (Bio-Rad) .................................................................................. 92
2.9 Statistical analysis .......................................................................................................... 93
Chapter 3 Isolation and characterisation of bioactive peptides derived from commercial Cheddar cheeses and fermented milk. ............................................................ 94
3.1 Introduction .................................................................................................................... 94
3.2 Materials and Methods ................................................................................................... 96
3.2.1 Cheddar cheeses and probiotic bacteria preparation ............................................... 96
3.2.2 Extraction of water-soluble peptides from Cheddar cheese .................................... 96
3.2.3 Proximate composition analysis of organic milk .................................................... 97
3.2.4 Extraction of organic milk protein .......................................................................... 97
3.2.5 Fermentation of organic milk protein ..................................................................... 97
3.2.5.1 Extraction of peptides from fermented organic milk protein ............................... 98
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3.2.6 Separation, fractionation and purification of peptides ............................................ 98
3.2.7 Identification of bioactive peptides derived from commercial Cheddar cheeses and fermented organic milk protein .................................................................... 99
3.2.7.1 Identification of peptide extracts with antimicrobial activity .............................. 99
3.2.7.2 Identification of peptide extracts with antioxidant activity ................................. 99
3.2.7.3 Identification of peptide extracts with ACE-inhibitory activity .......................... 99
3.3 Results .......................................................................................................................... 100
3.3.1 Proximate composition of the Cheddar cheeses ................................................... 100
3.3.1.1 Proximate composition of lite organic milk ....................................................... 100
3.3.2 Separation, fractionation and characterisation of cheese peptides ........................ 101
3.3.3 Separation, fractionation and characterisation of fermented peptide extracts ........................................................................................................................... 101
3.3.4 Screening for bioactive peptides ........................................................................... 104
3.3.4.1 Antimicrobial activity of Cheddar cheese extracts ............................................ 104
3.3.4.2 Antimicrobial activity of fermented milk protein extracts ................................. 113
3.3.4.3 Antioxidant activity of Cheddar cheese extracts ................................................ 114
3.3.4.4 Antioxidant activity of fermented peptide extracts ............................................ 116
3.3.4.5 ACE-inhibitory activity of Cheddar cheese extracts .......................................... 118
3.3.4.6 ACE-inhibitory activity of fermented peptide extracts ...................................... 127
3.3.5 Structure of ACE-inhibitory peptides by Mass Spectrometry and MASCOT database searching ........................................................................................ 128
3.4 Discussion .................................................................................................................... 134
3.5 Conclusions .................................................................................................................. 139
Chapter 4 Isolation and characterisation of bioactive peptides formed during enzymatic hydrolysis of organic milk protein. .................................................................. 142
4.1 Introduction .................................................................................................................. 142
4.2 Materials and Methods ................................................................................................. 143
4.2.1 Proximate composition of organic milk ................................................................ 143
4.2.2 Extraction of milk protein ..................................................................................... 143
4.2.3 Enzymatic hydrolysis of milk protein ................................................................... 143
4.2.4 Preparation of peptide extracts for RP-HPLC, Biorad protein assays and SDS-PAGE. .................................................................................................................... 144
4.2.5 Separation, fractionation and purification of peptides .......................................... 145
4.2.6 Identification of bioactive peptides derived from enzymatic hydrolysis of organic milk protein. ...................................................................................................... 145
4.2.6.1 Identification of peptide extracts with antimicrobial activity ............................ 145
4.2.6.2 Identification of peptide extracts with ACE-inhibitory activity ........................ 145
4.2.6.3 Identification of peptide extracts with antioxidant activity ............................... 146
4.3 Results .......................................................................................................................... 146
4.3.1 Proximate composition analysis of lite organic milk ............................................ 146
4.3.2 Screening for bioactive peptides ........................................................................... 146
4.3.2.1 Antimicrobial activity of hydrolysates ............................................................... 146
4.3.2.2 Antioxidant activity of hydrolysates .................................................................. 152
4.3.2.3 Antihypertensive activity of hydrolysates .......................................................... 154
4.3.3 Structure of Antimicrobial and ACE-inhibitory peptides by Mass Spectrometry and MASCOT database searching ........................................................... 160
4.4 Discussion .................................................................................................................... 170
4.5 Conclusions .................................................................................................................. 174
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Chapter 5 Bioactivity and NMR studies of selected bioactive peptides derived from organic cheese and milk ............................................................................................. 176
5.1 Introduction .................................................................................................................. 176
5.2 Materials and methods ................................................................................................. 177
5.2.1 Materials ................................................................................................................ 177
5.2.2 Determination of ACE-inhibitory activity of selected peptides ............................ 177
5.2.2.1 Stability of selected peptides against ACE ........................................................ 177
5.2.3 Stability of selected peptides against gastrointestinal enzymes ............................ 178
5.2.4 NMR Studies on selected peptides ........................................................................ 178
5.2.4.1 NMR data acquisition and processing ............................................................... 178
5.2.4.1.1 1D-NMR experiments ..................................................................................... 178
5.2.4.1.2 2D- Total Correlation Spectroscopy (TOCSY) experiments .......................... 178
5.2.4.1.3 2D- Rotating Frame Overhauser Effect Spectroscopy (ROESY) experiments .................................................................................................................... 179
5.2.4.2 NMR data analysis ............................................................................................. 179
5.2.4.2.1 Proton assignment ........................................................................................... 179
5.2.4.2.2 Sequential assignment ..................................................................................... 180
5.2.4.2.3 Chemical shift index (CSI) based structure analysis ...................................... 180
5.2.4.2.4 NOE based structure determination ................................................................ 181
5.3 Results .......................................................................................................................... 181
5.3.1 ACE-inhibitory activity of selected bioactive peptides ........................................ 181
5.3.2 Stability of selected peptides against gastrointestinal enzymes ............................ 182
5.3.3 NMR studies on selected peptides ........................................................................ 185
5.3.3.1 NMR-based structural analysis of selected peptides in water ............................ 185
5.3.3.2 Proton assignments ............................................................................................ 185
5.3.3.3 Sequential assignment ........................................................................................ 191
5.3.3.4 Chemical Shift Index (CSI) based structure analysis ......................................... 193
5.4 Discussion .................................................................................................................... 199
5.5 Conclusions .................................................................................................................. 203
Chapter 6 Conclusions and Future Research .................................................................... 205
Appendices ............................................................................................................................ 211
References ............................................................................................................................. 229
ix
LIST OF FIGURES
Figure 1.1 Organic milk processing 28 Figure 1.2 Pathways ACE utilises: Renin-Angiotensin System (RAS)
and the Kinin Nitric Oxide System (KNOS) 60
Figure 3.1 Proximate composition of lite organic milk 101 Figure 3.2 Separation of fermented peptide extracts by gel
electrophoresis 103
Figure 3.3 Average percentage inhibition of bacteria by cheese peptides.
105
Figure 3.4 Inhibition of bacteria by MWCO fractionated non-organic cheese peptide fractions
107
Figure 3.5 Inhibition of bacteria by MWCO fractionated organic cheese peptide fractions
109
Figure 3.6 Inhibition of B. cereus by cheese fractions 111 Figure 3.7 Inhibition of B. cereus by fractionated cheese fractions. 112 Figure 3.8 Inhibition of E. coli by fermented peptide extracts. 114 Figure 3.9 Inhibition of DPPH by MWCO Cheddar cheese peptide
extracts. 115
Figure 3.10 Inhibition of DPPH by fermented peptide extracts. 117 Figure 3.11 Inhibition of ACE by whole Cheddar cheese peptide
extracts. 119
Figure 3.12 Inhibition of ACE by Cheddar cheese peptide fractions. 120 Figure 3.13 Inhibition of ACE by fractionated organic Cheddar cheese
extracts 122
Figure 3.14 Inhibition of ACE by organic Cheddar cheese fractions. 124 Figure 3.15 Inhibition of ACE by organic Cheddar cheese fractions. 126 Figure 3.16 Inhibition of ACE by fermented milk peptide extracts. 127 Figure 3.17 RP-HPLC chromatogram of <5EF2A-6. 129 Figure 3.18 A. Summed mass chromatogram at mass 634.3497. B. Total
ion count chromatogram of sample 5EF2A-6. 131
Figure 3.19 Summed mass chromatogram at mass 692.877 corresponding to tridecapeptide FFVAPFPEVEKEK.
132
Figure 4.1 Inhibition of E. coli by hydrolysates. 148 Figure 4.2 Inhibition of B. cereus by fractionated hydrolysates. 149 Figure 4.3 Inhibition of S. aureus by fractionated hydrolysates. 150 Figure 4.4 Inhibition of S. aureus by 5F10.5S fractions. 151 Figure 4.5 Inhibition of DPPH by MWCO fractions. 153 Figure 4.6 Inhibition of ACE by MWCO hydrolysates. 155 Figure 4.7 Inhibition of ACE by MWCO hydrolysates. 157 Figure 4.8 Inhibition of ACE by fractionated hydrolysates. 158 Figure 4.9 Inhibition of ACE by MWCO hydrolysates. 159 Figure 4.10 Inhibition of ACE by MWCO hydrolysates. 160 Figure 4.11 RP-HPLC of chromatogram of ACE-inhibitory fraction
5F0.51I. 162
Figure 4.12 RP-HPLC chromatogram of ACE-inhibitory fraction 163
x
5P0.53IF2B. Figure 4.13 Summed mass chromatogram at 1055.48 corresponding to
decapeptide DIPNPIGSEN. 164
Figure 4.14 Summed mass chromatogram at 707.8406 corresponding to dodecapeptide AVPYPQRDMPIQ.
165
Figure 5.1 Inhibition of ACE by synthesised peptides. 182 Figure 5.2 Stability of ACE-inhibitory peptides against gastrointestinal
enzymes pepsin and pancreatin. 184
Figure 5.3 Total correlation spectrum (TOCSY) (HN-Hα region) of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 90 ms mixing time.
187
Figure 5.4 Expansion of Total correlation spectrum (TOCSY) (HN-Hα region) of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 90 ms mixing time.
188
Figure 5.5 Total correlation spectrum (TOCSY) (HN-Hα region) of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water at 25ºC and 90 ms mixing time.
190
Figure 5.6 Rotating frame nuclear Overhouser effect spectrum (ROESY) (HN-Hα region) of peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 450 ms mixing time.
192
Figure 5.7 (a) Chemical shift index (CSI) of Hα of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 450 ms mixing time.*
195
Figure 5.7 (b) Hα chemical shift difference plot of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP).
195
Figure 5.8 Chemical shift index (CSI) of Hα of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water at 25ºC and 450 ms mixing time.*
196
Figure 5.9 (a) Chemical shift index (CSI) of Hα of the peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in water at 25ºC and 450 ms mixing time.*
198
Figure 5.9 (b) Hα chemical shift difference plot of peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ)
198
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LIST OF TABLES
Table 1.1 Compositions of differently processed bovine milk 11 Table 1.2 Bioactivities and commercial applications of milk proteins 14 Table 1.3 Proposed health benefits of selected probiotic bacteria 22 Table 1.4 Bioactivity of main peptide types 37 Table 1.5 Examples of proteolytic probiotic bacteria, health benefits and
peptide bioactivity. 40
Table 1.6 Selected antimicrobial peptides isolated from bovine milk 48 Table 1.7 Selected antitumour peptides isolated from bovine milk 51 Table 1.8 Selected antioxidant peptides isolated from bovine milk 54 Table 1.9 Selected antihypertensive peptides derived from bovine milk 58 Table 1.10 Selected immunomodulatory peptides isolated from bovine milk 63 Table 1.11 Selected antiviral, mineral binding, opioid and other bioactive
peptides derived from bovine milk 68
Table 2.1 Preparation for ACE-inhibitory assay 87 Table 2.2 Gel preparation for SDS-PAGE 91 Table 3.1 Nutritional information for Cheddar cheeses (%) 100 Table 3.2 Mass spectrometry database search results for selected ACE-
inhibitory fractions isolated from organic cheese 133
Table 4.1 Optimum temperature and pH of enzymes used to hydrolyse milk protein
144
Table 4.2 Summary of peptides identified from various bioactive hydrolysate fractions by Mass spectrometry
160
Table 5.1 Concentrations required to inhibit 50% of ACE activity (µM). 182 Table 5.2 The chemical shifts (δ in ppm) of Tyr-Leu-Gly-Tyr-Leu-Glu-
Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water. 188
Table 5.3 The chemical shifts (δ in ppm) of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water
190
Table 5.4 The chemical shifts (δ in ppm) of the peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in water
191
Table 5.5 Sequential NOE connectivities of HN (i) to Hα (i-1) of the amino acid residues of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP)
192
Table 5.6 Sequential NOE connectivities of HN (i) to Hα (i-1) of the amino acid residues of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1).
193
Table 5.7 Sequential NOE connectivities of HN (i) to Hα (i-1) of the amino acid residues of the peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2).
193
Table 5.8 Chemical shift index (CSI) results of peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 450 ms mixing time.*
194
Table 5.9 Chemical shift index (CSI) results of decapeptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water at 25ºC and 450 ms mixing time.*
196
xiii
Table 5.10 Chemical shift index (CSI) results of dodecapeptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in water at 25ºC and 450 ms mixing time.
197
xiv
LIST OF ABBREVIATIONS
% Percent °C degrees Celsius A Absorbance ACE Angiotensin-I-converting enzyme Ala Alanine Arg Arginine ATP Adenosine triphosphate B. longum Bifidobacterium longum BSA Bovine Serum Albumin Ca2+ Calcium two plus CCK cholecystokinin cfu/mL colony-forming units per millilitre CHO Carbohydrate CO2 Carbon dioxide CP1 Cheese peptide 1 CP2 Cheese peptide 2 CPPs Casophosphopeptides DNA Deoxyribonucleic acid DPPH 1, 1-diphenyl-2-picrylhydrazyl E. Enterococcus ELISA Enzyme-Linked ImmunoSorbent Assay FIDs Free induction decay/s g grams GMOs Genetically Modified Organisms GMP Glycomacropeptide HA Hippuric Acid HCl Hydrochloric Acid HHL Hippuryl-Histydyl-Leucine HL Histydyl-Leucine HPLC High-Performance Liquid Chromatography Ig Immunoglobulin Ile Isoleucine kDa Kilodalton kJ Kilojoule L. Lactobacillus LAB Lactic Acid Bacteria LF Lactoferrin LP Lactoperoxidase M Molar MALDI-TOF Matrix-assisted Laser Desorption/Ionisation- Time of Flight Met Methionine mg milligrams min minute mL millilitre
xv
MP Milk peptide MQ Milli-Q MTT (4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide m/z mass to charge ratio nm nanometres NMR Nuclear Magnetic Resonance N-terminal amino-terminal Pro Proline RNA Ribonucleic acid RP-HPLC Reverse Phase-High Performance Liquid Chromatography rpm revolutions per minute S. Staphylococcus SHR Spontaneously Hypertensive Rats sp. Species Stds Standards Subsp. subspecies v/v volume to volume Val Valine x g g-force α alpha α-La alpha-lactoalbumin く beta く-Lg beta-lactoglobulin δ delta μ mu µL microlitre μM micromolar к kappa
xvi
LIST OF APPENDICES Appendix 1 Proximate composition analysis methods and data 212 Appendix 2 Gradient programs used for separation of peptides 215 Appendix 3 Molecular weight data and gels for cheese peptide extracts 216
xvii
LIST OF AMINO ACIDS: STRUCTURES AND CODES
Amino acid One letter code Three letter code Structure Alanine A Ala Neutral, nonpolar side chains Arginine R Arg Basic, polar side chains Asparagine N Asn Neutral, polar side chains Aspartate D Asp Acidic, polar side chains Cysteine C Cys Neutral, polar side chains Glutamate E Glu Acidic, polar side chains Glutamine Q Gln Neutral, polar side chains Glycine G Gly Neutral, nonpolar side chains Histidine H His Basic, polar side chains Isoleucine I Ile Neutral, nonpolar side chains Leucine L Leu Neutral, nonpolar side chains Lysine K Lys Basic, polar side chains Methionine M Met Neutral, nonpolar side chains Phenylalanine F Phe Neutral, nonpolar side chains,
aromatic, fluorescent Proline P Pro Neutral, nonpolar side chains Serine S Ser Neutral, polar side chains Threonine T Thr Neutral, polar side chains Tryptophan W Trp Neutral, polar side chains,
aromatic, fluorescent Tyrosine Y Tyr Neutral, polar side chains,
aromatic, fluorescent Valine V Val Neutral, nonpolar side chains
1
ABSTRACT
This research investigated the presence of antimicrobial, antihypertensive and
antioxidant peptides derived from fermented milk protein, hydrolysed milk protein as
well as various Cheddar cheese peptide extracts. In Chapter one, the introduction and
literature review, background information on known bioactive peptides are given.
Bioactive peptides are specific fragments of protein that have a positive impact on
health. They can be derived from fermentation and/or hydrolysis of protein and have
been shown to have various properties including antimicrobial, antihypertensive,
antioxidant, immunomodulatory, mineral-binding and opioid. Currently, the reported
literature has identified bioactive peptides obtained from fermented milk protein
predominantly by Lactobacillus helveticus and this research studied the use of other
probiotic bacteria to derive bioactive peptides. Similarly, the previous literature has
investigated the presence of bioactive peptides after hydrolysis using digestive
enzymes such as trypsin, chymotrypsin (rennin) and pepsin. This study used enzymes
derived from various plant and animal sources to hydrolyse milk protein and then
investigated if any bioactive peptides have been obtained. Also, the literature on
bioactive peptides derived from cheese is minimal therefore the presence of bioactive
peptides in five Australian Cheddar cheeses was investigated. The literature
pertaining to bioactive peptides derived from milk via hydrolysis using digestive
enzymes is vast and shows the variety of peptides that can be derived when milk is
used as the substrate. This food has been shown to contain the most active and potent
bioactive peptides to date particularly antihypertensive peptides. The discovery of
2
novel bioactive peptides could potentially lead to the production of functional foods
containing bioactive peptides or as use as food ingredients in various food substrates
The general screening process included screening of the whole extracts, then the
most bioactive extracts were separated by centrifugation using molecular-weight cut-
off (MWCO) membranes (5 kDa and 10 kDa) and subsequently the most bioactive
samples were fractionated by RP-HPLC, if deemed appropriate. The extracts were
screened for antioxidant activity against the free radical DPPH, antimicrobial activity
against three bacteria: E. coli, B. cereus and S. aureus and ACE-inhibitory activity
against the angiotensin-I-converting enzyme (ACE).
The first results chapter focuses on the identification of bioactive peptides derived
from fermented foods including various Cheddar cheeses and fermented milk
protein. The Cheddar cheese extracts showed low antioxidant activity (<20%) and
good antimicrobial activity against B. cereus (44.25% at 1.16 mg/mL). The most
ACE-inhibitory fraction derived from Cheddar cheese E contained two known
peptides with strong ACE-inhibitory activity YLGYLEQLLR and
FFVAPFPEVEKEK. The fraction acted like a substrate against ACE. The peptide
YLGYLEQLLR was synthesised (GenScript USA Inc.Piscataway, NJ, USA).
The soluble and insoluble protein fractions of organic lite milk were extracted by
acid precipitation and subsequently underwent fermentation using four different
probiotic bacteria namely L. acidophilus, L. casei, L. rhamnosus and L. helveticus.
Preliminary analysis was undertaken however, the RP-HPLC chromatograms showed
poor intensity and number of peptides in each extract and therefore analysis was not
3
continued. Subsequently, the use of various exogenous plant and animal enzymes
were used to hydrolyse the milk protein to determine if they produced bioactive
peptides.
The second results chapter discusses the use of various plant and animal enzymes to
derive bioactive peptides. The milk protein was extracted by acid precipitation and
hydrolysed separately by five enzymes at their optimum conditions for 1, 3 and 5
hours. The antimicrobial activity of the fractionated hydrolysates (by RP-HPLC) was
strongest against S. aureus (69.35% at 0.009 mg/ml). This active fraction was
analysed by Electron-Spray-Ionisation-Quadruple-Time of flight-Tandem Mass
Spectrometry and MASCOT database searching to determine the peptides in that
fraction, subsequently, 11 peptides were identified. The antioxidant activity of the
hydrolysates was low (<30% inhibition). The ACE-inhibitory activity was strong.
Various fractions were analysed by Mass spectrometry after several fractionations
and bioactivity screening assays by RP-HPLC and two novel peptide sequences were
synthesised DIPNPIGSEN and AVPYPQRDMPIQ (GenScript USA Inc.Piscataway,
NJ, USA).
The final results chapter examines three synthesised peptide’s ACE-inhibitory
activity and stability against ACE as well their gastrointestinal stability (using pepsin
and pancreatin). Their structure by NMR is also determined and relationship to their
bioactivity discussed. Chapter six discusses the results of the thesis, conclusions and
future directions.
4
Chapter 1 Introduction and Literature Review
Note: Parts of this chapter are taken from my publication ‘PRITCHARD, S. R. &
KAILASAPATHY, K. 2011. Chemical, Physical and Functional Characteristics of
Dairy Ingredients. In: CHANDAN, R. C. & KILARA, A. (eds.) Dairy Ingredients
for Food Processing. New Jersey: Wiley Blackwell’.
1.1 Background
Bioactive peptides are defined as specific protein fragments that have a positive
impact on body functions or conditions and may ultimately influence health (Kitts
and Weiler 2003). They usually range from two to twenty amino acids in length and
have been derived from various plant and animal sources including milk, cheese,
yoghurt, fish, soybean and kefir. Peptides derived from milk, in particular, have the
greatest potential to be used commercially. Bioactive peptides derived from milk
have been shown to have various properties including antimicrobial,
antihypertensive, opioid, antioxidant, antithrombiotic and mineral-binding.
The functional food and nutraceutical industries are becoming more important in
today’s society as consumers are increasingly more health conscious; consequently,
these industries are rapidly expanding. The nutraceutical industry was worth $117.3
billion globally in 2007 and is projected to be worth $176.6 billion in 2013. Also, the
consumption of fermented milk products has increased from 138 million tonnes to
143 million tonnes in two years in Australia (International Dairy Federation 2008).
Furthermore, the Australian organic food industry’s retail value is approximately 600
5
billion dollars with reports of 10 to 30% growth per annum since 2004 (Biological
Farmers of Australia 2008). Products containing bioactive peptides have been
marketed by the functional food industry in recent years, and this research will
possibly enable the industry to expand their range of products and consumer market
further, as consumers of organic foods avoid products that have utilised synthetic
fertilisers, pesticides, growth promoters or additives in their production and
processing (Kouba 2003).
Proteolytic probiotic bacteria have been used to produce peptides from milk that
were shown to have antitumour, immunostimulatory, opioid and antihypertensive or
ACE-inhibitory bioactivity (Rokka et al. 1997, Le Blanc et al. 2002, Savijoki et al.
2006, Donkor et al. 2007a, Quirós et al. 2007). However, there have been few studies
that have used probiotic bacteria other than L. helveticus and there are no studies that
have used organic milk to derive bioactive peptides. This study aims to use various
probiotic bacteria to possibly derive bioactive peptides including L. acidophilus, L.
casei, L. rhamnosus and L. helveticus.
Furthermore, various digestive enzymes including trypsin, chemotrypsin, pepsin and
chymosin have been used to derive bioactive peptides from milk (Meisel and
Fitzgerald 2003, Yamamoto et al. 2003, Korhonen 2009b). However, there are few
studies that have used enzymes derived from plant or animals such as flavourzyme,
papain, bromelain or fromase. Therefore, this study aims to compare the peptides
derived from enzymatic hydrolysis of milk protein and determine if novel bioactive
peptides are present.
6
1.2 Aim
The aim of this research is to isolate, characterise and compare the peptides formed
during the fermentation and hydrolysis of organic milk and Cheddar cheeses and
determine if they have bioactive properties.
1.3 Objectives
Extract, analyse and compare the peptides derived from fermented
organic milk, and commercial non-organic and organic cheese.
Extract, analyse and compare the peptides formed after enzymatic
hydrolysis of organic milk.
Investigate the properties of the peptides formed for potential bioactivity
(i.e. antimicrobial, antioxidant, ACE-inhibitory) using various screening
methods and analytical techniques.
Characterise selected bioactive peptides by NMR and ESI-Q-TOF MS-
MS
1.4 Significance and justification
Milk is a known food source of potent bioactive peptides with various properties
including antihypertensive, antimicrobial, antioxidant, opioid and antithrombiotic.
Many bioactive peptides have been derived from milk via enzymatic hydrolysis
using digestive enzymes, and to a lesser extent some proteolytic bacterial or animal
enzymes and probiotic bacteria particularly L. helveticus (Yamamoto et al. 1994b,
De Moreno De Leblanc et al. 2005, Pihlanto et al. 2009).
7
This study will examine bioactive peptides derived from various animal and plant
enzymes including rennin from calf stomach, fromase, papain, bromelain and
flavourzyme as well as using fermentation of organic milk by probiotic bacteria L.
acidophilus, L. casei, L. rhamnosus and L. helveticus. The use of these enzymes or
probiotic bacteria to derive bioactive peptides has not been largely reported and this
study aims to observe whether or not bioactive peptides are derived using these
enzymes.
Furthermore, the use of organic milk to obtain bioactive peptides has not been
reported in the literature therefore organic milk was the substrate chosen to derive the
bioactive peptides due to suggestions that it is healthier than non-organic milk and
growing consumer awareness of its potential health benefits. Organic milk contains
higher levels of omega three fatty-acids and conjugated linoleic acids than
conventional milk (Bloksma et al. 2008) and immunological studies revealed that
organically farmed cows are in better health than conventionally farmed cows
(Bloksma et al. 2008). The organic food industry is estimated to generate over one
billion dollars in sales by the end of 2010 (Biological Farmers of Australia 2010).
The discovery of novel bioactive peptides derived from organic milk could expand
the functional food market in Australia, which was estimated to be worth $117.3
billion globally in 2007 (Anon 2008b).
The functional food and nutraceutical industries also provide products containing
probiotic bacteria that when administered in adequate amounts confer physiological
benefits to their host (Food and Agriculture Organisation of the United Nations and
8
the World Health Organisation 2001), which can include alleviating gastrointestinal
tract diseases including diarrhoea and constipation, hypertension (Hata et al. 1996)
and infections with Helicobacter pylori (Wang et al. 2004). Probiotic bacteria have
been shown to produce bioactive peptides (Sutas et al. 1996, Gobbetti et al. 2000) via
fermentation in non-organic milk. The global probiotic market was worth 14.9 billion
in 2007 and is projected to be valued at 19.6 billion by 2013 (Anon 2008a). The
discovery of novel and biologically potent peptides could provide more therapeutic
benefits to consumers of organic milk products and other consumers.
1.5 Milk constituents
The major constituents of milk include water, protein, fat, carbohydrates (mainly
lactose) and trace elements (Fox 2009). The constituents of milk are the same for all
mammals, however; the concentration of each constituent varies depending on the
species. Water is the main constituent in milk totalling 79-90%. Milk fat makes up
approximately 3.5-4.5% of the milk constituents and is mostly contained in fat
globules, which are composed of about 98% triglycerides, 0.2-1% phospholipids,
0.2-0.4% sterols, fatty acids, vitamins A, D, E and K and enzymes. The protein in
milk makes up approximately 3-4% of milk constituents. There are two major protein
classes of milk protein: casein and whey. The major carbohydrate in milk lactose
makes up 4-5% of the total milk composition and 0.7-0.8% trace elements, which
include salts, minerals and vitamins.
1.5.1 Types of bovine milk
The main type of milk consumed by humans is bovine milk. The processing of milk
varies depending on the desired outcome. The concentration and properties of the
9
constituents in milk depend on several factors including the breed, species,
nutritional and lactation stage and diet of the animal (Fox 2003, Chandan 2006,
Pritchard and Kailasapathy 2011).
The composition of the milk varies particularly in relation to the concentration of fat,
and the types of fatty acid residues present between different species and breeds.
Milk from Guernsey and Jersey cows have been shown to have higher fat content
when compared with the Holstein cow breed (Jensen 1995). Similarly, the White
Thari cow breed produced higher amounts of saturated fatty acids than the Red
Sindhi cow breed and lower concentrations of mono-unsaturated fatty acids,
polyunsaturated fatty acids and conjugated linoleic acids (Talpur et al. 2006).
The composition of the diet and form in which it is delivered to cows has been
shown to have an effect on composition and milk yield. High fat and/or low
roughage diets have been shown to reduce the fat content of milk. Overall, the
influence of diet on protein and lactose content in the milk has been minimal.
Seasonal and regional changes have been shown to influence changes in diet
especially severe heat (Jensen 1995). However, generally slight but well defined
variations are present in both the fat and solid-not-fat components of milk over the
course of a year. Also, the lactation stage of the cow influences the milk yield and
the concentrations of lactose, fat and protein in milk. Lactose and fat concentrations
increase as lactation progresses (Jensen 1995).
10
The way the milk is processed influences the concentration of constituents in milk
(Table 1.1). For example, skim milk has lower fat and vitamin content compared
with whole milk. Furthermore, milk can be fortified with iron, vitamin D and omega-
3 fatty acids. Other modifications can include adding flavouring, adding culture or
evaporating the milk (Varnam and Sutherland 2001b).
Table 1.1 Compositions of processed bovine milk
Milk Type Water (g)
Energy (kJ)
Protein (g)
Total Fat (g)
Omega 3 (mg)
Sugars (g)
Cholesterol (mg)
Folate (μg)
Thiamin (mg)
Vitamin C (mg)
Retinol (μg)
Iron (mg)
Milk 87.5 278 3.3 4.0 6 4.7 13 7 0.03 1 36 0 (Pure Organic) Full cream cow’s Organic Milk
- 288 3.2 4.1 - 4.8 - - - - - -
Fortified Milk (Added Iron)
87.5 279 3.4 4.0 6 4.7 13 7 0.03 1 36 0.6
UHT Milk 87.4 269 3.5 3.7 0 4.5 11 7 0.03 1 48 - Skim Milk 90.7 142 3.6 0.1 0 4.8 3 5 0.04 1 0 -
All values expressed per 100g edible portion. Adapted from: (Food Standards Australia and New Zealand 2006, Family Health Network 2009).
10
11
12
1.5.2 Types of milk protein
Proteins are complex macromolecules made up of various amino acids that are
covalently bonded via peptide bonds (Rosenberg 1996). There are four levels of
protein structure. The primary level is the sequence of amino acids, secondary is the
regular local structural arrays (i.e beta sheets, alpha helices), tertiary: the
intramolecular arrangement of the secondary structure units in relation to each other
and quaternary the stiochiometry and spatial arrangement of the protein subunits
(Rosenberg 1996).
Milk proteins have been studied for over two-hundred years (Fox and Mcsweeney
2003). The protein content of milk is influenced by nutritional, physiological and
genetic factors including the type of forage consumed (Erasmus et al. 2001). Milk is
mainly composed of two types of protein: whey and casein. Casein is divided into
four types αs1-, αs2-, く-, and к-casein (Walstra et al. 1999) and constitutes about 80%
of the total protein in milk. Whey proteins include く-lactoglobulin, α-lactoalbumin,
bovine serum albumin, immunoglobulins particularly IgG, IgM and IgA, lactoferrin
and transferrin that constitute approximately 20% of total protein in milk (Zayas
1997). Whey is rich in essential, branched and sulphur containing amino acids
including methionine, valine, leucine, isoleucine, cysteine and asparagine (Smithers
2008).
Each protein has known bioactivities and several are used in commercial products
(Clare and Swaisgood 2000, Korhonen and Pihlanto 2003, Korhonen and Pihlanto
13
2006, Korhonen 2009a) (Table 1.2) and utilised as edible films, gels and capsules in
various food products including fruit juices, desserts, meats, dairy products and
carbonated beverages (Phillips et al. 1994, Zayas 1997, Singh and Ye 2009).
Table 1.2 Bioactivities and commercial applications of milk proteins
Milk Protein Bioactivities Commercial Applications References
く-casein, α-casein, к-casein Immunomodulatory, mineral carriers
(Gill et al. 2000, Vasiljevic and Shah 2007)
く-lactoglobulin (く-Lg) antiviral, pathogen adhesion prevention, antitumour, antioxidant, immunomodulatory
high-protein based beverages, soft drinks
(Zayas 1997, Gill et al. 2000, Korhonen 2009a)
α-lactoalbumin (α-La) Immunomodulatory, antitumour
high-protein based beverages, soft drinks
(Zayas 1997, Gill et al. 2000)
Bovine serum albumin (BSA) antitumour, antimutagenic high-protein based beverages, soft drinks
(Zayas 1997, Madureira et al. 2007)
Immunoglobulins (Ig) Immune activity Commercial products against rotavirus and traveller’s diarrhoea
(Vasiljevic and Shah 2007, Korhonen 2009a)
Lactoferrin (LF) antimicrobial, antitumour, antioxidative, anti-inflammatory, immunomodulatory, antiviral
Commercially used in USA to prevent viral infections, pathogen contamination of raw meat
(Lonnerdal 2003, Pellegrini 2003, Korhonen 2009a)
Lactoperoxidase (LP) antimicrobial, antifungal, antiviral
(Madureira et al. 2007)
Adapted from Madureira et al (2007)
14
15
1.5.2.1 Proteins: Separation and analysis
Proteins are complex macromolecules made up of various amino acids that are
covalently bonded via peptide bonds (Rosenberg 1996). There are four levels of
protein structure. The primary level is the sequence of amino acids. There are 20
main amino acids that make up a protein. The secondary is the regular local
structural arrays such as beta-sheets, beta turns, random coils and alpha helices
(Banga 2006). The alpha helices usually contain 10-15 amino acid residues and the
beta-sheets usually 3-10 residues not including proline (Banga 2006).
Structures such as く-bends, く-turns and hairpins connect anti-parallel strands and
maintain the globular shape of proteins (Banga 2006). The tertiary structure is the
intramolecular arrangement of the secondary structure units in relation to each other
and quaternary structure is the stiochiometry and spatial arrangement of the protein
subunits (Rosenberg 1996, Banga 2006). The subunits are held together by various
forces including hydrophobic and hydrogen bonding and Van Der Waals forces
(Banga 2006). More hydrophobic regions tend to be on the surface of the protein
molecule (Banga 2006).
There are various methods to separate proteins including by precipitation, adsorption
by chromatography, and by size (Nielsen 2010). Precipitation methods include acid
precipitation, isoelectric precipitation and salting out. Chromatography methods
commonly used include ion-exchange chromatography, size-exclusion
chromatography, high performance liquid chromatography and affinity
16
chromatography (Nielsen 2010). Separation by size utilises techniques including
dialysis, gel electrophoresis and membrane processes including reverse osmosis,
nanofiltration, ultrafiltration and microfiltration (Nielsen 2010).
Gel electrophoresis was first used by Tiselius in 1937 to separate alpha, beta and
gamma human serum albumin proteins (Rosenberg 1996). Nowadays, this technique
is commonly used to separate proteins and peptides as per Laemmli, 1970. This
technique uses acrylamide mixed with bisacrylamide to form a cross-linking network
that polymerises when ammonium persulfate is added. The concentration of
acrylamide determines the pore size with the larger proteins/peptides moving faster
when an electric field is applied. The proteins are denatured using sodium dodecyl
sulphate (SDS) and/or 2-metacapaethanol making the proteins negatively charged
and able to be separated by size (Rosenberg 1996). Other types of gel electrophoresis
include native PAGE and 2D gel electrophoresis.
Bromophenol blue is used as a dye agent to track the gel separation. The gels once
complete then can be stained with various reagents including Coomassie brilliant
blue G-250, which binds to basic and aromatic amino acids resulting in blue bands
on the gel (Rosenberg 1996). Other stains include silver staining and fluorescent
stains such as SYPRO ruby and flamingo pink.
Methods used to determine protein or peptide concentration include colorimetric
assays such as the Bradford (Bio-rad) assay, biuret reaction or the σ-phthaldialdhyde
reaction. The Bradford assay is commonly used to measure the amount of protein in
17
a mixture by the use of Coomassie blue. This dye binds to aromatic amino acids such
as tryptophan, tyrosine and phenylalanine. It is measured at absorbance of 595 nm
and compared with a standard curve of a protein such as bovine serum albumin
(BSA).
Various techniques have been used to analyse proteins or peptides including mass
spectrometry (MS), Nuclear magnetic resonance (NMR), circular diachroism (CD),
linear diachroism (LD) and infra-red spectroscopy (IR). The identification of proteins
is usually carried out by mass spectrometry or Edman degradation followed by
synthesis.
Mass spectrometry is a technique that is used to characterise a variety of molecules.
The mass spectrometry system consists of various components namely the sample
inlet, ion source, mass analyser, detector and vacuum, control and data systems. The
analytes are ionised and separated. There are several methods of ionisation including
Matrix-assisted laser desorption/ionisation (MALDI) and electron-spray ionisation
(ESI). The MALDI ionisation technique dissolves the sample in a UV-absorbant
compound (alpha-cyano-4-hydroxycinnamic acid widely used for peptides), forming
crystals. UV laser lines are used to vapourise the matrix (Lang 2009). The large
molecules are pulled into the vacuum of the mass spectrometer and ionised via
proton exchange reactions with other molecules driven by their gas-phase basicity.
Electrospray ionisation involves dissolving the sample in an acidic solution that is
sprayed directly into the mass spectrometry outlet by a small needle. When high
voltage is applied the droplets are dispersed from the tip of the needle, pronated then
18
analysed. Various analysers are available including time-of-flight (TOF), quadrupole
(Q) and ion trap (IT). Time-of-flight analyser’s accelerate ions in electric field using
high voltage.
The ions enter the field free region and travel at velocities inversely proportional to
the mass to charge ratio (m/z), which is used to calculate m/z of ion (Lang 2009).
Quadrupole consists of four parallel metal rods, opposing rods produce electrostatic
field which ions are influenced by and detected. Ion trap has a trapping region where
ions are trapped and detected by changes in the electrostatic field dependant on their
m/z ratio (Lang 2009).
Tandem mass spectrometry is another tool used which has multiple mass analysers
for example ESI-MS-MS (Lang 2009). Mass spectrometry used in conjunction with
liquid chromatography was initiated over 30 years ago and is a highly useful
technique allowing simultaneously separation of molecules and their analysis (Dass
2007). Ultra-high performance chromatography is a technique that separates various
types of molecules using solvents at very low range concentrations (picomoles-
nanomoles).
Circular diachroism (CD) is a spectrometry method that can be used to determine the
secondary and tertiary structural changes of peptides and proteins (Banga 2006). This
technique uses circularly polarised light to determine the conformation of amino
acids in a pure protein or peptide sample. Similarly, infra-red spectroscopy uses
19
vibrational bond energy to determine peptide or protein conformation in regards to
secondary structure (Banga 2006).
1.6 Functional Foods and Nutraceuticals
A functional food is defined as ‘foods similar in appearance to conventional foods
that are consumed as part of a normal diet and have demonstrated physiological
benefits and/or reduce the risk of chronic disease beyond basic nutritional functions’
(Hsieh and Ofori 2010). Fermented foods are incorporated under functional foods.
These foods are fermented by lactic acid bacteria have been shown to modulate the
immune system, alleviate constipation, promote bowel regularity and cure
gastrointestinal infections (Tamang 2010). Examples of fermented functional foods
include yoghurt and acidophilus milk.
A nutraceutical is defined ‘a food or part of a food that provides medicinal and health
benefits including the prevention and/or treatment of a disease’ and they have been
derived from both plant and animal materials. They contribute to the prevention of
various diseases including hypertension, cardiovascular disease, obesity and type II
diabetes (Bagchi et al. 2010).
The functional food and nutraceutical industries are rapidly growing as consumers
are becoming more health conscious. In Australia, the consumption of fermented
milk products has increased from 138 million tonnes to 143 million tonnes in two
years in Australia (International Dairy Federation 2008).
20
1.7 Probiotic bacteria
Probiotic bacteria are defined as ‘live microorganisms that when administered in
adequate amounts confer physiological benefits to their host’ (Food and Agriculture
Organisation of the United Nations and the World Health Organisation 2001).
Probiotic bacteria are classified by strict guidelines which include that the bacteria
must be of human origin, non-mutagenic, non-pathogenic, must confer health
benefits. The health benefits are strain specific (Table 1.3). For example,
Lactobacillus acidophilus has been shown to reduce diarrhoea, breast and colon
cancer (Lambert and Hull, 1996; Tavan et al., 2002).
Probiotic bacteria are predominantly lactic acid bacteria that are Gram-positive,
fermentatative, non-spore forming, non-motile, acid tolerant microorganisms that can
ferment foods by heterofermentation or homofermentation (Hutkins 2006).
Homofermenters include L. lactis, S. thermophilus, L. helveticus and Pediococcus sp.
which metabolise hexoses via enzymes of the Embden-Meyerhoff pathway (EMP)
yielding two moles of pyruvate and ATP per mole of hexose with more than 90% of
the substrate converted to lactic acid (Hutkins 2006). Heterofermenters including
Oenococcus oeni and Leuconostoc lactis use the phosphoketolase pathway to
metabolise hexoses resulting in production of lactate, acetate, ethanol, CO2 and ATP
per hexose (Hutkins 2006).
Lactic acid bacteria are multiple amino acid autotrophs requiring between 4-14
amino acids (Chopin 1993). In milk, casein contains all the necessary amino acids for
growth of lactic acid bacteria, but only <1% of the casein amino acids are utilised
21
(Venema et al. 1996). Also, lactic acid bacteria have been shown to inhibit the
growth of pathogenic bacteria in the gastrointestinal tract by various mechanisms
including bacteriocin production and rapid acid production resulting in a decrease in
pH (Meyer and Brandsina 2005).
The recommended dosage of probiotic bacteria required to be beneficial to the host is
greater than one million colony forming units per millilitre of product (Shah 2007)
because the minimum therapeutic dose per day is suggested to be 108-109 cells
(Kailasapathy and Chin 2000). However, during food processing several factors
affect the viability of the bacteria including oxygen levels, pH, temperature and
osmotic pressure (De Angelis et al. 2004). Probiotic bacteria are used in the
fermentation of several products including milk, cheese, yoghurt and sauerkraut
(Shah 2007).
22
Table 1.3. Proposed health benefits of selected probiotic bacteria
Probiotic bacteria Claimed health benefit/s References L. acidophilus and Bifidobacterium
reduce genotoxic activity, breast and colon cancer treat upper gastrointestinal tract diseases reduce retroviral diarrhoea
(Lambert and Hull 1996, Tavan et al. 2002)
L. rhamnosus GG Reduce inflammation (Isolauri et al. 2000, Parvez et al. 2006)
L. helveticus and Saccharomyces cervisiae
Reduce hypertension (Hata et al. 1996)
Several suppress or eradicate Helicobacter pylori infections
(Midolo et al. 1995, Coconnier et al. 1998, Wang et al. 2004)
23
1.7.1 Lactobacillus genus
The Lactobacillus genus was discovered by Martinus W. Beijernick in 1901.
Lactobacillus, meaning “milk rodlet”, consists of various species including L.
delbrueckii subsp. bulgaricus, L. animalis and L. gasseri (De Vos et al. 2009).
Bacteria from this genus can range from long and slender cells to coccobacilli,
commonly in chains. They are Gram-positive, non-spore forming, fermentative and
facultatively anaerobic. Also, these bacteria are catalyse and cytochrome negative
and are extremely fastidious requiring complex nutrients such as amino acids, fatty
acids, vitamins, nucleic acid derivatives and peptides (De Vos et al. 2009).
Lactobacilli grow optimally at 30°C-40°C and prefer acidic conditions from pH 5.5-
6.2 (De Vos et al. 2009) and are found in various foodstuffs including dairy, grain,
meat, fish, beer, wine, fruit, pickled vegetables and sourdough (Vasiljevic and Shah
2008). These bacteria are found as normal flora of the mouth, gastrointestinal tract
and vagina (De Vos et al. 2009).
The Gram-positive cell walls contain peptidoglycan (predominantly the Lys-D-Asp
type) and wall-bound teichoic acid. Also, in several Lactobacillus species surface-
layers (S-layers) and extracellular polysaccharides (EPS) have been detected (De
Vos et al. 2009). S-layers have been detected in L. acidophilus, L. helveticus, L.
gasseri and L. casei ( all-J Skel nen and Palva β00 ). Heteropolysaccharides
have been identified in various species including L. rhamnosus, L. casei, L.
acidophilus and L. helveticus (Vuyst and Degeest 1999).
24
Lactobacillus species, along with lactic acid bacteria in general, possess the ability to
inhibit the growth of competing microorganisms through various ways including
their ability to reduce pH by the production of lactic acid, acetic acid and
hypothianite formed by the reaction of hydrogen peroxide and thiocyanate which is
catalysed by lactoperoxidase in milk and bacteriocins (De Vos et al. 2009).
1.7.1.1 Lactobacillus acidophilus
Lactobacillus acidophilus (acid-loving milk rodlet) were discovered by Moro in
1900; however it was then called Bacillus acidophilus (Hansen and Mocquot 1970,
De Vos et al. 2009). They are obligately homofermentative organisms that are
phenogenetically a part of the L. delbrueckii group. They have G+C content between
34-37 mol % and have D and L-type lactic acid isomers. They ferment carbohydrates
including starch; however, they do not ferment mannitol (De Vos et al. 2009). They
are non-motile rods with rounded ends ranging from 0.6-0.9µm in width and 1.5-
6µm in length. These organisms require calcium pantothenate, folic acid, niacin and
riboflavin for growth.
1.7.1.2 Lactobacillus casei
Lactobacillus casei (cheese milk rodlet) was discovered by Sigurd Orla-Jensen in
1916; however, it was then known as Streptobacterium casei). They are non-motile
rods with square ends that typically occur in chains. The cells range from 0.7-1.1 µm
in width and 2-4 µm in length. Riboflavin, folic acid, pyridoxal or pyridoxamine,
calcium pantothenate and niacin are essential for their growth. They are facultative
heterofermentative organisms that are phenogenetically unique. They have a higher
G+C content than L. acidophilus of 45-47 mol % and have L-lactic acid isomers
25
only. They do not ferment carbohydrates including arabinose, meliboiose and xylose
(De Vos et al. 2009).
1.7.1.3 Lactobacillus helveticus
Lactobacillus helveticus (Swiss milk rodlet) was discovered in 1919 by Sigurd Orla-
Jensen and it was then known as Thermobacterium helveticum (De Vos et al. 2009).
They are obligately homofermentative organisms that are phenogenetically related to
the L. delbrueckii group. They have a G+C content of 37-40 mol% and have D- and
L- lactic acid isomers They ferment several carbohydrates including galactose,
lactose, maltose, mannose and trehalose (De Vos et al. 2009). They are non-motile
rods ranging from 0.7-0.9 µm in width and can be up to 6 µm long. They occur
singly or in chains and appear on lactose agar as small, greyish, viscid colonies (De
Vos et al. 2009). Lactobacillus helveticus strains have been shown to have high
proteolytic activity towards predominantly casein proteins, especially, く-casein
(Jensen et al. 2009).
1.7.1.4 Lactobacillus rhamnosus (also known as L. casei subsp. rhamnosus)
L. rhamnosus (pertaining to rhamnose milk rodlet) was discovered by Hansen in
1968. The cells are non-motile ranging between 0.8-1 µm in width to 2-4 µm in
length typically with square ends. They do not hydrolyse arginine and are urease
negative. They are facultatively heterofermentative organisms that are
phenogenetically unique. They have a G+C content of 45-47 mol%, similar to L.
casei, and have L-lactic acid isomers only. They ferment most carbohydrates except
xylose, melibiose and raffinose (De Vos et al. 2009).
26
1.8 Milk production and organic farming
Milk production has increased globally in the last decade. Between 1997 and 2007,
world milk production has increased by ββ% (or 1ββ million tonnes). Cow’s milk
represents approximately 84% of the total milk production, followed by buffalo milk
estimated to be 13% (International Dairy Federation 2008). The statistics of global
milk production of other animal species besides bovine and buffalo are rarely
reported; however, it is estimated that annually 14 million tonnes of goat milk, 9
million tonnes of sheep milk and 1.4 million tonnes of camel milk are produced
(International Dairy Federation 2008).
Since 2005 milk production in Australia has decreased due to higher feed costs and
reduced herd sizes. In 2005, 10089 million litres of cow’s milk was produced
compared with an estimated 9480 million tonnes in 2012 (Dairy Australia 2012).
In Australia, organic milk production is strictly regulated. The organic farmers
follow regulations according to the NASAA organic standard (National Association
for Sustainable Agriculture Australia Limited 2012) which incorporates all standards
for organic produce including milk production and dairy cattle management.
The use of synthetic fertilisers, pesticides, growth promoters or the use of additives
derived from genetically modified organisms (GMO’s) is strictly prohibited (Kouba
2003, Biological Farmers of Australia 2006). Also, strict cleaning procedures restrict
the use of ammonium, bleach and hypochlorite products as cleaning agents,
therefore, the use of biodegradable, low-toxic agents are recommended.
27
Furthermore, organic farming standards prohibit regular and routine use of antibiotic
treatments and vaccinations. If stock require their use they should be segregated from
milking stock and their milk should be discarded for specific periods because traces
of any antibiotic residues in processed milk is strictly prohibited (Biological Farmers
of Australia 2006, Chambers and Surapat 2007).
The processing of organic milk involves decreaming, homogenisation,
standardisation, and pasteurisation followed immediately by cooling (Spreer 1995,
Ahmed and Wangsai 2007)(Figure 1.1). Decreaming uses centrifugation to
completely or partially remove the milk fat. Following that, homogenisation is
carried out. This is the mechanical process of shearing milk fat globules via pressure
reducing the size of fat globules and reducing separation of the cream portion of the
product (Chandan 2006). Standardisation involves ensuring the fat and protein
concentration is uniform across all milk batches, and bactofugation is carried out to
remove spore-forming bacilli and other pathogens from the milk. Pasteurisation
destroys vegetative microorganisms by thermally inactivating them at temperatures
lower than 100°C (usually 72°C for 15 seconds) (Damodaran and Paraf 1997). This
procedure eliminates around 95% of microorganisms in milk (Spreer 1995, Chandan
2006).
28
Figure 1.1. Organic milk processing. Adapted from Ahmed and Wangsai (2007) *
should adhere to standards for organic milk production as per NASAA standard.
Organic fed cow*
Decreaming Homogenisation
Pasteurisation
Cooling Incubation Cooling
Fermentation
Packaged
Packaged Fermented
Milk
Adhere to strict cleaning standards
Organic Milk
29
1.9 Fermentation and proteolysis by lactic acid bacteria
Fermentation is defined as an energy yielding process where organic compounds are
metabolised, usually under anaerobic or microaerophilic conditions, to simpler
components without the involvement of an exogenous electron acceptor
(International Food Information Service 2005). It is used in a variety of products
including cheese, milk, sauerkraut and yoghurt (Shah 2007)
Starter cultures are used to accelerate fermentation in dairy products include
Lactobacillus lactis, L. cremoris, S. thermophilus, L. delbreuckii subspecies
bulgaricus, L. acidophilus, Bifidobacterium and Propionibacterium freudenreichii
(Ray and Bhunia 2008). The optimum dosage of starter culture in a product is 2-3%
with the culture prepared prior to being added to the milk. Firstly, the medium is heat
treated (90-95°C for 30-45 min) to destroy bacteriophages, and then cooled prior to
inoculation. Additionally to the starter culture, adjunct cultures are incorporated into
dairy products especially Lactobacillus and Bifidobacterium species due to their
known probiotic effects.
Proteolysis occurs when lactic acid bacteria ferment dairy products consequently
hydrolysing the peptide linkages of proteins by their cell envelope proteinases and
intracellular peptidases (Walstra et al. 1999, Hayes et al. 2007a) and also converting
lactose to lactic acid (Jelen et al. 2003); therefore, various amino acid sequences are
produced. The rate of proteolysis is dependent on the species (Korhonen et al. 1998)
due to the variety peptidases between species (Walstra et al. 1999), temperature and
30
fermentation time (Østlie et al. 2005). Various proteolytic microorganisms including
L. lactis, Enterococcus faecalis, Lactobacillus sp. and Bifidobacterium (Ray and
Bhunia 2008) have been shown to produce bioactive peptides (Table 1.4). The
production of bioactive peptides is enhanced by using mixed populations of bacteria
that are synergistic and highly proteolytic to carry out fermentation (Haque and
Chand 2006) and also using strains with similar optimum growth conditions (Ray
and Bhunia 2008).
The proteolytic system of lactic acid bacteria is well characterised particularly
Lactococcus lactis. It consists of an extracellular, cell-envelope bound, serine-
proteinases (PrtP) that degrade protein into oligopeptides, which are further degraded
by a number of intracellular peptidases including endopeptidases, aminopeptidases,
tripeptidases and dipeptidases (Venema et al. 1996, Haque and Chand 2006) into
amino acids and shorter peptides that are excreted into the environment (Ray and
Bhunia 2008).
The peptides are transported via several peptide transport systems depending on their
size. There are specific systems for amino acids, oligopeptides and two di- and tri-
peptide systems (Salminen et al. 2004, Hayes et al. 2007a, Stanton et al. 2008). There
are few reports on the specific proteinases and peptidases of lactic acid bacteria
responsible for bioactive peptide release (Hayes et al. 2007a).
There are few studies that have examined the proteolytic activity of different lactic
acid bacteria in milk. Eight strains of lactic acid bacteria were analysed for their
31
proteolytic activity in reconstituted skimmed milk. After twenty four hours of
fermentation the proteolytic activity of L. casei L26 was highest, followed by S.
thermophilus 1342, L. bulgaricus 1466, L. acidophilus strains, and the
Bifidobacterium strains (Donkor et al. 2007b). Another study examining the
proteolytic activity of eight strains of lactic acid bacteria in soy milk showed L.
delbrueckii subsp. bulgaricus Lb 1466 had the highest proteolytic activity followed
by B. lactis B94, L. casei L26, L. acidophilus LA 4962, L. acidophilus L10, L. casei
Lc279, B. longum BI 536 and S. thermophilus St1342 (Donkor et al. 2007a). It
appears from these few studies that the probiotic bacteria with the highest proteolytic
activity are L. casei and L. delbrueckii subsp. bulgaricus Lb 1466 and
Bifidobacterium in several studies have displayed low proteolytic activity compared
to the other bacterial species (Novik et al. 2006).
Proteolytic activity in yoghurt has also been examined. The proteolytic profiles of
nine strains of S. thermophilus, six strains of Lactobacillus delbrueckii subsp.
bulgaricus, 14 strains of L. acidophilus and 13 strains of Bifidobacterium species
were analysed and they indicated overall that S. thermophilus had the greatest
proteolytic activity, followed by L. acidophilus, L. delbrueckii subsp. bulgaricus and
Bifidobacterium. The proteolytic activity of Bifidobacterium was much lower than
the other bacteria (Shihata and Shah 2000).
Similarly, a study on the viability and proteolytic activity in yoghurt fermented to
different pH levels showed the control batch containing only starter cultures L.
delbrueckii subsp. bulgaricus and S. thermophilus showed lower levels of proteolytic
32
activity when compared to the batch containing the starter cultures and probiotic
bacteria (Donkor 2007).
1.9.1 Cheddar cheese fermentation and processing
The cheese fermentation process varies with the type of cheese. There are over 1400
different types of cheese that vary in coagulation type, ripening method and texture.
Cheddar cheese is the most widely available cheese. It was first made in Cheddar,
Somerset, England (Vondra 1978). It is manufactured on the largest scale worldwide
and uses either whole or skim milk. The milk is pasteurised and then cooled to 30ºC.
Mesophilic starter cultures are added usually L. lactis ssp. cremoris and/or L. lactis
ssp. lactis. The milk is coagulated by addition of rennet incubating at 30-31ºC for 45-
50 minutes until acidity reaches 0.1-0.14% lactic acid (Singh and Cadwallader 2008).
The curd is cut, scalded then stirred at 39-40ºC for 45 to 60 minutes. The whey is
drained from the curd. The curd is then cheddared (stretched and matted), milled, dry
salted, moulded and pressed. Salting controls the metabolism of lactose and pH
which in turn affects the rate of maturation and cheese quality. The cheese is left to
ripen (Varnam and Sutherland 2001a).
During ripening, the milk protein undergoes proteolysis due to various enzymes
including the coagulant rennin, indigenous milk enzymes such as plasmin and
cathespin, starter and non-starter bacterial enzymes:- both peptidases and proteinases
(Fernandez De Palencia et al. 1997, Singh and Cadwallader 2008). Proteolysis results
in the production of various small peptides and amino acids.
33
1.10 Enzymes and enzymatic hydrolysis of food proteins
Enzymes are proteins present in living cells that act as biological catalysts (Bugg
2004). They are large macromolecules ranging from five to five thousand
kilodaltons, however, are typically between twenty to one hundred kilodaltons (Bugg
2004). They contain an active site that makes up 10-20% of the total enzyme volume,
which is usually a hydrophobic cleft containing amino acid side chains. The active
site binds to a specific substrate through various interactions including hydrogen
bonding, electrostatic, non-polar or hydrophobic interactions and the enzyme-
substrate complex forms leading to product formation (Bugg 2004). Several factors
affect the rate of enzymatic reactions including pH, temperature and substrate
concentration (Mathewson 1998).
The activity of the enzyme can be inhibited by various mechanisms including
competitive, non-competitive, uncompetitive and partial inhibition (Bugg 2004).
Competitive inhibition restricts enzyme activity by the product molecule binding to
the active site of the enzyme, blocking the enzyme from turnover. Non-competitive
inhibition is where a molecule binds to the enzyme at a site distinct from the active
site; however, blocking product formation and with uncompetitive inhibition, the
inhibitor binds to the enzyme-substrate complex that blocks product formation (Bugg
2004). Partial inhibition enables product formation at a reduced rate.
Enzymatic hydrolysis is the breaking of chemical bond or bonds through a water-
mediated decomposition mechanism (Mathewson 1998). Specifically, enzymes that
hydrolyse proteins are known as proteases or peptidases (Mathewson 1998).
34
Peptidases hydrolyse amide bonds of the protein. There are several types of
peptidases including exopeptidases and endopeptidases. Exopeptidases hydrolyse
single polymer units whereas endopeptidases hydrolyse peptide bonds along the
peptide chain (Mathewson 1998). Several classes of proteases exist including serine,
thiol, sulfhydryl, metallo and acid. Each protease contains the particular group in its
active site (Mathewson 1998). Serine proteases include trypsin that has been used in
milk to derive bioactive peptides with various properties (Otani and Suzuki 2003,
Ferreira et al. 2007) while acid proteases such as pepsin and chymosin have also
been used with milk (Miyauchi et al. 1997, Kitts and Weiler 2003, Clifton et al.
2009).
1.10.1 Flavourzyme (Protease from Aspergillus oryzae)
Flavourzyme is a commercial enzyme that consists of a fungal peptidase and
protease complex produced by submerged fermentation of a selected strain of
Aspergillus oryzae. It has both endoprotease and exopeptidase activities. It optimum
activity is a pH 5-7 and its optimum temperature is at 50°C.
1.10.2 Bromelain from pineapple stem (E.C 3.4.22.32)
Bromelain is an enzyme extracted from the pineapple stem (Ananas comosus) and
has broad specificity for protein cleavage; however, it has strong preference for Z-
arg-arg-/-NHMec amongst small molecule substrates (Hatano et al. 2002). Its
optimum activity is at a pH range between 4.5-5.5 and an optimum temperature of
50°C (Swiss Institute of Bioinformatics 2010).
35
1.10.3 Papain from papaya latex (E.C 3.4.22.2)
This enzyme, also known as papainase or payaya proteinase 1, is derived from paw
paw (Caraya papaya). It consists of a single polypeptide chain with three disulfide
bridges and a sulfhydryl group and is 23406 daltons (Sigma-Aldrich). The enzyme is
found in the milky latex of green, unripe payaya fruits and has broad specificity of
peptide bonds, preferably hydrolysing amino acids with large side chains at the P2
position. It is a cysteine protease that cleaves protein bonds of basic amino acids
including leucine and glycine. Its optimum activity is in the pH range of 6.0-7.0 and
the optimum temperature of 65°C.
1.10.4 Fromase 750 XLG
Fromase 750 XLG is a commercial enzyme produced by DSM Food Specialties. It is
a microbial coagulant derived from a fermentation process using Rhizomucor meihei.
Its optimum activity is at the pH range of 4.5-5.5 and optimum temperature of 37°C.
1.10.5 Rennin from calf stomach (E.C 3.4.23.4)
Rennin, also known as chymosin, can be derived from various animal sources
including calf stomach as this enzyme is responsible for the degradation of protein in
the stomach. Commercial chymosin is produced via fermentation using a variety of
recombinant microorganisms including the fungi Aspergillus niger or Kluyveromyces
lactis (Hannigan et al. 2009).
36
It has broad specificity similar to pn A and clots milk rapidly via cleavage of a
single 105-Ser-Phe-]-Met-Ala-108 bond in the kappa-casein chain. Its optimum
activity is at pH 3-4 and at 37°C (Swiss Institute of Bioinformatics 2010).
1.11 Bioactive peptides
Bioactive peptides are specific protein fragments that have a positive impact on body
functions or conditions and may ultimately influence health (Kitts and Weiler 2003).
They influence numerous biological processes including evoking behavioural,
neurological, hormonal, gastrointestinal and nutritional responses (Clare and
Swaisgood 2000). They range from two to twenty amino acids and many have
multifunctional properties (Rutherfurd-Markwick and Moughan 2005, Korhonen
2009b) that may include opioid, and ACE-inhibitory activities (Table 1.4). They
have been derived from proteins in fermented dairy products such as yoghurt, sour
milk, kefir, and different cheese types (Fitzgerald and Murray 2006, Korhonen
2009a) and plant and animal proteins (Rutherfurd-Markwick and Moughan 2005).
However, milk proteins to date have been shown to provide the greatest source of
biologically active peptides (Rutherfurd-Markwick and Moughan 2005).
37
Table 1.4. Bioactivity of main peptide types
Bioactive Peptide Group
Protein Precursor
Bioactivity Systems Affected
Casomorphins く- and α- casein
Opioid agonists, ACE-inhibitory,
Immunomodulatory
Nervous, Cardiovascular
Immune α-Lactorphin α-Lactalbumin
(α-La) Opioid agonists, ACE-inhibitory
Nervous, Cardiovascular
く-Lactorphin く-Lactoglobulin
(く-Lg)
Opioid agonists, ACE-inhibitory, smooth muscle
contraction (Ileum)
Nervous, Cardiovascular,
Digestive
Lactoferroxins Lactoferrin Opioid antagonists Nervous
Casoxins к-casein Opioid antagonists, ACE-inhibitory,
some smooth muscle contraction
Nervous, Cardiovascular,
Digestive
Casokinins く- and α- casein
Antihypertensive, immunomodulatory,
cytomodulatory
Immune, Cardiovascular
Casoplatelins к-casein, Transferrin
Antithrombiotic Cardiovascular
Immunopeptides く- and α-casein
Immunomodulatory Immune
Phosphopeptides く- and α-casein
Mineral carriers Digestive
Lactoferricin Lactoferrin Antimicrobial, immunomodulatory
Immune, Digestive
Adapted from Korhonen et al.(1998) and Meisel (2004).
38
Bioactive peptides are inactive within the protein molecule and can be released in
three ways: enzymatic hydrolysis by digestive enzymes including pepsin, trypsin and
chymotrypsin; fermentation of milk with proteolytic starter cultures; or proteolysis
by enzymes derived from microorganisms or plants (Korhonen 2009a). Digestive
enzymes such as pepsin, alcalase, thermolysin, subtilisin, trypsin and chymotrypsin
have been used to liberate antihypertensive, calcium-binding phosphopeptides
(CPPs), as well as antibacterial (López-Fandiño et al. 2006), antioxidative (Pihlanto
2006), immunomodulatory and opioid peptides (Teschemacher 2003) both from
casein and whey protein fractions (Meisel and Fitzgerald 2003, Fitzgerald et al. 2004,
Korhonen 2009a, Korhonen 2009b).
The amount and activity of bioactive peptides produced from fermentation is
dependent on several factors including the type of starter cultures used, product type,
fermentation time and storage conditions (Korhonen, 2009a). Fermentation of milk
using various lactic acid bacteria including L. helveticus, L. GG, L. delbrueckii
subsp. bulgaricus, E. faecalis and L. acidophilus has resulted in the production of
bioactive peptides (Rokka et al. 1997, Gobbetti et al. 2002, Seppo et al. 2003,
Donkor et al. 2007a, Quirós et al. 2007) (Table 1.5). Milk fermented with L.
helveticus strains has been shown to have antihypertensive, antitumour and
immunomodulatory properties. Thirty nine hypertensive patients that consumed
Evolus milk fermented with L. helveticus had reduced blood pressure when
compared with the control milk which contained a mixed Lactococcus sp. culture
(Seppo et al. 2003).
39
Furthermore, milk fermented with L. helveticus has produced immunostimulatory
peptides that increased the production of immunoglobulin A-producing cells in the
intestines and decrease the size of fibrosarcomas (Le Blanc et al. 2002). Another
study showed that milk fermented with L. helveticus had antitumour and
immunomodulatory effects where the production of IgA and CD4+ positive cells
increased in the mammary glands of the mice (De Moreno De Leblanc et al. 2005).
The use of enzymes derived from microorganisms and plants to hydrolyse milk
proteins has produced bioactive peptides. A casein hydrolysate prepared using an
Aspergillus oryzae protease has exhibited ACE-inhibitory activity in a clinical trial
(Mizuno et al. 2005). Also, peptides derived from milk protein hydrolysed with three
microbial proteases all exhibited antioxidative activity (Hogan et al. 2009).
Antihypertensive peptides have been isolated from milk hydrolysed with an
extracellular proteinase of L. helveticus CP790 (Yamamoto et al. 1994a, Maeno et al.
1996).
The bioactive peptides derived from cheese are influenced by the maturation stage of
the cheese. It has been shown that the concentration of bioactive peptides increases
with cheese maturation (Meisel 1997, Ryhänen et al. 2001, Phelan et al. 2009).
Bioactive peptides have also been derived from milk protein using a combination of
the three techniques such as microbial fermentation using Streptococcus
thermophilus and L. bulgaricus as well as enzymatic hydrolysis using Flavourzyme,
40
which is a protease (Tsai et al. 2008), that resulted in the production of
antihypertensive peptides.
Futhermore, peptides derived from milk have been shown to have antithrombiotic
activity, inhibit αく fibril formation in vitro, which is associated with Alzheimer’s
disease (Bennett et al. 2009) and to reduce stress-related symptoms in women (Kim
et al. 2006).
Table 1.5. Examples of proteolytic probiotic bacteria, health benefits and peptide
bioactivity.
Adapted from Savijoki et al., 2006
Type of proteolytic probiotic bacteria
Bioactivity Reference
E. faecalis CECT 5728, 5726, 5827
Antihypertensive (Quirós et al., 2007)
L. acidophilus LAFTI L10 B. longum BI 536
Antihypertensive (Donkor, 2007)
L. helveticus LBK-16H Antihypertensive (Seppo et al., 2003) L. helveticus R389 Immunostimulatory,
Antitumour (Le Blanc et al., 2002; de Moreno de Le Blanc et al.,
2005) L. helveticus CP790 Antihypertensive (Yamamoto et al.,
1994a; Gobbetti et al., 2002)
L. bulgaricus SS1, L. lactis supsp. cremoris FT4
Antihypertensive (Gobbetti et al., 2000)
L. GG and pepsin Immunostimulatory, Opioid, ACE
inhibitory
(Rokka et al., 1997; Savijoki et al.,
2006)
41
1.11.1 Techniques used to isolate and characterise bioactive peptides
The isolation of bioactive peptides from milk has been carried out using various
methods including ultrafiltration, acid and isoelectric precipitation and several types
of chromatography (Nakai and Modler 1996, Korhonen and Pihlanto 2006).
The characterisation of bioactive peptides has been carried out using a variety of
techniques including two-dimensional gel electrophoresis, reverse phase-high
performance liquid chromatography (RP-HPLC) and matrix-assisted laser
desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. These
techniques have been used to investigate the degradation of milk proteins by
different enzymes (Mamone et al. 2003, Manso et al. 2005).
High performance liquid chromatography (HPLC) in conjunction with tandem mass
spectrometry has been used to identify biologically active peptides by milk
fermentation particularly angiotensin-I-converting enzyme (ACE) inhibitory peptides
(Hernández-Ledesma et al. 2004). Also, confocal microscopy and freeze-fracture
transmission electron microscopy have been used to examine the mode of action of
antimicrobial milk peptides (Van Der Kraan et al. 2004).
Nuclear magnetic resonance (NMR) is used to determine the structure of organic
molecules and biomolecules in solution (Jacobsen 2007). NMR could be used to
characterise bioactive peptides. Few studies have been undertaken to examine the
structure of bioactive peptides derived from milk; however, natural bioactive
42
peptides have been characterised by this technique (Daffre et al. 2008) as well as
some of the milk proteins (Léonil et al. 2000).
Peptides are synthesised by solid-phase peptide synthesis which involves four stages
anchoring, deprotection, coupling reaction and cleavage (Howl 2005). Peptide
synthesis allows the bioactivity and potentially the mechanism of a purified peptide
to be investigated in vivo or in vitro.
1.11.2 The gastrointestinal tract, peptide stability and absorption
The oral route of entry for peptides is problematic as various barriers limit the
absorption of the whole peptide into the bloodstream. Extracellular and intracellular
barriers are present. Proteinases and peptidases present in the stomach and the
gastrointestinal tract (GIT) are barriers to overcome. The stomach contains aspartic
proteinases such as pepsin and is also a highly acidic environment with a pH of 2-3.
The rapid change in pH from the stomach to the duodenum (pH 6-8) can cause the
peptide or protein to precipitate. Also, there are several pancreatic enzymes that may
lead to the breakdown of the peptide via endopeptidases, exopeptidases and
carboxypeptidase A (Madureira et al. 2007). The brush border membrane also
contains exopeptidases that digest polypeptides into tripeptides and dipeptides
(Banga 2006).
Peptides and proteins move across the epithelia via two routes: carrier-mediated
transfer and the paracellular diffusion route (Banga 2006, Phelan et al. 2009).
Carrier-mediated transfers of peptide or proteins are from the apical to basolateral
43
surface of the epithelial cells via specific uptake mechanisms of the cell or sequential
portioning events (Banga 2006). The paracellular route involves transfer between
adjacent cells. If this occurs the peptide or protein will not be degraded by
intracellular proteases (Banga 2006).
The absorption of bioactive proteins and peptides occurs in the gastrointestinal tract
(GIT). The GIT is lined with mucus that contains various molecules including
glycoproteins and bicarbonate ions. The mucus also contains about 20-40 villi/mm2
and they are covered by columnar epithelium. The upper part of the villi contain
capillary blood vessels. The space between the mucus and apical surface of the
epithelial layer known as the submucosal space contains blood vessels, nerves and
lymphatic ducts. Peptides, drugs and other small molecules cross the epithelial cells
via capillary entrance of the portal venous system, which results in rapid delivery to
the systemic circulation via the liver leading to metabolic breakdown of the
substance before entering the bloodstream or via the lymphatic lacteal, which
bypasses the liver leading to slow delivery of the molecule (Banga 2006).
The stability of bioactive peptides is paramount to ensure that the bioactivity is
maintained over a desired time period. Peptide stability is affected by both chemical
and physical interaction (Banga 2006). Chemical interactions can include hydrolysis,
oxidation, deamination, disulfide exchange, く-elimination and racemisation.
Oxidation is a major cause of peptide degradation as several amino acids may
undergo oxidation including methionine, cysteine, histidine, tryptophan and tyrosine
(Banga 2006). Aspartate and proline residues are most susceptible to hydrolysis and
44
asparagine and glutamine are most likely deamidinise to aspartate and glutamate
(Banga 2006). Amino acids in the D-form are more resistant to proteolytic enzymes
and may contribute to increased stability of proteins or peptides (Banga 2006).
The stability of bioactive peptides can be improved by encapsulation. Many different
techniques have been used to encapsulate peptides particularly for pharmaceutical
use including nanoencapsulation, liposomes, and micelles (Martins et al. 2007).
Nanoencapsulation is used in the pharmaceutical industry to protect and enhance the
benefits of drugs. It has been found in vivo that peptides degrade rapidly (Rutherfurd-
Markwick and Moughan, 2005) in the bloodstream. However, nanoencapsulated
peptides will be better protected. Also, nanoencapsulation could ensure the stability
and controlled release of the bioactive peptides in the gastrointestinal tract
(Champagne and Fustier 2007) because the peptides will be better protected and
therefore, delivered unmodified to the desired target in the body.
Other techniques used to improve the oral delivery of peptides include site-specific
delivery, chemical modifications and bioadhesive polymers (Banga 2006). Various
methods have been developed to study oral absorption capabilities of peptides
including the use of intestinal segments, diffusion cells, monolayer cultures, in vivo
studies, and the use of brush border membrane vesicles to determine intestinal
transport (Banga 2006). In vitro digestion models are widely used to study the
digestibility, structural changes and release of peptides (Hur et al. 2011). The
efficiency of delivery systems can be determined using suitable in vitro digestion
models. Cell culture models using predominantly Caco-2 cells have been utilised to
45
study digestibility of food components including peptides (Sienkiewicz-Szlapka et al.
2009).
1.11.3 Antimicrobial peptides
Antimicrobial peptides either eradicate or suppress the growth of microorganisms.
They have been derived from a variety of milk proteins including く-lactoglobulin,
αs1-casein, α-lactalbumin and κ-casein and several inhibit Gram-positive and Gram-
negative microorganisms (Table 1.6).
Isradicin and lactoferricin are two antimicrobial peptides that have been extensively
studied in vivo and in vitro. Isradicin has been shown in vivo to exert protective
effects against a range of pathogens including Listeria monocytogenes and
Staphylococcus aureus in mice and against S. aureus in rabbits, guinea pigs, and
sheep (Lopez-Exposito and Recio 2008).
sracidin was the first antimicrobial peptide derived from αs1-casein by chymosin
digestion (Hill et al. 1974, Lopez-Exposito and Recio 2008). It was shown to inhibit
the growth of lactobacilli in vitro and other Gram-positive bacteria at high
concentrations (0.1-1 mg/ml) (Hill et al., 1974; Lopez-Exposito and Recio, 2008).
Lactoferricin, a bioactive peptide derived from lactoferrin, has been shown to have
antimicrobial activity against both Gram-positive and Gram-negative bacteria, fungi
and parasites (Bellamy et al. 1992, Lopez-Exposito and Recio 2008). Several in vivo
studies have been undertaken to examine the effects of lactoferricin. It has been
46
reported to have protective effects against Staphylococcus aureus and infections
caused by Toxoplasma gondii (Bellamy et al. 1992, Tanaka et al. 1995, Wakabayashi
et al. 1996, Isamida et al. 1998, Recio and Visser 1999a). The in vivo properties of
lactoferricin are controversial, as it has been shown that the addition of five percent
cow’s milk or increasing the concentration of mucin reduced the antimicrobial
effects (Jones et al. 1994, Rutherfurd-Markwick and Moughan 2005). Furthermore,
lactoferrampin, also derived from lactoferrin, has shown antibacterial activity against
Bacillus subtilis, E. coli and Pseudomonas aeruginosa (Van Der Kraan et al. 2004,
Lopez-Exposito and Recio 2008) and tryptic or chymotryptic digestion of bovine α-
Lactalbumin and く-Lactoglobulin revealed several peptide fragments with moderate
antimicrobial activity against Gram-positive bacteria (Pellegrini et al. 1999,
Pellegrini et al. 2001, Lopez-Exposito and Recio 2008). Further, peptides derived
from pepsin hydrolysis of bovine αs2-casein f (183-207) and f (164-179) have shown
inhibitory effects on a broad spectrum of both Gram-positive and Gram-negative
microorganisms (Recio and Visser, 1999a; Lopez-Exposito and Recio, 2008).
Peptides have also shown inhibitory effects of Listeria innocua that have been
derived from αs2-casein f (164-207) (Mccann et al. 2006)
The mode of action of antimicrobial peptides has been extensively investigated
(Floris et al. 2003), and it has been shown that an amphiphilic, mostly α-helical
formation, and an overall net positive charge is proposed to initiate the interaction
with the bacterial surface and to enter the membrane (Floris et al., 2003).
47
Cationic peptides are thought to inhibit Gram-negative bacteria through a variety of
mechanisms including interacting with the lipopolysaccharides and electrostatic
interactions with the negatively charged lipid head groups in the membrane leading
to leakage of essential nutrients (Pritchard and Kailasapathy 2011).
Table 1.6. Selected antimicrobial peptides isolated from bovine milk
Peptide Isolation Successfully Inhibited
Multifunctional Properties
References
sracidin αs1-casein f(1-23) Chymosin digestion
Several microorganisms in vivo and in vitro.
(Hill et al. 1974)
Lactoferrin B f(18-36) f(17-41/42)
Pepsin or chymosin digestion
Several Gram-positive and Gram-negative bacteria.
(Bellamy et al. 1992, Recio and Visser 1999b)
Lactoferricin f(17-41) Bovine LF digested with pepsin or chymosin
Several Gram-positive and Gram-negative bacteria, viruses, fungi and parasites.
Antitumour, immunomodulatory, anti-inflammatory, antiviral
(Bellamy et al. 1992, Tanaka et al. 1995, Wakabayashi et al. 1996, Isamida et al. 1998)
Lactoferrampin f(268-284) B. subtilis E. coli P. aeruginosa
(Van Der Kraan et al. 2004)
αs2-casein f(164-179) αs2-casein f(183-207)
Bovine αs2-casein digested with pepsin
Several Gram-positive and Gram-negative bacteria
Growth Promoter (Recio and Visser 1999a)
48
Table 1.6. Selected antimicrobial peptides isolated from bovine milk (cont.).
Peptide Isolation Successfully Inhibited
Multifunctional Properties
References
к-casein f(106-169) (Kappacin)
Bovine casein digested with chymosin
S. mutans, P.gingivalis, E.coli
Bifidogenic, Immunomodulatory
(Malkoski et al. 2001)
к-casein f(18-24) к-casein f(30-32) к-casein f(139-146)
Bovine к-casein digested with pepsin
Several Gram-positive and Gram-negative bacteria
None reported (López-Expósito et al. 2007)
LF f(1-48) LF f(1-47) LF f(277-288) LF f(267-285) LF f(267-288)
Bovine LF digested with pepsin
Micrococcus flavus
None reported (Recio and Visser 1999a)
α-La f(1-5) α-La f(17-31)S-S(109-114) α-La f(61-68)S-S(75-80)
Bovine α-La digested with chymotrypsin
Several Gram-positive bacteria
None reported (Pellegrini et al. 1999)
く-Lg f(15-20) く-Lg f(25-40) く-Lg f(78-83) く-Lg f(92-100)
Bovine く-Lg digested with trypsin
Several Gram-positive bacteria
None reported (Pellegrini et al. 2001)
Adapted from Floris et al. (2003), Lopez-Exposito et al. ( 2007), Korhonen et al. (2009b) 49
50
1.11.4 Antitumour peptides
Antitumour peptides have been shown to inhibit the proliferation of tumours. They
have been derived from both whey and casein proteins and have been shown to cause
apoptosis and necrosis in several animal and human cancer cell lines (Table 1.7).
There have been numerous in vitro studies, but few in vivo studies. It has been
suggested that opioid peptides may be involved in the mechanism to exert the
antitumour activity (Lopez-Exposito and Recio 2008).
Several peptides derived from lactoferricin and く-casein cause apoptosis in the
human leukemic cell line HL-60 (Hata et al. 1998, Roy et al. 2002). Cytotoxic
activity of lactoferricin has been shown against neuroblastoma, human leukemic and
carcinoma cell lines, and to inhibit xenografts in vivo (Mader et al. 2005, Eliassen et
al. 2006, Lopez-Exposito and Recio 2008). Also, α-casein f(90-95), f(90-96) and く-
casomorphin f(1-5) have inhibited the proliferation of human prostate cancer cell
lines (Kampa et al. 1997), and peptides derived from αs1-casein by trypsin digestion
have been shown to cause necrosis in various types of leukemic B and T cell lines
(Otani and Suzuki, 2003).
Several antitumour peptides exhibit multifunctional properties including
lactoferricin, which also has immunomodulatory, anti-inflammatory and antiviral
activity (Mader et al., β00 ; Eliassen et al., β006) and く-casomorphin-7 that exhibits
antihypertensive, immunomodulatory, opioid and cytomodulatory activity (Hata et
al., 1998).
Table 1.7. Selected antitumour peptides isolated from bovine milk
Peptides Isolation Successfully Inhibited
Multifunctional Properties
References
Lactoferricin pepsin digestion of lactoferrin
human neuroblastoma, leukemic and carcinoma cell lines and xenograft.
antimicrobial, immunomodulatory, anti-inflammatory, antiviral
(Mader et al. 2005, Eliassen et al. 2006, Freiburghaus et al. 2009)
く-lactoferrin f(17-38) く-lactoferrin f(1-16) く-lactoferrin f(45-48)
pepsin digestion of lactoferrin
apoptosis in human leukemic cell line HL-60
(Roy et al. 2002)
く-casomorphin-7 also known as く-casein f(1-25)4P
derived from く-casein
apoptosis in cell line HL-60
antihypertensive, immunomodulatory, opioid, cytomodulatory
(Hata et al. 1998)
αs1-casein f(1-3) αs1-casein f(101-103) αs1-casein f(104-105)
trypsin digestion of αs1-casein
necrosis of animal leukemic B and T cell lines
(Otani and Suzuki 2003)
α-casein f(90-95) α-casein f(90-96) く-casomorphin f(1-5)
inhibit proliferation of human prostate cancer cell lines
(Kampa et al. 1997)
κ-casein f(17-21) also known as κ-casecidin
cytotoxic activity towards mammalian cell lines
antimicrobial (Matin and Otani 2002)
Adapted from Lopez-Exposito and Recio (2008)
51
52
1.11.5 Antioxidant peptides
Antioxidant peptides have been found to inhibit the formation of free radicals and to
scavenge free radicals or hydrogen peroxide and other peroxides (Pihlanto 2006).
They have been derived predominantly from casein proteins in milk (Table 1.8).
There are various antioxidant mechanisms that have been proposed to inhibit free
radicals including chain breaking, acceptor, donor, peroxide decomposer, metal
deactivator and UV absorber (Scott 1997). UV absorbers and chain-breaking donors
include phenols such as aromatic and amine compounds including the amino acids
tryphophan, tyrosine and phenylalanine, absorbic acid (vitamin C) and tocophenol
(vitamin E) (Scott 1997).
A free radical is defined as any atom or molecule that possesses an unpaired electron.
Major free radical species are oxygen free radicals (OFRs) that include the
superoxide free radical, hydroxyl free radical and lipid and other peroxyradicals
(Punchard and Kelly 1996). Oxygen free radicals are potentially toxic to cells
because they are highly reactive and combine with enzymes, receptors and ion
pumps, which causes oxidation directly, consequently, altering the cells normal
function (Punchard and Kelly 1996). This is known as oxidative stress, which may
lead to DNA damage leading to tumour formation (Halliwell and Gutteridge 1999).
However, oxygen free radicals are used in normal metabolic processes such as the
reduction of oxygen to water. Cellular damage is inhibited usually by natural
antioxidants such as tocophenol (vitamin E) and ascorbic acid (vitamin C), which
scavenge the free radicals in cells. Ascorbic acid is situated in the cytosolic region of
53
the cell and tocophenol in the membrane. Other natural antioxidants include
glutathionines and superoxide dismutase (Punchard and Kelly 1996).
Various methods have been used to assess the antioxidant activity of peptides
including the use of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), the
oxygen radical absorbance capacity (ORAC) assay, the free radical azobis (2-
amidinopropane) dihydrochloride (ABAP) and other reactive oxygen species
(Pihlanto 2006). The first three methods are conducted under non-physiological
conditions; however, the use of reactive oxygen species in vivo can determine the
presence of antioxidant peptides in intact cells. Also, biomarkers to determine the
oxidative damage of cellular DNA have been developed and measured using
fluorescence imaging techniques (Pihlanto 2006, Tikekar et al. 2011).
Antioxidant peptides extracted from bovine milk include αs1-casein f(144-149) that
has been shown to have radical scavenging activity against superoxides and1,1-
diphenyl-2-picrylhydrazyl (DPPH) (Suetsuna et al. 2000), く-casein f(177-183); f
(168-176) and f(170-176) have been shown to have DPPH radical scavenging
activity (Kudoh et al. 2001, Rival et al. 2001, Pihlanto 2006) (Table 1.8) and to
inhibit non-enzymatic and enzymatic lipid peroxidation. Also, κ-casein f(96-106)
derived by fermentation of milk with Lactobacillus delbreukii subsp. bulgaricus has
been shown to have DPPH radical scavenging activity in vitro (Kudoh et al. 2001).
Acid whey permeate has been shown to have antioxidant activity by inhibiting iron-
catalysed oxidation by 90% (Colbert and Decker 1991).
54
The mechanism by which peptides have antioxidant activity could be due to the
presence of phenolic amino acids such as tyrosine or phenylalanine or due to the
presence of Leu-Leu-Pro-His-His within the peptide sequence (Kitts and Weiler
2003). Understanding the relationship between the peptide sequence and antioxidant
activity could lead to the formation of extremely effective antioxidant peptides that
could potentially be used in various food applications.
Table 1.8. Selected antioxidant peptides isolated from bovine milk
Peptides Isolation Antioxidative activity
References
αs1-casein f(144-149) Pepsin digestion of casein
DPPH Radical scavenging activity
(Suetsuna et al. 2000)
く-casein f(98-105) く-casein f(169-176) く-casein f(177-183) く-casein f(170-176)
Trypsin digestion of casein
Inhibition of enzymatic and non-enzymatic lipid peroxidation DPPH radical scavenging activity
(Rival et al. 2001, Kitts and Weiler 2003)
κ-casein f(96-106) Fermentation with Lactobacillus delbrueckii subsp. bulgaricus
DPPH radical scavenging activity
(Kudoh et al. 2001)
く-lactoglobulin f(19-29) く-lactoglobulin f(58-61) く-lactoglobulin f(95-101)
Corolase-PP Oxygen radical absorbance capacity (ORAC)
(Hernandez-Ledesma et al. 2005) (Contreras et al. 2011)
Adapted from Pihlanto (2006)
55
1.11.6 Antihypertensive / ACE-inhibitory Peptides
Antihypertensive peptides have been shown to reduce hypertension in vivo and in
vitro. They are the most extensively studied peptides from milk (Korhonen 2009a).
They have been derived from both casein and whey protein by fermentation or
proteolysis by digestive enzymes (Table 1.9). Antihypertensive activity is measured
in vitro usually by measuring the inhibition of the angiotensin-I converting enzyme
(ACE).
The ACE is a chloride-dependant metallopeptidase that is located in somatic and
male germinal cells (Pina and Roque 2009). Zinc is a co-factor of this enzyme. There
are three isoforms of ACE: somatic, germinal and an ACE 2 homologue. Somatic
ACE is composed of two homologous domains (N and C-terminal) that each have a
functional site and distinct physiological and functional properties. The C-domain is
the dominant ACE-converting site (Pina and Roque 2009).This enzyme converts
angiotensin I to angiotensin II via the removal of the C-terminal dipeptide His-Leu
(HL) and also removes the C-terminal dipeptide HL from bradykinin, which results
in the regulation of blood pressure and fluid balance (Meng and Berecek 2001). It is
part of both the Renin-Angiotensin System and the Kinin-Nitric Oxide system that
control sodium balance, body fluid volumes and arterial pressure via membrane-
bound receptors located on different body tissues including the brain, heart, lungs,
liver, pancreas, intestine and vascular epithelial cells (Hall 2001, Fitzgerald et al.
2004).
56
The determination of ACE-inhibitory activity has utilised a variety of methods
including spectophotometry, bioassays, flourometric assays, and HPLC (Meng and
Berecek 2001). These methods detect the presence of hippuric acid (HA) and
histidyl-leucine (HL), which results from the hydrolysis of the ACE-specific
substrate hippuryl-histidyl-leucine (HHL) by ACE (Meng and Berecek 2001, Wu et
al. 2002). The method of Cushman and Cheung (1971) is the most utilised (Cushman
and Cheung 1971, Wu et al. 2002).
Several studies have been undertaken both in vivo and in vitro examining the effect
of antihypertensive peptides derived from milk protein (Table 1.9) (Nakamura et al.
1995b, Mullally et al. 1997, Seppo et al. 2003, Yamamoto et al. 2003, López-
Fandiño et al. 2006). The in vivo studies have used spontaneously hypertensive rats
(Nakamura et al. 1995b, Chen et al. 2007, Tsai et al. 2008) and several clinical trials
have also been undertaken. Nakamura et al (1995) showed that milk containing the
peptides Val-Pro-Pro and Ile-Pro-Pro reduced the systolic blood pressure of
spontaneously hypertensive rats from 6 to 8 hours after administration. Similarly, the
effect of daily consumption of milk containing the same antihypertensive peptides by
hypertensive patients was examined over a 21 week period, and showed that these
peptides exhibited a blood pressure-lowering effect (Seppo et al., 2003). Milk
fermented with five lactic acid bacteria strains followed by hydrolysis with a
microbial protease has been shown to increase ACE-inhibitory activity of
hydrolysates compared with milk fermented with only the lactic acid bacteria in vitro
and the two tripeptides isolated were shown to reduce hypertension in spontaneously
hypertensive rats after eight weeks oral administration (Chen et al., 2007). Also,
fermentation of cheese whey and skimmed milk with various lactic acid bacteria has
57
resulted in several antihypertensive fragments being isolated (Pihlanto-Leppälä et al.,
1998).
Several antihypertensive peptides have been derived from digestion with trypsin and
peptidases of casein including αs1-casein f(23-34), αs1-casein f(142-147), αs1-casein
f(157-164), αs1-casein f(194-199) and く-casein f(60-66) (Maruyama et al., 1985;
Maruyama et al., 1987; Miesel, 2004; Pihlanto-Leppala et al., 1998; Yamamoto et
al., 1994a; Korhonen, 2009b). Antihypertensive peptides derived from casein
digestion using pepsin showed potent antihypertensive activity as well as antioxidant
activity (Contreras et al. 2009).
The structure of most documented ACE-inhibitory peptides usually contains proline
residues or hydrophobic amino acids at the carboxyl terminal end. Proline is known
to be resistant to degradation by digestive enzymes and may pass from small
intestine to bloodstream (Yamamoto et al. 2003). Antihypertensive peptides bind to
ACE via the C-terminal tripeptide residues (López-Fandiño et al. 2006, Wu et al.
2006, Pina and Roque 2009). The binding affinity of antihypertensive peptides
including VPP and IPP has been shown to similar to that of ACE-inhibitory drugs
such as Lisinopril and Captopril (Pina and Roque 2009).
58
Table 1.9. Selected antihypertensive peptides derived from bovine milk
Peptide Fragment Isolation Multifunctional Properties
References
αs1-casokinin αs1-casein f(23-34)
From trypsin and peptidase digestion of αs1-casein
(Maruyama et al. 1985)
αs1-immunocasokinin
αs1-casein f(194-199)
Trypsin immunomodulatory (Maruyama et al. 1987, Meisel 2004)
αs1-casokinin αs1-casein f(142-147), f(194-199), f(157-164)
Fermentation (Pihlanto-Leppälä et al. 1998)
casein hydrolysate Lactobacillus helveticus proteinase and trypsin
(Yamamoto et al. 1994a)
α-La く-Lg く-casein
f(105-110) f(9-14) f(15-20) f(108-113) f(177-183) f(193-198)
Fermentation (Pihlanto-Leppälä et al. 1998)
く-casomorphin-7 く-casein f(60-66)
Pepsin digestion
Opioid, Immunomodulatory
αs1-casein αs1-casein αs2-casein
f(90-94) f(143-149) f(89-95)
Pepsin digestion
Antioxidant (Contreras et al. 2009)
α-La く-casein
f(24-26) f(58-76) f(59-76) f(192-196)
Thermolysin (Otte et al. 2007)
Adapted from Korhonen (2009b).
1.11.6.1 Role of ACE and hypertension
Hypertension or high blood pressure is defined as systolic blood pressure (SBP)
above 140 mm Hg and/or diastolic blood pressure (DBP) above 90 mm Hg
(Copstead and Banasik 2000, Majumder and Wu 2011). It influences various other
disorders including stroke, renal failure and coronary heart disease (Copstead and
59
Banasik 2000). Several factors influence the onset of hypertension including race,
obesity, sodium intake and diabetes (Copstead and Banasik 2000).
Food-derived antihypertensive peptides have been identified in various foods
including milk, soybean, fish, cheese and egg to be potentially used as a preventative
for hypertension (Fitzgerald et al. 2004, Majumder and Wu 2011). The Angiotensin
I-converting enzyme is key regulatory enzyme involved in two systems namely the
Renin-Angiotensin System (RAS) and the Kinin-Nitric Oxide System
(KNOS)(Figure 1.2). The ACE converts Angiotensin-I, a decapeptide to
Angiotensin-II an octopeptide and also inactivates bradykinin (Majumder and Wu
2011). Inhibition of ACE results in a reduction in high blood pressure. Several
synthetic drugs are available that have proven ACE-inhibitory properties including
captopril. However, these drugs can have adverse side effects including dry cough,
renal failure and hypotension (Majumder and Wu 2011). Food-derived
antihypertensive peptides are reported to be safer and have less side effects
(Majumder and Wu 2011).
60
Figure 1.2. Pathways ACE utilises: Renin-Angiotensin System (RAS) and the Kinin Nitric Oxide System (KNOS)
Adapted from Fitzgerald et al, 2004.
1.11.7 Other bioactive peptides
Various other types of bioactive peptides have been derived from milk including
immunomodulatory, antiviral, opioid and mineral binding peptides.
Immunomodulatory peptides either suppress or stimulate the immune system. They
have been derived from casein and whey proteins (Table 1.10). Immunomodulatory
peptides shown to stimulate the immune system include isracidins,
glycomacropeptides and く-casein fragments.
Angiotensinogen
Angiotensin 3
Angiotensin 1
Angiotensin 2
Angiotensin 4
renin prorenin
kallikrein
ACE
aminopeptidase N aminopeptidase A
Kininogen
Kallidin
Bradykinin
active fragment
く-receptor binds to bradykinin increasing Ca2+, which simulates nitric oxide synthase
L-arginine converts to nitric oxide
ACE
KNOS Pathway RAS Pathway
61
sracidins derived from αs1-casein have been shown to increase phagocytosis,
increase the production of IgG, IgM and antibody-forming cells, increase cell-
mediated immunity in mice (Lopez-Exposito and Recio 2008) and increase the
proliferation of lymphocytes (Maruyama et al. 1987, Meisel 1997, Gill et al. 2000).
Glycomacropeptides derived from κ-casein have been shown to increase cell
proliferation and phagocytic activity of a human macrophage-like cell line U937 (Li
and Mine 2004). Also, peptides generated by fermentation of milk with Lactobacillus
were shown to upregulate interleukin 4 and interferon-gamma production of blood
peripheral mononuclear cells (Sutas et al. 1996, Baldi et al. 2005) and く-casein
fragments have shown mitogenic activity towards mouse spleen cells and Payer’s
patch cells (Hata et al. 1998). Also, three specific peptide fractions isolated from
milk fermented with L. helveticus were shown to increase IgA (+) B cells in the gut
of mice (Le Blanc et al. 2002) and lactoferrin has been shown to enhance the
proliferation of B cells and immunoglobulin production of Peyer’s patch cells and
splenocytes (Miyauchi et al. 1997).
mmunomodulatory peptides shown to suppress the immune system include αs1-
casein fragments (59-79), f(1-3), f(101-103) and f(104-105) which have shown
cytotoxic activity because they cause necrosis to healthy mouse T and B cells, and
human leukemic cell lines. These peptide fragments, isolated by digestion using
trypsin, may contribute to the development of the neonate’s immune system by
stimulating necrotic cell death, or have a possible role in the prevention of
pathogenic infections (Otani and Suzuki 2003).
62
Peptides hydrolysed by Lactobacillus rhamnosus GG have been shown to suppress
T-cell activation in vitro (Pessi et al. 2001) and к-caseinoglycopeptide f(106-169)
has been shown to decrease proliferation of lymphocytes (Gill et al. 2000) and
peptides derived from trypsin digestion of αs1-casein have been shown to inhibit the
proliferation of splenocytes and Payer’s patch cells induced by concanavalin-A (Hata
et al. 1998).
Several immunomodulatory peptides suppress and stimulate the immune system
including peptides derived from く-casein such as く-casomorphin, く-casomorphin-7
and く-casokinin-10 that have been shown to stimulate lymphocyte proliferation at
high concentrations and decrease lymphocyte proliferation at low concentrations
(Kayser and Meisel 1996, Gill et al. 2000). Also, immunomodulatory peptides
derived from a Lactobacillus paracasei peptidase hydrolysate were reported to
repress lymphocyte proliferation, upregulate interleukin-10 production, downregulate
interferon-gamma and interleukin-4 production (Prioult et al. 2004). Three peptides
isolated from hydrolysates of く-casein using actinase E have potent chemotactic
activity because they induce macrophage migration and activation (Kitazawa et al.
2007).
Table 1.10. Selected immunomodulatory peptides isolated from bovine milk
Peptide Fragment Isolation Immunomodulatory effect References Stimulate components of the immune system2
Lactoferrin pepsin hydrolysate
Pepsin Enhance lymphocyte proliferation, and immunoglobulin production
(Miyauchi et al., 1997)
Glycomacropeptides
Pepsin and trypsin digestion Increase proliferation and phagocytosis of U937
(Li and Mine, 2004)
Isracidins αs1-casein f(1-23) αs1-casein f(90-96) αs1-casein f(90-95) αs1-casein f(194-199)
Chymosin digestion Trypsin digestion
Increase phagocytosis, Increase proliferation of lymphocytes Promote antibody formation and increase phagocytosis in vitro, reduce Klebsiella pnumoniae infection in vivo.
(Maruyama et al., 1987) (Gill et al. 2000)
Suppress components of the immune system κ-caseinoglycopeptide κ-casein f(106-169) Chymosin Decrease proliferation of
lymphocytes (Gill et al., 2000)
Isracidins αs1-casein f(1-25) αs1-casein f(59-79) αs1-casein f(1-3) αs1-casein f(101-103) αs1-casein f(104-105)
Trypsin digestion Cytotoxic towards healthy T and B cells
(Hata et al., 1998; Otani and Suzuki, 2003)
63
Table 1.10 Selected immunomodulatory peptides isolated from bovine milk (cont.)
Peptide Fragment Isolation Immunomodulatory effect References Casein peptides Fermentation
with Lactobacillus rhamnosus GG
Suppress T-cell activation (Pessi et al., 2001)
Upregulate and downregulate components of the immune system く-lactoglobulin derived peptides
Lactobacillus paracasei peptidase, acidic tryptic-chemotryptic digestion
Repress lymphocyte proliferation, upregulate interleukin-10 production, Downregulate interferon-け and interleukin-4.
(Prioult et al., 2004)
Casein peptides Fermentation with Lactobacillus casei GG enzymes
Upregulate interleukin-4 interferon –け production
(Sutas et al., 1996)
く-casomorphin く-casomorphin-7 く-casokinin-10
F(60-66) F(193-202)
Stimulate lymphocyte proliferation high concentrations, reduce at low concentrations
(Kayser and Meisel, 1996)
く-casein f(63-68) く-casein f(191-193)
Promote antibody formation, increase phagocytosis
(Gill et al. 2000)
64
65
Antiviral peptides are derived mainly from く-lactoglobulin. Peptides derived from く-
lactoglobulin have been shown to have antiviral effects (Korhonen, 2009a).
Lactoferricin, derived from lactoferrin, has been shown to have antiviral activity
against feline calicivirus, adenovirus, human cytomegalovirus and human simplex
viruses types 1 and 2 (Pan et al. 2006) (Table 1.11). Several modified bovine
peptides have also demonstrated antiviral activity (Floris et al. 2003).
Opioid peptides have been derived from both whey and casein proteins, mainly く-
casein and く-lactoglobulin. Opioid peptides can be antagonists that are ligands,
which bind to receptors without causing a cellular response; however, they inhibit the
agonist binding to the receptor that would enable a cellular response (Teschemacher
2003). Opioid peptides that exhibit agonistic activity include casomorphins, α-
lactophorin, く-lactophorin and serophin, which exhibit morphine-like effects. Opioid
peptides exhibiting antagonistic activity include lactoferroxins and casoxins, which
are able to suppress the activity of enkephalins (Brantl et al. 1981, Meisel 1986,
Antila et al. 1991, Tani et al. 1994, Rutherfurd-Markwick and Moughan 2005).
Casoxins, く-lactophins and α-lactophins bind to the μ-type receptor, whereas
exophorins bind to δ-opioid receptor (Ortiz-Chao et al. 2009). The structure of opioid
peptides commonly has an N-terminal tyrosine residue (Ortiz-Chao et al. 2009).
Casomorphins have several bioactive effects including modulating social behaviour
(Panskeep et al. 1984, Paroli 1988) in animals and infants, exerting antidiarrheal
action (Daniel et al. 1990, Clare and Swaisgood 2000) and affecting gastrointestinal
tract function by inhibiting gastric emptying and intestinal motility that consequently,
slows the passage of digestive contents in the tract (Daniel et al. 1990, Schanbacher
66
et al. 1998). They have also been shown to have depressive effects on the central
nervous system (Hedner and Hedner 1987, Rutherfurd-Markwick and Moughan
2005).
There are three main types of opioid receptors that the peptides bind to which are δ, μ
and κ (Teschemacher 2003) and they have all been identified by molecular cloning
(Pan 2003). They are located in the nervous, endocrine, gastrointestinal and immune
systems (Fitzgerald and Meisel 2000, Teschemacher 2003). The μ-receptor is
responsible for emotional behaviour and intestinal motility suppression, the к-
receptor for sedation and food intake regulation and the δ-receptor for emotional
behaviour (Rutherfurd-Markwick and Moughan, 2005).
Mineral-binding peptides are collectively called caseinophosphopeptides, as they are
derived from digests of casein (Rutherfurd-Markwick and Moughan, 2005). They are
able to chelate minerals, particularly calcium, and have been shown to recalcify
dental enamel (Schupbach et al. 1996, Rutherfurd-Markwick and Moughan 2005).
They have been detected in the gastrointestinal tract of animals fed both intact casein
and pure く-casein (Kitts et al. 1992, Rutherfurd-Markwick and Moughan 2005) and
in the ileum of adult humans (Meisel et al. 2001). A к-casein fragment called
casoplatelin is able to inhibit blood platelet aggregation, and casopiastrin also a к-
casein fragment inhibits fibrinogen binding (Fiat et al. 1989, Rutherfurd-Markwick
and Moughan 2005). Also, peptides isolated from whey protein concentrates have
been shown to have iron-binding abilities and antigenic properties (Kim et al. 2007).
67
Peptides have been shown to exhibit hypocholesterolemic activity in vivo,
specifically derived from く-lactoglobulin f (71-75) and f (149-159) in mice and rats
(Nagaoka et al. 2001, Chatterton et al. 2006).
Table 1.11. Selected antiviral, mineral binding, opioid and other bioactive peptides derived from bovine milk
Bioactivity Peptide Fragment Isolation Multifunctional Properties
References
Antiviral Lactoferricin Antitumour, immunomodulatory, anti-inflammatory, antimicrobial
(Tanaka et al. 1995, Wakabayashi et al. 2003, Eliassen et al. 2006, Pan et al. 2006)
Caseinophosphopeptides Caseinophosphopeptide αs1-casein f(59-79)5P く-casein f(1-25)
Trypsin digestion
immunomodulatory, antioxidative
(Fitzgerald 1998, Hata et al. 1998, Pihlanto 2006)
αs1-casein f(43-59) Trypsin digestion
(Schlimme and Meisel 1995)
OPIOID AGONISTS Opioid く-casomorphin-7 く-casein f(60-66) Trypsin
digestion of く-casein
ACE-inhibitory, immodulatory, cytomodulatory
(Brantl et al. 1981)
く-casomorphin-11 く-casein f(60-70) く-casein by fermentation, pepsin and trypsin
ACE-inhibitory (Meisel 1986)
Table 1.11 Selected antiviral, mineral binding, opioid and other bioactive peptides derived from bovine milk (cont.)
68
Bioactivity Peptide Fragment Isolation Multifunctional Properties
References
αs1-casein exorphin αs1-casein f(90-96) αs1-casein f(90-95) αs1-casein f(91-96)
Binds to δ-receptors only
Antitumour (Loukas et al. 1983, Loukas et al. 1990, Pihlanto et al. 1994, Kampa et al. 1997)
α-lactorphin く-lactorphin
α-lactoalbumin f(50-53) く-lactoglobulin f(102-105)
Pepsin and trypsin Pepsin
ACE-inhibitory ACE-inhibitory, smooth muscle contraction, artery relaxation
(Antila et al. 1991, Tani et al. 1994)
OPIOID ANTAGONISTS Casoxins
к-casein f(33-38) trypsin or pepsin digestion of к-casein
Bind to µ-receptors and κ-receptors.
ACE-inhibitory (Antila et al. 1991, Meisel and Fitzgerald 2000, Meisel 2004)
Antithrombiotic PO174, PO220 Whey protein hydrolysates
(Bennett et al. 2009)
κ-casein fragments (Fiat et al. 1989)
Adapted from FitzGerald (1998); Pihlanto (2006), Rutherford-Markwick et al., (2005).
69
70
1.11.8 Commercial bioactive peptide applications and production
problems
Commercial products containing bioactive peptides already have been introduced
worldwide (Hartmann and Miesel 2007, Korhonen 2009a) including Calpis® and
Evolus® which are based on antihypertensive tripeptides Val-Pro-Pro and Ile-Pro-
Pro and are derived from к- and く-casein (Korhonen 2009a). These peptides have
IC50 values of 9 µmol/L and 5 µmol/l, respectively. Other products containing
antihypertensive peptides include C12 peptides (DMV, Netherlands) and BioZate
(Davisco, USA) (López-Fandiño et al. 2006). Further, casein and whey hydrolysates
are being used in products including chewing gum, pastilles, capsules and
confectionary (Korhonen 2009b) because of their excellent gelling properties (Fox
and Kelly 2004).
Caseinophosphopeptides have the ability to chelate minerals particularly calcium and
therefore, have the ability to inhibit the formation of caries by recalcifying dental
enamel. They have been incorporated into pharmaceutical and dietary supplements.
It has also been suggested that they may be used as supplements in bread, cakes,
beverages, soft drink and toothpaste due to their ability to be resistant to proteolysis
(Rutherfurd-Markwick and Moughan, 2005) and glycomacropeptides (GMP) have
been incorporated into high-protein based beverages, soft drinks, chewing gum and
toothpaste, as they have been found to inhibit cariogenic bacteria (Zayas 1997,
Pellegrini 2003, Rutherfurd-Markwick and Moughan 2005).
71
Antimicrobial peptides could be used to prevent infection in gel or film matrices
(López-Fandiño et al. 2006) or be used in foods as food ingredients that inhibit
bacterial growth.
The large-scale production of products containing bioactive peptides has been
limited due to the lack of suitable large scale technologies; however, membrane-
separation techniques provide the best technology to enrich peptides of a particular
molecular weight range (Kitts and Weiler 2003, Korhonen 2009b). Current trends
include genetic cloning and the expression of bioactive peptides via the use of
bacterial and fungal vectors to increase the production of antimicrobial and
antihypertensive peptides.
1.12 Pathogenic bacteria
Various pathogenic bacteria have been used to screen extracts for potential
antimicrobial activity. Selected strains are detailed below.
1.12.1 Escherichia coli ATCC 8739
E. (named after Theodor Escherich) coli (of the colon) were discovered by Migula in
1895 and named Bacillus coli. It was then renamed by Castelleni and Chalmers in
1919 (De Vos et al. 2009). E. coli are straight cylindrical Gram-negative rods
measuring 1.1-1.5 µm wide and between 2-6 µm long. They occur singly or in pairs
and can be motile. They occur naturally in the lower intestines of healthy warm-
blooded animals and can occur as intestinal or extraintestinal pathogens of humans
and animals (De Vos et al. 2009).
72
At least seven classes of pathogenic E. coli have been identified including
verocytotoxin-producing E. coli, enterotoxigenic, enteroinvasive, enteropathogenic
and enteroaggregative (White and Mcdermott 2009). Most types of pathogenic E.
coli cause food poisoning via ingestion of toxins through contaminated water or
food. Verocytotoxin-producing E. coli secrete verocytotoxin that causes severe
abdominal pain and bloody diarrhoea which can lead to haemolytic ureamic
syndrome, haemolytic anaemia or thrombiotic thrombocytopenic purpura. As little as
ten verocytotoxin-producing E. coli can cause infection (White and Mcdermott
2009). Enterotoxigenic E. coli cause Traveller’s diarrhoea and diarrhoea in infants
lasting about 3-4 days after ingestion. Enteroinvasive E. coli destroys lower intestinal
cells causing bloody diarrhoea and fever. Enteropathogenic and enteroaggregative E.
coli both cause watery diarrhoea in children and infants. Enteraggregative E. coli
causes acute persistant diarrhoea lasting more than 14 days (White and Mcdermott
2009).
1.12.2 Bacillus cereus ATCC 11778
B. (rodlet) cereus (wax-coloured) was discovered by Frankland and Frankland in
1887. They are facultative anaerobic, motile, Gram-positive rods that occur singly, in
pairs and chains. They usually are between 1-1.2µm in width and between 3-5µm
long. B. cereus have endospores which are commonly found in soil and if ingested
can result in food poisoning (De Vos et al. 2009).
1.12.2.1 Bacillus cereus and food poisoning
B. cereus produce endospores that when ingested causes food poisoning due to their
high resistance to adverse environmental conditions. The endospores are able to
73
withstand 121°C for 90 minutes. There are two types of syndromes caused by B.
cereus: emetic and diarrhoeal. Emetic syndrome symptoms include nausea, vomiting
and diarrhoea usually occurring from one to six hours after ingestion of cereulide
toxin in contaminated cooked rice (Gillespie 2007). This syndrome lasts between 12
and 24 hours. The diarrhoeal syndrome presents with abdominal pain, rectal
tenesmus and diarrhoea usually after 8-16 hours ingestion of the contaminated food
containing a high-molecular weight enterotoxin that are contained in cooked meat,
vegetables, soups or desserts. The syndrome usually last about 24 hours (Gillespie
2007).
1.12.3 Staphylococcus aureus ATCC 6538
S. aureus (meaning golden) was characterised by Sir Alexander Ogston in 1880, who
referred to them as ‘micrococci’ and then subsequently isolated by Rosenbach in
1884 (De Vos et al. 2009, Schneewind and Missiakas 2009). S. aureus are non-
motile, non-spore-forming, facultative anaerobic Gram-positive cocci that occur in
pairs, singly or in clusters (De Vos et al. 2009). S. aureus is a versatile pathogen that
causes food poisoning, hospital-associated infections, toxic shock syndrome,
endocarditis, necrotizing fasciitis and other skin and soft tissue infections (Otto
2009). The cause of these diseases is through enterotoxin production. Nineteen
enterotoxins have been identified in relation to S. aureus infections (Gillespie 2007).
1.12.4 Streptococcus mutans
The Streptococcus (pliant grain) genus was discovered by Rosenbach in 1884 and the
species mutans (changing) was isolated by Clarke in 1924 from carious human teeth
(Marsh and Martin 2009). S. mutans are Gram-positive, facultatively anaerobic
74
coccoid that occur in pairs or short-medium chains. They are approximately 0.5-0.75
µm in diameter and can form rods in acidic conditions ranging from 1.5-3 µm in
length.
1.12.4.1 Streptococcus mutans and dental caries
S. mutans is the pathogen predominantly responsible for plaque formation leading to
dental caries in the oral cavity (Jaykas et al. 2009, Marsh and Martin 2009). Dental
caries are defined as localised destruction of tissues of the tooth by bacterial
fermentation of dietary carbohydrates (Marsh and Martin 2009). Poor oral hygiene
and high sugar consumption promotes enrichment of streptococcal bacterial
populations which are tolerant of slightly acidic pH (~5), which is generated by
homolactic fermentations of carbohydrates by the microbial environment (Jaykas et
al. 2009). S. mutans also has the ability to secrete a large array of bacteriocins which
could potentially inhibit the bacterial competition (Jaykas et al. 2009).
1.13 Scope of this of study
In this research, organic milk was used as the substrate to derive potential bioactive
peptides. Organic milk was chosen as the substrate because of its potential marketing
implications and not because it varies in protein concentration from non-organic milk
or may derive different types of bioactive peptides. This study is not a comparison
between peptides derived from organic and non-organic milk. This study uses
enzymes and bacteria to derive bioactive peptides that previously have not been
vastly used in the reported literature.
75
Chapter β General Methods
This chapter outlines the materials and methods used repeatedly throughout this
research. These methods incorporate various techniques including bacterial cell
culture, media preparation, buffer preparation, and methods of bioactivity analysis.
2.1 Bacterial cultures and growth
Probiotic bacteria were obtained and used to ferment organic milk. Lactobacillus
acidophilus LAFTI L10 and L. helveticus was obtained from DSM Food Specialties,
Moorebank, Australia, L. casei 2603 and L. rhamnosus 2625 were obtained from the
CSIRO starter culture collection (CSCC), Highett, Australia. All initial freeze-dried
cultures were propagated in MRS broth (10% v/v) anaerobically at 37ºC (anaerobic
jar containing AneroGen satchet (Oxoid, Adelaide, Australia)) for 48 hours.
Various microorganisms were used to determine the antimicrobial activity of the
peptide extracts. Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538,
Bacillus cereus ATCC 11778 and Streptococcus mutans were obtained from the
University of Western Sydney culture collection, Hawkesbury Campus, Australia.
E. coli was subcultured on Difco Luria-Bertani broth (BD, North Ryde, Australia)
and S. aureus, S.mutans and B. cereus were sub-cultured on Difco Brain Heart
Infusion (BD, North Ryde, New South Wales, Australia). All strains were incubated
aerobically at 37°C for approximately 18 hours (10% v/v). All cultures were
passaged twice prior to experimentation and maintained in McCartney bottles.
76
2.2 Chemicals, media, stock solutions, buffers and reagents
All chemicals used were reagent grade except RP-HPLC reagents which were HPLC
grade. They were obtained from Sigma-Aldrich (Castle Hill, New South Wales,
Australia), Bio-Rad Laboratories (Gladesville, New South Wales, Australia),
Thermofisher scientific (LOMB Scientific) (Scoresby, Victoria, Australia).
All chemical solutions, media, stock solutions, buffers and reagents were prepared
using Milli-Q water (Millipore, Bedford, MA, USA). Various analytical balances
were used for weighing chemicals AND HR-200 (AND Co. Ltd., Tokyo, Japan),
AA-200 (Denver Instruments, New York, USA) and AND HF 3000G (AND Co. Ltd,
Tokyo, Japan). When necessary, the measurement of pH was conducted using the pH
meter (Inolab, WTW, Weilheim, Germany) after calibration as per the
manufacturer’s instructions.
2.2.1 Chemicals
2.2.1.1 RP-HPLC solvents
Acetonitrile containing 0.1% TFA
100% HPLC grade acetonitrile was prepared containing 0.1% trifluroacetic acid
(TFA) (v/v). The solution was degassed using a sonicator (Unisonic Australia Pty
Ltd) before use on the RP-HPLC system. This solvent was used as solvent B during
fractionation and analysis.
77
50% Methanol containing 0.1% TFA
50% HPLC grade methanol was prepared by mixing equal volumes of Milli-Q water
and 100% methanol. Concentrated TFA (98%) was then added with a final
concentration of 0.1% TFA (v/v). The solution was degassed using a sonicator
(Unisonic Australia Pty Ltd) before use on the RP-HPLC system and used as solvent
A during ACE-inhibitory analysis.
0.1% TFA solution
Milli-Q water containing 0.1% TFA was prepared, degassed using a sonicator
(Unisonic Australia Pty Ltd) and used as solvent A on the RP-HPLC system. It was
used for fractionation and analysis.
Methanol, THF containing 50mM sodium acetate pH 5.9
Solution of 19% methanol, 1% tetrahydrofuran (THF) in Milli-Q water containing
0.05M sodium acetate (NaAc), adjusted to pH 5.9 by addition of 1M acetic acid was
prepared, degassed using a sonicator (Unisonic Australia Pty Ltd) and used to
analyse the enzyme kinetics of ACE-inhibitory peptides.
2.2.2 Bacterial Media
All media used was supplied by Oxoid (Oxoid Australia Pty Ltd, Thebarton, South
Australia, Australia) and BD (North Ryde, New South Wales, Australia). All media
was prepared by manufacturers instructions then autoclaved at 121ºC for 15 min
using Siltec HC2 (MK1-94) autoclave (Siltex Australia Pty Ltd, East Benleigh,
Victoria, Australia). Sterilised media was stored at 4ºC prior to use.
78
de Mann, Rogosa and Sharpe broth and agar (MRS)
MRS broth was prepared by dissolving 26g of MRS broth powder in Milli-Q water
(500 ml).
MRS agar was prepared by dissolving 31 g of MRS agar powder in Milli-Q water
(500 ml). The media was autoclaved at 121ºC for 15 minutes. Once cooled MRS agar
media was poured into sterile petri dishes (Greiner Bio One, Interpath Services Pty
Ltd, West Heidelberg, Victoria, Australia). The agar plates were stored at 4 ºC until
used.
Luria broth (LB)
Luria broth was prepared by dissolving 3.88g of Luria powder in Milli-Q water (250
ml). The media was autoclaved 121ºC for 15 min and stored at 4ºC until use.
Brain heart infusion broth (BHI)
Brain heart infusion (BHI) broth was prepared by dissolving 9.25g of brain heart
infusion powder in Milli-Q water (250 ml). The media was autoclaved 121ºC for 15
min and stored at 4ºC until use.
Bacterial preservation media
A 1:1 ratio of glycerol and double strength LB, MRS or BHI broth was mixed and
autoclaved 121ºC for 15 minutes. This media was used to preserve bacterial stocks at
-40ºC.
79
Tetracycline and tryptone
The antibiotic tetracycline made up as a stock solution (64.48 mg/ml) was used in
this study as a positive control in antimicrobial assays. Tryptone was used as a
substitute to peptide solutions as a negative control in antimicrobial assays.
2.2.3 Stock solutions
Bovine serum albumin (BSA)
Lyophilised BSA was diluted using 20 mL Milli-Q water (1.47 mg/mL) and stored at
-20ºC until further use. The stock solution was used to prepare standard solutions
required for the Bradford protein assay (Bio-Rad). Standard solutions contained
between 5-100 µg/mL BSA.
1U/mL Angiotensin-I-converting enzyme (ACE) solution
The ACE from rabbit lung was obtained from Sigma-Aldrich. It was used for the
ACE-inhibitory assays. The solution was prepared by dissolving 2U ACE in 2 mL in
0.01M potassium phosphate buffer, pH 7.0 containing 0.5M NaCl, 0.1 mL dispensed
into vials and stored at -20ºC until use.
5 mM Hippuryl-Histydyl Leucine (HHL)
5 mM HHL was prepared freshly by dissolving 0.0215 g HHL in 50 mM HEPES
buffer containing 300 mM NaCl, pH 8.3 to a final volume of 10 mL. This solution
was used as the substrate for the ACE-inhibitory assay.
80
0.5 mg/mL Hippuric acid
Stock solution of 0.5 mg/mL of hippuric acid was prepared by dissolving 5 mg
hippuric acid in 10 mL 50% methanol in Milli-Q water. This solution was used to
prepared standards of hippuric acid for the ACE-inhibitory assay.
0.1% bromophenol blue
0.1% bromophenol blue was prepared by dissolving 0.1 g bromophenol blue in 100
mL Milli-Q water then vacuum filtered through No. 1 Whatman filter paper and
stored at 4ºC until use.
10% SDS
10% SDS was prepared by adding 1 g SDS to 9 mL Milli-Q water. It was protected
from light and stored at room temperature until use. It was used in gel preparation.
2.2.4 Buffers
50 mM HEPES buffer containing NaCl, pH 8.3 for ACE-inhibitory assay
50 mM HEPES buffer was prepared by was prepared by dissolving 13.01 g HEPES
(4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt) and 17.53 g NaCl
in Milli-Q water to make 1000 mL final volume. The pH was adjusted to 8.3 by
addition of 1 M HCl. This buffer was used to dissolve HHL for the ACE inhibitory
assay.
81
Destaining buffer for gel electrophoresis
Gel destaining buffer was prepared by mixing 10% acetic acid with 40% methanol
and mill-Q water. It was stored at room temperature until used.
Staining buffer for gel electrophoresis
Coomassie brilliant blue was prepared by dissolving 1.2 g coomassie brilliant blue in
1L gel staining buffer and then vacuum filtered through No. 1 Whatman filter paper.
10X running buffer for gel electrophoresis
The 10X running buffer for gel electrophoresis was prepared by dissolving 30.2 g
Tris-base, 144 g glycine and 10 g SDS in 1L Milli-Q water. After preparation it was
stored at room temperature. The buffer for gel electrophoresis was prepared by
adding 50 mL of the 10X buffer to 450 mL Milli-Q water.
Laemmli (sample) buffer for gel electrophoresis
The sample buffer contained 0.125M Trisma-HCl, 10% sodium dedocyl sulphate
(SDS), 10% 2-mercaptoethanol, 20% glycerol and 0.004% bromophenol blue. It was
prepared by mixing 8.33 mL 1.5M Tris [Hydroxymethyl] aminomethane-HCl pH
8.8, 20 mL glycerol and 50 mL 20% SDS. The pH was adjusted 6.75 with
concentrated HCl followed by addition of 0.04 mL 10% bromophenol blue. Aliquots
of 900 µL sample buffer were stored at -20ºC until used. Prior to use, the Laemmli
buffer was defrosted and 100 µL of 2-mercaptoethanol was added and mixed.
82
2.2.5 Reagents
10% Ammonium persulphate (APS)
Ammonium persulphate (10% v/v) was prepared by dissolving 10 g APS in 100 mL
Milli-Q water. The solution was aliquoted into 1 mL vials and stored at -20ºC until
used. APS was used as a polymerising agent in gel electrophoresis.
0.5 McFarland Standard
The 0.5 McFarland standard was prepared by adding 0.5 mL 0.048M BaCl2 into 99.5
mL 1% H2SO4. It was stored at 4ºC and was used to standardise concentrations of
bacterial cultures prior to determination of antimicrobial activity.
2.3 Analytical Instruments
2.3.1 Shimadzu Reverse Phase High Performance Liquid
Chromatography
The reverse-phase HPLC system used in this study consisted of a DGU-20A5
Prominence degasser, SIL-20A Prominence autosampler unit, LC-20AT Prominence
liquid chromatography solvent delivery system, RID-10A refractive index detector
(not used), SPC-M20A diode array detector equipped with temperature control, RF-
10AXL fluorescence detector (not used) and FRC-10A fraction collector (Shimadzu
Scientific Instruments (Oceania) Pty Ltd, Mt. Waverly, Victoria, Australia). The
column used for this study was the Altima C18 analytical column (Altech, 5 µm, 4.6
x 250 mm).
83
2.3.2 Bio-Rad Benchmark Plus Microplate Spectrophotometer
The Bio-Rad benchmark plus multiplate spectrophotometer (BioRad Laboratories,
Gladesville, New South Wales, Australia) was used for determination of protein
content, as well as antimicrobial and antioxidant assays.
2.3.3 Bio-Rad Gel Electrophoresis Unit
The BioRad Mini Protean 3 system was used for gel electrophoresis. It was coupled
with a PowerPac 300 (BioRad Laboratories, Gladesville, New South Wales,
Australia). Gel electrophoresis was conducted using 0.75 mm or 1.5 mm spacer
plates with 5, 10 or 15 well combs.
2.3.4 Freeze-dryer
The Alpha 1-4 Christ freeze dryer (B. Braun biotech international, Germany) was
used attached to the Adixen (Alcatel) Pascal 2005 C1 vacuum pump. The freeze
dryer was used to remove liquid from peptide extracts.
2.3.5 Autoclaving and sterilisation
Two autoclaves were used to sterilise consumables and contaminated waste. The
Siltec HC2 (MK1-94) autoclave (Siltex Australia Pty Ltd, East Benleigh, Victoria,
Australia) was used for media preparation.
2.3.6 Quadruple Time-of-Flight Liquid Chromatography-Electronspray
Ionisation- Tandem Mass Spectrometer (QToF-LC-ESI-MS/MS)
Peptides were analysed using liquid chromatography interfaced to tandem mass
spectrometry (LC-MS/MS) and identified by Mascot database searching. The
84
peptides were trapped on a nanoAcquity Trap Symmetry C18 column (180 µM x 20
mm) (Waters) followed by separation on the analytical column nanoAcquity BEH
C18 (150 µM x 100 mm). The eluted peptides were ionised using a nanoelectrospray
ion source (Waters) equipped with PicoTip spray tips (New Objectives). Then
peptides were fragmented automatically using data-directed analysis. A mass
spectrometry survey scan was taken. MS/MS data was searched by the Mascot
algorithm against the SwissProt database using UNITE from the APCG (Ludwig,
Melbourne, Australia). Protein and peptide identifications were obtained from the
Mascot search and accessed through the UNITE software.
2.3.7 Nuclear Magnetic Resonance spectrometer (NMR)
The Bruker Avance 500 MHz Nuclear Magnetic Resonance (NMR) spectrometer
(Bruker BioSpin, Alexandria, New South Wales, Australia) was used for NMR
studies.
2.4 Bioactivity analysis general overview
Various screening assays were used to determine whether the peptide extracts had
bioactivity. Three types of bioactivity were analysed: antimicrobial, antioxidant and
ACE-inhibitory activities. Antimicrobial activity was measured against three
pathogenic bacteria. Antioxidant activity was measured against a free radical DPPH
and ACE-inhibitory activity was determined by measuring the amount of hippuric
acid produced after the peptide extract was exposed to the ACE and its substrate
HHL. All experiments were conducted in triplicate (except ACE-inhibition analysis)
with two replications totalling six observations. For the ACE analysis, at least four
observations per sample were obtained.
85
2.5 Bioactive Screening Assays
2.5.1 Antimicrobial assay
Three bacteria Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538,
Bacillus cereus ATCC 11778 were used to screen the peptide extracts for
antimicrobial activity. The 0.5 McFarland standard was prepared and used to
approximate the concentration of the bacteria (1.5 x 108 cfu/mL). Blank and positive
controls were obtained using tetracycline (64.2 mg/L), and negative control used
tryptone with the bacterial suspension.
All preparations were carried out in triplicate on a 96-well plate and the absorbance
at 595 nm was read after 24 hours incubation. 70 µL of each peptide extract was
added to 1γ0 μL bacterial culture.
Percentage of inhibition was determined by:
(ASample – ANegative Control)
(APositive Control – ANegative Control)
2.5.2 ACE-inhibitory assay
ACE-inhibitory activity was measured as a determination of potential
antihypertensive activity with all peptide extracts. This assay used a combined
method of Nakumara et al (1995) and Tsai et al (2008) with some modifications.
This method uses the angiotensin-I-converting enzyme (ACE) and the substrate
Hippuryl-Histidyl-Leucine (HHL) which produces hippuric acid. The amount of
x 100
86
hippuric acid produced was used to determine antihypertensive activity. Briefly, 5
mM Hippuryl-Histidyl-Leucine (HHL) was dissolved in 50 mM HEPES buffer
(containing 0.3 M NaCl pH 8.3) (see Section 2.2.3). The HHL solution was filtered
using a 0.β μm membrane filter with syringe (see Table β.1 for the preparation of
ACE-inhibitory vials).
Then 150 µL of the 5 mM HHL solution was added to 38 µL of peptide extract (A)
or the blank containing 38 µL milli -Q water (B) or the control containing 15 µL
Milli-Q water instead of ACE (C). The solutions were preincubated for three minutes
at 37°C. Then 15 µL 100 mU/mL ACE was added to the necessary tubes and the
mixture was incubated at 37°C for 30 minutes. The reaction was stopped by adding
188 µL 1M HCl and vortexed.
The mixture was separated using the RP-HPLC on an analytical column (see section
2.3.1) with a 0.4 mL/min flow rate in an isocratic condition of 50% methanol (v/v)
containing 0.1% TFA for 22 minutes. Pure hippuric acid was run as a standard using
the same program. The absorbance was detected at 228 nm.
The height and retention time of the hippuric acid was measured using class-VP 7.3
software and the percentage of inhibition of ACE was determined by the height of
hippuric acid peak in the control sample (C) (ACE, HHL), sample containing
inhibitor (A) (ACE, HHL and peptide) and sample without ACE (Blank B) (HHL,
peptide) as follows:
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Inhibition (%) = (B-A)/(B-C) x 100
Where: A is the absorbance in the presence of ACE and peptide sample
B is the absorbance without peptide fraction
C is the absorbance without ACE
The extent of the inhibitory activity is expressed as a concentration of
peptide/fraction that inhibits 50% of ACE activity (IC50).
Table 2.1. Preparation for ACE-inhibitory assay
Reagents Volume (µL)
A B C
Milli-Q Water - 38 15 Inhibitor/Peptide 38 - 38 5 mM HHL 150 150 150
Preincubate at 37ºC for 3 min 100 mU/mL ACE 15 15 -
Incubate 37ºC for 30 min 1M HCl 188 188 188
2.5.2.1 ACE-inhibitory peptide: stability assay
This assay followed the same method as for the determination of ACE-inhibitory
peptides except that the peptides were mixed with ACE (100 mU/mL) first and pre-
incubated at 37ºC for 3 hours (Fujita and Yoshikawa 1999) followed by the addition
of HHL (5 mM) and incubated for 1 hour at 37ºC. The reaction was stopped by
addition of 1M HCl and the liberated hippuric acid was measured by RP-HPLC as
described in Section 2.5.2.
88
The pre-incubated ACE results were compared with the post-incubated ACE results
(see method in Section 2.5.2). The samples were classified as inhibitor, substrate or
prodrug-type inhibitors. If classified as an inhibitor the IC50 value remained the same
showing that the peptide retained activity or as a substrate if the IC50 value of the
pre-incubated assay was higher because the ACE enzyme hydrolysed the peptide and
activity was lost or as a prodrug-type inhibitor if the IC50 value of the pre-incubated
assay was lower because ACE hydrolysed the peptides to the true inhibitors and the
activity improved. Prodrug-type inhibitors are thought to exert longer lasting ACE
activity in vivo (Fujita and Yoshikawa 1999).
2.5.2.2 ACE-inhibitory peptides: gastrointestinal stability assay
The gastrointestinal stability assay was used to determine the potential stability of
ACE-inhibitory peptides when exposed to digestive enzymes such as pepsin and
pancreatin (which contains chymotrypsin and trypsin).
For this assay, pepsin (Sigma-Aldrich, Castle Hill, New South Wales, Australia) was
prepared to a ratio of 1:50 (0.7 mg/mL) to peptide (1 mg/mL). Peptide prepared in
1M HCl (200 µL) was mixed with 4 µL pepsin solution and incubated for 1.5 hours
at 37ºC (Ruiz et al. 2004, Quirós et al. 2009). The mixture was boiled for 15 minutes
to cease enzyme activity. The pH was adjusted to 7.6 using NaOH. 80 µL of the
mixture was removed and the ACE-inhibitory assay was conducted using peptide-
enzyme mixture as per Section 2.5.2.
89
Pancreatin (Sigma-Aldrich, Castle Hill, New South Wales, Australia) was prepared
to a ratio of 1:25 (0.5 mg/mL) to peptide (1 mg/mL). Peptide-pepsin mixture (120
µL) was added to 9.4 µL pancreatin solution and incubated for 4 hours at 37ºC. The
mixture was again boiled for 15 minutes to cease enzyme activity. The ACE-
inhibitory assay was conducted using the remaining peptide-enzyme mixture as per
Section 2.5.2.
2.5.3 Antioxidant assay
The free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to determine if the
peptide extracts exhibited antioxidant activity. Antioxidative activity of the peptide
extracts was determined by modified method of Apostolidis et al (2007).
Briefly, 3 mL 60 μM DPPH in ethanol was mixed with β 0 µL peptide extract
(Apostolidis et al. 2007). A control of 250 µL Mill-Q water and 3 mL 60 µM DPPH
was also set up. Each solution in 1 mL portion was centrifuged at 9200 rpm for 2
minutes. The absorbance was read at 517 nm using the Bio-Rad Microplate
Spectrophotometer (Bio-Rad Laboratories, Gladesville, New South Wales,
Australia). The percentage of inhibition was calculated by:
2.6 Fractionation and purification of selected bioactive peptides
Identified active peptide extracts or hydrolysates were fractionated by centrifugation
using molecular-weight cut-off membranes (5kDa and 10kDa) (Sartorius,
x 100 AControl – AExtract
AControl
90
Dandenong South, Victoria, Australia). The bioactivity assays were repeated using
the molecular weight cut-off (MWCO) fractions and then selected active fractions
were further separated by RP-HPLC. These fractions were collected, lyophilised and
resuspended in Milli-Q water. Lyophilisation was undertaken using the Alpha 1-4
Christ freeze dryer (B. Braun biotech international, Germany) attached to the Adixen
(Alcatel) Pascal 2005 C1 vacuum pump. The samples were freeze-dried usually
overnight or until dried. Fractionation by RP-HPLC followed by lyophilisation and
resuspension was continued until fractions consisted of only a few peaks.
Subsequently, mass spectrometry analysis was undertaken on selected fractions.
2.7 SDS-PAGE reagents, preparation and casting
Gels were prepared containing 12.5% or 15% Bis-acrylamide solution with 4% bis-
acrylamide stacking gel (Laemmli 1970). All reagents were premade and purchased
from Bio-Rad except 10% SDS and the buffers (see section 2.2.4 for buffer
preparation).
The protein fractions and hydrolysates were prepared for gel electrophoresis by
mixing 100 µL of each sample with 100 µL Laemmli (sample) buffer. All prepared
samples were placed into a boiling water bath for 1.5 minutes and then placed in an
ice bath. Table 2.2 shows the preparation of 12.5% or 15% separation gels and 4%
stacking gels.
91
Table 2.2 Gel preparation for SDS-PAGE
Reagent 15% separation gel (mL)
12.5% separation gel (mL)
4% stacking gel (mL)
2% Bis-40% acrylamide solution
7.65 6.39 670 µL
2% Bis solution 4.86 3.48 365 µL 1.5M Tris-HCl (pH 8.8)
5.25 5.25 -
0.5M Tris-HCl (pH - - 1.75 Milli-Q water 3.63 5.58 4.09 10% SDS 210 µL 210 µL 70 µL 10% APS 210 µL 210 µL 70 µL TEMED 21 µL 21 µL 7 µL Total Volume 21 mL 21 mL 7 mL
The mixture was cast between two glass plates (1.5 mm) and once set the 4%
stacking gel mixture was prepared as per Table 2.2. The stacking gel mixture was
cast on top of the separation gel. A 10- or 15-well comb was inserted into the
stacking gel to produce 10 or 15 wells. After polymerisation, the well comb was
removed carefully and the wells were washed with running buffer (see Section 2.2.4
for preparation instructions) prior to sample loading.
The gels were loaded with peptide and broad/high range SDS-PAGE protein
standards (Bio-Rad Laboratories, Gladesville, New South Wales, Australia) on the
first and last wells, respectively. Selected samples were loaded (between 5-15 µL) in
the remaining wells. The gels were run on the Bio-Rad Mini Protean 4 System.
Approximately 450 mL of running buffer was added to the inner and outer chambers
of the gel reservoir. The Bio-Rad power pac 300 was used to supply the electrical
current which was set at 40 mA. The gels were run until the tracking dye reached the
bottom of the gel (approximately 1.5 hours).
92
After completion, the gels were removed carefully from the glass plates and
transferred into a staining container. The gels were stained with Coomassie Brilliant
Blue R-250 solution (see preparation 2.2.4). After staining overnight, the gels were
destained with destaining buffer (see preparation 2.2.4) for up to 5 hours until sharp
bands were visualised.
2.7.1 Gel imaging
Preliminary gels were scanned using the Canon CanoScan Lide500F and the
CanoScan Toolbox 4.9 software. All scans were at 600 dpi and in colour (multiscan).
They were automatically transported into Photostudio software. The molecular
weight of the bands was measured using Labworks Software 4.5 (Ultra-Violet
Products Ltd, Cambridge, United Kingdom).
Later gels were scanned using the Bio-Rad Gel Doc +XR system using the
colourimetric protocol, white epi-illumination and a standard filter. Molecular weight
analysis was conducted using the Image Lab software by the linear regression
method. Images were cropped, if necessary.
2.8 Bradford protein assay (Bio-Rad)
The measurement of protein content was determined by the Bradford protein assay
(Bio-Rad, Bradford 1976). The microassay procedure for microtitre plates was
followed according to the method of Bio-Rad. A standard curve was produced using
bovine serum albumin (BSA; ranging from 5 µg/mL to 40 µg/mL) and the Bio-Rad
protein dye concentrate (160 µL of sample: 40 µL dye). The samples were prepared
either diluted or undiluted and mixed with the protein dye concentrate (160 µL of
93
sample: 40 µL dye). The plate was incubated at room temperature for about 15
minutes before the absorbance was read at 595 nm using the Bio-Rad microplate
spectrophotometer (Bio-Rad laboratories, Gladesville, New South Wales, Australia).
All determinations were carried out in triplicate.
2.9 Statistical analysis
The mean, standard deviation and standard error of the mean were calculated for
each set of observations using Microsoft Office Excel, 2007. All further statistical
analysis including ANOVA and regression analysis was determined using PASW
SPSS Statistics 18.0.
94
Chapter γ Isolation and characterisation of bioactive
peptides derived from commercial Cheddar cheeses and
fermented milk.
Note: Sections of this chapter are taken from my publications ‘Pritchard S.R.,
Phillips M., Kailasapathy K., 2010. Identification of bioactive peptides in
commercial Cheddar cheese. Food Research International. 43, 1545-1 48’and
‘Pritchard S.R., Phillips M., Kailasapathy K., 2010. Identification of bioactive
peptides in commercial Australian organic cheddar cheeses. Australian Journal of
Dairy Technology. 65, 170-17γ’.
3.1 Introduction
Fermented foods are broadly defined as foods that are fermented by microorganisms
predominantly yeast or lactic acid bacteria, that results in desirable biochemical
changes to the food substrate (Tamang 2010). Examples of fermented foods include
fermented dairy products such as buttermilk, acidophilus milk, yoghurt, kefir and
cheese; fermented vegetables and meat products including sauerkraut, fermented
cucumbers, carrots or olives, bacon, ham, jerky or salami (Mayo et al. 2010, Tamang
2010).
Some fermented foods, such as yoghurt and cheese, may also be classified as
functional foods as they have been shown to provide health benefits beyond their
nutritional content. A fermented functional food provides a physiological benefit that
enhances overall health, helps to prevent or treat disease or improves physiological
95
or mental performance via an added functional ingredient, processing modification or
biotechnology (Shah 2001, Kailasapathy 2010).
Fermented milk derived bioactive peptides have been shown to have various
characteristics including ACE-inhibitory and immunomodulatory properties. Milk
has been fermented primarily using Lactobacillus helveticus to derive these peptides;
(Yamamoto et al. 1999, Le Blanc et al. 2002, Seppo et al. 2003, Matar et al. 2008)
however, Enterococcus faecalis (Miguel et al. 2006, Muguerza et al. 2006, Quirós et
al. 2006) and Lactobacillus lactis (Otte et al. 2011) have also been used.
Furthermore, some cheese varieties have been shown to contain bioactive peptides
that have cytomodulatory, ACE-inhibitory and antioxidant properties. They have
been derived from various cheeses including Cheddar (Ong and Shah 2008),
Mozzarella (De Simone et al. 2009), Swiss (Bütikofer et al. 2008), semi-hard (Ardö
et al. 2009), caprine, ovine (Silva et al. 2006) and Gouda cheese (Saito et al. 2000).
ACE-inhibitory peptides have been isolated from Cheddar, Swiss, semi-hard, ovine,
gouda and caprine cheeses (Saito et al. 2000, Silva et al. 2006, Bütikofer et al. 2008,
Ardö et al. 2009). Antioxidant peptides have been isolated from semi-hard, caprine
and ovine cheeses (Silva et al. 2006, Ardö et al. 2009).
This study aims to use various species of Lactobacillus to derive and identify
bioactive peptides from fermented milk and also examine the presence of bioactive
peptides in various organic and non-organic Cheddar cheeses.
96
3.2 Materials and Methods
3.2.1 Cheddar cheeses and probiotic bacteria preparation
The five commercially available Cheddar cheeses used in this study included three
non-organic Cheddar cheeses and two organic Cheddar cheeses. They were obtained
from the supermarket before being examined for the presence of bioactive peptides.
The probiotic bacteria used in this study were Lactobacillus acidophilus LAFTI L10
and Lactobacillus helveticus (DSM Food Specialties Australia Pty Ltd, Moorebank,
Australia), Lactobacillus casei subspecies casei 2603 and Lactobacillus rhamnosus
2625 (CSIRO starter culture collection (CSCC), Highett, Victoria, Australia). All
strains were grown anaerobically (10% v/v) in duplicate in MRS broth (Oxoid,
Adelaide, Australia) at 37°C using AnaeroGen satchets (Oxoid, Adelaide, Australia).
All strains were subcultured at least twice before commencement of experiment after
18 hours incubation at 37°C.
3.2.2 Extraction of water-soluble peptides from Cheddar cheese
The peptides from each cheese were extracted as per the method of Verdini et al.
(2004) with modifications. First, duplicate tubes each containing 100 g of each
Cheddar cheese were homogenised with 300 mL of distilled water. Tubes were
placed into a 40°C waterbath with 100 rpm shaking for 1 hour (Verdini et al. 2004).
Then the tubes were centrifuged at 4250 g, 4°C for 30 minutes. The supernatant was
filtered through Whatman No. 42 filter paper and then through 0.β μm membrane
filter. All extracts were stored at -20°C until further use.
97
3.2.3 Proximate composition analysis of organic milk
The total solids and moisture, fat, ash and protein content of organic milk was
determined using standard methods (see Appendix 1) (Horwitz 1975, Pearson 1976).
3.2.4 Extraction of organic milk protein
The milk protein from 40 mL sterilised (autoclaved 121°C for 20 min) organic milk
was extracted by acid precipitation using 1M HCl at pH 4.6. The tubes were
centrifuged at 5000g and 4ºC for 30 minutes. The supernatant was decanted into a
new tube and the pellet was washed by centrifugation 5000g for 10 minutes at 4ºC
with 30 mL Milli-Q water.
The supernatant was separated using a Vivaspin 20 centrifugal concentrator with
molecular weight cut off (MWCO) membrane of 10 kDa (Sartorius, Melbourne,
Australia) by centrifugation at 7000g and 4°C for 30 minutes. The retentate was
washed with 10 mL milli-Q water by centrifugation. The permeate was discarded.
The washed retentate was transferred into a new tube.
The pellet supernatant was removed and 20 mL 100 mM sodium phosphate buffer
was added and tube vortexed. The pH was adjusted to pH 7.
3.2.5 Fermentation of organic milk protein
After growth of bacteria for 18 hours (see Section 3.3.1) the bacterial cells were
harvested by centrifugation at 10000g and 4ºC for 30 minutes. The supernatant was
removed and the pellet was washed with Milli-Q water by centrifugation. The
98
supernatant was removed and 10 mL Milli-Q water added. Bacteria culture (10%
v/v) was added to each tube containing extracted milk protein. All tubes were
incubated for 24 hours at 37°C with 100 rpm shaking. The growth of bacteria was
measured by spread plating in triplicate after 24 hours.
3.2.5.1 Extraction of peptides from fermented organic milk protein
After 24 hours incubation the pH was adjusted to 7. All samples were subjected to
boiling water for 10 minutes to inactivate bacterial enzymes. All tubes were
centrifuged at 5000g and 4°C for 30 minutes. The supernatant was filtered through
0.2 µm syringe membrane filters and stored at -40°C until further use.
3.2.6 Separation, fractionation and purification of peptides
Approximately 10 mL of each peptide extract was fractionated using Vivaspin 20
centrifugal concentrators with molecular-weight cut-off membranes (5 kDa and10
kDa) (Sartorius, Melbourne, Australia) by centrifugation at 13000g, 4°C for 30
minutes.
The whole peptide extracts and the fractionated peptide extracts from the organic
cheeses were separated by an Alltima amino C18 column (Grace Davidson
Discovery Science, Deerfield, USA) using RP-HPLC (Shimadzu Scientific,
Melbourne, Australia). Solvent A was 0.1% TFA in Milli-Q water and solvent B was
0.1% TFA in acetonitrile (LOMB Scientific, Taren Point, NSW, Australia). For each
sample, 50 µL was injected and run with a linear gradient 0.2% to 60% of solvent B
up to 60 min followed by 0.2% of solvent B from 61 to 71 min at a flow rate of 1 mL
per minute at room temperature.
99
The fractions were collected from 0-20 minutes, 20-40 minutes and 40-60 minutes,
respectively. A list of gradient programs for separation of particular fractions is
shown in Appendix 2. The fractions were collected into three 50 mL tubes prior to
freeze-drying.
3.2.7 Identification of bioactive peptides derived from commercial
Cheddar cheeses and fermented organic milk protein
3.2.7.1 Identification of peptide extracts with antimicrobial activity
Preliminary screening of water-soluble peptide extracts for antimicrobial activity was
carried out as per Section 2.5.1. Both the whole and fractionated extracts (by MWCO
membranes) were used in the analysis of antimicrobial activity against E. coli, B.
cereus and S. aureus. The MWCO extracts exhibiting the greatest inhibition of
bacteria were subsequently fractionated by RP-HPLC as per the method described in
Section 2.3.2.
3.2.7.2 Identification of peptide extracts with antioxidant activity
Preliminary screening of water-soluble peptide extracts with antioxidant activity was
carried out as per Section 2.5.3. Both the whole and fractionated extracts (by MWCO
membranes) were used in the analysis of antioxidant activity.
3.2.7.3 Identification of peptide extracts with ACE-inhibitory activity
Preliminary screening of peptide extracts with ACE-inhibitory activity was carried
out as per Section 2.5.2. Both the whole and fractionated extracts (by MWCO
membranes) were used for the analysis of ACE-inhibitory activity.
100
3.3 Results
3.3.1 Proximate composition of the Cheddar cheeses
The proximate composition of various Cheddar cheeses used in this study is shown
in Table 3.1. All nutritional information was obtained from cheese labels. Cheddar
cheese A contained the most protein per 100g (30 g) followed closely by organic
Cheddar cheese D (25.4 g) and organic Cheddar cheese E (25.4 g), Cheddar cheese B
(24.3 g) and Cheddar cheese C (24.1 g).
Table 3.1. Nutritional information for Cheddar cheeses (%)
Cheddar Cheese
A B C D E
Protein 30 24.3 24.1 25.4 25.4 Fat 23.6 35.2 33.9 35.2 35.2 CHO <1 <1 <1 1.4 <1
3.3.1.1 Proximate composition of lite organic milk
The proximate composition of lite organic milk is shown in Figure 3.1. The amount
of total protein in the milk is 3.33%. The amount of fat is 1.02% and ash 0.71% (See
Appendix 1).
101
Figure 3.1. Proximate composition of lite organic milk (n = 12, 6, 6, 5 ± SEM). Total
moisture 90.48%.
3.3.2 Separation, fractionation and characterisation of cheese peptides
The concentration of peptide was determined by weighing after freeze-drying
(CHRIST Alpha 1-4, B. Braun Biotech International) overnight. A 12.5% SDS-
PAGE gel was run to confirm if the peptides had been extracted as per Laemmli
method (1970) (data in Appendix 3) (see Section 2.7).
3.3.3 Separation, fractionation and characterisation of fermented peptide
extracts
The whole fermented protein extracts were fractionated by MWCO membranes only
because the concentration of peptide in these samples was low compared with the
hydrolysates (see Chapter 4), which would otherwise be difficult to fractionate. The
Bradford protein assay was carried out on the samples to determine the concentration
9.52
0.71
3.33
1.02
0
2
4
6
8
10
12
Total Solids Ash Protein Fat
Per
cent
age
(%)
Total Solids
Ash
Protein
Fat
102
of peptides. Gel electrophoresis was also conducted to determine degree of
hydrolysis.
Separation of the fermented peptide extracts is shown in Figure 3.2. It shows
hydrolysis with all samples compared with control protein lanes (7, 9 and 14).
Figure 3.2 Separation of fermented peptide extracts by gel electrophoresis. M: Peptide Stds; 2: empty; 3: L. acidophilus SPF; 4: L. casei SPF; 5:L. rhamnosus SPF; 6: L. helveticus SPF; 7: Ctl SPF; 8: Empty; 9: Ctl IPF; 10: L. acidophilus IPF; 11: L. casei IPF; 12:L. rhamnosus IPF; 13: L. helveticus IPF; 14: Ctl IPF; 15: Protein Stds.
M 2 3 4 5 6 7 8 9 10 11 12 13 14 M
103
104
3.3.4 Screening for bioactive peptides
Peptide extracts obtained from fermented milk protein, organic and non-organic
cheeses were screened for antimicrobial, antioxidant and ACE-inhibitory activity. All
data collected is tabulated in Appendices 4, 5, 6, 7 and 8.
3.3.4.1 Antimicrobial activity of Cheddar cheese extracts
The whole peptide extracts were screened for potential antimicrobial activity against
three bacteria strains: E. coli, B. cereus and S. aureus. Figure 3.3 shows the average
percentage inhibition of the bacteria by both the organic and non-organic cheese
peptide extracts. B. cereus was inhibited the greatest by the non-organic Cheddar
cheese A extract (45.5% ±SEM 1.44 with 3.5 mg peptide fraction/mL). The non-
organic cheese peptides showed higher inhibition overall compared with the organic
cheese peptides. E. coli and S. aureus were inhibited the greatest by the non-organic
Cheddar cheese C peptide extract (32.85% ± 3.94 and 12.11 ± 0.32, respectively with
8.4 mg peptide fraction/mL). The results for inhibition of E. coli by the cheese
extracts were not significantly different (P>0.05) to each other. Cheddar cheese E
showed the greatest inhibition against E. coli (E: 28.39% ± 3.86 with 15.75 mg
peptide fraction/mL), followed by B. cereus (D: 17.24% ±2.22 with 15.29 mg
peptide fraction/mL) and then S. aureus (E: 6.08 ±3.3).
-10
0
10
20
30
40
50
60
A B C D E
Per
cent
age
of in
ihib
itio
n (%
)
Type of cheese peptide extract
E.coli
B. cereus
S. aureus
Figure 3.3. Average percentage inhibition of bacteria by Cheddar cheese peptides. (n = 6 ±SEM).
* Significantly different compared to all other B. cereus results (P<0.05). < Significantly different to non-organic cheese extracts that inhibit S. aureus (P<0.05).
*
< <
105
106
All extracts were fractionated using MWCO membranes (5 kDa and 10 kDa) by
centrifugation and reanalysed for antimicrobial activity. Abridged results are shown
in Figure 3.4 and Figure 3.5.
The average percentage inhibition of the bacteria by non-organic Cheddar cheese
extract fractions after being further separated by MWCO membranes (5 kDa and 10
kDa) is shown in Figure 3.4. Overall, B. cereus was inhibited the greatest by the non-
organic Cheddar cheese A fraction containing peptides greater than 10 kDa (35.70%
± SEM 5.65 with 16.52 mg peptide fraction/mL). E. coli was also inhibited the most
by that fraction (21.93% ±4.91 with 16.52 mg peptide fraction/mL). All extracts
showed relatively low inhibition against S. aureus similar to the non-fractionated
extracts and were not significantly different to each other (P>0.05).
-30
-20
-10
0
10
20
30
40
50
A5 A10 A10+ B5 B10 B10+ C5 C10 C10+
% In
hibitio
n
Type of non-organic cheese fraction
E. coli
B. cereus
S. aureus
Figure 3.4 Inhibition of bacteria by MWCO fractionated non-organic cheese peptide fractions (n = 6±SEM).
Notes: A: Cheddar cheese A; B: Cheddar cheese B; C: Cheddar cheese C; 5 - <5 kDa; 10- >5 kDa but <10 kDa; +10- >10 kDa peptides. * Significantly different to all other extracts that inhibit E. coli. < Significantly different to each other (all inhibit B. cereus).
*
* <
< <
< < *
107
108
The average percentage of inhibition of bacteria by organic cheese extracts is shown
in Figure 3.5 (n = 9 ±SEM). Overall, the fraction containing peptides between 5 kDa
and 10 kDa from organic Cheddar cheese D had the highest inhibition against B.
cereus (D10) (34.57% ±12.79 with 11.79 mg peptide fraction/mL). The fraction
containing peptides greater than 10 kDa from organic Cheddar cheese D had the
greatest inhibition against E. coli (E10+) (28% ± 5.63 with 21.59 mg peptide
fraction/mL). The inhibition of S. aureus by organic Cheddar cheese extracts was
negligible and consistent with those of whole organic Cheddar cheese extracts. The
results for S. aureus were not significantly different to each other (P>0.05).
-20
-10
0
10
20
30
40
50
<5D <10D +10D <5E <10E +10E
% In
hibi
tion
Type of organic cheese peptide fraction
E. coli
B. cereus
S. aureus
Figure 3.5 Inhibition of bacteria by MWCO fractionated organic cheese peptide fractions (n = 9 ±SEM). Notes: D: Cheddar cheese D; E: Cheddar cheese E; <5 - <5 kDa; <10- >5 kDa but <10 kDa; >10- >10 kDa. * Significantly different to >10D (E, coli). < Significantly different to all extracts inhibiting B. cereus.
<
* *
*
109
110
Only the four MWCO fractions from non-organic and organic Cheddar cheese with
the highest activity against B. cereus were fractionated using RP-HPLC into three
fractions each. Subsequently, these fractions were freeze-dried (see Sections 2.3.4
and 2.6) and resuspended in Milli-Q water. Their antimicrobial activity against B.
cereus was analysed and results are shown in Figure 3.6.
Figure 3.6 shows the average inhibition of B. cereus by fractionated cheese fractions
(n = 6 ±SEM). Overall, 10AF1 (38.3% ± 4.5 with 2.82 mg peptide fraction/mL)
inhibited B. cereus the greatest, followed closely by 5AF1 (37.8% ± 4.56 with 1.28
mg peptide fraction/mL) however no significant differences (P>0.05) were observed
between samples.
The <5 kDa Cheddar cheese A fraction 1 (5AF1) sample was fractioned again by
RP-HPLC and reanalysed. The results are given in Figure 3.7. Fraction 1A contained
peptides less than 5 kDa from Cheddar cheese A inhibited B. cereus by 44.25% ±
8.24 (1.16 mg peptide fraction/mL). This result was significantly different (P<0.05)
compared with the other fractions.
0
5
10
15
20
25
30
35
40
45
<5A
F1
<5A
F2
<5A
F3
<10
EF
1
<10
EF
2
<10
EF
3
>10
AF
4
<10
AF
1
<10
AF
2
<10
AF
3
<10
DF
1
<10
DF
2
<10
DF
3
<10
DF
4
>10
AF
1
>10
AF
2
>10
AF
3
>10
DF
1
>10
DF
2
>10
DF
3
>10
DF
4%
Inhi
biti
on
Cheese peptide fraction
Figure 3.6 Inhibition of B. cereus by cheese fractions (n = 6 ±SEM). Notes: A: Cheddar cheese A; D: Cheddar cheese D; E: Cheddar cheese E; <5 - <5 kDa; <10- >5 kDa but <10 kDa; >10- >10 kDa. F – fraction. No significant differences observed (P>0.05) .
111
0
10
20
30
40
50
60
<5AF1A <5AF1B <5AF1C
% Inh
ibiitio
n
Type of fraction
Figure 3.7 Inhibition of B. cereus by fractionated cheese fractions (n = 6 ±SEM). Notes: A: Cheddar cheese A; F: fraction; 5: <5 kDa peptides. * Significantly different to all other extracts
*
112
113
3.3.4.2 Antimicrobial activity of fermented milk protein extracts
The fermented peptide extracts were screened for antimicrobial activity against each
bacteria. The MWCO fraction containing peptides derived from fermention with
L. rhamnosus exhibited good activity against B. cereus (42.9% ±SEM 4.8 with 0.15
mg peptide fraction/mL) and S. aureus (35% ±7.62 with 0.07 mg peptide
fraction/mL). The activity against E. coli is shown in Figure 3.8 (n = 6 ±SEM).
E. coli was inhibited the greatest overall by soluble peptide fractions derived from
fermentation of L. casei. The most inhibitory fraction contained peptides greater than
10 kDa (65.69% ±12.83 0.30 mg/mL) followed by the peptides between 5 kDa and
10 kDa (64.48% ±10.57 0.0079 mg/mL). No significant differences were observed
between these two samples.
Compared with the hydrolysates (see Chapter 4) the inhibition of bacteria is low.
Also, the hydrolysis of the fermented protein has resulted in comparatively few
peptides and subsequent fractionation would be difficult. Therefore, screening of the
antimicrobial properties of the fermented peptide extracts was not continued.
.
114
0102030405060708090
<5LCS <10LCS >10LCS
% In
hibi
tion
Type of fermented extract
Figure 3.8. Inhibition of E. coli by fermented peptide extracts (n = 6 ±SEM). No significant differences observed. Notes: S: soluble peptide extract; LC: Lactobacillus casei; 5: <5 kDa; 10: >5 kDa <10 kDa; 10: >10 kDa.
3.3.4.3 Antioxidant activity of Cheddar cheese extracts
The antioxidant activity of the extracts was very low (<20%). Results were not
significantly different to each other (P>0.05). The average percentage of inhibition of
DPPH by cheese peptide fractions after separation by MWCO membranes (5 kDa
and 10 kDa) (n = 3±SEM) is shown in Figure 3.9. The inhibition is greatest in the
organic Cheddar cheese extracts compared with the non-organic Cheddar cheese
extracts. The extract showing the greatest inhibition of DPPH was the extract
containing peptides less than 10 kDa from organic Cheddar cheese E (18.22% ±4.52
with 1.74 mg peptide fraction/mL). The inhibition of DPPH by the MWCO Cheddar
cheese peptide extracts was low and the separation of the peptides was not continued
further
0
5
10
15
20
25
A5 A10
A10+
B5 B10
B10+
C5 C10
C10+
D5 D10
D10+
E5 E10
E10+%
Inhib
iton
Type of cheese extract
Figure 3.9. Inhibition of DPPH by MWCO Cheddar cheese peptide extracts (n = 3 ±SEM). * Significantly different to E10 (P<0.05). Notes: 5: <5 kDa; 10: <10 kDa; 10+: >10 kDa; A: Cheddar cheese A; B: Cheddar cheese B; C: Cheddar cheese C; D: Cheddar cheese D; E: Cheddar cheese E
*
115
116
3.3.4.4 Antioxidant activity of fermented peptide extracts
The whole fermented peptide extracts were screened for antioxidant activity against
DPPH. The whole extracts exhibiting the highest activity against DPPH were
separated by MWCO membranes and reanalysed. The results are shown in Figure
3.10.
The extract that exhibited the highest antioxidant activity contained peptides less
than 10 kDa fermented by L. acidophilus (56.27% ±0.92 SEM with 0.002 mg peptide
fraction/mL). The activity of the fermented peptide extracts compared with the
hydrolysates (see Chapter 4) was relatively low.
Figure 3.10. Inhibition of DPPH by fermented peptide extracts (n = 6 ±SEM). * Significantly different to all other samples except 20LCS (P<0.05). Notes: AA: Absorbic acid; 5: <5 kDa; 10: >5<10 kDa; 20: >10 kDa; LA: Lactobacillus acidophilus; LC: Lactobacillus casei; LR: Lactobacillus rhamnosus; S: soluble peptide extract.
-20
0
20
40
60
80
100
AA
5LA
S
10LA
S
20LA
S
5LC
S
10LC
S
20LC
S
5LR
S
10LR
S
20LR
S%
Inh
ibit
ion
Type of fermented peptide extract
*
117
118
3.3.4.5 ACE-inhibitory activity of Cheddar cheese extracts
Figure 3.11 shows the ACE-inhibitory activity of the whole Cheddar cheese extracts.
Overall the Cheddar cheese A peptide extract had the best ACE-inhibitory activity
(IC50: 0.078 mg peptide fraction/mL ±0.0095 SEM). All extracts were separated by
MWCO membranes and reanalysed. The results are shown in Figure 3.11.
0
0.1
0.2
0.3
0.4
0.5
A B C D E
% In
hbiti
on
Type of cheese peptide extract
Figure 3.11. Inhibition of ACE by whole Cheddar cheese peptide extracts (n = 6 ±SEM). Notes: A-C: Non-organic Cheddar cheese C; D-E: Organic Cheddar cheese * Significantly different to C (P<0.05).
The inhibition of ACE by Cheddar cheese peptide extracts fractionated by MWCO
membranes is shown in Figure 3.12. Overall, non-organic Cheddar cheese extracts
had better activity when compared to organic Cheddar cheese extracts. The extract
that exhibited the best ACE-inhibitory activity contained peptides <5 kDa from
Cheddar cheese A (A5) (inhibition 0.064 mg peptide fraction/mL ±0.0028) followed
by peptides >5 kDa <10 kDa from Cheddar cheese A (A10) (0.087 mg peptide
fraction/mL ±0.0053). The organic Cheddar cheese extract exhibiting the best ACE-
*
*
119
inhibitory activity was the fraction containing peptides less than 5 kDa from organic
Cheddar cheese E (E5) (0.114 mg peptide fraction/mL ±0.018). The three organic
Cheddar cheese fractions and the three non-organic Cheddar cheese fractions that
exhibited the lowest IC50 concentrations to inhibit ACE were fractionated by RP-
HPLC and reanalysed.
Figure 3.12. Inhibition of ACE by Cheddar cheese peptide fractions (n = 6 ± SEM).
Notes: Notes: A-C: Non-organic Cheddar cheese C; D-E: Organic Cheddar cheese. 5: <5 kDa; 10: >5<10 kDa; +10: >10 kDa. * Significantly different to <5A (P<0.05).
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0.400
0.450
A5
A1
0
A1
0+
B5
B10
B10
+
C5
C10
C10
+
D5
D1
0
D1
0+
E5
E1
0
E1
0+
IC50
(m
g/m
l)
Type of Cheddar cheese fraction
*
* *
120
121
The ACE-inhibitory activity of organic Cheddar cheese extracts after fractionation
by RP-HPLC is shown in Figure 3.13. The non-organic Cheddar cheese fractions
showed strong activity; however, results are not shown because subsequent
fractionation was not continued. Only selected organic Cheddar cheese extracts were
fractionated by RP-HPLC and reanalysed.
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
5EF1
5EF2
5EF3
10EF1
10EF2
10EF3
5DF1
5DF2
5DF3IC
50 (m
g/ml
)
Organic cheese fraction
Figure 3.13. Inhibition of ACE by fractionated organic Cheddar cheese extracts (n = 6 ±SEM). Notes: 5: <5 kDa; 10: >5<10 kDa; D-E: Organic Cheddar cheese; F: fraction. * Significantly different to 5EF3 (P<0.05).
*
*
122
123
Overall, the fraction that showed the strongest activity against ACE was fraction 3
containing peptides less than 5 kDa from organic Cheddar cheese D (5DF3) (IC50:
0.101 mg peptide fraction/mL ±0.038) followed by fraction 2 containing peptides
less than 5 kDa from organic Cheddar cheese D (5DF2)(IC50: 0.212 mg/mL ±0.028)
and fraction 2 containing peptides less than 5 kDa from organic Cheddar cheese E
(5EF2) (IC50: 0.268 mg/mL ±0.065). Fraction 3 from organic Cheddar cheese D
containing peptides less than 5 kDa (5EF3) showed few peaks on the chromatogram
and was not further analysed. Therefore, the two fractions (5DF2 and 5EF2) as well
as the third fraction of organic Cheddar cheese E (5EF3) was analysed for ACE-
inhibitory activity. The three fractions that inhibited ACE with the lowest
concentrations were fractionated further and reanalysed. The results are shown in
Figure 3.14.
-1
0
1
2
3
4
5
6
7
5EF2A
5EF2B
5EF2C
5EF3A
5EF3B
5EF3C
5DF2A
5DF2B
5DF2CIC
50 (m
g/ml
)
Cheese peptide fraction
Figure 3.14. Inhibition of ACE by organic Cheddar cheese fractions (n = 4 ±SEM). No significant differences observed between samples (P>0.05). Notes: <5: <5 kDa; D-E: Organic Cheddar cheese; F: Fraction.
124
125
The fraction exhibiting the strongest and most consistent activity against ACE was
fraction 2A containing peptides less than 5 kDa from organic Cheddar cheese E
(labelled as 5EF2A) (IC50: 0.36 mg peptide fraction/mL ±0.09). This fraction was
separated into 6 fractions by RP-HPLC and reanalysed for ACE-inhibitory activity
and stability (see Section 2.5.2.1 for method). The results are shown in Figure 3.15.
The sixth fraction containing peptides less than 5 kDa from organic Cheddar cheese
E (5EF2A6) showed the highest ACE-inhibitory activity having an IC50 value of 0.04
±0.01 mg/mL. It acts as a substrate meaning that the ACE binds to it and then the
ACE-inhibitory activity is decreased (see Section 2.5.2.1 for further explanation).
The 5EF2A and the subsequent fraction 5EF2A6 were analysed by Mass
Spectrometry to determine the peptide/s in the fractions. See Section 3.4.5.
Figure 3.15. Inhibition of ACE by organic Cheddar cheese fractions (n = 4 ±SEM). * Significantly different to 5EF2A3 (postincubated) (P<0.05).
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
1 2 3 4 5 6
IC50
(m
g/m
l)
5EF2A Fraction
Preincubated
Postincubated
*
126
127
3.3.4.6 ACE-inhibitory activity of fermented peptide extracts
The whole extracts were screened for ACE-inhibitory activity and the fraction
exhibiting the strongest activity was separated by MWCO membranes and
reanalysed. The results are shown in Figure 3.16. The extract exhibiting the strongest
ACE-inhibitory activity was the fraction containing peptides greater than 5 kDa and
less than 10 kDa derived from fermented by L. casei (10LCS) (IC50: 0.0019 mg
peptide fraction/mL) closely followed by peptides less than 5 kDa derived from the
soluble peptide fraction fermented by L. casei (5LCS)(IC50: 0.0021 mg/mL).
Fractionation was not continued due to the very low concentration of peptides in
fractions for the fermented extracts.
Figure 3.16. Inhibition of ACE by fermented milk peptide extracts (n = 3 ±SEM). * Significantly different to all other samples (P<0.05). 5: <5 kDa; 10: >5<10 kDa; 20: >10 kDa; LC: L. casei; S: Soluble fraction.
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
5LCS 10LCS 20LCS
IC50
(m
g/m
l)
Type of fermented peptide extract
*
128
3.3.5 Structure of ACE-inhibitory peptides by Mass Spectrometry and
MASCOT database searching
Electron Spray Ionisation- Quadruple- Time of flight- Tandem Mass Spectrometry
(ESI-Q-TOF-MS-MS) was undertaken to identify the molecular weight and amino
acid sequences of the ACE-inhibitory fractions 5EF2A and 5EF2A-6 (see Section
2.3.9 for method). The ACE-inhibitory fraction 5EF2A contained nine peptides
derived from αs1- and く-casein according to MASCOT database searching. The
subsequent fraction 5EF2A-6 had the highest ACE-inhibitory activity and when
analysed by Mass Spectrometry and MASCOT database searching it contained two
known ACE-inhibitory peptides derived from αs1-casein as shown in Table 3.2. The
peptide indicated in bold was synthesised (GenScript USA Inc.Piscataway, NJ, USA)
and reanalysed for ACE-inhibitory activity and its structure-activity relationship
elucidated by NMR (Chapter 5).
The RP-HPLC chromatogram (Figure 3.17) shows the location of fraction 6 in the
sample 5EF2A. This fraction was collected, freeze-dried and analysed by ESI-Q-
TOF-MS-MS.
Figure 3.17. RP-HPLC chromatogram of <5EF2A-6. This fraction was collected for Mass Spectrometry analysis and analysed.
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
3000
3250
3500
Spectrum Max Plot5DOF2A610/01/2011 4:58:00 PM5DOF2A6.dat
Fraction 6
129
130
Figure 3.18 shows the summed mass chromatogram at 634.3497 (decapeptide
YLGYLEQLLR) and also the total ion count chromatogram. The peptide
YLGYLEQLLR had an observed m/z ratio at 634.3497 and a molecular weight of
1266.6972 which means that has a net charge of 2+. Similarly, the tridecapeptide
FFVAPFPEVEKEK had an observed m/z ratio of 692.877 and a molecular weight of
1383.74 (net charge +2). The summed mass chromatogram at 692.877 is shown in
Figure 3.19.
Figure 3.18. A. Summed mass chromatogram at mass 634.3497. B. Total ion count chromatogram of sample 5EF2A-6.
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00
%
0
100
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00
%
0
100
T 1: TOF MS ES+ 634.35
93640.13
21.97
21.80 34.2733.57
46.48
45.00
41.62 45.20
48.26
T 1: TOF MS ES+ TIC
8.31e540.49
30.65
21.9720.6837.8934.8134.42
46.48
40.66
41.22
41.62
42.15
45.6844.9943.9842.54
48.26
A
B
131
Figure 3.19. Summed mass chromatogram at mass 692.877 corresponding to tridecapeptide FFVAPFPEVEKEK.
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00
%
0
100
RP_110530_stephanie_T 1: TOF MS ES+ 692.8772.15e3
40.39
46.45
45.9345.64
44.68
132
Table 3.2. Mass spectrometry database search results for selected ACE-inhibitory fractions isolated from organic cheese.
Sample Protein precursor Peptide sequence Molecular Weight Known activity? Preparation: References 5EF2A αs1-casein f (197-203) D.IPNPIGS.E 696.38 Novel - - f (197-204) D.IPNPIGSE.N 825.42 Novel - - f (197-205) D.IPNPIGSEN.S 939.45 Novel - - f (91-100) R.YLGYLEQLLR.L 1266.70 Stress relieving; opioid Trypsin
hydrolysis (Loukas et al.
1983, Lecouvey et al. 1997)
f (25-36) F.VAPFPEVFGKEK.V 1346.72 ACE-inhibitory; Antimicrobial;
Trypsin hydrolysis
(Maruyama et al. 1985,
Rizzello et al. 2005, Mills et
al. 2011) く-casein f (81-86) T.PVVVPP.F 606.37 ACE-inhibitory Fermentation by
L. helveticus (Nakamura et al.
1995a) f (185-190) K.VLPVPQ.K 651.40 ACE-inhibitory L. helveticus
protease (Maeno et al.
1996) f (67-73) P.FAQTQSL.V 793.40 Novel - f (80-87) Q.TPVVVPPF.L 854.49 ACE-inhibitory Proteinase K (Fitzgerald et al.
2004, Saito 2008)
5EF2A-6 αs1-casein f (91-100)
R.YLGYLEQLLR.L 1266.70 Stress relieving; opioid Trypsin hydrolysis
(Loukas et al. 1983, Lecouvey
et al. 1997) f (23-36) R.FFVAPFPEVFGKEK 1383.72 ACE-inhibitory;
antimicrobial Trypsin
hydrolysis (Maruyama et
al. 1985, Rizzello et al. 2005, Mills et
al. 2011) *Reference may correspond to peptide with sequence contained with other amino acids also or shorter peptide fragment
133
134
3.4 Discussion
These results have shown that peptides contained in fermented milk, organic and
non-organic Cheddar cheeses do exhibit various bioactivities. The antioxidant
activity exhibited in peptide extracts from both organic and non-organic Cheddar
cheeses was low (<20% inhibition) and the results were not significantly different
from each other (P>0.05). However, the antimicrobial activity (44.25% 1.16 mg/mL
against B. cereus) and ACE-inhibitory activity (IC50: 0.04 mg/mL) exhibited by
Cheddar cheese peptide extracts was strong.
The antimicrobial activity of Cheddar cheese peptide fractions was strongest against
B. cereus compared with E. coli and S. aureus at all screening stages. The non-
organic Cheddar cheese peptide fractions consistently inhibited B. cereus the highest.
The final screening showed that fraction <5 kDa Cheddar cheese A fraction 1A
(<5AF1A) inhibited B. cereus by 44.25% ±8.24 at 1.16 mg/mL.
There are very few studies that have examined the antimicrobial activity of cheese
peptide extracts. The study by Rizzello et al. (2005) showed antimicrobial activity
against various other bacteria including E. coli, Listeria innocua and Salmonella spp.
(Rizzello et al. 2005, Losito et al. 2006). However, this study examined the presence
of antimicrobial peptides from 13 Italian cheese varieties rather than Cheddar cheese
(Rizzello et al. 2005, Losito et al. 2006). Several Italian cheeses showed
antimicrobial activity against various bacteria including Pecorino Romano (MIC 20-
110 µg/mL) and Calprino del Piemonte (25-90 µg/mL) (Rizzello et al. 2005). The
135
concentrations of Cheddar cheese peptides required to inhibit the bacteria was
relatively high in this research compared with the Italian cheese study.
The antioxidant activity of the Cheddar cheese peptide extracts in this research were
relatively low (<20% inhibition of DPPH) compared with other studies that have
examined the antioxidant activity of peptides derived from cheese (Apostolidis et al.
2007, Gupta et al. 2009). The antioxidant activities of both the organic and non-
organic Cheddar cheese peptide extracts were similar. Gupta et al. (2009) reported
that the antioxidant properties of water-soluble cheddar cheese peptide extracts were
dependant on the ripening stage of the cheese. At the fourth month of ripening DPPH
was inhibited greatest by the peptide extracts (60% inhibition at 10 mg/mL. Further,
the study by Apostolidis et al, (2007) showed that the water-soluble extracts obtained
from Cabbot Plain, a Cheddar cheese, showed about 30% inhibition of DPPH (150
µg/g peptide), which compared with this study, is greater inhibition using the same
ratio of DPPH to peptide extract.
The non-organic Cheddar cheese peptide extracts exhibited good ACE-inhibitory
activity (Cheddar cheese A IC50: 0.064 mg/mL) compared with the organic Cheddar
cheeses (Cheddar cheese E IC50: 0.114 mg/mL) which was also consistent after
separation by MWCO membranes. However, after fractionation by RP-HPLC the
Cheddar cheese A fractions showed poor ACE-inhibitory activity compared with the
organic Cheddar cheese E fractions. After subsequent screening and fractionation by
RP-HPLC the fraction showing the greatest ACE-inhibitory was derived from
organic Cheddar cheese and contained two peptides. Both peptides are derived from
136
αs1-casein: f (91-100) YLGYLEQLLR (MW: 1266.70) and FFVAPFPEVFGKEK
(MW: 1383.72).
Previously, both peptides have been derived by trypsin hydrolysis of casein (Ingredia
Nutritional 2007, Mills et al. 2011). The peptide YLGYLEQLLR (αs1-casein f (91-
100)) is used as a commercially available and patented food ingredient that has been
proven to have stress relieving properties (Ingredia Nutritional 2007). It is registered
as Lactium (also marketed as CSPHP Prodiet F200) and was manufactured in France
by Ingredia. In vivo studies and clinical trials have shown that it reduces stress in rats
and humans (Kim et al. 2006). The studies also showed that this peptide has strong
affinity for GABAA receptors, which relieve anxiety similar to benzodiapines such as
Valium and Xanax (Lecouvey et al. 1997, Ingredia Nutritional 2007). However, the
manufacturers claim that the peptide does not have antihypertensive effects because
it does not modulate ambulatory blood pressure (Ingredia Nutritional 2007).
Interestingly, this study finds that the fraction 2A6 containing peptides less than 5
kDa from organic Cheddar cheese E (5EF2A-6) fraction does inhibit ACE; however,
this could be due to the presence of the peptide FFVAPFPEVFGKEK, which is a
known ACE-inhibitory peptide (or fragments within the peptide sequence)
(Maruyama et al. 1985, Rizzello et al. 2005, Arena et al. 2010, Mills et al. 2011).
Also, other research has identified similar peptide sequences that have opioid activity
(Loukas et al. 1983). The peptide RYLGYLE (αS1-casein f (90-96)) derived by
pepsin hydrolysis has been shown to inhibit adenylate cyclase in neuroblastoma x
glioma hybrid cell membranes and inhibit electrically stimulated contractions of
137
mouse vas deferens with an IC50 value of 1.2 µM (Loukas et al. 1983). Loukas et al.
(1983) claimed the opioid activity is lost without the arginine amino acid residue
prior to the amino-terminal tyrosine (Loukas et al. 1983). However Ingredia claims
that the peptide YLGYLEQLLR has similar activity to benzodiazepines that bind to
GABAA receptors enhancing the inhibitory effect of GABA on the central nervous
system. It has been shown that the peptide YLGYLEQLLR reduces stress-related
symptoms in women (Kim et al. 2006).
Unpublished work by Lu et al., 2010 (poster presentation at the JAM, 2010, Denver,
Colorado) on the production of bioactive peptides in various US Cheddar cheeses of
different ages indicated that various ACE-inhibitory peptides were contained in the
cheese at various ripening times. These included peptides derived from αs1-casein f
(23-β7) FF AP (evident at 180 and β70 days ripening period), αs2-casein f (24-32)
F APFPE F (evident after 7β0 days ripening) and く-casein f (84-86) VPP (evident
at all ripening times) and f (80-90) TPVVVPPFLQP (evident after 180-720 days
ripening) (Lu et al. 2010). These peptides have known ACE-inhibitory activity and
are similar to peptides contained in the fractions identified in this study. The study
showed that bioactive peptide production depends on ripening stage which is in
agreement with other studies (Ong and Shah 2008, Gupta et al. 2009). The peptide
FVAPFPEVF was also identified by Ong and Shah (2008) in Cheddar cheese
fermented with L. acidophilus LAFTI L10 after 168 days ripening period at 4ºC. A
similar peptide sequence FVAPFPEVFG has been shown to have antimicrobial
activity against various Gram-negative bacteria including E. coli (Rizzello et al.
2005). Furthermore, the peptide TPVVVPPFLQP has been derived using proteinase
138
K (IC50 749 µM) and has been shown to have ACE-inhibitory activity (Fitzgerald et
al. 2004, Saito 2008)
Another peptide FF APFPE FGK (αS1-casein f (23-34)) is a known ACE-
inhibitory peptide (IC50: 18 µM) (Maruyama et al. 1985, Yamamoto et al. 2003,
Saito 2008, Mills et al. 2011). It is used commercially as a food ingredient as well as
being used in soft drinks (Mills et al. 2011) in Japan and the US. It has been derived
by trypsin hydrolysis (Tauzin et al. 2002, Murray and Fitzgerald 2007, Saito 2008).
The peptides PVVVPP and TPVVVPPF identified in this study (Table 4.2) contain
the peptide sequence VPP which is known as a potent ACE-inhibitory peptide with
an IC50 value of 5 µM (Nakamura et al. 1995a, Seppo et al. 2003). It has been derived
via fermentation using L. helveticus in previous studies and has also been identified
in various cheeses (Dionysius et al. 2000, Bütikofer et al. 2008, Lu et al. 2010). The
peptide VPP is used commercially in several fermented milk products including
Calpis and Evolus (Nakamura et al. 1995a, Korhonen 2009b)
The peptide KVLPVP, which is similar to VLPVPQ identified in this study (Table
4.2) has been shown to have ACE-inhibitory activity (Maeno et al. 1996). It has been
derived by use of L. helveticus protease and also produced synthetically.
Several other investigations have revealed potential ACE-inhibitory peptides in
cheese (Haileselassie et al. 1999, Ryhänen et al. 2001, Gómez-Ruiz et al. 2002,
Bütikofer et al. 2008, Ong and Shah 2008, Tonouchi et al. 2008). It is clear that there
139
are an abundance of ACE-inhibitory peptides in various types of cheese dependent
on ripening times, enzymes and/or fermentation methods used. During the
manufacture of organic Cheddar cheese E non-animal rennet is the enzyme used.
Potentially, this could be microbial-derived rennet like enzymes such as
Flavourzyme derived from Aspergillus oryzae (Novozymes) or Hannilase derived
from Mucor miehei (Chr. Hansen).
The concentration of peptide derived from fermented milk protein to inhibit DPPH,
bacteria and ACE was substantially lower than the hydrolysates derived in
subsequent studies (see Chapter 4) and Cheddar cheese extracts, which demonstrates
better bioactivity. However, the RP-HPLC chromatograms displayed very low
intensity and amount of peptides in each extract when compared to the Cheddar
cheese and hydrolysates therefore, fractionation by RP-HPLC of the fermented
peptide extracts was not undertaken due to the small amount of peptides in each
fermented extract. This could be due to the slower proteolytic enzymes of
Lactobacillus species other than L. helveticus or that the enzymes used in Cheddar
cheese manufacture and the hydrolysates have broader specificity or better affinity
for casein protein (Novik et al. 2006, Donkor et al. 2007b, Donkor et al. 2007a,
Jensen et al. 2009).
3.5 Conclusions
Fermented milk protein peptide extracts showed good bioactivity; however, the slow
proteolytic activity of the bacterial enzymes resulted in low concentration of peptides
140
compared with the Cheddar cheese extracts and hydrolysates (see Chapter 4)
therefore screening was not continued.
Bioactive peptides were present in non-organic and organic Cheddar cheeses.
Peptides in organic and non-organic cheeses were shown to have various properties
including antioxidant, antimicrobial and ACE-inhibitory. The antimicrobial and
antioxidant activity of Cheddar cheese peptide extracts was relatively low compared
with other cheese peptide extracts in other studies (Rizzello et al. 2005, Gupta et al.
2009). The ACE-inhibitory peptides that have been identified in this study are used
commercially in various functional food products such as Lactium and C12 peption
(Mills et al. 2011). The peptide YLGYLEQLLR has been identified to have stress-
relieving properties but not ACE-inhibitory (Ingredia Nutritional 2007). The peptide
FFVAPFPEVFGK has been shown to have antihypertensive activity.
The novel peptides identified in this study may alleviate hypertension and could be
potentially used as an ACE-inhibitory/antihypertensive marketed product or a food
ingredient like Lactium (containing YLGYLEQLLR) that is marketed as an
organically derived peptide that relieves stress.
There have been a few novel peptides derived from Cheddar cheese that are a
consequence of starter and non-starter bacterial enzymes, rennet and ripening time.
The use of enzymes derived from animal and plant sources could provide variations
in the peptides derived from milk proteins therefore several animal and plant
141
enzymes were used to hydrolyse milk protein in Chapter 4 to potentially produce
novel bioactive peptides.
142
Chapter 4 Isolation and characterisation of bioactive
peptides formed during enzymatic hydrolysis of organic
milk protein.
4.1 Introduction
Bioactive peptides have been derived from milk protein predominantly using
digestive enzymes such as trypsin, chemotrypsin and pepsin. Various types of
bioactive peptides have been identified including ACE-inhibitory, antimicrobial,
antioxidant, antithrombiotic, mineral binding, opioid, antitumour and
immunomodulatory (Floris et al. 2003, Pihlanto 2006, Contreras et al. 2009,
Korhonen 2009b, Jacquot et al. 2010, Rousseau-Ralliard et al. 2010).
Few studies have derived bioactive peptides via the use of plant and animal enzymes
such as Flavourzyme (a protease from Aspergillus oryzae) (Mizuno et al. 2005),
Coralase PP (Hernandez-Ledesma et al. 2005, Contreras et al. 2011) or various
microbial proteases including alkaline protease from Bacillus licheniformis and
extracellular protease from Lactobacillus helveticus (Yamamoto et al. 1994a, Maeno
et al. 1996, Tsai et al. 2008, Hogan et al. 2009).
Therefore, in this study various enzymes derived from plant and animal sources were
used to hydrolyse milk protein to potentially derive bioactive peptides. The enzymes
used included Papain (from Papaya fruit), Bromelain (from pineapple stem),
Fromase (from the fungus Rhizomucor meihei), Flavourzyme (protease from the
143
fungus Aspergillus oryzae) and Rennin (from calf stomach). These enzymes have
different active sites and specificities that could result in the discovery of novel
bioactive peptides and could provide the nutraceutical and functional food industries
with a new food ingredient or product.
4.2 Materials and Methods
4.2.1 Proximate composition of organic milk
The proximate composition of organic milk methods and raw data are shown in
Appendix 1. Results are shown in Section 3.3.1.1 (Figure 3.1).
4.2.2 Extraction of milk protein
The protein was separated by acid precipitation into soluble and insoluble fractions.
Briefly, 40 mL of ‘You love coles’ lite organic milk was adjusted to pH 4.6 by using
1M HCl in duplicate. The tubes were centrifuged 5000g for 10 minutes. The
supernatant was placed into a new tube. The pellet was washed using 30 mL Milli- Q
water by centrifugation 5000g at 4°C for 10 minutes. The supernatant was discarded
and the pellet was homogenised in 30 mL 100 mM sodium phosphate buffer. The
supernatant of the pellet (insoluble fraction) and the supernatant (soluble fraction)
were filtered through No. 42 Whatman filter paper followed by 0.2 µm membrane
syringe filters (Sartorius, Melbourne, Australia). All extracts were stored at -80ºC
until use.
4.2.3 Enzymatic hydrolysis of milk protein
Five enzymes were used to hydrolyse the organic milk protein namely Papain,
Bromelain, Flavourzyme, Rennin from calf stomach and Fromase. Two different
144
concentrations of enzyme (0.5% and 1% enzyme to protein) were added to 40 mL of
‘You love coles’ organic lite milk. The pH was adjusted to the optimum range for
each enzyme then incubated at their optimum temperature for 1, 3 and 5 hours with
100 rpm shaking (See Table 4.1).
Table 4.1: Optimum temperature and pH of enzymes used to hydrolyse milk protein
* adjusted using 1M HCl to optimum Ph
4.2.4 Preparation of peptide extracts for RP-HPLC, Biorad protein assays
and SDS-PAGE.
The samples were prepared for RP-HPLC by placing 1 mL into a 2 mL HPLC vial
after filtration using a 0.2 µm membrane syringe filter (Sartorius, Melbourne,
Australia). The Bradford protein assay was carried out in triplicate (see Section 2.8)
using standards of Bovine Serum Albumin (Bio-Rad).
The samples were prepared for SDS-PAGE as per the Laemmli method (see Section
2.7). After running of the gels, samples were stored at -80°C.
Enzyme Optimum pH (actual pH)
Optimum Temperature
Bromelain from pineapple stem
4.5-5.5* (~5) 55°C (50°C)
Flavourzyme 5-7* (5.7-6) 50°C Fromase 3.5-7* (~3.5) 37°C Papain 4-7* (~5-6) 65°C Rennin from calf stomach 3.4* 37°C
145
4.2.5 Separation, fractionation and purification of peptides
The hydrolysates showing the highest bioactivity were separated by MWCO
membranes (7000 g for 30 minutes) and reanalysed. The MWCO fractions exhibiting
the highest activity were then fractionated by RP-HPLC as per Section 2.6.
4.2.6 Identification of bioactive peptides derived from enzymatic
hydrolysis of organic milk protein.
The peptide extracts were subjected to various screening assays to determine if they
contained potentially bioactive peptides including antimicrobial, antioxidant and
ACE-inhibitory peptides.
4.2.6.1 Identification of peptide extracts with antimicrobial activity
Preliminary screening of the hydrolysates for antimicrobial activity was carried out
as per Section 2.5.1. The whole and fractionated extracts (by MWCO membranes)
were analysed for antimicrobial activity against E. coli, B. cereus and S. aureus. The
MWCO extracts exhibiting the greatest inhibition of bacteria were subsequently
fractionated by RP-HPLC as per the method described in Section 2.6.
4.2.6.2 Identification of peptide extracts with ACE-inhibitory activity
Preliminary screening of the hydrolysates with ACE-inhibitory activity was carried
out as per Section 2.5.2. The whole and fractionated extracts (by MWCO
membranes) were analysed for ACE-inhibitory activity. The extracts exhibiting the
greatest inhibition of ACE were subsequently fractionated by RP-HPLC as per
Section 2.6.
146
4.2.6.3 Identification of peptide extracts with antioxidant activity
Preliminary screening of the hydrolysates with antioxidant activity was carried out as
per Section 2.5.3. The whole and fractionated extracts (by MWCO membranes) were
analysed for antioxidant activity. The extracts exhibiting the greatest inhibition of
DPPH were subsequently fractionated by RP-HPLC as per Section 2.6.
4.3 Results
4.3.1 Proximate composition analysis of lite organic milk
The proximate composition of milk is shown in Section 3.1.
4.3.2 Screening for bioactive peptides
The hydrolysates were screened for the presence of antioxidant, ACE-inhibitory and
antimicrobial peptides. The whole hydrolysates and hydrolysates fractionated by
MWCO membranes were analysed for each bioactivity. Subsequently, if substantial
bioactivity was observed the samples with the highest activity were fractionated by
RP-HPLC and reanalysed.
4.3.2.1 Antimicrobial activity of hydrolysates
The whole and fractionated hydrolysates were screened for antimicrobial activity
against E. coli, B. cereus and S. aureus. The results after fractionation by RP-HPLC
are shown in Figures 4.1, 4.2 and 4.3.
The fraction exhibiting the highest activity against E. coli was fraction 2 derived
from Bromelain hydrolysis for 5 hours (using 1% enzyme to protein) of the soluble
147
protein fraction (10B15SF2). It contained peptides between 5 and 10 kDa (21.88%
±SEM 1.41 at 0.013 mg peptide fraction/mL).
Figure 4.2 shows the inhibition of B. cereus by fractionated hydrolysates. The
insoluble fraction that inhibited B. cereus the most was fraction 3 containing peptides
greater than 10 kDa derived by bromelain hydrolysis (0.5% enzyme to protein) for 5
hours (20B0.55IF3) (35.44% ±6.45 at 0.007 mg/mL), however no significant
differences were observed between samples (P>0.05).
The inhibition of S. aureus by the fractionated hydrolysates is shown in Figure 4.3.
The fraction that inhibited S. aureus the greatest was fraction 1 that contained
peptides less than 5 kDa derived from Flavourzyme hydrolysis (0.5% enzyme to
protein) of the soluble protein fraction for 1 hour (5F0.51SF1) (69.35% ±3.02 SEM
at 0.009 mg/mL).
0
5
10
15
20
25
5F30.5SF1
5F30.5SF2
5F30.5SF3
20R11SF1
20R11SF2
20R11SF3
10B15SF1
10B15SF2
10B15SF3%
Inhib
ition
Type of hydrosylate
Figure 4.1. Inhibition of E. coli by hydrolysates (n = 6 ±SEM). Notes: 5: <5 kDa; 10: >5 <10 kDa; 20: >10 kDa; F: Flavourzyme; R: Rennin from calf stomach; B: Bromelain; 3: 3 hours hydrolysis; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; 1: 1% enzyme to protein; SF: Soluble fraction. * significantly different compared to 10B15SF2 (P<0.05).
* * *
148
05
1015202530354045
5B0.55IF1
5B0.55IF2
5B0.55IF3
20B0.55IF1
20B0.55IF2
20B0.55IF3
10R0.53IF1
10R0.53IF2
10R0.53IF3%
Inhib
ition
Type of hydrolysate
Figure 4.2. Inhibition of B. cereus by fractionated hydrolysates (n=6 ±SEM). Notes: 5: <5 kDa; 20: >10 kDa; 10: >5 <10 kDa; R: Rennin from calf stomach; B: Bromelain; 3: 3 hours hydrolysis; 5: 5 hour hydrolysis; 0.5: 0.5% enzyme to protein; IF: Insoluble fraction. No significant differences observed (P>0.05).
149
01020304050607080
5F10.5SF1
5F10.5SF2
5F10.5SF3
5F0.53SF1
5F0.53SF2
5F0.53SF3
10F13SF1
10F13SF2
10F13SF3%
Inhib
ition
Type of hydrolysate
Figure 4.3. Inhibition of S. aureus by fractionated hydrolysates (n=6 ±SEM). Notes: 5: <5 kDa; 10: >5 <10 kDa; F: Flavourzyme; 3: 3 hours hydrolysis; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; 1: 1% enzyme to protein; SF: Soluble fraction. All samples significantly different to 5F10.5SF1 except 10F13SF1 (P<0.05).
150
151
The fraction that inhibited S. aureus the greatest 5F10.5SF1 was separated and
reanalysed against all three bacteria. The results for inhibition against S. aureus are
shown in Figure 4.4.
The fraction that inhibited S. aureus the greatest was fraction 1C containing peptides
less than 5 kDa derived from Flavourzyme hydrolysis (0.5% enzyme to protein) of
the soluble protein fraction for one hour (5F0.51SF1C) (38.05% ±3.89 at 0.51
mg/mL) however no significant differences were observed between fractions and
subsequently separation was not continued. The MWCO fraction (5F10.5S) was
analysed by Mass Spectrometry to determine the peptide sequences (See Section
4.4.4).
Figure 4.4.Inhibition of S. aureus by 5F10.5S fractions (n=6 ±SEM). Notes: 5: <5 kDa; F: Flavourzyme; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; S: soluble fraction. No significant differences observed (P>0.05)
0
5
10
15
20
25
30
35
40
45
1A 1B 1C 1D
% I
nhib
itio
n
5F10.5S Fraction
152
4.3.2.2 Antioxidant activity of hydrolysates
The antioxidant activity of the hydrolysates was measured by the inhibition of the
free radical DPPH. The MWCO fraction showing the highest antioxidant activity
was the fraction containing peptides greater than 10 kDa from the insoluble fraction
hydrolysed by Flavourzyme for 5 hours (1% enzyme to protein) (83.36% ± 0.66)
(Figure 4.5). The three fractions with the highest activity were fractionated further by
RP-HPLC.
After fractionation by RP-HPLC, all extracts showed low antioxidant activity and the
higher activity of the MWCO fractions was attributed to the Flavourzyme enzyme.
All other hydrolysates showed low inhibition of DPPH (<30%) therefore
fractionation was not continued.
Figure 4.5. Inhibition of DPPH by MWCO fractions (n=6 ±SEM). Notes: 5: <5 kDa; 20: >10 kDa; 10: >5 <10 kDa; F: Flavourzyme; 5: 5 hours hydrolysis; 3: 3 hours hydrolysis; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; 1: 1% enzyme to protein; S: soluble fraction; I: insoluble fraction; AA: Absorbic acid. * Significantly different to AA (P<0.05).
0
10
20
30
40
50
60
70
80
90
5F11S
10F11S
20F11S
5F13S
10F13S
20F13S
5F15S
10F15S
20F15S
5F11I
10F11I
20F11I
5F13I
10F13I
20F13I
5F15I
10F15I
20F15I
AA
% I
nhib
itio
n
Type of hydrolysate
* *
*
*
153
154
4.3.2.3 Antihypertensive activity of hydrolysates
All hydrolysates were analysed for ACE-inhibitory activity. The results for the
molecular-weight cut off fractions are shown in Figures 4.6 (soluble fractions) and
4.7 (insoluble fractions). The soluble fraction that inhibited ACE the greatest
contained peptides less than 5 kDa derived from Flavourzyme hydrolysis (0.5%
enzyme to protein) for 1 hour (5F0.51S) (IC50: 0.014 ±0.001 mg peptide/mL)
followed by the soluble fraction containing peptides less than 5 kDa derived by
Fromase hydrolysis for 1 hour (0.5% enzyme to protein) for 1 hour (5FR0.51S)
(IC50: 0.0201 ±0.003 mg peptide/mL) and then the soluble fraction containing
peptides less than 5 kDa derived from Papain hydrolysis (1% enzyme to protein) for
5 hours (5P15S) (IC50: 0.0312 ±0.011 mg peptide/mL). These three fractions were
separated by RP-HPLC and reanalysed (Figure 4.8).
Figure 4.6. Inhibition of ACE by soluble hydrolysate fractions. Notes: (n=6 ±SEM). 5: <5 kDa; 20: >10 kDa; 10: >5 <10 kDa; F: Flavourzyme; FR:Fromase; P: Papain; 5: 5 hours hydrolysis 3: 3 hours hydrolysis; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; 1: 1% enzyme to protein; S: soluble fraction. * Significant different to 20FR10.5S and 20P15S.
-0.500
-0.400
-0.300
-0.200
-0.100
0.000
0.100
0.200
0.300
5FR
10.5S
10FR
10.5S
20FR
10.5S
5FR
30.5S
10FR
30.5S
20FR
30.5S
5F10.5S
10F10.5S
20F10.5S
5P15S
10P15S
20P15S
IC50
(m
g/m
l)
Type of hydrolysate
*
155
156
The insoluble fractions that inhibited ACE the greatest was the fraction containing
peptides less than 5 kDa derived from Flavourzyme hydrolysis (0.5% enzyme to
protein) for 3 hour (5F0.53I) (IC50: 0.024 ±0.002 mg peptide/mL) followed by the
fraction containing peptides less than 5 kDa derived by Flavourzyme hydrolysis
(0.5% enzyme to protein) for 1 hour (5F0.51I) (IC50: 0.0529 ±0.011 mg peptide/mL)
and then the fraction containing peptides less than 5 kDa derived from Papain
hydrolysis (0.5% enzyme to protein) for 3 hours (5P0.53I) (IC50: 0.0721 ±0.017 mg
peptide/mL). These three MWCO fractions were separated by RP-HPLC and
reanalysed (Figure 4.7).
The inhibition of ACE by the RP-HPLC fractions is shown in Figure 4.8. Various
fractions were analysed by ESI-Q-TOF-MS-MS including Fraction 2 containing
insoluble peptides derived from Flavourzyme hydrolysis (0.5% enzyme to protein)
for 1 hour (5F0.51IF2) (IC50: 0.093 ±0.006 mg peptide/mL), Fraction 2 containing
insoluble peptides less than 5 kDa derived from Papain hydrolysis (0.5% enzyme to
protein) for 3 hours (5P0.53IF2) (IC50: 0.073 ±0.008 mg peptide/mL), and Fraction 2
containing soluble peptides less than 5 kDa derived from Papain hydrolysis (1%
enzyme to protein) for 5 hours (5P15SF2) (IC50: 0.019 ±0.012 mg peptide/mL). The
fraction 5F10.5IF2 contained three known ACE-inhibitory peptides, 5P0.53IF2
contained 85 peptides: 68 with known bioactivity, 17 potentially novel and 5P15SF2
contained 13 peptides, 7 potentially novel (some results shown in Table 4.1). The
fraction 5P0.53IF2 was chosen to be further fractionated and reanalysed.
Figure 4.7. Inhibition of ACE by insoluble hydrolysate fractions. Notes: (n=6 ±SEM). 5: <5 kDa; 20: >10 kDa; 10: >5 <10 kDa; F: Flavourzyme; P: Papain; 3: 3 hours hydrolysis; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; I: insoluble fraction. *Significantly different (P<0.05) to sample 20F0.51I.
0.00
0.10
0.20
0.30
0.40
0.50
5P0.53I
10P0.53I
20P0.53I
5F0.53I
10F0.53I
20F0.53I
5F0.51I
10F0.51I
20F0.51I
IC50
(m
g/m
l)
Type of hydrolysate
* *
*
*
*
157
Figure 4.8. Inhibition of ACE by fractionated hydrolysates. Notes: (n=6 ±SEM). 5: <5 kDa; 20: >10 kDa; 10: >5 <10 kDa; F: Flavourzyme; FR:Fromase; P: Papain; 5: 5 hours hydrolysis 3: 3 hours hydrolysis; 1: 1 hour hydrolysis; 0.5: 0.5% enzyme to protein; 1: 1% enzyme to protein; I: Insoluble fraction; S: soluble fraction. indicates selected fractions to separate further. No significant differences observed between samples (P<0.05).
-0.600
-0.400
-0.200
0.000
0.200
0.400
0.600
0.800
5P0.53IF
2
5P0.53IF
3
5F0.51IF
1
5F0.51IF
2
5F0.51IF
3
5F0.53IF
1
5F0.53IF
2
5F0.53IF
3
AC
EH
HLC
AP
5F10.5S
F1
5F10.5S
F2
5F10.5S
F3
5FR
10.5SF
1
5FR
10.5SF
2
5FR
10.5SF
3
5P15S
F1
5P15S
F2
5P15S
F3
IC50
(m
g/m
l)
Type of hydrolysate fraction
158
159
The fraction 5P0.53IF2 was separated into four fractions and reanalysed for ACE-
inhibitory activity (Figure 4.9) and stability against ACE (Figure 4.10). The most
ACE-inhibitory fraction was fraction 2A (5P0.53IF2A).
Figure 4.9. Inhibition of ACE by fractionated hydrolysates (n=6 ±SEM). * Significantly different to 5P0.53IF2A. Notes: 5: <5 kDa; P: Papain; 3: 3 hours hydrolysis; 0.5: 0.5% enzyme to protein; I: insoluble; F: Fraction.
The stability of the peptide fractions against ACE were measured by the ACE-
stability assay (see Section 2.5.2.1 for the method) (Figure 4.10). The fractions 2A,
2C and 2D had substrate-like activity meaning that ACE hydrolysed the peptide
fractions into smaller less active peptides, whereas fraction 2B acted as a pro-drug
type inhibitor meaning that ACE hydrolysed the peptides to their true inhibitors
(Fujita and Yoshikawa 1999). Fraction 2B in the normal assay had an IC50 value of
0.056 ±0.004 mg peptide/mL (postincubated ACE) whereas preincubated ACE had
an IC50 value of 0.051 ±0.002 mg peptide/mL. The pro-drug type fraction was
analysed by Mass Spectrometry (see Section 4.3.3).
0.000
0.010
0.020
0.030
0.040
0.050
0.060
5P0.53IF2A 5P0.53IF2B 5P0.53IF2C 5P0.53IF2D
IC50
(m
g/m
l)
Type of fraction
*
160
Figure 4.10. Stability of the peptide fractions against ACE (n=6 ±SEM). Notes: 5: <5 kDa; P: Papain; 3: 3 hours hydrolysis; 0.5: 0.5% enzyme to protein; I: insoluble fraction; F: Fraction. * significantly different to post-incubated ACE; ^ significantly different to pre-incubated ACE samples.
4.3.3 Structure of Antimicrobial and ACE-inhibitory peptides by Mass
Spectrometry and MASCOT database searching
Electron Spray Ionisation- Quadruple- Time of flight- Tandem Mass Spectrometry
(ESI-Q-TOF-MS-MS) was undertaken to identify the molecular weight and amino
acid sequences of peptides contained in the antimicrobial fraction 5F10.5S and the
ACE-inhibitory fractions 5P0.53IF2B and 5F0.51IF2 (see Section 2.3.9 for method).
The antimicrobial fraction F10. S contained 11 peptides derived from αs1- and く-
casein according to MASCOT database searching. The ACE-inhibitory fraction
P0. γ FβB contained γβ peptides derived from κ-, αs1- and く-casein and the ACE-
inhibitory fraction 5F0.51IF2 contained three known ACE-inhibitory peptides
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
5P0.53IF2A 5P0.53IF2B 5P0.53IF2C 5P0.53IF2D
IC50
(m
g/m
l)
Type of fraction
Post-incubated ACE Pre-incubated ACE
*
^
161
derived from く-casein (Table 4.2). The peptides indicated in bold were synthesised
(GenScript USA Inc., Piscataway, NJ, USA) and reanalysed for ACE-inhibitory
activity and their structure-activity relationships elucidated by NMR (Chapter 5).
The RP-HPLC chromatogram for Fraction 2 containing peptides less than 5 kDa
derived from Flavourzyme hydrolysis (0.5% enzyme to protein) of the insoluble
protein fraction for 1 hour (5F0.51IF2) is shown in Figure 4.11. This fraction was
collected and analysed by ESI-Q-TOF-MS-MS (Table 4.2).
The RP-HPLC chromatogram for Fraction 2B containing peptides less than 5 kDa
derived from Papain (0.5% enzyme to protein) hydrolysis of the insoluble protein
fraction for three hours is shown in Figure 4.12. This fraction was analysed by ESI-
Q-TOF-MS-MS (Table 4.2).
Figure 4.11. RP-HPLC of chromatogram of ACE-inhibitory fraction 5F0.51I that was collected and analysed by Mass Spectrometry (see Table 4.2).
Minutes
20 21 22 23 24 25 26 27 28 29 30
0
500
1000
1500
2000
2500
3000
3500
Spectrum Max Plot5F10.5CF215/02/2011 2:59:27 PM5F10.5CF2.dat
Fraction 2
162
Figure 4.12. RP-HPLC chromatogram of ACE-inhibitory fraction 5P0.53IF2B. This fraction was collected and analysed by Mass Spectrometry (See Table 4.2).
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
-250
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
2750
3000Spectrum Max Plot5P0.53CF2B1/06/2011 12:08:00 PM5P0.53CF2B1-06-2011 12-08-00 PM.dat
Fraction 2B
163
164
The summed mass chromatogram corresponding to the decapeptide DIPNPIGSEN is
shown in Figure 4.13. This decapeptide has an observed m/z ratio of 1055.48 and a
molecular weight of 1054.4931 and is a singly charged molecule.
Figure 4.13. Summed mass chromatogram at 1055.48 corresponding to decapeptide DIPNPIGSEN.
The summed mass chromatogram corresponding to the dodecapeptide
AVPYPQRDMPIQ is shown in Figure 4.14. This dodecapeptide has an observed m/z
ratio of 707.8406 and a molecular weight of 1413.6667 (net charge of 2+).
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
%
0
100
Y_DDA_1 1: TOF MS ES+ 1055.484
3.02e326.30
26.50
29.0633.96
165
Figure 4.14. Summed mass chromatogram at 707.8406 corresponding to dodecapeptide AVPYPQRDMPIQ.
x10 dilution
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00
%
0
100
C 1: TOF MS ES+ 707.841
18225.14
Table 4.2. Summary of peptides identified from various bioactive hydrolysate fractions by Mass Spectrometry
* Reference may correspond to peptide with sequence contained with other amino acids also or shorter peptide fragment # Each peptide shown with adjacent amino acid residues corresponding to parent protein.
Sample Protein Precursor
Peptide sequence Molecular Weight
Known activity? Derived previously by: References
5F10.5S Antimicrobial activity
αS1-casein f (197-203)
D.IPNPIGS.E 696.3806 Novel sequence, no known activity.
- -
f (201-209) P.IGSENSGKI.T 903.4661 Novel sequence, no known activity.
- -
f (196-205) S.DIPNPIGSE.N 940.4502 Novel sequence, no known activity.
- -
f (196-206) S.DIPNPIGSEN.S 1054.4931 Peptide partly identified to be antimicrobial
Fermentation by L. acidophilus (Hayes et al. 2006)*
く-casein f (129-133) K.YPVEP.F 603.2904 Chemotactic Actinase E (Kitazawa et al. 2007) f (218-224) R.GPFPIIV.- 741.4425 ACE-inhibitory Trypsin (Cadee and Mallee 2010) f (75-81)
く-casomorphin-7 V.YPFPGPI.P 789.4061 ACE-inhibitory, Opioid,
Immunomodulatory, cytomodulatory
Pepsin, trypsin (Brantl et al. 1981,
Kayser and Meisel 1996,
Meisel and Fitzgerald
2000) f (75-82)
く-casomorphin-8 V.YPFPGPIP.N 886.4589 ACE-inhibitory, opioid Synthesised (Sakaguchi et al. 2003)
f (195-203) Q.EPVLGPVRG.P 922.5306 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b) f (126-133) P.FPKYPVEP.F 975.5066 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b) f (124-133) E.MPFPKYPVEP.F 1203.5998 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b) 5F10.5IF2 ACE-inhibitory activity
く-casein f (195-203) Q.EPVLGPVRG.P 922.5306 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b)
166
Table 4.2. Summary of peptides identified from various bioactive hydrolysate fractions by Mass Spectrometry (cont.). Sample Protein Precursor Peptide sequence Molecular Weight Known activity? Derived previously by: References f (126-133) P.FPKYPVEP.F 975.5066 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b) f (124-133) E.MPFPKYPVEP.F 1203.5398 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b) 5P0.53IF2B ACE-inhibitory activity
く-casein f (75-79) I.PPLTQ.T 554.3064 Novel sequence, no known activity
- -
f (169-175) K.VLPVPQ.K 651.3956 ACE-inhibitory, Antioxidant.
Fermentation by L. helveticus proteases, L. rhamnosus fermentation and gastrointestinal digestion.
(Yamamoto et al.
1994b, Rival et al. 2001,
Hernández-Ledesma et
al. 2004)* f (74-79) N.IPPLTQ.T 667.3905 ACE-inhibitory Fermentation by L.
delbrueckii subsp. bulgaricus (Gobbetti et al. 2000)*
f (103-108) F.LQPEVM.G 715.3575 Novel sequence, no known activity
- -
f (144-149) T.DVENLH.L 725.3344 Novel sequence, no known activity
- -
f (183-188) Q.RDMPIQ.A 758.3745 ACE-inhibitory Fermentation by L. rhamnosus
(Hernández-Ledesma et al. 2004)
f (73-79) Q.NIPPLTQ.T 781.4334 ACE-inhibitory Fermentation by L. delbrueckii subsp. bulgaricus
(Gobbetti et al. 2000)*
f (168-175) Q.SKVLPVPQ.K 866.5226 ACE-inhibitory L. helveticus proteinase (Yamamoto et al.
1994a, Hayes et al.
2007b)
* Reference may correspond to peptide with sequence contained with other amino acids also or shorter peptide fragment # Each peptide shown with adjacent amino acid residues corresponding to parent protein
167
Table 4.2. Summary of peptides identified from various bioactive hydrolysate fractions by Mass Spectrometry (cont.) Sample Protein Precursor Peptide sequence Molecular Weight Known activity? Derived previously by: References f (124-131) Q.SLTLTDVE.N 876.4440 Immunogenic Commercially fermented milk (Hernández-
Ledesma 2005,
Kumar and Wong
2007) f (166-175) L.SQSKVLPVPQ.K 1081.6132 ACE-inhibitory Fermentation by L. animalis (Hayes et al.
2007b) f (16-25) A.RELEELNVPG.E 1154.5931 Novel sequence, no known
activity - -
f (166-176) L.SQSKVLPVPQK.A 1209.7081 ACE-inhibitory Fermentation by L. animalis (Hayes et al. 2007b)*
f (193-203) A.VPYPQRDMPIQ.A 1342.6703 No known activity; patent applied for.
- (Kruzel 2009)*
f (193-203) A.VPYPQRDMPIQ.A 1358.6653 No known activity; patent applied for.
- (Kruzel 2009)*
f (192-203) K.AVPYPQRDMPIQ.A 1413.7075 Part of sequence identified, No known activity; patent applied for.
- (Kruzel 2009)*
αs1-casein f (33-37) N.ENLLR.F 643.3653 Novel sequence, no known activity
- -
f (109-114) Q.LEIVPN.S 683.3854 Sequence identified previously, no known activity
Fermentation by six L. helveticus strains
(Jensen et al. 2009)
f (171-177) Q.LDAYPSG.A 721.3283 Novel sequence, no known activity
- -
f (173-179) Q.YTDAPSF.S 799.3388 ACE-inhibitory Porcine pepsin A (Contreras et al. 2009)
f (41-47) V.APFPEVF`.G 805.4010 Inhibits DPP-IV - (Boots 2005) * Reference may correspond to peptide with sequence contained with other amino acids also or shorter peptide fragment # Each peptide shown with adjacent amino acid residues corresponding to parent protein.
168
Table 4.2. Summary of peptides identified from various bioactive hydrolysate fractions by Mass Spectrometry (cont.)
Sample Protein Precursor
Peptide sequence Molecular Weight
Known activity? Derived previously by:
References
f (195-202) F.SDIPNPIG.S 811.4076 Novel sequence, no known activity
- -
f (173-180) Q.YTDAPSFS.D 886.3709 ACE-inhibitory Pepsin (Contreras et al. 2009)*
f (171-180) G.TQYTDAPSFS.D 1115.4771 ACE-inhibitory Pepsin (Contreras et al. 2009)*
κ-casein f (190-195)
R.SPAQIL.Q 627.3592 Novel sequence, no known activity
- -
f (25-29) K.YIPIQ.Y 632.3533 Antioxidant Pepsin, Trypsin and Chymotrypsin
( -Ruiz et al.
2008) f (61-65) G.LNYYQ.Q 699.3228 Novel sequence, no known
activity - -
f (60-65) Y.GLNYYQ.Q 756.3442 Novel sequence, no known activity
- -
f (176-184) E.SPPEINTVQ.V 983.4924 ACE-inhibitory Fermentation (Gobbetti et al. 2000)
* Reference may correspond to peptide with sequence contained with other amino acids also or shorter peptide fragment # Each peptide shown with adjacent amino acid residues corresponding to parent protein. The peptides DIPNPIGSEN and AVPYPQRDMPIQ were synthesised (GenScript USA Inc., Piscataway, NJ, USA). Their ACE-inhibitory
activity was reanalysed and structure-activity relationship determined by NMR studies (see Chapter 5).
169
170
4.4 Discussion
These results have shown that the hydrolysate fractions from the different proteases
used in this study do exhibit various bioactivities. The hydrolysate fractions showed
low antioxidant activity (<30% inhibition) except for Flavourzyme hydrolysates
(>80%), which were attributed to the enzyme. The antimicrobial activity was greatest
against S. aureus particularly by Flavourzyme hydrolysates. Papain and Flavourzyme
hydrolysates derived from the insoluble fraction had the greatest ACE-inhibitory
activity. Several fractions were analysed by ESI-Q-TOF-MS-MS and MASCOT
database searching and several novel and known peptides were identified.
Antimicrobial activity overall was highest against S. aureus (5F0.51SF1: 69.35%
±3.02 at 0.009 mg/mL) followed by B. cereus (20B0.55IF3: 35.44% ±6.45 at 0.007
mg/mL) and then E. coli (10B15S: 21.88% ±1.41 at 0.013 mg/mL). The fraction
5F0.51S was analysed by Mass Spectrometry and MASCOT database searching and
contained 11 peptides (see Table 4.1).
The fraction contained peptides less than 5 kDa derived by Flavourzyme hydrolysis
of the soluble protein fraction (0.5% enzyme to protein) for one hour (5F0.51S). This
fraction contained four peptides derived from αs1-casein and seven peptides derived
from く-casein. The peptides derived from αs1-casein f(197-203) corresponding to the
amino acid sequence IPNPIGS, f(201-209) corresponding to IGSENSGKI, f(196-
205) corresponding to DIPNPIGSE and f(196-206) corresponding to DIPNPIGSEN
are all novel sequences. Hayes et al (2006) identified a similar peptide
171
SDIPNPIGSENSEK (also known as Caseicin C) that was derived from αs1-casein f
(195-208) by fermentation with L. acidophilus and it was shown to have very low
antimicrobial activity against L. innocua (Hayes et al. 2006). Generally,
antimicrobial peptides contain mostly alpha-helices, have an overall net positive
charge and are amphipathic (Floris et al. 2003). They are proposed to inhibit bacteria
by interrupting the cytoplasmic membrane via two mechanisms: peptides bundles
form transmembrane pores (barrel-stave) or high concentrations of the peptide cover
the membrane leading to permeabilisation (carpet) (Floris et al. 2003)
The peptides derived from く-casein have all been previously reported and have been
shown to be ACE-inhibitory (sequences GPFPIIV, YPFPGPI, YPFPGPIP,
EPVLGPVRG, FPKYPVEP and MPFPKYPVEP) (Fitzgerald and Meisel 2000,
Sakaguchi et al. 2003, Hayes et al. 2007b, Cadee and Mallee 2010). The peptide
corresponding to く-casein f (129-133) YPVEP has been shown to induce macrophage
chemotaxis (Kitazawa et al. 2007). This peptide was derived by actinase E digestion
of く-casein. The peptides f(75-81) YPFPGPI and f(75-82) YPFPGPIP are also
known as く-casomorphin-7 and く-casomorphin-8, respectively. They have been
derived by pepsin and trypsin hydrolysis and both have been shown to have opioid
and ACE-inhibitory activity (Brantl et al. 1981, Meisel and Fitzgerald 2000).
The peptides f(210-218) EPVLGPVRG, f(126-133) FPKYPVEP and f(124-133)
MPFPKYPVEP are all ACE-inhibitory and have been derived by fermentation by L.
animalis (Hayes et al. 2007b). The fraction 5F10.5IF2 was also shown to contain the
three peptides. It was derived by Flavourzyme hydrolysis (0.5% enzyme to protein)
172
of the insoluble protein fraction for one hour and was fraction number two. It was
shown to have strong ACE-inhibitory activity (IC50: 0.093 ±0.006 mg peptide/mL),
which is in agreement with the study by Hayes et al (2007b).
The hydrolysates exhibited good ACE-inhibitory activity with both the soluble and
insoluble fractions. The activity of the MWCO soluble fractions was slightly better
overall compared with the MWCO insoluble fractions. Flavourzyme and Papain
hydrolysates consistently had the best activity when comparing all five enzymes at
each stage of fractionation. Several fractions were analysed by Mass Spectrometry
and their peptide sequences identified by MASCOT database searching including
5F10.5IF2 and 5P0.53IF2B.
The fraction containing peptides less than 5 kDa derived from Papain hydrolysis of
the insoluble fraction (number 2B) (0.5% enzyme to protein) for three hours was
shown to contain 14 peptides derived from く-casein (6 novel), 8 peptides derived
from αs1-casein (4 novel) and peptides from κ-casein (3 novel). The novel peptides
derived from く-casein include PPLTQ, LQPEVM, DVENLH, RELEELNVPE,
PYPQRDMP Q, A PYPQRDMP Q, from αs1-casein: ENLLR, LEIVPN,
LDAYPSG, SD PNP G and from κ-casein: SPAQIL, LNYYQ and GLNYYQ.
Previously, the peptides RDMPIQ, SKVLPVPQ (IC50: 39 µM), and SQSKVLPVPQ
(derived from fraction 5P0.5IF2B have all been derived by fermentation and have
been shown to be ACE-inhibitory (Yamamoto et al. 1994a, Hernández-Ledesma et
al. 2004, Hayes et al. 2007b). Similar sequences SQSKVLPVPQK (MW:
173
1209.7081), VLPVPQ, IPPLTQ and NIPPLTQ were also identified in this fraction
and are ACE-inhibitory (Gobbetti et al. 2000, Hayes et al. 2007b). They have all
been derived by fermentation in other studies. The peptide SLTLTDVE (MW:
876.4440) has been shown to have immunogenic properties and was isolated from
commercially fermented milk (Hernández-Ledesma 2005, Kumar and Wong 2007).
It is listed in a patent application (Kumar and Wong 2007).
The peptide A PYPQRDMP Q (MW: 141γ.707 ) derived from く-casein has not
been reported as bioactive. However, the peptide sequence VPYPQRDMPIQ (MW:
1358.6653) is listed in a very general patent for the therapeutic use of peptides
(Kruzel 2009) in relation to central nervous system disorders and related diseases.
The peptide AVPYPQRDMPIQ was synthesised and reanalysed for ACE-inhibitory
activity and structure-bioactivity relationship determined by NMR (see Chapter 5).
The peptides derived from αs1-casein YTDAPSF, YTDAPSFS and TQYTDAPSFS
have been isolated previously by pepsin digestion and have been shown to be ACE-
inhibitory (Contreras et al. 2009). Two peptides derived from κ-casein YIPIQ and
SPPEINTVQ have been shown to have antioxidant and ACE-inhibitory activities,
respectively (Gobbetti et al. 2000, G Mez-Ruiz et al. 2008).
ACE-inhibitory peptides usually contain hydrophobic (aromatic or branched chain
aliphatic) residues at the three C-terminal end positions (such as tyrosine, tryptophan,
leucine, isoleucine, valine) and N-terminal also contains branched chain aliphatic
amino acids (Jauregi 2008).
174
All of the peptides identified in active fractions have been derived from casein rather
than whey proteins. This may be due to the difficulty of proteolytic enzymes to
access whey proteins as they are globular and have randomly distributed hydrophilic
and hydrophobic amino acids compared with casein proteins that are flexible and
therefore hydrolysed relatively easily as they contain distinct hydrophilic and
hydrophobic regions.
The enzymes flavourzyme and papain hydrolysed casein proteins to produce the
most bioactive fractions overall. Flavourzyme consists of a fungal peptidase and
proteinase complex that has both exo- amd endo-peptidase activity therefore it has
broad specificity of active sites that it hydrolyses. Similarly, papain hydrolyses a
broad range of active sites but prefers to cleave basic amino acids such as leucine and
glycine.
4.5 Conclusions
Enzymatic hydrolysis of milk protein to yield novel bioactive peptides has been
effective particularly in identifying potentially novel amino acid sequences that are
ACE-inhibitory. Time constraints and research funds are limitations of this work that
have restricted the scope of the peptides that could be investigated for their bioactive
potential. However, a substantial amount of data was obtained showing that
particular enzymes derived novel sequences which could be potentially investigated
in the future. Overall, enzymatic hydrolysis resulted in greater yield of peptides with
175
stronger antimicrobial and ACE-inhibitory activities being identified compared with
peptides derived by milk fermentation (Chapter 3).
176
Chapter 5 Bioactivity and NMR studies of selected bioactive
peptides derived from organic cheese and milk
5.1 Introduction
In Chapter 4, bioactive peptides derived from milk proteins have been shown to have
antihypertensive and antimicrobial activity. Currently, there are no reported studies
that have examined the structure-activity relationship of milk peptides with NMR or
similar techniques. Determining the structure-activity relationship of milk peptides
could provide knowledge to discover peptides with stronger bioactivity.
Fractions containing the peptides YLGYLEQLLR, DIPNPIGSEN and
AVPYPQRMDPIQ were shown in Chapters 3 and 4 to have ACE-inhibitory activity.
These peptides were synthesised due to their novelty and bioactivity and then their
ACE-inhibitory activity and their stability against various gastrointestinal enzymes
and ACE were examined.
Nuclear magnetic resonance (NMR) spectroscopy is a technique that measures the
interaction of magnetic nuclei with an external magnetic field to determine resonance
frequency. In this study, the structures of various bioactive peptides were determined
using 1D- and 2D-proton NMR spectroscopic techniques. Examining the structure-
activity relationship of various peptides by NMR can provide insight into the
possible mechanism of action of peptides that is responsible for their observed
activity. Furthermore, determining their structures can provide secondary structural
177
elements that may form building blocks for the possible development of bioactive
peptides with enhanced activity.
5.2 Materials and methods
5.2.1 Materials
The peptides Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP),
Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) and Ala-Val-Pro-
Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) were purchased
from Genscript (GenScript USA Inc.Piscataway, NJ, USA) and used for analyses of
ACE-inhibitory activity and stability against gastrointestinal enzymes as well as
NMR studies. Deuterium dioxide (DO2) (Sigma-Aldrich, Castle Hill, NSW,
Australia) was used for the NMR experiments. All other chemicals used for ACE-
inhibitory experiments were the same as described in Sections 2.5.2, 2.5.2.1 and
2.5.2.2.
5.2.2 Determination of ACE-inhibitory activity of selected peptides
Analysis for ACE-inhibitory activity was carried out as per Section 2.5.2. A stock of
1 mg/mL in Milli-Q water of each peptide was prepared before preparing the ACE-
inhibitory reaction mixture.
5.2.2.1 Stability of selected peptides against ACE
Analysis of the peptide stability against ACE was carried out as per Section 2.5.2.1. .
A stock of 1 mg/mL in Milli-Q water of each peptide was prepared before preparing
the reaction mixture.
178
5.2.3 Stability of selected peptides against gastrointestinal enzymes
Analysis for stability against the gastrointestinal enzyme degradation was carried out
as per the method described in Section 2.5.2.2. A stock of 1 mg/mL in Milli-Q water
of each peptide was prepared before preparing the ACE-inhibitory reaction mixture.
5.2.4 NMR Studies on selected peptides
Individual peptide solutions for NMR experiments were prepared by dissolving each
peptide powder in a mixture of 90% H2O and 10% D2O to obtain a final
concentration of 3 mM. These solutions were used to analyse the structure of each
peptide in water.
5.2.4.1 NMR data acquisition and processing
Standard homonuclear proton one dimensional (1D) and two-dimensional (2D) NMR
experiments were conducted to assign protons and determine structures. All
experiments were performed using a Bruker Avance 500 MHz NMR spectrometer
(Bruker BioSpin, Alexandria, NSW, Australia). All spectra were recorded at 25ºC.
5.2.4.1.1 1D-NMR experiments
One-dimensional 1H NMR spectra were recorded with water suppression by pre-
saturation and also by watergate sequence. The FID was zero-filled to 4K points
before Fourier transformation.
5.2.4.1.2 2D- Total Correlation Spectroscopy (TOCSY) experiments
All TOCSY spectra were recorded with mixing times, tm , of 90 ms and 120 ms to
ensure the magnetisation transfer throughout the coupled spin networks. The spectra
179
were collected with 256 x 1024 data points. The numbers of transients collected for
each FID were 16 and the FIDs were zero-filled to 1024 points in F1 dimension and
to 4096 data points in F2 dimension before Fourier transformation.
5.2.4.1.3 2D- Rotating Frame Overhauser Effect Spectroscopy (ROESY)
experiments
All ROESY spectra were recorded with mixing times, tm, of 300 ms, 450 ms and 600
ms. The spectra were collected with 256 x 1024 data points. The numbers of
transients collected for each FID were 32, and the FIDs were zero-filled to 1024
points in F1 dimension and to 4096 data points in F2 dimension before Fourier
transformation.
5.2.4.2 NMR data analysis
The NMR spectra were analysed to assign protons, assign correct peptide sequences,
to examine secondary structure via CSI analysis and NOE-based structure
determination
5.2.4.2.1 Proton assignment
The assignments of all protons in Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg
(YLGYLEQLLR) (CP), Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN)
(MP1) and Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu
(AVPYPQRDMPIQ) (MP2) in water were performed mainly by examining the 2D-
TOCSY cross-peaks. TOCSY provided total correlation of proton spin systems in
each amino acid residue starting from HN.
180
5.2.4.2.2 Sequential assignment
The sequential assignment of Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg
(YLGYLEQLLR) (CP), Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN)
(MP1) and Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu
(AVPYPQRDMPIQ) (MP2) in water was carried out by examining the strong
rotating frame Overhauser effect (ROE) cross-peaks between HN (i) to Hα (i-1).
5.2.4.2.3 Chemical shift index (CSI) based structure analysis
The structure of each peptide was determined by the chemical shift index (CSI)
based method (Wishart et al., 1992). It was carried out by comparing the Hα chemical
shifts of each amino acid residue within each peptide with the random coil chemical
shift reference values. n doing this comparison, a CS mark of ‘1’ is given if the Hα
is greater (by 0.1 ppm) than the random coil chemical shift reference; ‘-1’ mark is
given if the Hα is less than the random coil reference; and ‘0’ is given if the chemical
shifts are the same as the random coil chemical shift reference value. Using these
marks, any ‘dense’ grouping of four or more ‘-1’ not interrupted by a ‘1’ is assigned
as an α-helix. Any ‘dense’ grouping of three or more ‘1’ not interrupted by a ‘-1’ is
assigned as a く-strand. All other regions are designated as random coil. In addition, a
local ‘density’ of non-zero chemical shift indices that exceeds 70% is required when
designing regions of helical or extended structure. All other regions that are not
identified as either helix or く-strand or regions where the local density of either ‘-1’
or ‘1’ fall below 70% are defined as ‘random coils’ (Wishart et al. 199β). The
deviation of experimental Hα chemical shift values from the corresponding random
181
coil values can also be plotted directly for secondary structure analysis of peptides
(Torres et al. 2003)
5.2.4.2.4 NOE based structure determination
Sequential assignments of the individual peptides have been determined from the
strong ROE connectivities between HN(i) and Hα (i-1) of each amino acid residue.
Long range ROE connectivities between the protons of ith amino acid residue and the
protons of (i + n)th residue provide the information regarding secondary structures of
peptides.
5.3 Results
5.3.1 ACE-inhibitory activity of selected bioactive peptides
The ACE-inhibitory activity of the peptides Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-
Leu-Arg (YLGYLEQLLR) (CP), Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn
(DIPNPIGSEN) (MP1) and Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu
(AVPYPQRDMPIQ) (MP2) are shown in Figure 5.1. The peptides were pre-
incubated with ACE to determine the stability of the peptide compared with post-
incubation. The type of inhibition was classified as either substrate, pro-drug or
inhibitor (see Section 2.5.3). MP1 (DIPNPIGSEN) is classified as an inhibitor (Post
IC50: 0.048 ±0.001 mg/mL; Pre IC50: 0.044 ±0.004 mg/mL) as the values are not
significantly different (P>0.05), MP2 (AVPYPQRDMPIQ) is classified as a pro-drug
type inhibitor (Post IC50: 0.221 ±0.022 mg/mL; Pre IC50: 0.049 ±0.006 mg/mL) and
CP (YLGYLEQLLR) is classified as a pro-drug type inhibitor (Post IC50: 0.058
±0.005 mg/mL; Pre IC50: 0.046 ±0.008 mg/mL).
182
Table 5.1 shows a comparison between peptide concentrations required to inhibit
ACE and their inhibitor type classification. The peptide AVPYPQRDMPIQ (MP2)
has the best ACE-inhibitory activity overall (34.66 µM) closely followed by the
peptide YLGYLEQLLR (CP) (36.32 µM) after pre-incubation.
Figure 5.1. Inhibition of ACE by synthesised peptides. Notes: (n = 6 ±SEM). MP1 (DIPNPIGSEN), MP2 (AVPYPQRDMPIQ), CP (YLGYLEQLLR). * Post-incubated ACE MP2 IC50 value significantly different to all other samples (P<0.05).
Table 5.1. Concentrations required to inhibit 50% of ACE activity (µM).
Peptide DIPNPIGSEN AVPYPQRDMPIQ YLGYLEQLLR Pre-incubated ACE 41.73 µM 34.66 µM 36.32 µM Post-incubated ACE 45.79 µM 156.33 µM 45.52 µM Type of inhibitor Substrate Pro-drug Pro-drug
5.3.2 Stability of selected peptides against gastrointestinal enzymes
The three peptides were subjected to the gastrointestinal enzymes pepsin and
pancreatin as a simulated gastrointestinal environment after which they were exposed
to the ACE enzyme and their ACE-inhibitory activity was determined (see section
2.3.2.2 for method).
0
0.05
0.1
0.15
0.2
0.25
0.3
MP1 MP2 CP
IC50
(m
g/m
l)
Synthesised Peptide
Post-incubated ACE
Pre-incubated ACE*
183
Figure 5.2 shows the effect of the gastrointestinal enzymes pepsin and pancreatin on
the peptides Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP),
Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) and Ala-Val-Pro-
Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in relation to
their stability against ACE. The cheese peptide YLGYLEQLLR (CP) had reduced
activity against the ACE enzyme after exposure to pepsin and pancreatin enzymes
compared with its activity after exposure to the ACE enzyme only (initial ACE-
inhibitory activity) however no significant differences were observed between
samples. Milk peptide DIPNPIGSEN (MP1) also lost its ability to inhibit the ACE
enzyme when exposed to pepsin but retained similar activity after exposure to
pancreatin compared with the ACE enzyme only. The peptide AVPYPQRDMPIQ
(MP2) after exposure to both pepsin and pancreatin showed better inhibition of the
ACE enzyme when compared to exposure to the ACE enzyme only.
Figure 5.2. Stability of ACE-inhibitory peptides against gastrointestinal enzymes pepsin and pancreatin. Notes: (n = 6). * not significantly different to normal MP2 value (P<0.05).
0
0.05
0.1
0.15
0.2
0.25
0.3
CP MP1 MP2
IC50
(m
g/m
l)
Peptide
NORMAL
PEPSIN
PANCREATIN*
184
185
5.3.3 NMR studies on selected peptides
The peptides Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP),
Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) and Ala-Val-Pro-
Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) were analysed
by Nuclear Magnetic Resonance (NMR) to determine their structures that may be
responsible for their biological activity.
5.3.3.1 NMR-based structural analysis of selected peptides in water
Data collected from the NMR experiments was used to determine the structures of
the three peptides Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR)
(CP), Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) and Ala-
Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2).
Chemical shift index (CSI) based approach (Wishart et al. 1992) was used for
secondary structure determination. Initially, the proton assignment of individual
residues was undertaken using 1D-proton and 2D-TOCSY NMR spectra. Chemical
shift index based analysis involved using the Hα chemical shift of the amino acid
residues and comparing them with the Hα random coil chemical shifts of the same
amino acid residues.
5.3.3.2 Proton assignments
Proton assignments were accomplished by a detailed analysis of TOCSY cross-
peaks. TOCSY data revealed the total correlation of the proton spin systems of each
amino acid residue starting from HN. This was used to identify the spin systems of
different residues in each peptide sequence and all of the protons were assigned.
186
Figures 5.3 and 5.4 show the TOCSY spectra with proton assignments marked for
the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP),
Figure 5.5 shows the TOCSY-based proton assignments for the peptide Asp-Ile-Pro-
Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1). Similar assignment strategy
was employed for the assignment of protons in dodecapeptide Ala-Val-Pro-Try-Pro-
Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2).
Figures 5.3 and 5.4 show the expansions of TOCSY spectrum (HN-Hα and side chain
proton region) for the peptide YLGYLEQLLR. Total correlations depicting all the
side chain proton assignments of the amino acid residues have been identified on
these spectra. The HN protons of the amino acid residues appeared in the following
order: leucine (Leu-2, L; 8.51 ppm), glutamine (Gln-7, Q; 8.23 ppm), arginine (Arg-
10, R; 8.15 ppm), glutamine (Glu-6, E; 8.10 ppm), leucine (Leu-8, L; 8.10 ppm),
leucine (Leu-5, L; 8.08 ppm), leucine (Leu-9, L; 8.09 ppm), glycine (Gly-3, G; 8.06
ppm). Table 5.2 shows the chemical shift values of all the protons assigned by the
above method for the decapeptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg
(YLGYLEQLLR).
187
Figure 5.3. Total correlation spectrum (TOCSY) (HN-Hα region) of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 90 ms mixing time.
188
Figure 5.4 Expansion of Total correlation spectrum (TOCSY) (HN-Hα region) of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 90 ms mixing time.
Table 5.2. The chemical shifts (δ in ppm) of Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water.
AAs* HN Hα Hく Hく’ Hけ Hけ’ Hけγ Hδ Hδ’ Ya - 4.22 3.10 - - - - 7.10b 6.82b
L 8.51 4.35 1.52 - - - - 0.90 0.86 G 8.06 3.90 3.86 - - - - - - Y 7.99 4.53 2.98 - - - - 7.10 6.82 L 8.08 4.20 1.54 1.50 1.40 - - 0.87 0.81 E 8.10 4.22 2.07 2.07 - 2.45 - - - Q 8.23 4.25 1.98 2.07 - 2.33 - - - L 8.10 4.34 1.60 1.63 - - - 0.86 0.91 L 8.09 4.32 1.60 - - - - 0.91 0.85 R 8.15 4.32 1.91 1.76 - 1.61 - - 3.19
* Obtained by analysis of the TOCSY and 1D-NMR spectra. a Not detected due to fast exchange of HN protons with water protons. b Aromatic amino acid residues
189
Figure 5.5 shows the expansion of the TOCSY spectrum (HN-Hα and side chain
proton region) for the peptide DIPNPIGSEN. Total correlations depicting all the side
chain proton assignments of the amino acids residues have been identified on these
spectra. The HN protons of the amino acid residues appeared in the following order:
isoleucine (Ile-6, I; 8.54 ppm), aspargine (Asn-4, N; 8.42 ppm), glutamine (Gln-9, E;
8.37 ppm), glycine (Gly-7, G; 8.36 ppm), asparagine (Asn-10, N; 8.32 ppm),
isoleucine (Ile-2, I; 8.15 ppm), serine (Ser-8; 8.14 ppm). Both proline residues have
been assigned from the aliphatic region of the TOCSY spectrum. Table 5.3 gives the
chemical shift values of all the protons assigned by the above method for the
decapeptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN). Table 5.4
gives the chemical shift values of all the protons assigned by the above method for
the dodecapeptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu
(AVPYPQRDMPIQ).
190
Figure 5.5. Total correlation spectrum (TOCSY) (HN-Hα region) of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water at 25ºC and 90 ms mixing time. Table 5.3. The chemical shifts (δ in ppm) of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water.
AAs HN Hα Hく Hく’ Hけ Hけ’ Hけγ Hδ Hδ’ Hδγ Da - 4.32 2.85 2.92 - - - - - - I 8.15 4.13 1.86 - 1.49 1.21 0.91 - - 0.84 Pa - 4.37 2.25 1.89 1.98 - - - 3.83 3.68 N 8.42 4.92 2.80 2.66 - - - - - - Pa - 4.42 2.44 1.93 1.99 - - - 3.79 3.70 I 8.54 4.51 1.87 - 1.48 1.16 0.95 - - 0.89 G 8.36 3.97 - - - - - - - - S 8.14 4.44 3.85 - - - - - - - E 8.37 4.40 2.18 1.96 2.50 2.47 - - - - N 8.32 4.67 2.85 2.77 - - - - - -
* Obtained by analysis of the TOCSY and 1D-NMR spectra. a Not detected due to fast exchange of HN protons with water protons.
191
Table 5.4. The chemical shifts (δ in ppm) of the peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in water.
AAs* HN Hα Hく Hく’ Hけ Hけ’ Hけγ Hδ Hδ’ Hδγ Hз Aa - 4.10a V 8.40 4.41 2.03 - 0.97 0.93 - - - - - Pa - 4.35s Y 8.06 4.50 2.87 3.03 2.96 - - - - - - Pa - 4.35a Q 8.35 4.30 2.11 1.96 2.37 2.30 - - - - - R 8.31 4.27 1.61 1.59 1.75 1.80 - 3.15 - - - D 8.47 4.66 2.87 2.76 - - - - - - - M 8.16 4.79 1.92 2.04 2.59 2.50 - - - - 2.13 Pa - 4.40a I 8.20 4.09 1.81 - 1.49 1.19 0.91 0.85 - - - Q 8.34 4.30 2.01 1.97 2.37 2.30 - - - - -
* Obtained by analysis of the TOCSY and 1D-NMR spectra. a Not detected due to fast exchange of HN protons with water protons.
5.3.3.3 Sequential assignment
The sequential assignment of the amino acids of each peptide was determined by
analyses of corresponding ROESY spectra. This was accomplished by recognising
the fact that the ROE cross-peaks between HN of every residue and Hα of the adjacent
residue will always be intense. Expansion of the ROESY spectrum of Tyr-Leu-Gly-
Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) is shown in Figure 5.6. This
was used to assign the sequential ROE connectivities (HN (i) to Hα (i-1)) of the
protons of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg
(YLGYLEQLLR) (CP). Similar assignment strategy was used to assign sequential
ROE connectivities for the decapeptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn
(DIPNPIGSEN) (MP1) and dodecapeptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-
Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2). Tables 5.5, 5.6 and 5.7 provide the
sequential connectivities for the amino acid residues of the three peptides.
192
Figure 5.6. Rotating frame nuclear Overhouser effect spectrum (ROESY) (HN-Hα region) of peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 450 ms mixing time.
Table 5.5 Sequential NOE connectivities of HN (i) to Hα (i-1) of the amino acid residues of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP).
Number Sequential NOE connectivities 1 HN (Leu-2) Hα (Tyr-1) 2 HN (Gly-3) Hα (Leu-2) 3 HN (Tyr-4) Hα (Gly-3) 4 HN (Leu-5) Hα (Tyr-4) 5 HN (Glu-6) Hα (Leu-5) 6 HN (Gln-7) Hα (Glu-6) 7 HN (Leu-8) Hα (Gln-7) 8 HN (Leu-9) Hα (Leu-8) 9 HN (Arg-10) Hα (Leu-9)
193
Table 5.6 Sequential NOE connectivities of HN (i) to Hα (i-1) of the amino acid residues of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1).
Number Sequential NOE connectivities 1 HN (Ile-2) Hα (Asp-1) 2 HN (Pro-3) Hα (Ile-2) 3 HN (Asn-4) Hα (Pro-3) 4 HN (Pro-5) Hα (Asn-4) 5 HN (Ile-6) Hα (Pro-5) 6 HN (Gly-7) Hα (Ile-6) 7 HN (Ser-8) Hα (Gly-7) 8 HN (Gln-9) Hα (Ser-8) 9 HN (Asn-10) Hα (Gln-9)
Table 5.7 Sequential NOE connectivities of HN (i) to Hα (i-1) of the amino acid residues of the peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2).
Number Sequential NOE connectivities 1 HN (Val-2) Hα (Ala-1) 2 HN (Pro-3) Hα (Val-2) 3 HN (Tyr-4) Hα (Pro-3) 4 HN (Pro-5) Hα (Tyr-4) 5 HN (Gln-6) Hα (Pro-5) 6 HN (Arg-7) Hα (Gln-6) 7 HN (Asp-8) Hα (Arg-7) 8 HN (Met-9) Hα (Asp-8) 9 HN (Pro-10) Hα (Met-9) 10 HN (Ile-11) Hα (Pro-10) 11 HN (Gln-12) Hα (Ile-11)
5.3.3.4 Chemical Shift Index (CSI) based structure analysis
The chemical shift index (CSI) calculations for each peptide are shown in Tables 5.8,
5.9 and 5.10. The chemical shift indices of Hα protons of decapeptide
YLGYLEQLLR, decapeptide DIPNPIGSEN and dodecapeptide AVPYPQRDMPIQ
in water are plotted in Figures 5.7, 5.8 and 5.9.
194
Chemical shift analysis of the peptide YLGYLEQLLR shows that the peptide does
not have an alpha helical or beta sheet structure but exhibits an extended structure
(Wishart, 1992). However, an examination of the modified chemical shift difference
plot given in Figure 5.7 (b) (Torres et al. 2003) reveals that this peptide has some
propensity to form helix in the region between residues three and seven. This was
further confirmed by the medium range ROE connectivity between isoleucine (Ile-6)
and Proline (Pro-3) in this peptide.
Table 5.8. Chemical shift index (CSI) results of peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 450 ms mixing time*.
Amino acid residuesa Hα coilb
Hα experiment Hα differencesc CSI valuesd
Tyrosine (Tyr,Y) 4.60 4.22 -0.38 -1 Leucine (Leu, L) 4.17 4.35 0.18 1 Glycine (Gly, G) 3.97 3.90 -0.07 0 Tyrosine (Tyr,Y) 4.60 4.53 -0.07 0 Leucine (Leu, L) 4.17 4.20 0.03 0 Glutamic acid (Glu, E) 4.29 4.22 -0.07 0 Glutamine (Gln, Q) 4.37 4.25 -0.12 -1 Leucine (Leu, L) 4.17 4.34 0.17 1 Leucine (Leu, L) 4.17 4.32 0.15 1 Arginine (Arg, R) 4.38 4.32 -0.06 0 * The experimental chemical shift values were obtained from the 2D TOCSY spectrum as per Wishart et al (2002). a Listed in the sequence of the peptide b The Hα chemical shift of leucine has been altered as per Wishart et al, 1992. c Obtained by subtraction of Hα coil from Hα experiment values. d Value of -1 given if experimental Hα is less than coil Hα; value of 1 given if the experimental H is greater than coil H; and value of 0 if the values are the same. Values must be greater than 0.1ppm or value of 0 is given (Wishart et al., 1992).
195
Figure 5.7 (a) Chemical shift index (CSI) of Hα of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP) in water at 25ºC and 450 ms mixing time.*
Figure 5.7 (b) Hα chemical shift difference plot of the peptide Tyr-Leu-Gly-Tyr-Leu-Glu-Gln-Leu-Leu-Arg (YLGYLEQLLR) (CP). Chemical shift analysis of DIPNPIGSEN shows that this peptide does not have any
alpha helical or beta sheet structure. This peptide has conformational freedom and
displays random conformational structure (Wishart, 1992).
-1.5
-1
-0.5
0
0.5
1
1.5
Y L G Y L E Q L L R
CSI
val
ues
Amino acid residues
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
Y L G Y L E Q L L R
Hα
Che
mm
ical
shf
t dev
iati
ons
Amino acid residues
196
Table 5.9. Chemical shift index (CSI) results of decapeptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water at 25ºC and 450 ms mixing time.*
Amino acid residuesa Hα coilb Hα experiment
Hα differencesc
CSI valuesd
Aspartic acid (Asp, D) 4.76 4.32 -0.44 -1 Isoleucine (Ile, I) 3.95 4.51 0.56 1 Proline (Pro, P) 4.44 4.37 -0.07 0 Asparagine (Asn, N) 4.75 4.92 0.17 1 Proline (Pro, P) 4.44 4.42 -0.02 0 Isoleucine (Ile, I) 3.95 4.13 0.18 1 Glycine (Gly, G) 3.97 3.97 0 0 Serine (Ser, S) 4.50 4.44 -0.06 0 Glutamate (Glu, E) 4.29 4.40 0.11 1 Asparagine (Asn, N) 4.75 4.67 -0.08 0 * The experimental chemical shift values were obtained from the 2D TOCSY spectrum. a Listed in the sequence of the peptide b The Hα chemical shift of leucine has been altered as per Wishart et al, 1992. c Obtained by subtraction of Hα coil from Hα experiment values. d Value of -1 given if experimental Hα is less than coil Hα; value of 1 given if the experimental H is greater than coil H; and value of 0 if the values are the same. Values must be greater than 0.1ppm or value of 0 is given (Wishart et al., 1992).
Figure 5.8. Chemical shift index (CSI) of Hα of the peptide Asp-Ile-Pro-Asn-Pro-Ile-Glu-Ser-Gln-Asn (DIPNPIGSEN) (MP1) in water at 25ºC and 450 ms mixing time.*
-1.5
-1
-0.5
0
0.5
1
1.5
D I P N P I G S E N
CSI
val
ues
Amino acid residues
197
Table 5.10. Chemical shift index (CSI) results of dodecapeptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in water at 25ºC and 450 ms mixing time.
Amino acid residuesa Hα coilb
Hα experiment
Hα differencesc CSI valuesd
Alanine (A, Ala) 4.35 4.10a -0.25 -1 Valine (V, Val) 3.95 4.41 0.46 1 Proline (P, Pro) 4.44 4.35s -0.09 -1 Tyrosine (Y, Tyr) 4.60 4.50 -0.10 0 Proline (P, Pro) 4.44 4.35a -0.09 -1 Glutamine (Q, Gln) 4.37 4.30 -0.07 0 Arginine (R, Arg) 4.38 4.27 -0.11 -1 Aspartic Acid (D, Asp) 4.76 4.66 -0.10 0 Methionine (M, Met) 4.52 4.79 0.27 1 Proline (P, Pro) 4.44 4.40a -0.04 -1 Isoleucine (I, Ile) 3.95 4.09 0.14 1 Glutamine (Q, Gln) 4.37 4.30 -0.07 -1
Chemical shift analysis (Figure 5.9 (a)) and the chemical shift difference analysis
(Figure 5.9 (b)) indicate that the dodecapeptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-
Met-Pro-Ile-Glu (AVPYPQRDMPIQ) displays strong propensity to form helix in the
region between residues three and eight. This is clearly seen in Figure 5.9b where the
Hα chemical shift differences are all negative for residues three to eight.
198
Figure 5.9 (a). Chemical shift index (CSI) of Hα of the peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ) (MP2) in water at 25ºC and 450 ms mixing time.*
Figure 5.9 (b). Hα chemical shift difference plot of peptide Ala-Val-Pro-Try-Pro-Gln-Arg-Asp-Met-Pro-Ile-Glu (AVPYPQRDMPIQ).
-1.5
-1
-0.5
0
0.5
1
1.5
A V P Y P Q R D M P I Q
CSI
val
ues
Amino acid residues
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
Hα
chem
ical
shi
ft d
evia
tion
s
Amino acid residues
199
5.4 Discussion
All of the synthesised peptides had strong ACE-inhibitory activity ranging between
34-41 µM after pre-incubation with ACE. The dodecapeptide AVPYPQRDMPIQ
had the lowest concentration of peptide required to inhibit ACE (34.66 µM) by 50%
indicating strong ACE-inhibitory activity. Several studies have reported ACE-
inhibitory peptides with similar IC50 values (Hernández-Ledesma et al. 2011) that
include: the peptide YKVPQL derived by digestion of αs1-casein using a L.
helveticus proteinase (22 µM), another peptide derived by pepsin hydrolysis derived
from αs2-casein f (89-95) YQKFPQY (20.1 µM) and a peptide derived from the
whey protein く-lactaglobulin by thermolysin hydrolysis LQKW (34.7 µM). Various
other studies have shown peptides with better ACE-inhibitory activity such as VPP
(5 µM) derived by fermentation with L. helveticus and RYLGY (0.71 µM) and also
peptides with weaker ACE-inhibitory activity such as FFVAPFPGVFGK (77 µM)
derived by trypsin hydrolysis and LLF (79.8 µM) derived by thermolysin hydrolysis
(Nakamura et al. 1995a, Seppo et al. 2003, Murray and Fitzgerald 2007, Contreras et
al. 2009, Hernández-Ledesma et al. 2011)
The ACE-inhibitory activity of the synthetised peptides can be further confirmed
using spontaneously hypertensive rats (SHRs). Unfortunately, due to time and cost
limitations this was not possible in this research. However, determining their stability
against various gastrointestinal enzymes including pancreatin (a mixture of enzymes
containing lipases, amylases and various proteases including chymotrypsin and
trypsin) and pepsin that mimics the gastrointestinal environment provides an
estimation of their prospective activity in vivo.
200
The peptides were pre-incubated with ACE to classify their type of inhibition to be
inhibitor, substrate or prodrug-type activity (Fujita and Yoshikawa 1999). The
peptides AVPYPQRDMPIQ and YLGYLEQLLR both acted as pro-drug type
inhibitors therefore being hydrolysed by ACE into smaller stronger inhibitory
peptides (the true inhibitors of ACE) than their primary structures. It may be
hypothesised that the propensity of these two peptides to form helical structures, as
evidenced by the NMR data, may be responsible for the pro-drug type activity of
these peptides. However, the peptide DIPNPIGSEN acted as a substrate-type
inhibitor meaning that the peptide is hydrolysed by ACE into less active or non-
active peptides (Fujita and Yoshikawa 1999, Li et al. 2004). It should be noted that
the NMR data of this peptide showed random conformational structures. Therefore,
the peptides AVPYPQRDMPIQ and YLGYLEQLLR may be contenders for further
study using spontaneously hypertensive rats (SHRs). Few studies have shown that
the peptides derived from food proteins display pro-drug activity. A study by Fujita
and Yoshikama showed that the peptide LKPNM derived from bonito, a fish species,
had pro-drug like activity against ACE (Fujita and Yoshikawa 1999).
The peptides YLGYLEQLLR (CP) and DIPNPIGSEN (MP1) either had unchanged
or lower ACE-inhibitory activity after exposure to pancreatin and pepsin enzymes
when compared with incubation with ACE only. However, the peptide
AVPYPQRDMPIQ had stronger activity against ACE after incubation with pepsin
and pancreatin. As per the ACE-inhibitory assay to classify their inhibitor type, this
assay further confirms that the peptide AVPYPQRDMPIQ has pro-drug type activity
because it is hydrolysed by the proteases pepsin, trypsin and chymotrypsin into
201
stronger ACE-inhibitory peptides. The inhibitory activity of the peptides
YLGYLEQLLR and DIPNPIGSEN remained unchanged compared with exposure to
ACE alone, suggesting that their activity in vivo possibly would be minimal.
The peptide YLGYLEQLLR was shown by NMR analysis to have weak propensity
to form helical structure (Figure 5.7 (b)). This was also confirmed by medium range
ROE connectivity between isoleucine (Ile-6) and proline (Pro-3) and also between
the isoleucine (Ile-2) and glycine (Gly-3). Other than this all other ROE
connectivities were linear between adjacent residues.
A study using circular diachroism (CD) showed that this peptide in an SDS
environment formed a amphipathic 310helical structure (Lecouvey et al. 1997).
Further NMR studies of this peptide in an SDS environment would be significant to
evaluate if the peptide has tendency to form stable secondary structure in the
presence of a membrane mimicking environment. The peptide DIPNPIGSEN
displayed random conformational structures as revealed by chemical shift analysis.
Chemical shift analysis of the dodecapeptide AVPYPQRDMPIQ showed a strong
propensity to form helical structure in the region between the residues three and
eight. NMR data also revealed that this peptide forms cis and trans isomers possibly
due to the presence of the proline residues in this sequence.
There are no reported studies on the structure-activity relationships of bioactive
peptides derived from milk protein that have been examined by NMR spectroscopy.
202
However, there have been a few studies that have used NMR spectroscopy to
determine the structure of milk proteins and peptides including kappa casein peptides
PP3 glycopeptide and kappa casein macropeptide (Belloque and Ramos 1999).
Generally, strong ACE-inhibitory peptides derived from milk protein have specific
characteristics. They may contain proline or hydrophobic residues such as
tryptophan, tyrosine or phenylalanine at their carboxyl terminal end which is where
the peptide is thought to bind to ACE (Meisel 1998, Saito 2008). Furthermore, the
proline residues are resistant to degradation by digestive enzymes and positively
charged amino acids arginine and lysine are thought to increase ACE-inhibitory
activity.
The peptides examined in this research have some of the above characteristics shown
by other studies on strong ACE-inhibitory peptides. The peptide YLGYLEQLLR
contains arginine residues in its C-terminal, the peptide DIPNPIGSEN contains two
proline residues in its sequence and the peptide AVPYPQRDMPIQ contains several
proline residues throughout the sequence including the C-terminal end.
Consequently, the peptide AVPYPQRDMPIQ has the strongest ACE-inhibitory
activity potentially due to the presence of several proline residues as well as the
positively charged arginine residue.
The NMR analysis of the dodecapeptide AVPYPQRDMPIQ showed strong
propensity to form helical structure in the region between residues three and eight. In
addition to the presence of proline and the positively charged arginine residues that
203
contribute to ACE-inhibitory activity, the helical structure of this peptide
(AVPYPQRDMPIQ) may be responsible for further enhancing its activity. The
ROESY spectrum of this peptide was complex, possibly due to the presence of cis-
trans isomers caused by the three proline residues in its sequence. Therefore, it was
not possible to undertake ROE-based structure analysis. It would be interesting to
conduct NMR experiments on this peptide in a membrane mimicking environment,
where it would be likely to display much stronger and stable secondary structure.
5.5 Conclusions
The peptides YLGYLEQLLR, DIPNPIGSEN and AVPYPQRDMPIQ were all
shown to be ACE-inhibitory. However, the peptides YLGYLEQLLR and
AVPYPQRDMPIQ were shown to have pro-drug like activity against ACE as they
were hydrolysed by ACE into more inhibitory peptides. Furthermore, the peptide
AVPYPQRDMPIQ was hydrolysed into stronger ACE-inhibitory peptides by the
gastrointestinal enzymes pepsin and pancreatin. Therefore, the peptide
AVPYPQRDMPIQ is the strongest ACE-inhibitory peptide derived from milk
protein that could be potentially useful in vivo.
Furthermore, the peptide AVPYPQRDMPIQ has various characteristics consistent
with the literature that are pertinent to strong ACE-inhibitory peptides including the
presence of proline residues throughout the structure and particularly at the C-
terminal end. The pbelresence of arginine residues may also contribute to its strong
ACE-inhibitory activity. The NMR analysis of this peptide showed strong propensity
204
to form helical structures in the region between the residues three and eight which
may be linked to its enhanced activity.
NMR analysis showed that the peptide DIPNPIGSEN had mostly random
conformational structures. However, the peptide YLGYLEQLLR showed some
propensity to form alpha helical structure.
205
Chapter 6 Conclusions and Future Research
This research investigated the presence of bioactive peptides in fermented organic
milk, commercial Cheddar cheeses and various hydrolysates. All samples were
screened for antioxidant activity against the free radical DPPH, antimicrobial activity
against Escheridia coli, Bacillus cereus and Staphylococcus aureus and inhibition of
the activity of the angiotensin-I-converting enzyme (ACE).
All the food proteins, when hydrolysed, produced peptides that had varying degrees
of bioactivity, particularly ACE-inhibitory activity while the antioxidant activity of
most samples was low (<20%). Enzymatic hydrolysis generally resulted in stronger
antimicrobial and ACE-inhibitory peptides identified compared with peptides
derived by fermentations.
The techniques used to derive bioactive peptides included screening a large volume
of samples then fractionating a smaller set of samples followed by identification via
mass spectrometry and NMR analysis. Potentially, these techniques could be used to
derive bioactive peptides from other protein sources. Interestingly, the large sample
set resulted in bioactive peptides being identified from all protein types- cheese
casein, fermented protein and hydrolysed protein. This could be due to the
accessibility of the enzymes to the casein protein where it is easily hydrolysed. The
use of different food proteins may not result in large concentrations of peptides or
indeed bioactive peptides generated such as in fish protein (Salampessy 2010). This
may be due to the biological implications of milk as it is full of bioactive nutrients
206
for the calf and therefore has a greater capacity to generate bioactive peptides than
other non-mammalian protein sources. However, the use of other types of milk may
result in larger concentrations of particular bioactive peptides being generated due to
the variation in casein and whey concentrations.
The water-soluble extracts from five commercial Cheddar cheeses examined in this
research showed low antioxidant activity (<20%), good antimicrobial activity
(44.25% 1.16 mg/mL against B. cereus) and strong ACE-inhibitory activity (IC50:
0.04 mg/mL). The ACE-inhibitory fraction derived from Cheddar cheese E was
analysed by mass spectrometry and shown to contain two peptides. Both peptides are
derived from αs1-casein: f(91-100) YLGYLEQLLR (MW: 1266.70) and
FFVAPFPEVFGKEK (MW: 1383.72). They have previously been used in
commercially available food ingredients known as Lactium and soft drinks in Japan
and the USA. The peptide YLGYLEQLLR was synthesised by Genscript (GenScript
USA Inc.Piscataway, NJ, USA) for further characterisation.
The organic milk protein was extracted and fermented separately using four probiotic
bacteria namely Lactobacillus acidophilus, L. casei, L. helveticus and L. rhamnosus
for 24 hours. The fermented milk protein peptide extracts showed good bioactivity;
however, the slow proteolytic activity of the bacterial enzymes resulted in low
concentration of peptides compared with the Cheddar cheese extracts and
hydrolysates therefore screening was not continued.
207
Five enzymes i.e. papain from papaya fruit, bromelain from pineapple stem, rennin
from calf stomach, Flavourzyme and Fromase were used to derive peptides from
organic milk protein. The results have shown that the hydrolysate fractions do exhibit
various bioactivities. The hydrolysate fractions showed low antioxidant activity
(<30% inhibition) except for Flavourzyme hydrolysates (>80%), which were
attributed to the enzyme. The antimicrobial activity was greatest against S. aureus
particularly by Flavourzyme hydrolysates. Papain and Flavourzyme hydrolysates
derived from the insoluble fraction had the greatest ACE-inhibitory activity. Several
fractions were analysed by Mass Spectrometry and their peptide sequences identified
by MASCOT database searching including 5F0.51S, 5F10.5IF2 and 5P0.53IF2B.
The hydrolysate fraction that had the best antimicrobial activity was fraction 1
containing 5 kDa peptides derived by Flavourzyme hydrolysis of the soluble protein
fraction (0.5% enzyme) for one hour (5F0.51SF1). It inhibited the growth of S.
aureus by 69.35% ±3.02 at 0.009 mg/mL. The fraction 5F0.51S, due to lack of peaks
in fraction 1, was analysed by Mass Spectrometry and MASCOT database searching
and contained 11 peptides.
The ACE-inhibitory fraction 5F10.5IF2 was shown to contain three peptides. It was
derived by Flavourzyme hydrolysis (0.5% enzyme to protein) of the insoluble protein
fraction for one hour and was from fraction number two. It was shown to have strong
ACE-inhibitory activity (IC50: 0.093 ±0.006 mg peptide/mL), which is in agreement
with the study by Hayes et al (2007b).
208
The ACE-inhibitory fraction containing peptides less than 5 kDa derived from
Papain hydrolysis of the insoluble fraction (number 2B; 0.5% enzyme to protein ratio
for three hours hydrolysis) was shown to contain 14 peptides derived from く-casein
(6 novel), 8 peptides derived from αs1-casein (4 novel) and peptides from κ-casein
(3 novel).
Three peptides were synthesised by Genscript (GenScript USA Inc.Piscataway, NJ,
USA). They were YLGYLEQLLR (shown to be potentially ACE-inhibitory and
derived from organic Cheddar cheese E), DIPNPIGSEN (derived from antimicrobial
hydrolysate 5F10.5S) and AVPYPQRDMPIQ (derived from ACE-inhibitory fraction
5P0.53IF2B) and their ACE-inhibitory activity was analysed, as well as stability to
the gastrointestinal enzyme pancreatin which contains pepsin and chymotrypsin. The
peptides YLGYLEQLLR, DIPNPIGSEN and AVPYPQRDMPIQ were all shown to
be ACE-inhibitory. However, the peptides YLGYLEQLLR and AVPYPQRDMPIQ
were shown to have pro-drug like activity against ACE as they were hydrolysed by
ACE into smaller, more inhibitory peptides. Furthermore, the peptide
AVPYPQRDMPIQ was hydrolysed into stronger ACE-inhibitory peptides by the
gastrointestinal enzyme pancreatin. Therefore, the peptide AVPYPQRDMPIQ is the
strongest ACE-inhibitory peptide derived from milk protein that could be potentially
useful in vivo.
The structure-activity relationship of all three peptides was determined using nuclear
magnetic resonance (NMR) studies. The peptides YLGYLEQLLR possibly
contained weak alpha helical structures as revealed by chemical shift index (CSI)
209
analysis. This was also confirmed by medium range ROE connectivity between
isoleucine (Ile-6) and proline (Pro-3) and also between the isoleucine (Ile-2) and
glycine (Gly-3). However, the peptide DIPNPIGSEN was shown by NMR analysis
to have random conformational structures. The chemical shift differences plot of the
peptide AVPYPQRDMPIQ showed strong propensity to form helical structures in
the region between the residues three and eight.
Future directions of this research could include conducting mixed fermentations
using synergistic bacteria with varying degrees of proteolytic activity that could
result in the production of larger amounts of peptides that may be potentially
bioactive. Also, research could include synthesising the novel peptides identified in
this study (Table 4.2) and screening them for various bioactivities including ACE-
inhibitory and antimicrobial against a larger set of bacteria. The use of confocal
microscopy could potentially elucidate the mechanisms of the peptides inhibiting the
bacteria. Furthermore, nuclear magnetic resonance (NMR) could be used to
determine the structure-activity relationship of the novel potentially ACE inhibitory
peptides FAQTQSL and IPNPIGSEN and also investigate their structures in a
membrane mimicking environment. The ACE-inhibitory peptides could be
investigated for their hypotensive activity in vivo using spontaneously hypotensive
rats (SHRs). Also, bacterial or yeast vectors could be used to amplify the bioactive
peptide sequences.
210
Other research concerning bioactive peptides could include investigating their taste
and flavour properties, role in food preservation, and their potential role in regulatory
functions of obesity and anorexia (Pellegrini 2003).
Various enzymes derived from plant, microbial or animal sources could be used to
derive a much wider variety of bioactive peptides. These and also the many bioactive
peptides identified but not characterised in this project could be investigated further
using NMR characterisation and circular diachroism (CD) analysis.
Globally, this research could provide the nutraceutical and functional food industries
with knowledge-based information on the peptides available via the use of various
enzymes and their potential uses in food ingredients or products.
211
Appendices
212
Appendix 1
Raw Proximate Composition Analysis Methods and Data
Ash Five porcelain crucibles with lids were labelled numerically (1-5) and placed into a muffle furnace (Ceramic Engineering Furnace Manufacturers, Sydney, Australia) at 525°C for thirty minutes, removed and placed into a desiccator, after ten minutes cooling, for thirty minutes (Pearson 1976). The crucibles with lids were weighed and approximately 5mL of ‘You love Coles’ lite organic milk placed into each crucible. The final weight was recorded. The crucibles were placed over a hot plate until milk became charred, and then placed into the muffle furnace overnight. The crucibles were removed and placed into desiccator until cooled. The crucibles were weighed. Percentage of ash was determined as below:
% ash = weight of ash x 100
weight of sample 1 Total Solids and Moisture content Twelve aluminium dishes were placed in an air oven (D + A Laboratory Services, Baulkham Hills, New South Wales, Australia) at 105°C for one hour then stored in a desiccator (Pearson 1976). The dishes were weighed before approximately 5mL of ‘You love Coles’ flite organic milk was added to the dishes and the final weights were recorded. The dishes were placed in the air oven overnight. Then they were removed and stored in the desiccator before being reweighed. The total solids percentage was calculated as per below:
(dish weight – (dry sample + dish weight) x 100 (dish weight – (wet sample + dish weight)
The total moisture content was calculated by 100 – total solids percentage. Nitrogen and Protein Analysis- Kjeltec System (Kjeldahl Method) The digestion block (2006 Digestor, FOSS Tecator, North Ryde, Australia) was preheated to 420°C. Three millilitres of milk was added to 5 digestion tubes. Two kjeltec catalyst tablets and 15 mL sulphuric acid was added to each tube as well as a blank tube (containing no milk) and mixed. The sample was digested until the solution was clear (approximately 40 minutes). The tubes were cooled and samples were distilled (2200 Kjeltec auto distillation, FOSS Tecator, North Ryde, Australia). Samples were titrated using 0.1 M HCl until grey end point is reached (Pearson 1976). The percentage of nitrogen was calculated as follows:
14.01 (sample titrant-blank titrant) x 0.1M sample weight x 10
213
The percentage of protein was calculated by nitrogen percentage times 6.38 (nitrogen conversion factor for milk). Fat Analysis- Babcock Method Approximately, 25.53mL of ‘You love Coles’ lite organic milk was weighed and made up to 200mL with distilled water. The final weight was recorded. 17.6mL was pipetted into 6 skim milk Babcock flasks before 1mL Zephiran was added and mixed. 17.6mL sulphuric acid (1.822 density ±0.005 g/mL at 20°C) was added slowly. The flasks were centrifuged for five minutes (Scientific apparatus, H. I Clements and Sons Pty. Ltd, Sydney, Australia) before 60°C distilled water was added up to neck of flask. The flasks were recentrifuged 2 minutes before 60°C distilled water was added up to second graduation mark, and recentrifuged for one minute. The flasks were placed in a 60°C water bath (Labec, Marrickville, Australia) for five minutes (Horwitz 1975). The fat percentage was read and fat content (g/100g) was calculated by:
Babcock fat reading x 100 Weight of milk
Results Ash
Rep Cruicible + lid
crucible + lid + milk
crucible + lid + ash
milk sample- blank
ash sample-blank ash/milk average S.D
1 28.6702 33.843 28.7073 5.1728 0.0371 0.717 0.710 0.016211 2 30.6783 35.8761 30.7137 5.1978 0.0354 0.681
5 30.3901 35.499 30.4267 5.1089 0.0366 0.716 6 30.9417 36.1004 30.9788 5.1587 0.0371 0.719 7 31.748 36.7777 33.4224 5.0297 1.6744 33.290 8 32.0891 37.1802 32.2997 5.0911 0.2106 4.137 9 31.823 37.1882 31.1319 5.3652 -0.6911 -12.881 10 32.2093 37.6854 33.1696 5.4761 0.9603 17.536 11 30.525 35.7237 30.4399 5.1987 -0.0851 -1.637 12 32.5003 37.6537 32.5372 5.1534 0.0369 0.716 13 32.2931 37.497 29.9252 5.2039 -2.3679 -45.502 14 29.0582 34.1375 29.9238 5.0793 0.8656 17.042
Moisture and Total Solids
Rep Pan Wgt Wet/Pan dry/pan Total Solids % Average S.D
1 46.9099 52.0857 47.4006 9.481 9.516 0.04019 2 46.5295 51.6691 47.0155 9.456
3 71.1513 76.2666 71.6363 9.481 4 36.7451 42.0821 37.2527 9.511 5 70.9685 76.1401 71.4594 9.492 6 68.9457 73.9659 69.422 9.488 7 37.8927 43.0642 38.3848 9.516 8 37.5956 42.9932 38.1092 9.515
214
9 41.4447 46.8088 41.9558 9.528 10 42.3543 47.5494 42.852 9.580 11 53.2019 58.3964 53.6993 9.576 12 50.8881 55.9267 51.3701 9.566
Protein Sample Titrant Blank %N %protein Average S.D 1 11.2 0.05 0.520705 3.322098 3.331632 0.020792 2 11.15 0.05 0.51837 3.307201
3 11.3 0.05 0.525375 3.351893 4 11.31 0.05 0.525842 3.354872 5 11.2 0.05 0.520705 3.322098
Fat
Rep Babcock reading
Final Weight
Fat content (g/100g) Average S.D
1 0.2 25.5399 0.783 0.848 0.505481 2 0.2
0.783
3 0
0.000 4 0.25
0.979
5 0.4
1.566 6 0.25
0.979
215
Appendix 2: Gradient Programs used for separation of peptides. First separation method for cheese extracts/fermented extracts/hydrolysates: Time (min) % Solvent B (ACN containing 0.1% TFA) 0.01 2% 60.00 60% 61.00 2% 76.00 2% Fractionated cheese extracts: As per above, collected with fraction collector 3 mL vials. Fractionation of ACE hydrolysates (modified from Verdini et al., 2004) Time (min) % Solvent B (ACN containing 0.1% TFA) 0.01 0% 0.04 lock fraction collector 10.00 0% 20.00 unlock 30.00 50% 33.00 lock 38.00 unlock 43.00 lock 44.00 50% 45.00 100% 55.00 100%
216
Appendix 3: SDS-PAGE molecular weight data and gels for cheese peptide extracts.
Gel 1: Non-organic Cheddar cheese peptide extracts (MWCO fractions)
Lane 1: Peptide standards Lane 2: Smaller than 5kDa Cheddar cheese B Lane 3: Smaller than10kDa Cheddar cheese B Lane 4: Larger than 10kDa Cheddar cheese B Lane 5: Empty Lane 6: Smaller than 5kDa Cheddar cheese C Lane 7: Smaller than10kDa Cheddar cheese C Lane 8: Larger than 10kDa Cheddar cheese C Lane 9: Empty Lane 10: Protein standard Abbreviations shown in brackets.
1 2 3 4 5 6 7 8 9 10
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Gel 2: Non-organic Cheddar cheese extracts (MWCO fractions and whole)
Lane 1: Peptide standards Lane 2: Smaller than 5kDa Cheddar cheese A Lane 3: Smaller than10kDa Cheddar cheese A Lane 4: Larger than 10kDa Cheddar cheese A Lane 5: Empty Lane 6: Empty Lane 7: Cheddar cheese A Lane 8: Cheddar cheese B Lane 9: Cheddar cheese C Lane 10: Protein standard Abbreviations shown in brackets. 5 µL loaded into each well on both gels. The molecular weights and amounts of peptide in each band were estimated using Labworks software.
1 2 3 4 5 6 7 8 9 10
218
Gel 1: Non-organic Cheddar cheese peptide extracts (MWCO fractions) Experiment Id: -1 Date/Time: 2009-03-27 12:35:49 Modified Date/Time: 2009-03-27 12:35:49 Title: Cheese Peptide Gel 1 Experimenter: steph Images: Description: Mol. Weight Standard: Broad range protein standard unstained Mol. Weight Unit: kDa Amount unit: Lanes: Lane 1 Lane 2 Lane 3 Lane 4 Lane 6 Lane 7 Lane 8 Lane 10 Peptide Standards 5DM 10DM 20DM 5NIM 10NIM 20NIM Protein Standard Rows (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) r1 250 .10247 r2 150 .01989 r3 100 .28760 r4 84.581 .524 74.697 1.591 75 .40648 r5 50 .70471 r6 37 .08749 r7 25 .20516 r8 20 .53645 r9 14.586 .502 15 .06111 r10 11.663 .871 13.043 .368 r11 10.724 1.683 11.183 2.227 10 1.3005 r12 8.455 1.382 8.338 .199 r13 6.130 1.031 6.574 1.403 6.5741 1.2777 r14 5.797 .863 5.879 1.182 r15 5.481 .853 5.559 .321 5.5781 .01042 Sum 5 5 5 5 In Lane 5 5 5 5
Only wells loaded are shown.
Gel 2: Non-organic Cheddar cheese extracts (MWCO fractions and whole) Experiment Id: -1
219
Date/Time: 2009-03-27 12:35:49 Modified Date/Time: 2009-03-27 12:35:49 Title: Experimenter: steph Images: Description: Mol. Weight Standard: Broad range protein standard unstained Mol. Weight Unit: kDa Amount unit: Lanes: Lane 1 Lane 2 Lane 3 Lane 4 Lane 7 Lane 8 Lane 9 Lane 10 Peptide standards 5CB 10CB 20CB CB DM NIM Protein Standards Rows (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) (mol.w.) (amount) r1 250 .14024 r2 150 .37560 r3 109.08 1.1336 109.47 1.0748 113.92 1.6031 r4 100 .43670 r5 75 .49780 r6 50 .34694 r7 37 .13234 r8 25 .34049 r9 20 .24154 r10 18.250 .68967 18.510 2.4286 18.415 2.0721 18.606 1.7540 r11 17.132 .10207 r12 16.165 1.0840 r13 15 1.7331 r14 5.4433 1.5631 6.0240 1.4378 1.6128 1.8531 1.1899 1.5408 1.7849 5 10 .75527 r15 .15670 1.6633 Sum 5 5 5 5 5 5 In Lane 5 5 5 5 5 5
Only wells loaded are shown.
220
Gel 3: Organic Cheddar cheese extracts
221
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223
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225
226
227
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