Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: 
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you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. A comparative study of immunomodulatory activity
of deer and cow milk proteins
A thesis
Submitted in partial fulfilment
of the requirements for the Degree of
PhD of Food Science
at
Lincoln University
by
Nelum Lawanya Opatha Vithana
Lincoln University
2012
i
Declaration
Parts of this thesis are submitted or accepted for publication and/or presented in advance of
submission of the thesis:
Publications
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton (2012).
Immunomodulatory activity of fresh and fermented red deer and cow milk. Food and
Bioprocess Technology Journal (submitted).
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton (2012). In vitro digestion
of red deer (Cervus elaphus) and cow (Bos taurus) milk. International Food Research
Journal. 19: 409-416.
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton (2011). The release of
peptides by in vitro digestion of fermented red deer (Cervus elaphus) and cow (Bos taurus)
milk. Proceedings of the Nutrition Society of New Zealand. Volume 35. (Full paper accepted)
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton (2011). The release of
peptides by in vitro digestion of fermented red deer (Cervus elaphus) and cow (Bos taurus)
milk. Proceedings of the Nutrition Society of Australia.35: 66. (Abstract)
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton (2010) Immunomodulatory
bioactives from red deer milk. Proceedings of the IDF International Dairy conference.
http://www.wds2010.com/delegates/posters/DST-Opatha%20Vithana.Pdf (Abstract)
Presentations and Posters
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton. The composition and
immunomodulatory properties of milk from red deer and cows. Annual Conference of the
Nutrition Society of New Zealand. 22nd November – 23rd December 2012. Auckland, New
Zealand.
ii
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton. The release of peptides by
in vitro digestion of fermented red deer (Cervus elaphus) and cow (Bos taurus) milk. Joint
Annual Conference of the Nutrition Society of New Zealand and the Nutrition Society of
Australia. 29th November – 2nd December 2011. Queenstown, New Zealand.
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton. Immunomodulatory
bioactives from red deer milk. Lincoln University Postgraduate Conference. 2nd – 3rd
September 2010. Lincoln University, New Zealand.
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton. Immunomodulatory
bioactives from red deer milk. IDF International Dairy conference. 4th – 11th November 2010.
Auckland, New Zealand.
N. L. Opatha Vithana, S. L. Mason, A. E. A. Bekhit, J. D. Morton. Immunomodulatory
bioactives from red deer milk. New Zealand Society for Biochemistry and Molecular
Biology: Canterbury Branch Regional Meeting. 21st September 2009. Lincoln University,
New Zealand.
iii
Abstract of a thesis submitted in partial fulfilment of the
requirements for the Degree of PhD of Food Science.
Abstract
A comparative study of immunomodulatory activity
of deer and cow milk proteins
by
Nelum Lawanya Opatha Vithana
The aim of this study was to compare the immunomodulatory activity of peptides derived
from deer and cow milk using in vitro lymphocyte proliferation assays and cytokine (IL-2
and INFγ) production by human peripheral blood mononuclear cells (PBMC). Bioactive
peptides were produced using in vitro digestion and lactic acid bacteria (LAB) fermentation.
Deer and cow milk were fermented using Lactobacillus delbrueckiisubspbulgaricus,
Streptococcus salivarius subsp thermophilus and Lactobacillus casei strain Shirota at 37oC
for 24 hours. Unfermented and fermented milk proteins were digested in two steps; imitating
both the human stomach (Pepsin, pH 2.5, 30 min) and the duodenum (Corolase PP, pH 7.5,
30 min) digestion. The production of peptides was quantified using the O-phthaldialdehyde
assay and the degradation patterns of the milk proteins were analysed by SDS-PAGE.
Immunomodulatory activity of each hydrolysate was carried out for 0.125, 0.25 and 0.5
mg/ml protein concentrations. All unfermented and fermented deer and cow milk digestions
were fractionated using Fast Protein Liquid Chromatography (FPLC) in order to separate and
identify the most active fractions.
Identification of peptides present in these
immunostimulating fractions were carried out using Liquid Chromatography–Mass
Spectrometry (LC-MS/MS) and database searching.
Deer milk contained 8.8 ± 0.13% protein which was twice the level found in cow milk (4.1 ±
0.02%). Peptide production was significantly higher in unfermented deer milk than
unfermented cow milk (P< 0.05). The commercial proteolytic enzymes degraded milk
proteins from deer more rapidly than those from cow. Deer milk fermentation gave higher
peptide production than cow milk fermentation. Following in vitro digestion peptide
production was significantly greater in deer milk ferment than cow milk ferment (P≤ 0.05).
iv
Lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (INFγ) production were
significantly greater in deer milk digestions than cow milk digestions (P ˂ 0.001)at the same
concentration. LAB fermentation of both deer and cow milk prior to in vitro digestion
increased (P < 0.001) lymphocyte proliferation. Fermentation with lactobacillus strains
increased lymphocyte proliferation and cytokine IL-2 and IFNγ production (P < 0.05) more
than with streptococcus. The immunostimulating effect was significantly greater (P ˂ 0.001)
at 0.125 mg/ml protein concentration than at higher protein concentrations.
The deer milk digests were more effective at increasing lymphocyte proliferation than the
cow milk digests (P < 0.001) at the same protein concentration. Proliferation was higher (P <
0.001) at 0.125 mg/mL protein than at higher protein concentrations.
FPLC fractions (11, 22 and 30) with high immunostimulatory activity from deer digests
(unfermented and fermented) showed higher lymphocyte proliferation and higher cytokine
IL-2 and IFNγ production than the correspondening fractions of cow digests when measured
in vitro with human PBMC.
Mixtures of different peptides with different protein origin have been identified in the above
FPLC fractions in both deer and cow milk digests which could be responsible for
theimmunostimulating effect. The main protein source was β casein for both deer and cow
fractions. Some differences in amino acid composition of β casein, α1 and α2 casein were
identified between deer milk than cow milk. Few previously identified immunostimulating
peptide sequences were found in tested FPLC fractions in cow digests. Some different
peptides were detected in deer fractions compared to corresponding cow fractions. No
previously identified immunostimulating peptides were detected in deer digests. Further
studies are required to further purify and identify unknown immunostimulating peptides. The
activity could be confirmed with synthesised peptides.
Keywords: deer milk, cow milk, milk proteins, peptides, in vitro digestion, fermentation,
immunomodulatory activity, human PBMC, lymphocyte proliferation, cytokine production,
FPLC, LC-MS/MS, database searching
v
Acknowledgements
I would like to thank my main supervisor Dr. Sue Mason for her patience, time,
guidance,knowledge and advice. I would also like to thank my associate supervisors Dr.
Aladin Bekhitand Prof. Jim Morton for their expertise, time and advice throughout the
project. Having access to such individuals with their specialized knowledge allowed the
project to flow smoothly. I also would like to acknowledge Dr. Richard Sedcole for helping
me with statistical analysis and for his expertise knowledge.
I acknowledge the support and cooperation of Martin Ridgway and Martin Wellby, Lincoln
University deer farm and Mr. Keith Neylon (Blue River Dairy) for the donation of sheep
milk.Additional acknowledgements go to Dr. Stefan Clerens from AgResearch who helped
with the mass spectrometric analysis.Also I would like to thank Karl, Omega, Nadia and all
other technical staff at Wine, Food and Molecular Biosciences Department that helped in
completing this project.
I would like to thank my loving husband Hashan, daughter Linara, parents and brothers for
their
support
and
guidance
throughout
the
doctoral
programme.
Additional
acknowledgements include fellow lab mates, postgraduate students and friends for their help
in surviving the undertaking of the doctoral programme. I would not have been able to
complete my doctorate successfully without help of all mentioned above.
This research could not have been conducted without the financial support of Lincoln
University. Also part of this project was funded by the Primary Partnership fund from Otago
University. I acknowledge their partnership in the project and would like to thank them for
their contribution.
vi
Table of Contents
Declaration……………………………………………………………………………………ii
Abstract ............................................................................................................................... iv
Acknowledgements............................................................................................................. ivi
Tabel of Content................................................................................................................. vii
List of Tables ........................................................................................................................ x
List of Figures...................................................................................................................... xi
Chapter 1 Introduction ........................................................................................................ 1
Chapter 2 Literature Review............................................................................................... 5
2.1 Milk.............................................................................................................................. 5
2.2 Milk Bioactives............................................................................................................. 5
2.3 Bioactive Peptides from Milk........................................................................................ 6
2.3.1 Immunomodulatory Bioactive Peptides from Milk............................................. 8
2.3.2 Immunomodulatory Bioactive Peptides from Fermented Milk ........................... 8
2.4 Overview of the Immune System ................................................................................ 13
2.5 Deer milk,Usage and Potential of Immunomodulating Bioactives ............................... 23
Chapter 3In vitroDigestion of Red Deer (Cervus elaphus) and Cow (Bos taurus) milk .. 26
3.1Introduction..................................................................................................................... 26
3.2Materials & Methods....................................................................................................... 27
3.2.1 Materials ........................................................................................................... 27
3.2.2 Preparation of Milk Samples ............................................................................. 27
3.2.3 Protein Content ................................................................................................. 28
3.2.4 Dry Matter Content ............................................................................................ 28
3.2.5 In vitro Digestion .............................................................................................. 28
3.2.6 Proteolysis Assesment Using OPA Assay.......................................................... 29
3.2.7 SDS-PAGE ....................................................................................................... 29
3.2.8 Quantification of Protein Bands......................................................................... 29
3.2.9 Statistical Analysis ............................................................................................ 30
3.3Results & Discussion....................................................................................................... 30
3.4Conclusion ...................................................................................................................... 38
Chapter 4 Fermentation of Deer and Cow Milk ............................................................... 39
4.1Introduction..................................................................................................................... 39
4.2 Materials & Methods...................................................................................................... 40
4.2.1 Materials ........................................................................................................... 40
4.2.2 Milk Fermentation............................................................................................. 40
4.2.3 In vitro Digestion .............................................................................................. 40
4.2.4 Proteolysis Assesment Using OPA Assay.......................................................... 41
4.2.5 SDS-PAGE ........................................................................................................ 41
4.2.6 Statistical Analysis ............................................................................................ 41
4.3 Results ........................................................................................................................ 41
4.4 Discussion...................................................................................................................... 50
4.5 Conclusion…………………………………………………………………………….....54
vii
Chapter 5 Immunomodulatory Activity of Deer and Cow Milks..................................... 55
5.1Introduction..................................................................................................................... 55
5.2Material & Methods ........................................................................................................ 55
5.2.1 Materials................................................................................................ 56
5.2.2 Collection &Preparation of Milk Samples .............................................. 56
5.2.3 Fermentation.......................................................................................... 56
5.2.4 In vitro Digestion ................................................................................... 57
5.2.5 BCA Protein Assay ................................................................................ 57
5.2.6 Isolation of human PBMC...................................................................... 57
5.2.7 In vitro Lymphocyte Proliferation Assay ............................................... 57
5.2.8 Cell Viability Test.................................................................................. 58
5.2.9 Cytokine Production…………………………………………………... 58
5.2.10 Statistical analysis…………………………………………………… 59
5.3Results & Discussion....................................................................................................... 59
5.4Conclusion ...................................................................................................................... 71
Chapter 6 Comparative Study of Deer Milk with Three other mammallian Milks ........ 73
6.1 Introduction................................................................................................................... 73
6.2 Materials & Methods..................................................................................................... 73
6.2.1 Materials................................................................................................ 73
6.2.2 Compositional analysis .......................................................................... 74
6.2.3 Determination of BC of Deer, Cow, Goat & Sheep Milk ........................ 75
6.2.4 In vitro Digestion of Deer, Cow, Goat & Sheep Milk ............................ 75
6.2.5 SDS-PAGE of Deer, Cow, Goat & Sheep Milk ...................................... 76
6.2.6 Immunomodulation Activity……………………………………………..........76
6.2.6.1 In vitro Lymphocyte Proliferation Assay............................................. 76
6.2.6.2 Cytokine Production............................................................................ 76
6.2.7 Statistical Analysis…………………………………………………………….76
6.3 Results ........................................................................................................................ 77
6.4 Discussion...................................................................................................................... 87
6.5 Conclusion…………..…………………………………………………………………...95
Chapter 7Fractionation of Immunomodulatory Bioactive Peptides of deer and Cow
Milk Ferments and Digests ................................................................................................ 96
7.1 Introduction................................................................................................................... 96
7.2 Materials & Methods..................................................................................................... 97
7.2.1 Materials................................................................................................ 97
7.2.2 Fractionation of Peptide by FPLC .......................................................... 97
7.2.3 In vitro Lymphocyte Proliferation Assay................................................ 98
7.2.4 Cytokine Production ............................................................................... 98
7.2.5 BCA Protein Assay ................................................................................ 98
7.2.6 Statistical Analysis…………………………………………………….....98
7.3 Results ........................................................................................................................ 99
7.4 Discussion.................................................................................................................... 109
7.5 Conclusion……………………………………………………………………………..113
viii
Chapter 8Identification of Peptides from Immunostimulating Fractions of Deer and
Cow Milk Digestions ........................................................................................................ 114
8.1 Introduction................................................................................................................. 114
8.2 Materials & Methods................................................................................................... 114
8.2.1 Materials.............................................................................................. 114
8.2.2 Liquid Chromotography-Mass Spectrometry........................................ 115
8.2.3Data Base Searching……………………………………………………115
8.3 Results & Discussion.................................................................................................... 115
8.4 Conclusion ................................................................................................................... 123
Chapter 9 General Discussion , Conclusion & Future Work ......................................... 124
References......................................................................................................................... 128
Appendix A The release of peptides by in vitro digestion of fermented red deer
(Cervuselphus) and cow (Bos taurus) milk. ........................................................................ 150
Appendix BIL-2 production by human peripheral blood mononuclear cells (PBMC)
stimulated by Con A with FPLC fraction 13 and 36 of unfermented and fermented deer and
cow milk in vitrodigests ..................................................................................................... 155
Appendix CDeer and cow milk digests FPLC fractions absorbance at 280 nm .................. 156
ix
List of Tables
Table 2.1
Table 2.2
Table 2.3
Table 2.4
Table 2.5
Table 3.1
Table 3.2
Table 4.1
Table 5.1
Table 5.2
Table 6.1
Table 6.2
Table 6.3
Table 6.4
Table 6.5
Table 7.1
Table 8.1
Table 8.2
Table 8.3
Immunomodulatory peptides derived from milk proteins ..................................... 9
Immunomodulatory peptides derived from fermented milk ................................ 11
Cytokine families............................................................................................... 20
Traditional products and uses of Reindeer milk. ................................................. 24
Gross composition (%) of milk of human & some domesticated mammals......... 25
Dry matter & protein content of red deer and cow milk. ..................................... 30
Protein content (%) remaining in raw milk before digestion (start), after stage
1 digested with pepsin at pH 2.5 for 30 min / 37 °C and after stage 2 digested
with CPP at pH 7.5 for 30 min / 37 °C.. ............................................................. 37
Remaining protein content (%) in digest of milk, Lactobacillus delbrueckii
subsp bulgaricus, Streptococcus salivarius subs thermophilus and
Lactobacillus casei strain Shirota fermented milk before and after in vitro
digested.............................................................................................................. 49
ANOVA Table for percent change in lymphocyte proliferation for deer and
cow unfermented and fermented. ....................................................................... 65
Proportion of viable cells compares to control sample after up to three days
incubation with different protein concentrations................................................. 67
Mean of proximate composition for deer, cow, sheep and goat milks and
concentration of milk proteins............................................................................ 78
Major and minor mineral contents of deer, cow, sheep and goat milk. ................ 79
Mineral composition of red deer early, middle and late milking. ........................ 80
Canonical variate analysis for the red deer minerals. .......................................... 82
ANOVA Table for % change in lymphocyte proliferation. ................................. 84
ANOVA Table for % change of lymphocyte proliferation for FPLC fractions
13, 24 and 36 of unfermented and fermented deer and cow milk digests at
0.125 mg/ml protein concentration................................................................... 107
Total number of identified peptides contained and source of protein in FPLC
fraction 11, 22 and 30 from unfermented and Lactobacillus delbrueckii subsp
bulgaricus fermented cow and deer in vitro digestions...................................... 116
Comparison of amino acid residues in milk protein of deer with cows giving
its specific position base on results of peptides found in FPLC fractions. ......... 118
Sequences and corresponding milk protein fragment of peptides contained in
FPLC fractions................................................................................................. 120
x
List of Figures
Figure 1.1 Structure of chapters and research experiments of the thesis................................. 4
Figure 2.1 Summarizing scheme of the possible mechanisms by which bioactive peptides
can be released from the precursor proteins by microbial fermentation and/or
gastrointestinal digestion...................................................................................... 7
Figure 2.2 Different biological activites of peptides. ............................................................. 7
Figure 2.3 Integrated human immune system. ..................................................................... 14
Figure 2.4 Recognition by the immune system.................................................................... 16
Figure 2.5 Immune response. .............................................................................................. 17
Figure 2.6 Distribution of lymphocytes to the different organs............................................ 18
Figure 2.7 Regulation of adaptive immunity by cytokines................................................... 20
Figure 3.1 pH during in vitro digestion of deer andcow milk............................................... 31
Figure 3.2 Peptide production during in vitrodigestion of deer and cow milk simulated
stomach and then simulated duodenum. ............................................................. 33
Figure 3.3 SDS-PAGE of defatted Deer Milk & Deer Milk digested with pepsin and CPP.. 34
Figure 3.4 SDS-PAGE of skimmed CowMilk & CowMilk digested with pepsin and CPP. . 35
Figure 3.5 In vitro digestion of casein (a), α-lactalbumin (b), immunoglobulin (c) in raw
milk from deer and cow by pepsin at pH 2.5 / 37 °C and CPP at pH 7.5 / 37
°C. ..................................................................................................................... 36
Figure 4.1 Growth of Lactobacillus delbrueckiisubsp bulgaricus, Streptococcus salivarius
subsp thermophilus in deer and cow milk at 37°C for 24 hr............................... 42
Figure 4.2 Growth of Lactobacillus casei strain Shirota in deer and cow milk at 37°C for
72hr. .................................................................................................................. 42
Figure 4.3 pH drop during fermentation of deer and cow milk at 37°C..………………...... .44
Figure 4.4 Peptide production measured by OPA assay during fermentation of deer and
cow milk at 37°C. .............................................................................................. 45
Figure 4.5 Peptide production during in vitro digestion of 24hr fermented deer and cow
milk. .................................................................................................................. 46
Figure 4.6 Peptide production after in vitro digestion of raw milk, 24hr fermented milk
and fermented digests for deer and cow milk...................................................... 47
Figure 4.7 SDS-PAGE of cow and deer milk following fermentation.................................. 48
Figure 5.1 Percent change of lymphocyte proliferation at a) 0.125mg/mL, b) 0.25mg/mL
and c) 0.5mg/mL protein level for in vitro digestions of deer and cow milk and
fermented milk................................................................................................... 60
Figure 5.2 Percent change of lymphocyte proliferation of digest deer andcow milk at
0.125, 0.25 and 0.5 mg/mL protein concentrations. ............................................ 62
Figure 5.3 Percent change of lymphocyte proliferation with ConA and without ConA for
different protein concentrations of digested a) Deer and b) Cow......................... 63
Figure 5.4 Percent change of lymphocyte proliferation with ConA) and without ConA for
different protein concentrations of digests of Lactobacillus delbrueckiisubsp
bulgaricus ferments of a) Deer and b) Cow. ....................................................... 64
Figure 5.5 Interleukin-2 production by human peripheral blood mononuclear cells at
different protein concentrations of deer) and cow milk digesions and
fermented milk digestions. ................................................................................. 68
Figure 5.6 IFNγ production by human peripheral blood mononuclear cells at different
protein concentration of deer and cow milk digest and fermented milk digests. .. 69
Figure 6.1 Canonical analysis for 15 minerals at four milking times.................................... 81
Figure 6.2 Buffering index of cow, sheep and goat milks compared with deer milk............. 83
xi
Figure 6.3 SDS- PAGE of Deer, Cow, Sheep and Goat milk before and after digestion ...... 84
Figure 6.4 Percentage change of lymphocyte proliferation at 0.125mg/ml,0.25mg/ml and
0.5mg/ml protein concentration for deer, cow, sheep and goat milk digestions. .. 85
Figure 6.5 Interleukin-2 production by Con A stimulated human peripheral blood
mononuclear cells. ............................................................................................. 86
Figure 6.6 IFNγ production by Con A stimulated human peripheral blood mononuclear
cells. .................................................................................................................. 86
Figure 7.1 RP-FPLC chromatograms of the unfermented and fermented deer and cow
milk after in vitro digest. .................................................................................. 101
Figure 7.2 Percentage change in lymphocyte proliferation of FPLC fraction 11, 22 and 30
of unfermented and fermented deer and cow milk in vitro digests. ................... 102
Figure 7.3 Interleukin-2 production by human peripheral blood mononuclear cells
stimulated by Con A treated with FPLC fraction 11, 22 and 30 of unfermented
and fermented deer and cow milk in vitro digests. ............................................ 104
Figure 7.4 IFNγ production by human peripheral blood mononuclear cells stimulated by
Con A treated with FPLC fraction 11, 22 and 30 of unfermented and
fermented deer and cow milk in vitro digests .............................................. 105
Figure 7.5 Percentagechange of lymphocyte proliferation with ConA and without ConA
for different protein concentrations of deer milk digests FPLC fraction 30. ...... 106
Figure 7.6 IL-2 and IFNγ production by human peripheral blood mononuclear cells
stimulated by Con A with FPLC fraction 24 of unfermented and fermented
deer and cow milk in vitro digests. ................................................................... 108
Figure 8.1 Localization of identified peptides in FPLC fraction 30 of unfermented cow
and deer milk digests and digests of Lactobacillus delbrueckiisubsp bulgaricus
ferment of cow and deer on cow β casein. ........................................................ 122
xii
Chapter 1
Introduction
Milk has been described as nature’s most complete food (Park, 2009). It provides essential
nutrients during neonatal development and plays an important protective role in development
of the immune system of new-born mammals (Inglingstad et al., 2010; Korhonen and
Pihlanto, 2006). Ruminant milk and milk products have also formed an integral part of
human diets since the earliest domestication of livestock (Cross &Gill, 2000). The nutritional
benefits of milk derived protein, vitamins and minerals have been promoted extensively by
commercial dairy enterprises, and the vast range of milk based products now available are
partly the result of the continued image held by consumers of milk as a ‘healthy’ product.
Research has identified milk as a product with benefits beyond its nutritional value (Cross
&Gill, 2000). The concept of milk as a functional food with bioactive peptides and one
which has direct and measurable influence on the health of its recipient, has increasingly
gained scientific creditability (Meisel, 1997; Mclntosh et al., 1998; Meance et al., 2003;
Korhonen, 2009). Bioactive peptides have been defined as specific protein fragments that
have an impact on body functions or conditions and may ultimately influence health (Kitts &
Weiler, 2003). Bioactive peptides exhibit opiate-like, mineral binding, immunomodulatory,
antimicrobial, antioxidant, antithrombotic, hypocholesterolemic, and antihypertensive actions
(Erdmann et al, 2008) and most contain 2–20 amino acid residues per molecule, although
some are larger than 20 amino acids (Erdmann et al., 2008). Such peptides are inactive within
the sequence of the parent protein and can be released by digestive enzymes during
gastrointestinal transit or by fermentation or ripening during food processing (Korhonen &
Pihlanto, 2003). Immunomodulatory peptides have been identified from milk and milk
products (Fabienne Parher, 1984; Laffineur et al., 1996; LeBlanc et al., 2002; Silva et al.,
2005; Erdmann et al., 2008; Korhonen, 2009; Nagpal et al., 2011; Perez Espitia et al., 2012).
Currently milk proteins are the most important commercial source of bioactive peptides
(Korhonen, 2009) and widely studied but information on peptides from milk of species other
than cow is very limited.
The proteins in milk from different species vary in concentration and amino acid
composition. Cow milk has a protein content of approximately 3.3% (Park & Haenlein,
1
2006), whereas red deer milk contains more protein, approximately 7.6% (Arman et al.,
1974). This suggests deer milk might produce more bioactive peptides than cow milk after
fermentation and digestion. Along the borders between Russia, Mongolia, and China there is
historical use of deer milk to cure digestive problems; heal wounds, frostbite and other
injuries (Park &Haenlein, 2006), which suggest immunomodulating bioactive properties of
deer milk. Extracts of deer velvet have been shown to induce proliferation of a variety of cell
lines and trials to develop novel anti-cancer products and products to treat specific
gastrointestinal conditions are underway (Gosh & Playford, 2003; Yang et al., 2009). In
2010, Liu et al. reported that hydrolysed red deer plasma was rich in bioactives with AEC
inhibitory activity (Angiotensin I-Converting Enzyme Inhibitors). There is an interest in
bioactive peptides from deer products (velvet, antler) and deer milk which could be a good
source of bioactive peptides and is not utilized presently. New Zealand has 1.2 million
farmed deer which is half of the world’s farmed deer population.They are used to
producemeat and velvet. Therefore, there is a large potential for isolation of
immunomodulatory peptides from deer milk for pharmaceutical or nutraceutical purposes.
Hypotheses
•
Deer milk proteins are a better source of immunomodulating bioactive peptides than
cow milk proteins.
•
Lactic acid bacteria (LAB) fermentation of milk prior to digestion with commercial
enzymes will improve the formation of bioactive peptides.
Project Aims
There are a number of studies and reviews on cow milk bioactives (Minervini et al., 2003;
Gobbetti, 2007; Korhonen, 2009; Picariello et al., 2010) and a few on bioactives from other
species such as sheep and goat (Silva et al., 2006; Papadimitriou et al., 2007; HernandezLedesma et al., 2011), camel (El-Agamy, 2009), buffalo (Pandya and Haenlein, 2009) and
yak (Mao et al., 2005). To the author’s knowledge there are no reported studies on red deer
milk bioactives. This PhD thesis aimed at investigating immunomodulatory bioactive
peptides from red deer milk and comparedthem to cow milk bioactivity.
2
The first objective of this study was to investigate the in vitro digestion of deer and cow milk
by commercial gastric and duodenal enzymes with the major focus being on comparison of
the degradation of protein to evaluate the differences in digestibility and peptide production.
Secondly deer and cow milks were fermented using three LAB cultures to compare peptide
production before and after in vitro digestion and to compare deer and cow milk proteins
digestibility.
The LAB fermentation and in vitro digestion of both deer and cow milks were tested for
immunomodulatory activity. Lymphocyte proliferation and cytokine production (IL-2 and
IFNγ) were determined using human peripheral blood mononuclear cells (PBMC).
The immunomodulatory activity of deer milk in vitro digests was compared with that of milk
digests from three species (cow, sheep and goat).
Finally immunomodulatory peptides of deer milk were fractionated by FPLC and their
sequences were determined by mass spectrometry and compared with those of cow milk.
Thesis Structure
Including this introduction, this thesis contains nine chapters. Chapter 2 is the literature
review which gives background information on which this project is based. Chapters 3 to 8
are experimental chapters prepared as journal papers. Chapter 9 provides the overall
conclusions from the project, future work and is followed by a complete list of references.
Figure 1.1 details how the experimental chapters are related and data that contribute to more
than one chapter.
3
Chapter 1: Introduction
(Importance, aims and structure of the project)
Chapter 2: Literature review
(Background information necessary for project)
Chapter 3
Peptide production &
digestibility of deer and cow
milk protein by in vitro
digestion
Chapter 4
Peptide production after LAB
Fermentation&in vitro
digestion of deer and cow milk
protein
Chapter 5
Immunomodulatory activity of
unfermented & fermented deer and cow milk
protein
Chapter 6
Immunomodulatory activity of
deer milk comparedwith three
other milk species (cow, sheep
and goat)
Chapter 7
Fractionated immunomodulatory peptides from deer and cow milk digests
(unfermented and fermented) by FPLC
Chapter 8
Identification ofpeptides in immune active fractions by LC-MS/MS and sequencing
Chapter 9: General discussion, conclusion and future work
Figure 1.1: Structure of chapters and research experiments of the thesis.Chapters shown in
boxes are experimental chapters.
4
Chapter 2
Literature Review
2.1 Milk
Milk is the substance that is produced to feed the mammalian infant. All mammals, from
mice to whales, produce milk for this purpose. Milk has been described as the most perfect
food in nature and it is balanced for most nutrients and often has a high caloric value (Park,
&Haenlein, 2006). It contains lactose, protein, lipid and minerals which all are required for
optimal health and growth. The components of milk provide critical nutritive elements,
immunological protection, and biologically active substances to both neonates and adults
(Shah, 2000).
Milk and milk products are consumed everywhere and in 2010 the world total milk
production was 713.6 million tonnes (FAO,
http://www.fao.org/docrep/012/ak341e/ak341e10.htm). Cowshave been the major source of
milk and dairy products in developed countries, especially in the Western world (Haenlein,
2001) but milks from other mammals such as goat, sheep, reindeer, buffalo, camel and yak
are also consumed (Park, 2009).
2.2 Milk Bioactives
Research over the last twenty years has identified milk as a product that has a commercial
value beyond its traditional nutritional value (Cross & Gill, 2000). In their native form, milk
proteins exert an appreciable range of physiological activities. The major role of milk
proteins is to supply amino acids and nitrogen to young mammals and constitute an important
part of dietary proteins for the adult. Intact milk proteins also have specific functions such as
micelle formation (Nagpal et al., 2011). Specific immunoglobulins in milk provide the first
line of defence to suckling neonates through passively acquired immunity. Also milk is good
source of several hormones and growth factors (Grosvenor et al., 1993). Other non-specific
antimicrobial milk factors including the iron-binding protein, lactoferrin, and several
enzymes such as lactoperoxidase and lysozyme prevent microbial proliferation (Florisa et al.,
2003).
5
Although the protein fraction of milk has been the most widely studied in terms of bioactive
compounds, there are other compounds that have shown physiological significance in milk
such as some milk sugars (lactose, oligosaccharides), calcium and lipid-based bioactive
compounds (Shah, 2000). Lactose has been reported to enhance calcium absorption and
lactulose which is a product of lactose, is used as a promoter of probiotic bacteria and its use
is now widespread in infant formula (Shah, 2000). Calcium plays a role in the regulation of
blood pressure and calcium phosphate binds bile salts in order to prevent their toxic effect,
thereby reducing the risk of colon cancer (Der Meer et al., 1991). The lipid-based bioactive
compounds in milk include fatty acids and the role of conjugated linoleic acid for inhibition
of cancer and atherosclerosis has been studied (Pariza et al., 2001).
2.3 Bioactive Peptides from Milk
In recent years, intense research interest has been focused on identifying biologically active
components in cow milk and characterising the mode by which mammalian physiological
functionsare modulated by these compounds (Fait et al., 1993; Korhonen, 2009). At present,
milk proteins are considered the most important source of bioactive peptides (Korhonen,
2009). Bioactive peptides have been defined as specific protein fragments that have a positive
impact on body functions or conditions and may ultimately influence health (Kitts & Weiler,
2003). They usually contain 2–20 amino acid residues per molecule, but in some cases they
may consist of more than 20 amino acids (Erdmann et al., 2008). Such peptides are inactive
within the sequence of the parent protein and become active only when they are released
from the precursor protein (Fig. 2.1.).
Different mechanisms can release the encrypted bioactive peptides from the precursor
proteins (Korhonen & Pihlanto, 2003):
a.) In vivo, during gastrointestinal digestion through the action of digestive enzymes or of
microbial enzymes of the intestinal flora;
b.) During milk processing (e.g. milk fermentation, cheese production) through the action of
microbial enzymes expressed by the microorganisms used as starter;
c.) During milk processing through the action of a single purified enzyme or a combination of
selected enzymes;
6
In many studies, combinations of (a) and (b) or (a) and (c), have proven to be effective in
generation of short functional peptides (Korhonen & Pihlanto, 2003).
Removed due to copy right
Moller, S. A., Danielsson, L. D., & Borrebaek, C. A. (1986). Concanavalin A-induced B-cell
proliferation mediated by allogeneically derived helper factors. Immunobiology, 57(3), 387393.
Fig 2.1. Summarizing scheme of the possible mechanisms by which bioactive peptides can be
released from the precursor proteins by microbial fermentation and/or gastrointestinal
digestion, from Möller at al.(2008).
The production and properties of milk protein-derived bioactive peptides have been reviewed
in many articles (Samacchi & Goggetti, 2000; Silva & Malcata, 2005; Korhonen & Pihlanto,
2006; Korhonen, 2009). Depending on the sequence of amino acids, these peptides can
exhibit diverse activities, including opiate-like, mineral binding, immunomodulatory,
antimicrobial, antioxidant, antithrombotic, hypocholesterolemic, and antihypertensive actions
(Erdmann et al., 2008). Many bioactive peptides are multifunctional and can exert more than
one of the effects (Fig. 2.2) mentioned (Korhonen, 2009; Pihlanto et al. 2011).
Removed due to copy right
Pihlanto, A., & John, W. F. (2011). Milk protein products | bioactive peptides. In
Encyclopedia of Dairy Sciences (Second Edition) (pp. 879-886). San Diego: Academic Press.
Retrieved from http://www.sciencedirect.com/science/article/pii/B9780123744074003514
Fig 2.2. Different biological activities of peptides from Pihlanto & John (2011).
7
2.3.1 Immunomodulatory Bioactive Peptides from Milk
Cow milk contains a number of potent peptides that affect the immune system via cellular
functions including cell proliferation and downstream immunological responses (Werner et
al., 1986; Coste & Tome, 1991; Fiat et al., 1993; Kayser & Meisel, 1996; Gill et al., 2000;
Cross &Gill, 2000; Phelan et al., 2009). A large number of studies have demonstrated that
hydrolysis of cow milk protein by digestive enzymes can produce biologically active peptides
including immunomodulatory peptides (Gill et al., 2000; Korhonen and Pihlanto, 2009) and
some of these are summarised in Table 2.1.
Immunomodulation can be defined as adjustment of the immune response to a desired level,
as in immunopotentiation, immunosuppression, or induction of immunologic tolerance.
Pepsin, trypsin and chymotrypsin have been shown to release a number of
immunomodulatory peptides both from caseins (α-, β-, and κ- casein) and whey proteins, e.g.
α- lactalbumin, β-lactoglobulin and glycomacropeptide (Gauthier et al., 2003; Gobbetti et al.,
2004; Phelan et al., 2009).
2.3.2 Immunomodulatory Bioactive Peptides from Fermented Milk
A number of reports (Table 2.2) have demonstrated up- and down-regulation of the immune
system by milk-derived immunomodulatory peptides from fermented milk products
(Korhonen & Pihlanto, 2006). Growing evidence drawn from epidemiological studies,
scientific reports and clinical data indicate that consumption of fermented dairy products
reduces the risk of certain types of cancer; including tumours of breast, colon, lung and
subcutaneously implanted fibrosarcomas (Matar et al., 2001; Matsuzaki et al., 1996; Biff et
al., 1997). The peptidic profile of milk proteins is significantly different after microbial
fermentation, suggesting that modification exerted by microbial proteolysis can be a potential
source of bioactive peptides (Matar et al., 1996). It is now well established that
physiologically active peptides are produced from milk protein during fermentation of milk
with lactic acid bacteria (Korhonen & Pihlanto, 2006). Lactic acid bacteria (LAB) utilize
milk proteins as their source of essential and growth-stimulating amino acids (Juillard et al.,
1998). These bacteria possess cell-envelope-located proteinases which degrade caseins into
oligopeptides, peptide transport systems, which internalize the released oligopeptides; and
intracellular peptidases, which hydrolyse the oligopeptides into smaller peptides or into
amino acids, which can then be used by the cell (Juillard et al., 1998).
8
Table 2.1. Immunomodulatory peptides derived from milk proteins
Peptide
Val-Glu-Pro-IlePro-Tyr
Source
Human
β casein
Hydrolysis
Trypsin
Immune effect
No effect on antibody formation
↑ Phagocytosis
↓ Klebsiella pneumoniae infection
Reference
Parker et al., 1984
Gly-Leu-Phe
Human casein
Trypsin
↑ Phagocytosis
↓ Klebsiella pneumoniae infection
Berthou et al., 1987
Leu-Leu-Tyr
Cow casein
Trypsin
↑ Phagocytosis
No effect on Klebsiella pneumoniae
infection
↓ Antibody formation
Berthou et al., 1987
Tyr-Gly
Cow κ - casein
↑ Human lymphocyte proliferation
Kayser & Meisel, 1996
Casein
hydrolysates
Casocidin-I
Cow casein
↑ IL-2 production
Phelan et at., 2009
Cow α2 - casein
synthetic peptides
corresponding to bioactive
sequences
Commercial enzyme
hydrolysis
Acid hydrolysis
Zucht et al., 1995
Goat α2 – Casein
fraction
Casein
hydrolysates
Beta-casomorphin
Goat casein
No hydrolysis
↓ growth of E. coli&Staphylococcus
carnosus
↑ Antibody formation (indirect ELISA)
Cow casein
Human gastric and duodenal ↓ Human lymphocyte proliferation
or commercial enzymes
Trypsin
↑ lymphocytes proliferation,
NK activity and neutrophil locomotion
Chymosin
Stimulation of phagocytosis
and immune responses
against bacterial infections
Casecidin αs1casein
f(1-23)
Cow β-casein
Cow αs1-casein
Haza et al., 1995
Eriksen et al., 2008
Migliore-Samour et al.,
1989
Lahow and Regelson,
1996
9
Isracidin
Cow αs1-casein
Chymosin
Cow κ casein
f(106-169)
Cow whey protein
hydrolysates
Lactoferrin
hydrolysates
Cow κ casein
Rennin
Cow whey proteins
Trypsin/ Chymotrypsin
Cow Lactoferrin
Pepsin
Cow & goat milk
whey protein
hydrolysates
Tyr-Gly-Gly
Cow & goat whey
protein
Human Gastric juice &
Human duodenal juice
Cow α-lactalbumin
synthetic peptides
corresponding to bioactive
sequences
Trypsin, Chymotrypsing &
Lactobacillus paracasei
NCC2461 Peptidases
Unsequenced
Cow β - Lactoglobulin
acidic
immunomodulatory
peptides
of less than
1000kDa in size
Stimulation of phagocytosis
and immune responses
against bacterial infections
Depression of mice lymphocytes
proliferation
↑ mice lymphocyte proliferation at
lower concentration (0.5 - 500µg/ml)
Stimulation of proliferation
and antibody production in
murine splenocytes and
↓ Human lymphocyte proliferation
Lahow and Regelson,
1996
Eriksen et al., 2008
↑ Human lymphocyte proliferation
Kayser & Meisel, 1996
↑ IL-10 production
↓lymphocyte proliferation
Prioult et al, 2004
Otani et al., 1992
Mercier et al., 2004
Miyauchi et al., 1997
10
Table 2.2. Immunomodulatory peptides derived from fermented milk
Fermentative
strain
Lactobacillus
helveticus R389
Lactobacillus
helveticus R389
Lactobacillus
helveticus L89
(In vitro digestion)
Lactobacillus
helveticus 5089
Lactobacillus
helveticus R389
Lactobacillus
helveticus R389
Lactobacillus
acidophilus
DPC6026
Lactic acid bacteria
(LAB)
Peptide Fraction
Immune effect
Reference
Size-exclusion HPLC Fraction п
Response following Escherichia coli O157:H7 infection
↑Intestinal IgA+ B cells
↑ Intestinal IgA secretion
↑ total serum IgA secretion
↑ Cytokine production (IL-4 & IFN-γ )
LeBlanc et al., 2004
Size-exclusion HPLC Fractions
↑Intestinal IgA+ B cells
↓ Fibrosarcoma Tumor
_
LeBlanc et al., 2004
↑ IFN-γ production
↓ IL-2 production
↑ lymphocyte proliferation
Laffineur et al., 1996
↑ IL-6 production
↑ IgA production
↑ IgA production
-↑ Phagocytosis
Vinderola et al., 2006
IKHQGLPQE, VLNENLLR,
SDIPNPIGSENSEK
-Antibacterial against Enterobacter sakazakii ATCC 12868
&Escherichia coli DPC5063
Hayes et al., 2006
Isolate from goat cheese
Antibacterial against the Gram-positive bacteria Bacillus
cereus, Clostridium sporogenes and Staphylococcus aureus;
and against the Enterobacteriaceae, Shigella sonnei and
Klebsiella pneumoniae.
Hernandez et al., 2005
Reverse-phase HPLC
- low molecular mass peptides
- YPVEPF
- HPHPHLSFM
β Casein ferment
Non-bacterial fraction
Fermented milk
Matar et al., 1996
Martha et al., 2001
11
Lactobacillus
helveticus PR4
Human β casein f184-210
Lactobacillus casei
strain Shirota
Dairy products
Lactobacillus
helveticus
Lactobacillus casei
strain Shirota
Fermented milk
Fermented drink
Antibacterial against Gram-positive and -negative bacteria,
including species of potential clinical interest, such as
Enterococcus faecium, Bacillus megaterium, Escherichia
coli, Listeria innocua, Salmonella spp., Yersinia
enterocolitica, and Staphylococcus aureus.
↑ IFN-γ production
↑ IL-1β
↑ TNF-α
↓ Tumour growth in rodents
↓ Bladder cancer in human
↓ Mammary tumour growth in mice
↓ IL-6 production
↓ natural killer (NK) activity
Minervini et al., 2003
Matsuzaki, 1998
Richid et al., 2006
Takeda & Okumura,
2007
12
The transport system of LAB enables them to internalize oligopeptides up to 18 amino acids
in length, although rarely above 10 amino acids (LeBlanc et al., 2002). This allows the longer
oligopeptides to be a source of bioactive peptides when further degraded by gastrointestinal
enzymes or by intracellular peptidases of LAB.
Lactobacillus helveticus fermented milk has demonstrated an immunomodulating effect on
lymphocyte proliferation in vitro (Laffineur et al., 1996). This strain is known to have high
proteolytic activity, causing the release of oligopeptides from milk proteins. Matar et al.
(2001) identified immunostimulatory peptides generated through fermentation of milk protein
using LAB, specifically Lactobacillus helveticus commonly used in the manufacture of
Swiss-type cheese and other fermented milk products. The immunostimulatory and antitumor
properties of peptidic fractions issued from milk fermented with Lactobacillus helveticus
R389 were also reported by LeBlanc et al. (2002).
2.4 Overview of the Immune System
Although the immunomodulation mechanism of bioactive peptides remains uncertain, it was
confirmed that immunomodulatory activity was related to lymphocyte proliferation, cytokine
and antibody synthesis (Gauthier et al., 2006; Yang et al., 2009; Hou et al., 2012). Therefore,
it is important to have an overview of the human immune system in order to gain a better
understanding of the potential immunomodulation mechanisms of bioactive peptides.
Immune system
The mammalian immune system has evolved to provide protection against infectious agents
called pathogens (Wood, 2006). Pathogens include infectious organisms, such as bacteria,
viruses and eukaryotic parasites, and foreign inanimate materials, for example toxins. The
immune system has historically been divided into innate and adaptive (specific) systems
(Turvey et al., 2010).
The human immune system can be simplistically viewed as consisting of three levels: (1)
anatomical and physiological barriers; (2) innate immunity; and (3) adaptive immunity (Fig
2.3). Failure in any of these systems will greatly increase susceptibility to infection (Turvey
et al., 2010).
13
Removed due to copy right
Turvey, S. E., & Broide, D. H. (2010). Innate immunity. Journal of Allergy and Clinical
Immunology, 125(2, Supplement 2), S24-S32.
Fig 2.3. Integrated human immune system from Turvey et al. (2010). The human defence
system can be simplistically viewed as consisting of 3 levels: (1) anatomical and
physiological barriers; (2) innate immunity; and (3) adaptive immunity. In common with
many classification systems, some elements are difficult to categorize. For example, natural
killer (NK), T cells and dendritic cells could be classified as being on the cusp of innate and
adaptive immunity rather than being firmly in one camp.
Innate immune system
The innate immune system includes:
• Cells (e.g. macrophages, neutrophils, mast cells)
• Soluble factors (e.g. the complement proteins, acute-phase proteins).
The innate immune system used to be regarded as a primitive defence system of limited
effectiveness, found in vertebrates and invertebrates. Cells and proteins of the innate immune
system can recognize certain molecules on the surface of pathogens (pathogen-associated
molecular patterns – PAMPs) as foreign. PAMPs are large molecules with a significant nonprotein component and examples include lipopolysaccharides, peptido-glycans, lipotechoic
acids and mannans. They are found only on microbes and are essential for microbial survival;
therefore they cannot stop manufacturing them as a way of avoiding recognition by the innate
immune system. The cell receptors that recognize PAMPs are called pattern-recognition
receptors and include toll-like receptors and lectins such as the mannose receptor. Soluble
14
factors recognizing PAMPs include mannose-binding lectin and C-reactive protein (Fig. 2.4)
(Wood, 2006).
There is a limit to the number of receptors that can evolve to recognize foreign PAMPs and,
given the quicker replication of pathogens compared with mammals, some pathogens will
evolve ways of escaping recognition by the innate immune system. For example, avirulent
streptococci are recognized and eliminated by the innate immune system and therefore do not
cause disease. However, virulent streptococci synthesize a waxy carbohydrate outer coat that
is not recognized by the innate immune system and therefore to eliminate them a specific
immune response is required(Wood, 2006).
Adaptive immune systems
The cellular components of the adaptive immune system are the lymphocytes (mainly T and
B lymphocytes). Lymphocyte precursors originate from the bone marrow and some
differentiate in the thymus into T lymphocytes which are responsible for cellular immunity.
They have receptors for antigen on their surface (Fig. 2.4). Other lymphocyte precursors
differentiate into B lymphocytes in the fetal liver and after birth in the bone marrow. B
lymphocytes are responsible for humoral immunity (immunity that is mediated by secreted
antibodies). They have antibody molecules on their surface (Fig. 2.4). After differentiation,
the lymphocytes migrate to the lymph nodes and other lymphoid tissue. Although
morphologically indistinguishable, the three types of lymphocyte perform different functions.
One of the main features distinguishing specificor adaptive immunity from innate immunity
is that specific immunity is not present before infection. Following initial infection with a
pathogen it takes about 4 days to produce antibodies against it.
The specific immune system is found only in vertebrates and was thought to be the main
defence mechanism. It is now known that the innate immune system does more than provide
the first line of defence and plays an important role in determining the type of specific
immune response that is mounted against infectious agents (Wood, 2006).
15
Removed due to copy right
Wood, P. J. (2006). The immune system: recognition of infectious agents. Anaesthesia &
Intensive Care Medicine, 7(6), 179-180.
Fig 2.4. Recognition by the immune system from Wood, (2006)
The simplified working procedure of our immune system is illustrated in Fig. 2.5. Specialized
antigen presenting cells (APCs) called macrophages circulate throughout the body and if they
encounter an antigen, they ingest and fragment them into antigenic peptides (I). The pieces of
these peptides are displayed on the cell surface by major histocompatibility complex (MHC)
molecules existing in the digesting APC. The presented MHC–peptide combination on the
cell surface is recognised by the T-cells causing them to be activated (II). Activated T-cells
secrete some chemicals as alert signals to other units in response to this recognition (III). Bcells, one of the units that respond to these signals, become activated with the recognition of
antigen by their antibodies occurring at the same time (IV). When activated, B-cells turn into
plasma cells that secrete antibodies which are bound on their surfaces (V). Secreted
antibodies bind the existing antigens and neutralize them signalling other components of
immune system to destroy the antigen–antibody complex (VI) (Polat et al., 2006).
16
Removed due to copy right
Perelson, A. S., & Oster, G. F. (1979). Theoretical studies of clonal selection: Minimal
antibody repertoire size and reliability of self-non-self discrimination. Journal of Theoretical
Biology, 81(4), 645-670.
Fig 2.5. Immune response from Perelson & Oster (1979).
Lymphocytes
The three major types of lymphocytes are T lymphocytes (or T cells), B lymphocytes (or B
cells) and natural killer cells (NK cells). T and B lymphocytes are the main self-defence
weapons of the adaptive immune system (Turvey et al., 2010). The T cells coordinate the
entire immune response and eliminate viruses hiding in infected cells and contribute to the
immune defences in a cell-mediated way and can be sub-grouped as follow. T helper cells
(Th cells) assist other white blood cells in immunologic processes, including maturation of B
cells into plasma cells and activation of cytotoxic T cells and macrophages, among other
functions. Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumour
cells, and are also implicated in transplant rejection. After the infection has resolved, another
subset of antigen-specific T cells persist and they are called Memory T cells. They quickly
expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus
providing the immune system with "memory" against past infections. Memory T cells
comprise two subtypes: central memory T cells (TCM cells) and effector memory T cells
(TEM cells). Finally, regulatory T cells (Treg cells), formerly known as suppressor T cells,
are crucial for the maintenance of immunological tolerance. Their major role is to shut down
17
T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus (Wood, 2006).
Natural killer cells (NK cells) are a type of cytotoxic lymphocyte. NK cells play a major role
in the rejection of tumours and cells infected by viruses. The cells kill by releasing the
proteins called perforin and granzyme that cause the target cell to die by apoptosis. Most of
the production of lymphocytes occurs during fetal and neonatal life, but there is a slow
continuous production of new lymphocytes from stem cells in adults (Turvey et al., 2010).
Lymphocytes reside in different organs of the human body. They circulate through the
primary lymphoid organs (thymus and bone marrow), the secondary lymphoid organs
(spleen, lymph nodes (LN), tonsils and Peyers patches (PP)) as well as non-lymphoid organs
such as blood, lung and liver (Fig. 2.6.). Especially in lymphoid organs, lymphocyte subsets
migrate and home to different compartments (Blum & Pabst, 2007).
Removed due to copy right
Blum, K. S., & Pabst, R. (2007). Lymphocyte numbers and subsets in the human blood: Do
they mirror the situation in all organs? Immunology Letters, 108(1), 45-51.
Fig. 2.6. Distribution of lymphocytes to the different organs from Blum and Pabst, (2007)
18
Cytokines
The components of the immune system act cooperatively to eliminate the infection. The
“communication” between the different parts is mediated by specialized chemical mediators,
called cytokines (Belardelli & Ferrantini, 2002). Cytokines are diverse and potent chemical
messengers secreted by the cells of the immune system. Over 80 cytokines have been
described and they play an important role in coordinating embryonic development, cell
growth and maturation, new blood vessel formation, wound repair and healing, the immune
response including acute phase reactions and septic shock, and destroying targeted cells
including cancer cells (Dunlop et al., 2000). Expression of cytokines and their receptors after
stimulation is an important indicator of mononuclear cell activation (Garibay-Escobar et al.,
2003)
Lymphocytes, including both T cells and B cells, secrete cytokines called lymphokines, while
the cytokines of monocytes and macrophages are called monokines. Many of these cytokines
are also known as interleukins because they serve as a messenger between leukocytes.
Binding to specific receptors on target cells, cytokines recruit many different subsets of the
immune system. Moreover, it is common for different cell types to secrete the same cytokine
or for a single cytokine to act on several cell types. Cytokines are redundant in their activity,
meaning that the same function can be stimulated by different cytokines.
Cytokines include several categories such as interleukins (IL-1, IL-2, IL-6, IL-10),
interferons (IFNα, IFNβ, IFNγ), tumor necrosis factor (TNFα), colony-stimulating factors
(CSF), growth factors and others (Dunlop et al., 2000). They may be grouped into various
sub-families (Table 2.3), although the nomenclature is somewhat arbitrary, having arisen in
different branches of biology.
19
Table 2.3. Cytokine families
Removed due to copy right
Hopkins, S. J. (2003). The pathophysiological role of cytokines. Legal Medicine, 5,
Supplement(0), S45-S57.
Reference: Hopkins, (2003)
A complex network of cytokines, simplistically outlined in Fig. 2.7, critically affects the
nature of the lymphocyte response.
Removed due to copy right
Hopkins, S. J. (2003). The pathophysiological role of cytokines. Legal Medicine, 5,
Supplement(0), S45-S57.
Fig. 2.7.Regulation of adaptive immunity by cytokines from Hopkins, (2003). Cytokine
production (solid arrows) regulates adaptive immunity at the level of antigen presentation (1),
clonal expansion (2), differentiation along alternate T-cell regulatory pathways (3), regulation
of B cell differentiation (4) and effector actions of T cells at the tissue level (5).
20
In particular, two important cytokines involved in the proliferation and activation of
lymphocytes are IL-2 and INFγ (Belardelli and Ferrantini, 2002). IL-2 is a T cell growth
factor produced by T helper 1 (Th1) and NK cells. As an autocrine and paracrine growth
factor, IL-2 induces proliferation and differentiation of T and B cells (Belardelli and
Ferrantini, 2002). IL-2 is responsible for stimulation of B cells for antibody synthesis. IL-2
stimulates the growth of NK cells and enhances the cytolytic function of these cells,
producing lymphokine-activated killer cells. IL-2 can also induce interferon INFγ secretion
by NK cells (Smyth et al., 2001).IL-2 is a major autocrine and/or paracrine T-cell growth
factor, produced primarily by CD4+ T cells and used for the treatment of some human
malignancies and priming of immune activity (Belardelli & Ferrantini, 2002). IFN-γ has
marked effects on cells of innate immunity, including macrophages, the activation state of
which is regulated mainly by INF-γ (Belardelli, 1995). The fact that IFN-γ is produced
typically during a Th1 immune response has led this cytokine to be considered as
characteristic of adaptive immunity. However, recent studies have reported IFN-γ production
by cells of the innate immune system, such as macrophages (Gessani & Belardelli, 1998) and
T cells (Frucht et al., 2001), suggesting that IFN-γ plays a role in bridging the innate and
adaptive branches of the immune response. IFN-γ is important in host defense against
intracellular pathogens, viruses, natural surveillance against cancer and IFN-γ can induce
marked antitumor effects in mouse models (Belardelli & Ferrantini, 2002).
In vitro lymphocyte proliferation assay as an immunomodulatory parameter
After activation by encounter with an antigen (or foreign protein), B and T lymphocytes start
to proliferate and rapidly expand their numbers, so that there are many lymphocytes available
which can recognize and fight the invading antigens (Zilman et al., 2010). Measurement of
lymphocyte proliferation is frequently used in clinical and experimental immunology as a
means of assessing lymphocyte activation and response to a variety of stimuli including
nonspecific mitogens, specific antigen, cytokines, and allergenic stimulation in mixed
lymphocyte reactions (De Fries & Mitsuhashi, 1995).
The lymphocyte proliferation assay measures the ability of lymphocytes placed in short-term
tissue culture to undergo a clonal proliferation when stimulated in vitro by a foreign
molecule, antigen or mitogen (Zhi-Jun et al., 1997; Wanger et al., 1999; Zilman et al., 2010).
21
Proliferation of lymphocytes in response to various stimuli has been assessed by several
means. Traditionally the most commonly used method assessed the incorporation of
radiolabeled nucleotides (3H-thymidine) into cells during cell division (Khan et al., 1986;
Fries & Mitsuhashi, 1995; Metcalf & Wiadrowski, 1966 and Eriksen et al., 2008). The
primary drawbacks of the 3H-thymidine incorporation assay are that it requires specialized
equipment, as well as the use of radioisotopes which present a hazard in the laboratory and
which pose increasing difficulties in their disposal (Fries & Mitsuhashi, 1995).
A number of alternative methodologies for measurement of lymphocyte proliferation have
been reported. Assays which measure total cell number by detecting reduction of tetrazolium
compounds, such as MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
by microbial enzymes, have been evaluated (Weichert et al., 1991). Methods which measure
incorporation of fluorescent dye into cellular DNA (Blaheta et al., 1991) and incorporation of
BrdU (5-bromodeoxyuridine) and subsequent detection by ELISA assay (Houng et al., 1991)
have also been reported.
The Alamar Blue assay was developed by Alamar Bioscience Inc (Fries and Mitsuhashi,
1995), as a reliable and sensitive alternative assay for determining lymphocyte proliferation
which is convenient, simple to perform and does not require the use of radioactive materials
(Ansar Ahmad et al., 1994; Zhi-Jun et al., 1997; Al-Nasiry et al., 2007; Jacquot et al., 2010).
An oxidized form of the Alamar Blue reagent is added to cells and this is reduced by
mitochondrial enzyme activity of the cells being tested. Reduction causes a change in colour
as well as a shift in fluorescence profile which can be quantitatively detected colorimetrically
or fluorimetrically (Zhi-Jun et al., 1997). Fries and Mitsuhashi, (1995) have demonstrated the
reliability of Alamar Blue assay in measuring human peripheral blood mononuclear cell
(PBMC) proliferation.
Cytokine production as immunomodulatory parameter
It has been well established that cytokine levels correlate with the severity of diseases and/or
clinical outcomes (Kurzrock et al., 1993; Tartour et al., 1994). Commercially available
ELISA kits (based on a sandwich technique using antibodies) make it convenient to measure
cytokines in biological samples and cell culture supernatants (Leduret al., 1995). The same
ELISA procedure has been used to test immunomoduatory activity of milk proteins
previously with human lymphocytes (Laffineur et al., 1996; Phelan et al., 2009) and murine
splenocytes (Saint-Sauveur et al., 2007).
22
2.5. Deer
milk,
usage
and
potential
of
immunomodulating
bioactives
Most of the studies and reviews on bioactive peptides are on cow milk (Minervini et al.,
2003; Gobbetti, 2007; Korhonen, 2009; Picariello et al., 2010) while only a few on milk from
different species such as sheep and goat (Silva et al., 2006; Papadimitriou et al., 2007;
Hernandez-Ledesma et al., 2011) are found In the mass production of bioactive peptides cow
milk is used as an ingredient for the manufacture of functional foods by chromatographic
purification (Choi et al., 2012). There is no reported study on deer milk bioactives which may
have this potential.
Knowledge of milk and milk bioactives of ruminants is mainly related to studies on bovids.
These are often taken to be typical of the sub-order as a whole, but it is interesting to extend
our knowledge to the milks of other families to see their value. Of these families, the cervids
are the largest and appear to have evolved earlier than the bovids. The cervids, especially the
reindeer (Rangifer tarandus L.) are herded but their milk and milk proteins have not been
studied in any detail (McDougall & Stewart, 1975). Reindeer pastoralism, where milking was
an integrated part of the farming activity, evolved at least two thousand years ago in the
Taiga region of eastern Siberia around Lake Baikal and spread to nearby ethnic groups
(Fondahl, 1989). The most intensive milking regime was developed along the borders
between Russia, Mongolia and China. The animal provided milk both for immediate fresh
consumption and processing. The milk was dried and processed into cheese, butter, and sour
cream, (Table 2.4.) as well as used medicinally. The milk was curdled and often mixed with
herbs such as Oxyria spp. and Angelica spp. (Park & Haenlein, 2006). Reindeer milk and its
products were highly priced and also used as a medical remedy to cure digestive problems
(the milk has antidiarrheic properties) and to heal wounds (Park & Haenlein, 2006). Fat
exuded by heat from reindeer cheese was also used to cure nursing pains, frostbites, and other
injuries. In addition, colostrum was used for children’s ailments (Fondahl, 1989).
23
Table2.4. Traditional Products and Uses of Reindeer milk (Park & Haenlein, 2006)
Removed due to copy right
Park, Y. W., & Haenlein, G. F. W. (Eds.). (2006). Handbook of Milk of Non-Bovine
Mammals. Iowa: Blackwell Publishing.
Red deer (Cervus elaphus L.) has become an experimental animal and increasing attention is
being given to methods of farming it (Gómez et al., 2002). Intensive farmed red deer
production for meat increased in France following the success of Scotland and New Zealand
where the first commercial flocks were created 20 years ago and deer hinds produce 120 to
200kg milk in 100 days depending on pasture quality and the milk contains on average 10 %
fat and 8 % protein which is twice the levels found in cow milk (Theriez, 1988). Milk
composition of Red deer and some domesticated mammals and human milk are shown in
Table 2.5.
24
Table 2.5. Gross Composition (%) of milk of human & some domesticated mammals
Species
Fat
Protein
Total solids
Human
4.1
1.3
12.3
Cow
3.6
3.3
12.3
Goat
3.8
3.5
12.2
Sheep
5.3
5.5
16.3
Deer (Red Deer)
10.3
7.8
23.9
Data from Emmett & Rogers, (1997) and Park & Haenlein, (2006)
Since milk protein is the precursor of several bioactive peptides, this may indicate that deer
milk might be rich in bioactives compared to bovine milk. So far there are no studies on deer
milk bioactives. Furthermore, the evidence of historical use of deer milk as a medical remedy
suggests there is potential to capture immunomodulatory bioactive peptides in deer milk and
utilize them more efficiently for certain age groups that require support for the immune
system.
25
Chapter 3
(Paper published in International Food Research Journal 2012,19 (4): 409-416
In vitro Digestion of Red Deer (Cervus elaphus) and Cow
(Bos taurus)milk
3.1. Introduction
The proteins in milk from different species vary in concentration and amino acid
composition. The protein content in milk from bovine species is approximately 3.3%,
whereas reindeer milk contains more protein, approximately 7.8% (Park & Haenlein, 2006).
Casein is the main protein component of bovine milk constituting about 80% of the total milk
proteins (Shah, 2000). The casein fraction consists of αs1-, αs2-, β- and κ-casein. The main
proteins in the whey fraction are β-lactoglobulin (β-lg), α-lactalbumin (α-la), serum albumin
(SA), immunoglobulins (Igs), lactoferrin (LF) and lysozyme (LZ) (Inglingstad et al., 2010).
The amino acid composition of casein and whey protein fractions varies in different species
and will affect their digestibility (Almaas et al., 2006).
Protein digestion starts in the stomach by the action of acid and pepsin and is followed by
intestinal digestion by trypsin, chymotrypsin and various carboxypeptidases and
aminopeptidases. Milk protein represents an important dietary source for humans, providing
that they are digested suitably.The high digestibility of milk protein (~ 95%), combined with
a superior amino acid composition for human requirements, makes milk a “high quality
protein” source (Bos et al., 1999). In vivo results of Dangin et al. (2002, 2003) led to the
concept of “slow” digested caseins and “fast” digested whey protein. In vivo studies with
either milk or purified milk proteins in healthy human showed that whey proteins were taken
up more rapidly in the upper jejunum than the casein (Mahe et al., 1995, 1996). However,
studies by Almaas et al., (2006) using human gastric and duodenal juices for the in vitro
digestion of cow and caprine milk proteins revealed that the whey proteins, α-lactalbumin
26
and β-lactoglobulin were very resistant to hydrolysis and that the caseins were degraded
faster.
This study investigates the in vitro digestion of deer and cow milk by commercial gastric and
duodenal enzymes with the major focus being on comparison of the degradation of protein to
evaluate the differences in digestibility and peptide production. No information on the
digestion of red deer milk, which is unique from other species in its composition (LandeteCastillejoset al., 2000), is available and only basic composition of red deer milk has been
reported. This information could potentially be useful in understanding functional roles in
animal nutrition and evaluating its potential for human use.
3.2. Materials & Methods
3.2.1. Materials
Oxytocin was purchased from Pharm Distributors (Auckland, New Zealand). Pepsin (66 units
/ mg solid) from porcine stomach mucosa, OPA (O-phthaldialdehyde), Leucine and TCA
(trichloroacetic acid) were purchased from Sigma (St. Louis, MO, USA) and Corolase PP
(CPP) (350 proteolyticU/mg) from pig pancreas was from AB Enzymes (Darmstadt,
Germany). Corolase PP is a mixture of trypsin, chymotrypsin and several amino and
carboxypeptidases. NuPAGE 4-12% Bis-Trisprecast gels were purchased from Invitrogen
USA. Bio-Rad Precision Plus Protein
TM
standard was used as a high molecular weight
marker.
3.2.2. Preparation of Milk Samples
Pooled deer milk was obtained from the Lincoln University deer farm at Lincoln (New
Zealand). Calves were weaned from twenty hinds which were then milked twice-daily.
Immediately prior to milking, each hind received an injection of 1 ml oxytocin (10 IU/ml) to
enable the ‘let down’ of milk. The hinds were milked in a side-loading crush, using a
commercial machine designed for milking sheep and goats. Approval for this work was given
by Lincoln University Animal Ethics Committee (# 377).Pooled cow milk was obtained from
Lincoln University Dairy Farm. Fat was removed by centrifugation at 4000 ×g for 30 min at
4°C. Defatted milk was freeze dried and stored at room temperature in airtight containers
until analysis.
27
3.2.3. Protein content
Total nitrogen (TN), non-protein N (NPN) and non-casein nitrogen (NCN) were measured
using the Kjeldahl method (Barbano et al., 1991). NPN was estimated after precipitation of
protein with 24% trichloracetic acid followed by centrifugation at 27,000 x g for 60 min. The
supernatant was filtered using Whatman No 1 filter paper and the filtrate was used for
Kjeldahl analysis. NCN was measured after precipitation of casein by adjusting the milk to
pH 4.6 with slow addition of 1M HCL while stirring and centrifugation at 10,000 ×g for 15
min at 200C. The supernatant was filtered and protein content was determined using the
Kjeldahl method. Total protein [(TN-NPN) x 6.38], casein protein [(TN-NCN) x 6.38] and
whey protein [(NCN-NPN) x 6.38] concentrations were calculated.
3.2.4. Dry matter content
Weights of deer and cow milk were taken before and after freeze drying of milk. Dried
weight was calculated as percentage of total milk weight.
3.2.5. In vitro Digestion
Defatted deer and cow milk samples (20 ml) were prepared in triplicate by rehydrating freeze
dried defatted milk in 26% and 12% rehydration ratio respectively. In vitro protein digestion
was performed using pepsin and CPP according to Eriksen et al. (2008). The procedure
mimics “normal digestion” in the human gastro-intestinal tract. The first incubation, which
mimics digestion in the stomach, was adjusted to pH 2.5 with 1M HCl, and used pepsin
(4mg/g milk protein) at 37°C for 30 min. The second incubation used CPP (4 mg/g milk
protein) at 37°C for 60 min after the pH was adjusted to 7.5 with 1M NaOH. Continuous
shaking (150 rpm) was maintained during digestion. The digestion of milk protein was
monitored by measuring pH, the production of amino-terminals by O-phthaldialdehyde
(OPA) assay andsodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Small samples were drawn for OPA assay (250 µl) and SDS-PAGE (1 ml) every 10 min of
digestion.
28
3.2.6. ProteolysisAssessment using OPA assay
The OPA assay followed the method of Church et al. (1983). The OPA reagent contained 2.5
ml of 20% (w/v) SDS, 25 ml of 100 mM sodium tetraborate, 40 mg OPA (previously
dissolved in 1 ml methanol), 100 µl of 2-mercaptoethanol and distilled water up to 50 ml.
Samples were incubated with 0.75 M TCA at a sample:TCA ratio of 1:3 (250 µl: 750 µl) at
4°C for 30 min and then centrifuged (4000 ×g for 10 min)to eliminate any interference of the
undigested protein fractions as suggested by Church et al. (1983). A 10 µl aliquot of the
supernatant was diluted by adding 140 µl H2O, 1 ml of OPA reagent was added and then the
tube was incubated at room temperature for 2 min. Absorbance at 340 nm was measured
using UNICAM 8625 UV/VISspectrophotometer. Leucine (Leu) was used for the
construction of a standard curve and the proteolytic activity was expressed as mM Leu
equivalent.
3.2.7. SDS -polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis was used to analyse the protein samples taken at different hydrolysis times
during digestion. SDS-PAGE was performed according to a standard protocol (Laemmli,
1970) using NuPAGE 4-12% Bis-Trisgels. Bio-Rad Precision Plus Protein
TM
standard was
used as a molecular weight marker. Samples were diluted to 2 mg /ml protein with 6x sample
buffer (30 µl)
containing 0.35 M Tris-HCl, 10.28% SDS, 30% glycerol, 0.012%
bromophenol blue and 5% mercaptoethanol. The volume was adjusted to 180 µl with water
and the sample was heated at 95°C for 4min. Samples (15 µl) and 10 µl of protein marker
were loaded into the wells. Electrophoresis was performed at a constant voltage of 200V for
40 min. Gels were fixed in 50% methanol and 7% acetic acid for 15 min with rocking and
then washed 3 times for 15 min with water. Gels were stained with Coomassie Brilliant Blue
(Gel Code Blue) and destained with continuous shaking in water overnight.
3.2.8. Quantification of protein bands
The intensities of protein bands in the gels were quantified (Inglingstad et al., 2010) by
ImageJ software (version 1.42 http://rsbweb.nih.gov/ij/). Gels were scanned using Coral
Photo Paint 12. Image rectangle was applied for background subtraction, and rolling ball
radius was fixed to 30 pixels. Bands of interest were marked in each lane to be able to
investigate the degradation pattern of the different proteins. In order to measure degradation,
the amount of intact protein remaining in the digested samples was calculated as a percentage
29
of its undigested counterpart. Three gels from each sample were quantified, and the results
are given as a mean value.
3.2.9.Statistical analysis
All experiments were carried out in triplicate and values are mean ± standard deviation. The
data were subjected to one way analysis of variance (ANOVA), followed by Tukey’s test to
determine the significant differences between samples at P < 0.05 level using Minitab
statistical software(version 16, Minitab Inc., USA).
3.3. Results and Discussion
Milk composition
The dry matter content and protein composition of deer and cow milk are shown in Table 3.1.
Dry matter content of deer milk was 25.7% while cow milk has 12.1% dry matter. Hence
deer milk had more than twice the dry matter of cow milk. Similarly total protein content in
deer milk is 8.8% which is more than twice that of cow milk (4.1%). In accordance with this
finding Arman et al.,(1973) showed total protein content of red deer has between 7.1 to 8.6
%.Total protein is the combination of casein and whey proteins. Deer milk has 8.7% casein
while cow milk has only 4.0% casein. Both deer and cow milk have very low amounts of
whey proteins which are 0.64% and 0.57% respectively. Therefore both milks have high
casein to whey protein ratio. The difference between total proteins detected and sum of casein
and whey proteinmay be due to experiment error.
Table 3.1. Dry matter & protein content of red deer and cow milk
Milk Type Dry matter (%) Total Protein (%) Casein (%) Whey protein (%)
Deer Milk 25.7 ± 0.76b
8.8 ± 0.13b
8.7 ± 0.13b
0.64 ± 0.002
Cow Milk
12.1 ± 0.01a
4.1 ± 0.02a
4.0 ± 0.02a
0.57 ± 0.004
Values are mean ± standard deviation (n= 3).
Means within the same column that have different letters (a-b) are statistically different (P <
0.05) as determined by Tukey’s test.
The milk from ruminants is characterized by high casein: whey protein ratio when compared
with other groups of mammals (McDougall and Stewart, 1975; Vincenzetti et al, 2008).
Among ruminants, most domesticated species are bovids and their milk is readily available
and economically important. Hence our knowledge of the milk proteins of ruminants is
30
mainly confined to bovids, but it would be useful to investigate the milk of other ruminant
families. The cervids are the largest of these families and appear to have evolved earlier than
the bovids (Young, 1962). Consistent with data of Arman et al., (1974) our results showed
that deer milk casein and whey contents (Table3.1) had a similar ratio to that found in other
ruminants.
In vitro Digestion
In humans, pepsin digestion and acid hydrolysis at pH of 1.5 – 2.5 are the first steps in the
gastrointestinal degradation of protein, followed by stomach emptying and further digestion
in the duodenum by pancreatic enzymes at pH ~ 7. To mimic in vivodigestion the present
work has used invitrodigestion of defatted deer and cow milk by commercial pepsin and the
duodenal enzymes trypsin and chymotrypsin. Pepsin is an aspartic protease which prefers to
cleave peptides at bonds with Phe, Tyr, Trp and Leu in position P1’ (Fujimoto et al., 2004).
Trypsin cleaves peptide bonds following a positively charged amino acid and chymotrypsin
cleaves peptides bonds following bulky hydrophobic amino acid residues (Antal et al., 2001).
Therefore digestion pattern with these enzymes will vary depending on the protein structure
of cow and deer milks and this could lead to differences in digestibility and digestive
products with different bioactivity.
8
7
pH
6
5
4
3
2
0
20
40
60
80
100
Digestion time (min)
Figure 3.1. pH during in vitro digestion of deer (
) and cow (
) milk.0 to 30 min
digestion is simulated stomach. At 30 min pH was adjusted to7.5. 30 to 90 min digestion is
simulated duodenum. Data are mean ± S.D. (n = 3).
31
The pH affects the activity of enzymes during in vivo digestion. The pH of stomach increases
rapidly from 2 (pH of gastric juice) to pH of the diet immediately following the consumption
of meal and then decreases progressively toward its initial value (Savalle et al., 1989). At the
beginning of digestion the pH was adjusted to 2.5 with 1 M HCl for the pepsin digestion. The
pH increase during digestion was greater (Fig 3.1) in cow milk than deer milk which could
indicate a higher buffering effect of deer milk. Milk acts as a complex buffer because it
contains carbon dioxide, protein, phosphate, citrate and a number of minor constituents
(Ismail et al., 1973). Goat milk has a higher buffering capacity due to its higher content of
major buffering components including minerals (Park, 1992). Deer milk had 1.1 ± 0.05 % ash
while cow milk had only 0.70 ± 0.04 % (n = 6). This finding was in accordance with mineral
composition analysis reported by Arman et al. (1974) which showed a 1.11 % ash content in
deer milk (average of 6 hinds in mid lactation). Our results showed (investigated further in
Chapter 6 Fig. 6.2) that deer milk had higher buffering effect than cow milk. This could be
due to high protein and mineral content and this high buffering quality of deer milk could
enhance its value for subject suffering from peptic ulcers and other gastric ailments. At 40
min (10 min into the second stage of digestion) deer milk showed only 0.8 unit pH drop
whereas the pH of cow milk decreased by 1.3 (Figure 3.1). Again the buffering effect of deer
milk could contribute to the observed differences. In deer milk, the pH change during the
start of second stage of digestion was higher than that for the first stage of digestion. It could
be a result of the higher digestibility of deer protein in simulated duodenum by trypsin and
chymotrypsin than pepsin. Cow milk caseins were more susceptible to hydrolysis by trypsin
than camel caseins, whereas camel caseins were more prone to hydrolysis by chymotrypsin
than cow casein (Salami et al., 2008). To examine the differences in digestibility of deer and
cow protein, peptide production and protein profile were evaluated using OPA assay and SDS
– PAGE using quantification by ImageJ software.
Proteolysis Assessment Using OPA Assay
In this study, the hydrolysis of milk protein was measured using a rapid, sensitive and simple
OPA-based spectrophotometric assay (Pescuma et al., 2008; Salami et al., 2008; Salami et
al., 2009). The peptide production from the in vitro digestion of milk proteins is shown in
figure 3.2. Deer milk produced more hydrolysed products (peptides and amino acids) than
cow milk which is consistent with deer milk containing twice the protein available in cow
milk. However, peptide production was not two times greater than cow milk. This could be
due to differences in protein structures and hence enzyme target sites in deer and cow milk
32
that may lead to formation of different peptides with different lengths. Kopf-Bolanz et al.
(2012) demonstrated the formation of different number of peptides with different length
during in vitro digestion of cow milk. Therefore the number of N terminals produced by deer
milk digestion might not be twice that of cow milk digestion.
Peptide production (mM)
0.5
0.4
0.3
0.2
0.1
0.0
0
20
40
60
80
100
Digestion time (min)
Figure 3.2. Peptide production during in vitro digestion of deer (
) and cow (
)
milk (up to 30 min: simulated stomach and then simulated duodenum). Peptide production
was measured using OPA assay after TCA precipitation. Data are mean ± S.D. (n = 3).
There was a gradual increase of peptide production during digestion with pepsin in the
simulated stomach for both milks and then a rapid increase in the simulated duodenum. This
was faster in deer milk than cow milk (Fig3.2). Peptide production of simulated duodenum
was significantly (P<0.05) higher than simulated stomach for both milks. The analysis of
hydrolysis of deer and cow milk (Fig 3.2) revealed greater production (P<0.05) of peptides
from deer milk than cow milk in simulated duodenum. Therefore hydrolytic activity was
higher in simulated duodenum in deer milk compare to cow milk. After 60min of digestion,
peptide production had plateaued. No new peptides were produced by extending the reaction
time. Therefore, for gel band quantification only 30min was used for simulated duodenum
digestion (stage 2).
SDS – polyacrylamide gel electrophoresis (SDS – PAGE)
SDS – polyacrylamidegels (4-12%) were used to analyse the milk proteins and digested
proteins based on a preliminary study of various concentrations (Appendix A). Inglingstad et
33
al.,(2010) used 12.5 % gel to separate milk protein and digests of cow, goat, horse and human
milk. The protein patterns on SDS – PAGE (Figs 3.3 and 3.4), illustrated the protein profile
of red deer (lane D of Fig 3.3) and cow (lane C of Fig 3.4) milk and these milks during
invitro digestion at 10 min intervals. Protein bands of deer milk were identified based on cow
proteins (Preliminary studies inAppendix A). The intensity of casein like bands reduced from
0 min to 30 min in digests of both milks. Deer casein like protein was totally digested after 40
min of digestion (10 min after second stage of digestion) whereas traces of casein (14%) were
still present in cow milk (Fig 3.3, 3.4 and 3.5 a). The casein band in cow milk was completely
digested after 50 min (Fig 3.4 and 3.5a). This result confirms that deer casein like protein was
digested more rapidly than cow casein. The gel results supported the higher hydrolysis
activity of deer milk than cow milk in simulated duodenum which was suggested by OPA
results. About half (49%) of intact major milk protein (casein) started to degrade at the
beginning of simulated duodenum digestion in deer milk while only about a quarter (27%) of
intact casein was digested during simulated duodenum in cow milk under the same conditions
of time, temperature and pH (Fig 3.3 and 3.4, Fig 3.5 a). The major protein components of
milk, the αs1- and β- caseins, contain covalently attached phosphate groups bound to residues
of serine and threonine (Medina et al., 1992). The bound phosphate groups influence many
functional properties of these proteins, including their digestibility, bioavailability of divalent
cations and immunogenicity (Tezcucano Molina et al., 2007).
MW:
kDa
250
150
100
75
50
37
25
20
15
10
M D D10 D20
IgHC
LF
SA
CN
β-lg
α-la
D30 D40 D50 D60 D90
Figure 3.3. SDS- PAGE (4-12%) of skimmed deer milk & deer milk digested with pepsin
and CPP.
Major bands; Immunoglobulins heavy chain (IgHC), lactoferrin (LF), serum
albumin (SA), casein (CN), β-lactoglobulin (β-lg) and α-lactalbumin (α-la). High molecular
weight markeris shown on left-hand side of the gel. The lanes are: Protein molecular
marker(M), Deer milk(D), Digest after10 min (D10), 20 min (D20), 30 min (D30) in
34
simulated stomach digestion and digest after 40 min (D40), 50 min(D50), 60 min(D60), 90
min(D90) (simulated duodenum digestion). Each contained had 2 mg/ µl protein.
MW:
kDa
250
150
100
75
50
37
25
20
15
10
MC
IgHC
LF
SA
CN
β-lg
α-la
C10 C20 C30 C40 C50 C60 C90
Figure 3.4. SDS- PAGE (4-12%) of skimmed cowmilk & cowmilk digested with pepsin and
CPP. The wells contain; Immunoglobulins heavy chain (IgNC), lactoferrin (LF), serum
albumin (SA), casein (CN), β-lactoglobulin (β-lg) and α-lactalbumin (α-la). High molecular
weight markeris shown on left-hand side of the gel. The lanes are: High molecular
marker(M), Cow milk(C), Digest after10min (C10), 20min (C20), 30min (C30) in simulated
stomach and digest after 40min (C40),50min(C50), 60min(C60), 90min(C90) (simulated
duodenum). Each lane contained 2 mg/ µl protein.
The specific protein pattern and in particular the physico-chemical and biological properties
of milk proteins may influence their digestibility (Salami et al., 2008). There is limited
information on red deer milk proteins. McDougall (1976) reported amino acid analysis of
deer milk β-lactoglobulin, adjusted to lysine = 15 residues (the number of residues is given
relative to 15 residues of lysine) showed that it contains one more residue of aspartic acid,
alanine and methionine and one less glutamic acid residue and two less leucine residues than
cow β-lactoglobulin. From this McDougall (1976) concluded there was only a small
difference in amino acid composition of β-lactoglobulin in deer milk compared to cow milk
and demonstrated the similarity by gel chromatography and electrophoretic methods. This
similarity of β-lactoglobulin in the two species could lead to similarities in digestibility of
this protein in milk. β-lactoglobulin is considered the dominant cow milk allergen and its
rigid spatial conformation exhibits high resistance to gastric digestion, which in part explain
its allergenicity (Prioult et al., 2005).
35
b)
120
120
100
100
80
80
60
60
% intact α−la
% intact Casein
a)
40
20
20
0
c)
40
0
0
10
20
30
40
50
60
0
Digestion time (min)
10
20
30
40
50
60
Digestion time (min)
120
100
% intact IG
80
60
40
20
0
0
10
20
30
40
50
60
Digestion time (min)
Figure 3.5.In vitro digestion of casein (a), α-lactalbumin (b), immunoglobulin (c) in raw
milk from deer (
) and cow (
) by pepsin at pH 2.5 / 37°C (1 step of digestion up
to 30 min) and CPP at pH 7.5 / 37°C (step 2 until 60 min). Values were obtained using
ImageJ of SDS-PAGE. Data are means ± S.D. (n = 3).
Our results confirmed that the digestibility of β-lactoglobulin is low in milk from both
species. After completein vitro digestion 54% and 55% of intact β-lactoglobulin was still
intact in deer and cow milk respectively.
With respect to digestion of whey proteins,
Inglingstad et al. (2010) reported that β-lactoglobulin and α-lactalbumin of cow and goat
were very resistant to human gastric and duodenal enzyme digestion, while horse milk
showed rapid duodenal degradation of β-lactoglobulin. Cow milk α-lactalbumin possesses 26
potential chymotrypsin-specific target sites and 13 trypsin-specific target sites in its primary
structure (Salami et al.,
2008).The digestibility of α-lactalbumin like protein was
significantly ( P<0.05) higher in deer milk than cow milk after total digestion (Table3.2). At
36
40 min of digestion there was only 14% α-lactalbumin like protein remaining undigested in
deer milk, where as 62% remained in cow milk (Fig. 3.5b). Present results are in accordance
with those of Pintado and Malcata (2000) who also found that cow milk α-lactalbumin was
resistant to hydrolysis by trypsin. β-lactoglobulin and α-lactalbumin are the major milk
allergen whey proteins (El-Ghaish et al., 2011; Wal, 1998). Significantly (P<0.05) lower αlactalbumin like proteinconcentration in deer milk digest than cow milk digest (Table 3.2)
may suggest less allergenic effect of deer milk than cow milk.
Table 3.2. Protein content (%) remaining in raw milk before digestion (start), after
stage 1 digested with pepsin at pH 2.5 for 30 min / 37 °C and after stage 2 digested with
CPP at pH 7.5 for 30 min / 37 °C. Values are obtained from SDS-PAGE using ImageJ
(n=3)
Cow
Deer
Start Stage 1 Stage 2
Start Stage 1 Stage 2
IgHC 100 10 ± 5* 1 ± 2*
100 53 ± 9* 21 ± 5*
LF
100 25 ± 2
0±1
100 32 ± 4
1±1
100 25 ± 3* 1 ± 1
SA
100 11 ± 6* 0 ± 0
CN
100 49 ± 9* 0 ± 0
100 27 ± 3* 1 ± 1
100 91 ± 4 55 ± 8
β-lg
100 94 ± 3 54 ± 5
α-la
100 79 ± 8 11 ± 2*
100 82 ± 10 27 ± 5*
S1 (stage 1) = simulated stomach
SA: serum albumin
S2 (stage 2) = simulated duodenum
CN: casein
IgCN: immunoglobulin heavy chain
β-lg: β-lactoglobulin
LF: lactoferrin
α-la: α-lactalbumin
* statistically different between deer & cow (P < 0.05) as determined by Tukey’s test
The other whey proteins such as lactoferrin and serum albumin were highly degraded by the
gastrointestinal enzymes. In this study, lactoferrin and serum albumin bands disappeared after
digestion of both milk samples (Fig 3.3 and 3.4). But for serum albumin like protein this was
significantly faster in deer milk than in cow milk, with only 11% intact serum albumin like
protein remaining after simulated stomach in deer while cow milk had 25% (Table3.2). Our
results showed that deer immunoglobulin is more susceptible to digestion by commercial
enzymes than cow milk. Deer immunoglobulin like protein was almost completely digested
(1% intact) after simulated stomach and duodenal digestion while 21% of cow milk
immunoglobulins remained (Fig 3.5c). Many in vitro studies have shown that cow
immunoglobulins are resistant to proteolysis by digestive enzymes and are not inactivated by
gastric acid (Korhonen et al., 2000; Hurley &Theil, 2011).
37
The results reveal that less intact protein is present after simulated stomach and duodenal
digestion of deer milk than cow milk (Table 3.2). The rate of hydrolysis was different
between proteins and between the two species as shown in Figures 3.3-3.5. Commercial
proteolytic enzymes degraded milk proteins from deer more rapidly than those from cow.
This suggests deer milk may also be more digestible in vivo than cow milk.
3.4. Conclusion
In vitro digestion using commercial proteolytic enzymes has provided new knowledge of deer
milk protein digestion. Commercial proteolytic enzymes degraded milk proteins from deer
milk more rapidly than those from cow. Most noticeable was the difference observed in
casein, α-lactalbumin and immunoglobulin. Deer and cow milk β-lactoglobulins were
resistant to both gastric digestion (simulated stomach) and simulated duodenal digestion. This
study provides better knowledge about the digestion of deer milk, compared with cow milk
and may reveal important issues with regard to the proteins in nutrition. The results obtained
may be relevant for development of easily digestable products for consumer groups with
special needs, such as infants, athletes and the elderly. However, as this is an in vitromodel
system, clinical studies are needed in order to confirm results.
Deer milk produced more peptides than cow milk after in vitro digestion using commercial
enzymes. This may mean that deer milk will have more bioactive peptides. The bioactivity of
the peptides produced is currently under investigation.
38
Chapter 4
(Paper from part of this work was accepted in Proceedings of the Nutrition Society of New
Zealand, 2011, Volume 35) (Appendix B)
Fermentation of Deer and Cow Milk
4.1. Introduction
Fermented milk products, in addition to providing energy and nutrients, also have biological
functions that may improve health upon ingestion. The health benefits of fermented milk
have been recognized and documented for centuries (Hitchins &McDonough., 1989).
Empirical observations and scientific demonstrations have concurred, strengthening this
evidence. The beneficial health effects of fermented dairy products may be the result of
bacterial metabolites, such as cell wall components (Furushiro et al., 1993), bacteriocins
(Hernandez et al., 2005) and the hydrolytic action of cell-free extracts containing proteinase /
peptidase activities on milk protein substrates (Pan et al., 2005). Casein and whey protein
derived peptides can be released from milk proteins during fermentation by various bacterial
strains. Moreover, the health benefits of fermented dairy products have been related to the
living microorganisms contained in the fermented products (Perdigon et al., 1991; Saucier et
al., 1992).
Chapter 3results showed that deer milk has 8.8 % protein which is twice as much as in the
cow milk. This could be a good substrate for proteolytic activity of LAB strains and may
produce more peptides which might have biological functions beneficial to health. LAB
utilize the lactose as their first food source producing lactic acid. They then hydrolyse
protein). The lactose content is not different between these milks.LAB can withstand the
increased acidity from organic acid production (Kandler, 1983
The aim of this study was to compare deer and cow milk during fermentation using three
LAB cultures in terms of peptide production before and after in vitro digestion and to
compare deer and cow milk protein digestibility. TheLactobacillus delbrueckiisubsp
bulgaricus and Streptococcus salivarius subsp thermophilus stains used in this study are
commonly used in commercial yogurt making (Tharmaraj and Shah, 2003) while
Lactobacillus casei strain Shirota is a probiotic strain (Yuki et al., 1999).
39
This is the first study to investigate the fermentation of red deer milk followed by in vitro
digestion. The information from the present work is potentially useful in understanding the
release of peptides in animal nutrition and to evaluate the potential use of deer milk products
as functional foods for human.
4.2. Material & Methods
4.2.1. Materials
Lactobacillus
delbrueckiisubsp
bulgaricus
(Ldb),
Streptococcus
salivarius
subsp
thermophilus (Sst) cultures were the kind gifts from Dr. Michelle McConnell, Department of
Microbiology and Immunology, Otago University (New Zealand) and Lactobacillus casei
strain Shirota (Lcs) was propagated from Yokuth (Australian dairy product).All other
materials were those stated in 3.2.1.
4.2.2. Milk Fermentatiom
Cultures (Lactobacillus delbrueckiisubsp bulgaricus (Ldb), Streptococcus salivarius subsp
thermophilus (Sst) and Lactobacilluscasei strain Shirota (Lcs) were propagated by weekly
transfers in skimmed cow milk(heated at 850C for 30 min to killed endogenous bacteria and
enzyme activity (Wang et al., 2009))and stored at 4°C after growing for 17 hr at 37°C in
closed containers (Matar et al., 1996; Pescuma et al., 2008).
Deer and cow milk samples (50 ml) were prepared in triplicate by rehydrating freeze dried
defatted milk in 26% and 12% rehydration ratios, respectively. Samples were heated at 850C
for 30 min (Wang et al., 2009). Prepared milk samples were inoculated withLactobacillus
delbrueckiisubsp
bulgaricus,
Streptococcus
salivarius
subsp
thermophilus
and
Lactobacilluscasei strain Shirota with 2% (vol/vol) inoculums separately and incubated at
37°C for 24 hr (Matar et al., 1996). Fermentation was carried out under aerobic conditions
(conical flask closed with aluminium foil) without pH control.A control milk sample without
any inoculum added to it was maintained in same condition.Lactobacillus delbrueckiisubsp
bulgaricus and Lactobacillus casei strain Shirota growth was determined by plating serial
40
dilutions on Lactobacilli MRS agar, incubating anaerobically at 37°C for 72 hr in Oxoid jars
and counting colony-forming units (cfu). Similarly Streptococcus salivarius subsp
thermophilus were plated in sheep blood agar anaerobically at 37°C for 48 hr. The fermented
milk solutions were used for in vitro digestion.
4.2.3. In vitro Digestion
The in vitro protein digestion was performed for unfermented and fermented milk in two
steps using Pepsin and CPP as described in chapter 3.
4.2.4 Proteolysis Assessment using OPA assay
Proteolytic activity was measured after fermentation and after in vitro digestion of
fermentations as described in chapter 3. TCA precipitation was done prior to OPA assay.
4.2.5. SDS -polyacrylamide gel electrophoresis (SDS-PAGE)
The sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed to
monitorthe protein degradation of deer and cow milks during fermentation and in vitro
digestion. SDS-PAGE of unfermented and fermented milk and digest of fermented milk
samples were done according to a standard protocol (Laemmli, 1970) using NuPAGE 4-12%
Bis-Trisgels.
The protein bands in the gels were quantified by ImageJ software (version 1.42) as described
in chapter 3.2.8.
4.2.6. Statistical analysis
All experiments were carried out in triplicate and the reported values are mean ± standard
deviation. The data were subjected to one way analysis of variance (ANOVA), followed by
Tukey’s test to determine the significant difference between means at P ˂0.05 level using
Minitab (version 16).
4.3. Results
Lactobacillus delbrueckiisubsp bulgaricus and Streptococcus salivarius subsp thermophilus
were able to grow in both milks at 37°C.Their growth plateaued after 12 hours (Fig. 4.1).
41
There was no growth shown in the control milk sample.In cow milk Streptococcus salivarius
subsp thermophilus population reached 107 CFU/ml, whereas it was 106 CFU/ml in deer milk.
The growth of Lactobacillus delbrueckiisubsp bulgaricus was also slightly lower in deer milk
than cow milk. Lactobacillus caei strain Shirota showed a similar growth pattern in both
milks (Fig.4.2). Lactobacillus casei strain Shirota grew slower than the other two organisms
reaching its highest level of 108 CFU/ml level after 24 hours fermentation.
Figure 4.1. Growth of Lactobacillus delbrueckiisubspbulgaricus (Ldb), Streptococcus
salivarius subsp thermophilus (Sst) in deer and cow milk at 37°C for 24 hr. Data are mean of
three independent fermentations ± S.D.
Figure 4.2. Growth of Lactobacillus casei strain Shirota (Lcs) in deerand cow milk at 37°C
for 72hr. Data are mean of three independent fermentations ± S.D.
42
The pH of deer milk was 6.1 whereas it was 6.5 for cow milk. Cow milk fermented by
Lactobacillus
delbrueckiisubsp
bulgaricus
and
Streptococcus
salivarius
subspthermophilusshowed a slight pH reduction from the beginning of fermentation and
started to decrease more rapidly after 6 hr of fermentation (Fig. 4.3 a and b). In deer milk, pH
declined only after 6 hr of fermentation for those two strains and the pH decrease was less
than in cow milk fermentation. After 24 hr of fermentation, the pH of cow milk was
significantly (P ˃ 0.05) lower than deer milk for all 3 stains. The pH fell below 4.6 for cow
milk at which pH the casein precipitated. Although the deer milk fermentation started at a
lower pH than cow milk, it did not fall below 4.6 for any of the three LAB fermentations
(Fig. 4.3). The pH drop for Lactobacillus casei strain Shirota fermentation was observed after
12hr of fermentation and reached a plateau after 44 hr for both milks whilst pH reached a
plateau after 17 hr for
Lactobacillus delbrueckiisubsp bulgaricus and Streptococcus
salivarius subsp thermophilus.
The proteolytic activity of the studied strains was significantly higher (P < 0.05) in deer milk
than cow milk for Lactobacillus casei strain Shirota and Lactobacillus delbrueckiisubsp
bulgaricus stains. There was no significant difference (P > 0.05) between deer and cow
ferments using Streptococcus salivarius subsp thermophilus. The highest production of
peptides was shown by Lactobacillus casei strain Shirota (Fig. 4.4) after 24 hr of
fermentation at 37°C.
43
Figure 4.3. pH drop during fermentation of deer and cow milk at 37°C with (a)
Lactobacillus delbrueckiisubsp bulgaricus (Ldb), (b) Streptococcus salivarius subsp
thermophilus (Sst), (c) Lactobacillus casei strain Shirota (Lcs). Data are means of three
independent fermentations ± S.D (SD values are too small to view).
44
Figure 4.4. Peptide production measured by OPA assay during fermentation of deer and cow
milk at 37°C with (a)Lactobacillus delbrueckiisubsp bulgaricus (Ldb), (b) Streptococcus
salivarius subsp thermophilus (Sst), (c) Lactobacillus casei strain Shirota (Lcs).Data are
means± S.D. (n = 3)
Once ferments were digested in in vitro system, significantly higher (P < 0.05) peptide
production was observed in deer milk than in cow milk for all 3 stains. The highest peptide
production was shown in digests of Lactobacillus casei strain Shirota fermented deer milk
(Fig 4.5).
45
Figure 4.5. Peptide production during in vitro digestion of 24hr fermented (Lactobacillus
delbrueckiisubsp bulgaricus, Streptococcus salivarius subsp thermophilus and Lactobacillus
casei strain Shirota) deer and cow milk - up to 30 min: simulated stomach (step 1) and 30 –
90 min simulated duodenum (step 2). Peptide production was measured after TCA
precipitation prior to OPA assay. Data are means of 3. SD not shown for clarity of graph.
Comparison of peptide production of fermented milk digests with milk digests and fermented
milk prior to digestion is shown in Figure 4.6. Digestion (with or without fermentation)
always produced significantly higher (P < 0.05) peptide levels compared to the level
produced from fermentation only for all 3 LAB strains in both deer and cow milk. Digests of
fermented milk always had higher concentrations of peptides than digests of raw milk. This
was significantly higher for Lactobacillus casei strain Shirota fermented deer milk and
ferments using all 3 stains in cow milk than the raw milk digestion. This means the
fermentation process of milk favoured an increase in the peptide production during digestion
(Fig. 4.6). This was clearer for Lactobacillus casei strain Shirota fermentation than other
strains and this showed higher level for deer milk than cow milk (Fig 4.6 c). There was no
significant difference (P > 0.05) in peptide production between the three strains fermentations
after in vitro digestion for both deer and cow milk.
46
Figure 4.6. Peptide production after in vitro digestion of raw milk (30min simulated stomach
+ 30min simulated duodenum) , 24hr fermented milk and fermented digests for deer ( ) and
cow( ) milk (a)Lactobacillus delbrueckiisubspbulgaricus (b)Streptococcus salivarius subsp
thermophilus (c)Lactobacilluscasei strain Shirota) -peptide production was measured after
TCA precipitation prior to OPA assay. Data are means± S.D. (n = 3)
The protein degradation of fermented milk by all 3 strains was monitored by SDS-PAGE and
compared to unfermented deer and cow milk (Fig. 4.7). After 24 hr of fermentation, casein
degradation was visible as indicated by the fading (Cf and Df lanes) of the original bands (C
and D lanes) on the electrophoresis gel (Fig. 4.7). After in vitro digestion, most of the milk
protein bands completely disappeared and new lower molecular mass bands could be seen on
the gel (Cfd and Dfd lanes).
47
(a)
(b)
(c)
IgHC
LF
SA
CN
β-lg
α-la
C Cf
Cfd
D Df Dfd
C Cf
Cfd
D
Df Dfd
M
C Cf Cfd
D Df Dfd
Figure 4.7. SDS-PAGE (4-12%) of fermented cow and deer milks by (a)Lactobacillus
delbrueckiisubsp bulgaricus, (b)Streptococcus salivarius subsp thermophilus and
(c)Lactobacillus casei strain Shirota at 37°C for 24 hr and in vitro digestion (30min
simulated stomach + 30min simulated duodenum). The lanes are (C) cow milk, (Cf) cow milk
ferment, (Cfd) cow milk ferment digest, (D) deer milk, (Df) deer milk ferment, (Dfd) deer milk
ferment digest, (M) high molecular marker.Each contained 2 mg/ µl protein.
Protein band intensities were quantified using ImageJ software to illustrate degradation of
protein after fermentation and in vitro digestion of ferment (Table 4.1). All the main proteins
started to degrade after fermentation for both milk. After 24 hr of fermentation, cow milk had
significantly higher (P<0.05) intact lactoferrin (LF) than deer milk. LF of cow milk was more
resistant to fermentation than that of deer milk for all 3 strains. Specifically in the
Lactobacillus casei strain Shirota ferment LF was still intact in cow milk while only 47%
remained for deer milk. Digestibility of β-lactoglobulin (β-lg) and α-lactalbumin (α-la) like
protein of deer milk was improved by fermentation. The results showed 55% intact β-lg and
11% intact α–la like protein after in vitro digestion of raw deer milk. Fifty one % of β-lg
remained intact for Lactobacillus delbrueckiisubsp bulgaricus and Streptococcus salivarius
subsp thermophilus fermented digest. At the same time Lactobacillus delbrueckiisubsp
bulgaricus ferment digest only had 1% intact α –la like protein in deer milk. Digestibility of
cow α –la also improved after fermentation and showed only 13% intact protein in
Lactobacillus delbrueckiisubsp bulgaricus ferment digest when it was 27% for unfermented
milk digest. There was a dramatic difference observed in digestion of immunoglobulin (Igs)
of cow milk. Intact protein level was 21% after in vitro digestion of unfermented milk and
decreased to 0 (no intact protein) after in vitro digestion of fermented milk for all 3 strains.
Intact β-lg in cow milk was higher (68%) inStreptococcus salivarius subsp thermophilus
ferment digest than unfermented milk digests (58%). These improvements or reductions of
digestibility of different protein may lead to formation of novel peptides during digestion of
fermented milk.
48
Table 4.1.
Remaining protein content (%) in in vitrodigests of milk;before and after in vitro digestion of Lactobacillus
delbrueckiisubspbulgaricus (Ldb), Streptococcus salivarius subsp thermophilus (Sst)and Lactobacillus casei strain Shirota (Lcs) fermented (24
hr at 37°C) milk (30min simulated stomach + 30min simulated duodenum). Values are obtained by ImageJ.
Lactobacillus delbrueckiisubsp bulgaricus
IgHC
LF
SA
CN
β-lg
α -la
Milk Digest
Deer Cow
1± 2 21±5
ND
1±1
ND
1±1
ND
1±1
54±5 55±8
11±2 27±5
Ferment
Deer Cow
71±4 74±3
42±5 63±4
62±7 63±6
76±8 82±4
85±2 91±7
87±2 90±3
Ferment
Digest
Deer Cow
ND 8±1
ND 2±1
ND 1±1
1±0
1±1
51±1 57±4
1±1 13±4
Streptococcus salivarius subspthermophilus
Ferments
Deer Cow
73±3 77±3
48±2 69±5
70±1 67±6
85±2 84±1
83±4 78±2
85±3 91±2
Ferment
Digest
Deer Cow
ND
ND
ND
ND
ND
ND
2±0
51±1
15±3
2±0
68±1
23±4
Lactobacillus caei strain Shirota
Ferment
Deer
62±1
47±3
77±1
83±2
87±1
92±2
Cow
64±2
101±1
68±1
85±1
86±1
90±1
Ferment
Digest
Deer Cow
ND
ND
1±1
2±1
1±0
ND
ND
ND
52±1
8±4
57±1
17±1
Data are means ± S.D. (n=3)
Not detected (ND)
49
4.4. Discussion
Lactobacillus delbrueckiisubsp bulgaricus,Streptococcus salivarius subsp thermophilus and
Lactobacillus casei strain Shirotaare widely used LAB in the dairy industry. Lactobacillus
delbrueckii subsp. bulgaricus is commonly used alongside Streptococcus salivarius subsp
thermophilus as a starter for yoghurt making and is also found in other naturally fermented
products. Lactobacillus casei strain Shirotahas been extensively studied and it is widely
available as functional food (e.g.Yakult, http://www.yakult.com.au/index.html).
The lower growth of fermentative organisms in deer milk may suggest some kind of
inhibitory effect of deer milk. There was a historical use of reindeer milk as a medical
remedy to heal wound, frostbites and other injuries (Park & Haenlein, 2006). Milk contains
several antimicrobial compounds, including lactoferrin, lactoperoxidase, and lysozyme,
which may be involved in protecting against bacterial growth. Milks of different species
contain different amounts of the various antimicrobial factors (Aimutis, 2004). Cow milk has
high lactoperoxidase, but low lactoferrin and lysozyme, while human breast milk has high
lactoferrin and lysozyme, but low lactoperoxidase (Aimutis, 2004). The antibacterial
properties of milk may be due to synergistic activity of naturally occurring proteins and
peptides, in addition to peptides generated from inactive protein precursors (Clare &
Swaisgood, 2000). Minervini et al. (2003) found an antibacterial peptide from hydrolysis of
human β casein, which had a wide spectrum of antibacterial activity, including activity
against Gram-positive and Gram-negative species. This peptide (β-CN f184-210) inhibited
the growth of Lactobacillus delbrueckiisubspbulgaricusB15. However the identity of the
factors responsible for these low microbial growths is unknown and further studies should be
done on the antibacterial effect of deer milk.
Deer milk is slightly more acidic (6.1 pH) than cow milk (6.5 pH). Differences in pH and
buffering capacity of fresh milk reflect compositional variation (McCarthy, 2002). After 24
hr of fermentation, deer milk showed significantly higher (P < 0.05) pH than cow milk for all
three strains and after fermentation, cow milk pH decreased below the isoelectric point of
casein resulting in precipitation of casein (Fig. 4.3). LAB growth in milk is low at higher pH
closer to 7 (Dat et al., 2011) than lower pH. The higher pH during later part of fermentation
in deer milk than cow milk may be a reason for the low growth in cultures in deer milk
compared to cow milk during fermentation. Lower pH drop of deer milk may be due to
several factors such as low bacterial growth resulting in low lactic acid formation or higher
buffering capacity of deer milk than cow milk. The ultimate pH of cheese results from the
50
lactic acid produced (from starter culture breaking down lactose) and is moderated by the
buffering capacity of the milk (Pendey et al., 2003). Buffering action of milk is due to the
presence of acid buffers (weak acid–conjugate base pairs) and base buffers (weak base–
conjugate acid pairs) (McCarthy, 2002). Milk contains many acidic and alkaline groups that
result in buffering action over a wide pH range. Principal buffer components in milk are
soluble phosphate, colloidal calcium phosphate, citrate, bicarbonate, casein, salt and a
number of minor constituents (Ismail et al., 1973; McCarthy, 2002). Goat milk has high
buffering capacity due to its high content of major buffering components including minerals
(Park, 1992). The mineral content of red deer milk is about two times that of cow milk
(Arman et al., 1974). Also deer milk has twice as much as casein as cow milk (Chapter 3).
This high casein and mineral content may lead to high buffering quality of deer milk and
could reduce the pH drop during fermentation compared to cow milk. However, further
studies should be carried out to confirm the buffering effect of deer milk.
Nielsen et al. (2009) reported that the total amount of peptides produced was significantly
affected by final fermentation pH using 4 species of LAB in cow milk. In that study more
peptides were produced at a final pH of 4.3 than 4.6. In the present study pH did not affect
the same way (more peptide from lower pH)since; milks are from two species of animals. In
this study high pH milk ferment from deer milk yielded more peptides than low pH cow milk
ferment. Lactobacillus delbrueckiisubsp bulgaricus and Lactobacillus caei strain Shirota
ferment of deer milk produced significantly (P < 0.05) higher peptide concentrations than in
cow milk ferments. Deer milk fermented with Lactobacillus casei strain Shirota produced
significantly (P < 0.05) higher peptide levels followed by Lactobacillusdelbrueckiisubsp
bulgaricus. Gobbetti et al. (2000) showed that during milk fermentation, probiotic strains
may produce several oligopeptides which generate bioactive peptides only after subsequent
digestion by pepsin and trypsin. Peptide bonds that are hidden within the native protein
structure might be exposed by the action of LAB enzymes, thereby allowing the release of
new peptides. In terms of peptide level there is no significant difference (P < 0.05) between
three strains for both milk type after in vitro digestion of ferments (Fig. 4.5).
Bioactive peptides are inactive within the sequence of the parent protein and can be released
by enzymatic hydrolysis by digestive enzymes and fermentation of milk. In many studies use
of both fermentation and digestion has proven more effective in generating biofunctional
peptides than fermentation or digestion alone (Korhonen & Pihlanto, 2007). Both milk
51
digestions (with or without fermentation) produced significantly higher (P < 0.05) peptide
level than fermentation alone for all three strains.
This proved that more peptides are
produced by digestion than fermentation. Mata et al., (1996) reported that cow milk
fermentation by Lactobacillus helveticus resulted in a very low degree of proteolysis. They
concluded that proteolysis during fermentation enhanced the release of peptides and may lead
to the formation of novel peptides during gastrointestinal digestion. A large number of studies
have demonstrated that hydrolysis of milk proteins by digestive enzymes, prominently
pepsin, trypsin and chymotrypsin, can produce biologically active peptides (Korhonen &
Pihlanto, 2006). When comparing the digestions with and without fermentation, deer and cow
milk acted in different ways. In deer milk only Lactobacillus casei strain Shirota produced
significantly higher (P < 0.05) peptide levels after digestion compared to unfermented digest,
whereas in cow milk ferment digest for all 3 strains produce significantly higher (P <0.05)
peptide levels than unfermented digest (Fig. 4.6). In contrast digestion of deer milk produces
more peptides than cow milk and was further improved by fermentation prior to digestion.
Highest peptide concentrations were observed in deer milk digest followed by fermentation
and there were no significant difference between LAB strains.
Proteolysis is considered to be one of the most important biochemical processes involved in
the manufacture of many fermented dairy products, irrespective of the contribution of the
proteolytic/peptidolytic enzymes of LAB to organoleptic properties of the final milk
products. The ability to produce extracellular proteinases is a very important feature of LAB.
They catalyse milk proteins, providing the amino acids essential for growth of LAB (Fira et
al., 2001). Proteolysis by microbial enzymes in yogurt is a desirable process for improving
milk digestibility and enhancing the nutritional quality of yogurt. It is accepted that the
proteolytic system of LAB degrades proteins and hence, changes the texture, the taste and the
aroma of fermented products (El-Ghaish et al., 2011). In this study, LAB fermentation
improved the digestibility of β-lg like protein of deer milk more than that of cow milk (Table
4.1). There was 54% intact β-lg like protein in deer milk digest without fermentation. It
decreased to 51% in digest of Lactobacillus delbrueckiisubsp bulgaricus and Streptococcus
salivarius subsp thermophilus deer ferment. β-lg is the main cause of milk allergies causing
about 80% of all cases in children and infants. β-lg is the major whey protein in milk and
dairy products and it is of particular interest since it is the only whey protein of cow milk
absent in human milk (El-Ghaish et al., 2011). The lower β-lg like protein level in ferment
digest in deer milk than cow ferment digest may lead to less allergenicity. Kleber et al.(2006)
52
studied the ability of some LAB strains to reduce the antigenicity of β-lg using skim milk and
sweet whey by indirect competitive ELISA, using polyclonal antibodies. They observed a
reduction of antigenicity of skim milk by 90% and sweet whey by 70% compared to the
initial value. α-lactalbumin (α –la) is another whey protein which has lesser extent of
allergenicity compared to β-lg (Wal, 1998). Intact α –la like protein of deer milk reduced to
1% in digests of
Lactobacillus delbrueckiisubspbulgaricus ferment and to 8% in
Lactobacillus casei strain Shirota ferment from 11% intact α–la of unfermented digest (Table
4.1). The digestibility of α–la in cow milk was also improved by fermentation with the above
two strains. There was always significantly low intact α–la like protein level in deer ferment
digest than cow ferment digests for all three LAB strains. Bu et al. (2010) found combined
strains of LAB reduced the antigenicity of cow skim milk α –la and β-lg. Therefore, our
results suggest fermented deer milk digest may have less allergenicity than fermented cow
milk digest. There was a significant improvement of digestibility of cow Igs after
fermentation by all tested LAB strains (Table 3). Intact Igslevel of cow unfermented digests
was 21% and this went down to 8% in Lactobacillus delbrueckiisubsp bulgaricus ferment
digest and 0% with other 2 strains. The milk Igsare relatively resistant to digestive enzymes
in the gastrointestinal tract but can be sensitive to a group of microbial enzymes (Marnila &
Korhone, 2011).
In this study lactoferrrin (LF) in cow milk was significantly (P < 0.05) more resistant to
fermentation than that in deer milk for all 3 strains. Lactoferrin is an 80 kDa iron-binding
glycoprotein of the transferrin family that is expressed in most biological fluids and is a
major component of the mammalian innate immune system (Gonzalez-Chavez et al., 2009).
By characterising the various peptides generated by hydrolysis of LF, the antimicrobial
activity of the peptides that have minimal variations in the amino acid sequence change was
found. For example, LFampin 268–284 and LFampin 265–284, differ in only three amino
acids (265Asp-Leu-267Ile) but exhibit different strength of antibacterial activity (Kraan et
al., 2006). Therefore different levels of hydrolysis in deer and cow milk fermentation may
form different peptides with different biological activity. Similar to present cow milk
fermentation results of Franco et al., (2010) indicated that LF structure does not seem to be
altered by the activity of commercial (yogurt) LAB bacteria. Hence, their study demonstrated
that yogurt is an excellent product for supplementation with LF though it should be added
after milk pasteurization because heat treatment could make LF more susceptible to
proteolysis by LAB. This work results suggest deer milk will be a better option than cow
53
milk for this purpose. In summary, digestibility of deer milk was higher than cow milk and
this was further improved by LAB fermentation prior to digestion.
4.5. Conclusion
Fermentation prior to digestion increased the production of peptides from and the digestibility
of major milk proteins from both deer and cow milk. Digestion of fermented deer milk
produced more peptides than digestion of fermented cow milk. There was no significant
difference in peptide level produced after digestion among three LAB strains used in this
experiment.
Digestibility of major milk proteins was higher in deer ferment than cow
ferment. The study demonstrates unique characteristics of deer milk which may prove useful
in understanding the potential use in human or animal nutrition and generate new information
which will help in understanding fawn nutrition. Immunomodulation activity of deer and
cow peptides will be examined in the following chapters.
54
Chapter 5
(Paper was prepared for submission toFood and Bioprocess Technology Journal)
Immunomodulatory Activity of Fresh and Fermented Red Deer and
Cow Milks
5.1. Introduction
Milk provides essential nutrients during neonatal development and plays an important
protective role in the development of the immune system of newborns (Inglingstad et al.,
2010). While milk contains components such as immunoglobulin that can modulate the
immune system, an increasing number of studies suggest that milk proteins, may also contain
biologically active peptides with immunomodulating properties within their primary
sequences (Kayser & Meisel 1996; Eriksen et al., 2008; Korhonen, 2009; Phelan et al.,
2009). The concept of developing nutraceuticals based on peptide-enriched fractions from
milk to target the immune system is attractive and is an area of active research (Bhat & Bhat,
2011).
The goal of this study was to investigate the in vitro immunomodulatory activity of
hydrolysed deer and cow milk protein obtained by in vitro digestion using pepsin and CPP,
with and without prior fermentation using three LAB strains [Lactobacillus delbrueckii subsp
bulgaricus (Ldb), Streptococcus salivarius subspthermophilus (Sst) and Lactobacillus casei
strain Shirota (Lcs)]. The effects of hydrolysates on lymphocyte proliferation and cytokine
production (IL2 and IFNγ) were determined.
5.2. Material & Methods
5.2.1. Materials
Oxytocin was purchased from Pharm Distributors (Auckland, New Zealand). Pepsin from
porcine stomach mucosa (66 units / mg solid), O-phthaldialdehyde (OPA), Leucine and
trichloroacetic acid (TCA) were purchased from Sigma (St. Louis, MO, USA) and Corolase
PP (CPP) (350 proteolyticU/mg) from pig pancreas was from AB Enzymes (Darmstadt,
55
Germany). Corolase PP is a mixture of trypsin, chymotrypsin and several amino- and
carboxypeptidases.
Ficoll-Paque PLUS was from GE Healthcare (Sweden) and Concanavalin A (Con A) and
Trypan blue were from GIBCO (Invitrogen, Grand Island, NY, USA). Complete RPMI
(RPMI media was developed by Royal Park Memorial Institute) was prepared from; Lglutamine supplemented RPMI 1640, 9 mL/L non-essential amino acid, 9 mL/L sodium
pyruvate, 10 mL/L Penstrep (GIBCO, Invitrogen, Grand Island, NY, USA ) and 10% heat
inactivated foetal calf serum (Bio International, New Zealand ). All cell culture plates were
sterilized individually packed, flat bottom 96-well cell culture plates from Costar (Corning,
NY, USA). Bovine serum albumin (BSA) was obtained from Sigma, New Zealand. Human
IL-2 and Human IFNγ enzyme-link immunosorbent assay (ELISA) Ready-SET-Go kits were
purchased from eBioscience (USA).
All incubations of cells were performed at 37°C in a humidified 5% CO2 incubator (Function
line BB15, BioLab Scientific LTD, Heraeus).
5.2.2. Collection and Preparation of Milk Samples
Pooled deer milk was obtained from the Lincoln University Deer Farm (Lincoln, New
Zealand) as described in previous work (Chapter 3.2.2). Approval for this work was given by
Lincoln University Animal Ethics Committee (# 377). Pooled cow milk was obtained from
Lincoln University Dairy Farm. Fat was removed after centrifugation at 4000 ×g for 30 min
at 4°C. Defatted milk was freeze dried and stored at room temperature in airtight containers
until analysis.
5.2.3. Fermentation
Cultures (Lactobacillus delbrueckii subsp bulgaricus (Ldb), Streptococcus salivarius subsp
thermophilus (Sst) and Lactobacillus casei strain Shirota (Lcs) were propagated by weekly
transfers in skimmed cow milk and stored at 4°C after growing for 17h at 37°Cin closed
container (as per chapter 4.2.2.)(Matar et al., 1996; Pescuma et al., 2008).
Deer and cow milk samples (50 ml) were prepared to the original concentration by
rehydrating freeze dried defatted milk in 26% and 12% rehydration ratio, respectively.
Samples were heated at 850C for 30 min (Wang et al., 2009). Prepared milk samples were
inoculated in triplicate with Ldb, Sst or Lcs strains with 2% (vol/vol) inoculums separately
and incubated at 37°C for 24 hr (Matar et al., 1996). Fermentation was carried out under
56
aerobic conditions without pH control and the resultant ferment was used for in vitro
digestion.
5.2.4. in vitro Digestion
Unfermented and fermented deer and cow milk samples (15 ml) were digested in triplicate. In
vitroprotein digestion was performed using pepsin and CPP according to Eriksen et al.
(2008). The procedure mimics “normal digestion” in the human gastro intestinal tract. In the
first incubation, which mimics digestion in the stomach, the sample was adjusted to pH 2.5
with 1M HCl and incubated with pepsin (~4mg/g milk protein) at 37°C for 30 min. The
second incubation, mimicking digestion in the small intestine, used CPP (~4 mg/g milk
protein) at 37°C for 60 min after the pH of the sample was adjusted to 7.5 with 1M NaOH.
Continuous shaking (150 rpm) was maintained during digestion. The hydolysates were used
for further analysis.
5.2.5. BCA Protein Assay
Protein concentration in each sample was determined using Bicinchoninic acid reagent
(Pierce, Rockford, Il., USA) according to the manufacturer’s instructions. Samples (10 µl)
were assayed in triplicate and compared to a standard curve generated using dilutions of 2
mg/mL Albumin Standard (Pierce, Rockford, Illinois, USA), to provide concentrations
ranging from 0.0625 mg/ml to 1 mg/ml. Samples were diluted before BCA assay whenever
needed.
5.2.6. Isolation of human peripheral blood mononuclear cells (PBMC)
Human peripheral blood mononuclear cells (PBMC) were isolated from whole blood
collected from a healthy donor (Lincoln University Human Ethics Committee approval #
2011-2) using Ficoll-Paque PLUS as per manufacturer’s instructions. The isolated PBMC
were adjusted to the appropriate cell concentration in complete RPMI.
5.2.7. In vitro lymphocyte proliferation assay
Lymphocyte proliferation assays followed the method of De Fries & Mitsuhashi,(1995) and
Zhi-Jun et al. (1997).Digested fresh and fermented milks were tested for their effects on
lymphocyte proliferation. The protein concentration of each sample was diluted to
0.125 mg/ml, 0.25 mg/ml and 0.5 mg/ml in complete RPMI. The positive control had no milk
57
proteins while the negative control contained BSA as in Eriksen et al. 2008 which used the
same condition with human PBMC.
A 100 µL cell suspension (105 cells/ml) was added to each well in a 96-well cell culture plate.
10 µL Con A (10 µg/ml) (Eriksen et al. 2008) was added to the wells to stimulate cells.
Triplicate 100 µl aliquots of each concentration of the milk protein samples were added to
wells. The plates were incubated at 37°C in a humidified 5% CO2 incubator for 48 hr. Alamar
Blue (20 µl) (Invitrogen, Camarillo, CA 93012) was added to each well and plates were
incubated at 37°C and measured after 24 hr. Fluorescence readings were determined at
wavelengths of 544 nm excitation and 590nm emission using a FLUOstar Omega (BMG
LABTECH) plate reader. Proliferation was expressed as the percentage increase of
fluorescence compared to the fluorescence of the positive control.
% Change in proliferation= Sample fluorescence reading – Positive control fluorescence reading x100
Positive control fluorescence reading
The effects of Con A and protein concentration on lymphocyte proliferation were also tested
using this assay procedure with and without Con A and by using different concentrations of
protein (10 to 640 µg/ml). Deer and cow milk hydrolysates with and without
Lactobacillusdelbrueckiisubsp bulgaricus fermentation were tested.
5.2.8. Cell viability test
The viability test was carried out according to Eriksen et al. (2008). Aliquots (100 µl) of
0.125 mg/ml, 0.25 mg/ml and 0.5 mg/ml deer and cow milk digestions and ferment digests
were added to 100 µl lymphocytes (105 cells/ml) in complete RPMI and incubated for up to 4
days. A 20 µl sample from each incubation was then mixed with 20 µl Trypan blue and
allowed to stand for 5 min at room temperature. A haemocytometer was used to count living
(unstained) and dead (stained) cells using a light microscope. Living (viable) cells were
expressed as a percentage of total cell number.
5.2.9.
Cytokine production
Cytokine production by PBMC was stimulated by Con A (Laffineur et al., 1996, Phelan et
al., 2009). PBMC (105 cells/ml) were supplemented with 10 µg/ml Con A in the presence or
absence of milk protein hydrolysates and incubated for 24 hr in 96-well plates with final
volume of 200 µl. The production of IL-2 and IFNγ were assessed using ELISA kits as per
the manufacturer’s instructions (Burrells et al., 1999). Absorbance was read at 450 nm
58
wavelength and cytokine concentrations were quantified using the standard curve (IL-2 and
IFNγ standard solutions in the kit).
5.2.10. Statistical analysis
Data were presented as the mean ± standard deviation of three independent experiments with
triplicate measurements (n = 3). The lymphocyte proliferation and cytokine production data
were subjected to factorial analysis of variance using GenStat 12.2 (GenStat Release 12.2,
VSN International Ltd, UK). Animal species (deer and cow), treatment (digests of
unfermented and fermented with three LAB strains) and protein concentrations (0.125, 0.25
and 0.5 mg/ml) were used as variable factors and interaction of all factors were tested.
Significant differences between means were determined by Tukey’s test and the level of
statistical significance was taken as P < 0.05. Cell viability and production of cytokines with
and without Con A data were subjected to one way analysis of variance (ANOVA), followed
by Tukey’s test to determine the significant differences between samples at P < 0.05 level
using Minitab statistical software (16 version, Minitab Inc., USA).
5.3. Results and discussion
Effect of milk protein samples on in vitro lymphocyte proliferation
The immunomodulatory activity of in vitro digestions of deer and cow milk and digests of
these milks after fermentation with three lactic acid bacteria commonly used in fermented
dairy products was investigated. Previous researchers have revealed both suppressive and
stimulating effects of cow milk proteins on in vitro lymphocyte proliferation (Cross &Gill,
2000; Gauthier et al., 2006). The degradation rate of milk protein and production of peptides
by LAB fermentation and subsequent in vitro digestion of deer and cow milk were previously
reported (Chapter 4, Opatha Vithana et al., 2011). The aims of the present study were to
investigate whether these digests exhibited immunomodulatory activity and whether
differences in such activity exist due to the milk source.
59
b)
50
50
40
40
*
*
30
% change in proliferation
% change in proliferation
a)
20
10
*
**
*
**
30
20
10
0
0
Milk digest
Ldb fer di
Sst fer di
Lcs fer di
Milk digest
0.125 mg/ml protein
Ldb fer di
Sst fer di
Lcs fer di
0.25 mg/ml protein
Deer
Cow
Deer
Cow
c)
50
*
**
% change in proliferation
40
*
*
30
*
*
20
10
0
Milk digest
Ldb fer di
Sst fer di
Lcs fer di
0.5 mg/ml protein
Deer
Cow
Figure 5.1. Percent change of lymphocyte proliferation of 10 µg/ml Con A stimulated human
PBMC at a) 0.125mg/ml, b) 0.25mg/ml and c) 0.5mg/ml protein level for in vitro digestions
of deer and cow milk, Lactobacillus delbrueckiisubsp bulgaricus (Ldb fer di), Streptococcus
salivarius subsp thermophilus (Sst fer di), Lactobacillus casei strain Shirota (Lcs fer di)
fermented milk. BSA (negative control) values for a,b & c were -1.5%, -5.7% & -10.8%
respectively. Data are means± S.D. (n = 3)
‫ ٭‬Deer significantly higher than cow P<0.01
‫ ٭٭‬Deer significantly higher than cow P<0.001
To test the effect on in vitro lymphocyte proliferation, three different concentrations (0.125,
0.25, 0.5 mg/ml) of milk protein digests were added to cultures of PBMCs which were
60
stimulated with 10 µg/ml Con A. De Fries & Mitsuhashi (1995) has shown use of Con A
stimulated human PBMC with Almar Blue assay as a reliable method to assess proliferation.
Results were evaluated as percentage change of proliferation compared to the control without
milk proteins. Positive values indicate proliferation had increased and negative values
indicate proliferation had decreased compared to the control. All samples caused significant
proliferation of lymphocytes (P < 0.05) except for unfermented cow milk digestions at 0.25
and 0.5 mg/ml protein concentration (Fig. 5.1) and BSA which was the negative control. BSA
significantly (P < 0.01) suppressed the proliferation at 0.5 mg/ml protein concentration. This
is in agreement with Eriksen et al., (2008) who showed a suppressive effect for BSA on
human PBMC proliferation. Also, in agreement with Eriksen et al., (2008), the highest
proliferation activity was found with 0.125 mg/ml protein concentration and the proliferation
activity was decreased with an increase in protein concentration (Fig. 5.1). Our results for
cow milk are consistent with the substantial body of evidence that indicates cow milk
contains a number of potent immunoregulatory peptides (Cross & Gill, 2000; Gauthier et al,
2006) while there are no published studies on deer milk immunomodulatory activity.
Lymphocyte proliferation was higher (P < 0.001) for deer milk protein than cow milk protein
(Table5.1). At 0.125 mg/ml protein concentration, deer milk digest stimulated the production
of significantly more lymphocytes (P < 0.01) than cow milk digest (Fig. 5.1). This suggests
that deer milk protein has higher immunomodulatory activity than cow milk protein at the
same protein concentration (Figure 5.1a, b and c). Reindeer milk has a long history in
Mongolian traditional medicine for curing digestive problems and healing wounds (Park
&Haenlein, 2006). Our findings suggest that the efficacy of deer milk as a medical remedy
may be the result of the immunomodulatory effects of peptides derived from deer milk
proteins. This result taken with previous results of a higher protein content of 8.8 %, which is
twice the level in cow milk (Chapter 3, Opatha Vithana et al., 2012), suggests deer milk
could be a good source to purify bioactive peptides.
Fermentation of the milk prior to digestion significantly increased effectiveness (P < 0.001)
at inducing lymphocyte proliferation than without fermentation (Table5.1) for both deer and
cow milks. This suggests fermentation prior to digestion increases the release of
immunomodulatory peptides from both deer and cow milk. There was no significant
difference (P > 0.05) between digested deer and cow ferments at the lower protein
concentration (0.125 mg/ml), although there was a significant difference in lymphocyte
proliferation between deer and cow milk digests without fermentation at the same
concentration (Fig. 5.1a and Fig. 5.2). Cow milk digests at higher protein concentrations
61
(0.25 and 0.5 mg/mL) did not significantly (P>0.05) enhance the lymphocyte proliferation;
however once it had been fermented with LAB (specifically with Sst) and digested, there was
significantly higher (P < 0.05) proliferation with all three concentrations (Fig. 5.1). This
suggests LAB fermentation of cow milk might generate more immunostimulative peptides
which counteract the immunosuppressive peptides generated by digestion. The type of
microorganism seems to be important in the net activity.
35
‫٭‬
%change inproliferation
30
‫٭‬
25
‫٭‬
20
15
‫٭‬
10
5
0
0.1
0.2
0.3
0.4
0.5
0.6
Protein (mg/ml)
Deer milk digests
Cow milk digests
Figure 5.2. Percent change of lymphocyte proliferation of digest deer (
) and cow
(
) milk at 0.125, 0.25 and 0.5 mg/ml protein concentrations. Data are means± S.D. (n =
3)
‫ ٭‬Significantly enhance proliferation compared to control (P < 0.001).
The results from the extended protein concentration trial (10 to 640 µg/ml) which was carried
out in the presence and absence of Con A for digests of unfermented milk and Lactobacillus
delbrueckii subsp bulgaricus fermented deer and cow milk supported the above conclusion of
improvement of immunostimulation effect by LAB fermentation (Fig. 5.3). When PBMCs
were not treated with Con A, there was no significant increase of proliferation by cow milk
digest. Once cow milk was fermented with Lactobacillus delbrueckiisubsp bulgaricus and
digested, it showed significantly higher (P< 0.05) proliferation without Con A at 40 and 80
µg/ml (Fig. 5.3) than unfermented. This evidence of the release of immunostimulatory
peptides during LAB fermentation of cow milk is in accordance with previous published
work (Matsuziki, 1998; LeBlanc et al., 2002 and 2004; Vinderola et al., 2007; Korhonen,
2009). This study represents the first documented evidence that peptides derived from deer
milk after digestion and LAB fermentation has potentially enhancethe proliferation of
lymphocytes.
62
a)
*
*
50
*
**
*
40
%change inproliferation
*
*
30
*
*
20
10
0
-10
0
100
200
300
400
500
600
700
500
600
700
Protein (µg/ml)
Deer milk digests with ConA
Deer milk digests without ConA
b)
30
*
*
%change inproliferation
20
*
*
10
0
-10
-20
0
100
200
300
400
Protein (µg/ml)
Cow milk digests with Con A
Cow milk digests without Con A
Figure 5.3. Percent change of lymphocyte proliferation with ConA (
) and without
ConA(
) for different protein concentrations of digested a)Deer and b) Cow. Data are
means± S.D. (n = 3).
‫٭‬Significantly enhance proliferation compared to control (P < 0.05).
63
a)
*
**
*
120
%changeinproliferation
100
80
*
*
*
*
60
40
*
*
*
*
*
*
20
0
-20
0
100
200
300
400
500
600
700
Protein (µg/ml)
DL with Con A
DL without Con A
b)
100
%changeinproliferation
80
*
*
**
*
*
*
*
60
*
*
*
*
40
20
0
-20
0
100
200
300
400
500
600
700
Protein (µg/ml)
CL with Con A
CL without Con A
Figure 5.4. Percent change of lymphocyte proliferation with ConA (
) and without
ConA(
) for different protein concentrations of digests of Lactobacillus
delbrueckiisubsp bulgaricus ferments of a) Deer (DL)and b) Cow (CL). Data are means± S.D. (n
= 3).
‫٭‬Significantly enhance proliferation compared to control (P < 0.05).
64
Fermentation
with
lactobacillus
strains
appeared
more
effective
at
producing
immunomodulatory peptides than with streptococcus. At 0.125 mg/ml protein concentration
digest of Lactobacillus casei strain Shirota ferment, deer milk showed significantly higher
(P < 0.05) proliferation compared with Streptococcus salivarius subps thermophilus but was
not significantly (P > 0.05) different from Lactobacillus delbrueckiisubsp bulgaricus
ferments(Fig. 5.1a).Lactobacillus casei strain Shirota ferment of cow milk produced
significantly higher lymphocyte proliferation than milk fermented with Streptococcus
salivarius subsp thermophilus (P< 0.05)and Lactobacillus delbrueckiisubsp bulgaricus (P <
0.001).
Table 5.1.ANOVA for percent change in lymphocyte proliferation for deer and cow
unfermented and fermented (Lactobacillus delbrueckiisubsp bulgaricus, Streptococcus
salivarius subspthermophilus, Lactobacillus casei strain Shirota) milk digests at three protein
concentrations (0.125 mg/ml, 0.25 mg/ml and 0.5 mg/ml).
Variable
Species (deer vs cow)
Unfermented vs fermented
Protein concentration
Species x fermentation
Species x protein concentration
Fermentation x protein concentration
Species x fermentation x protein concentration
d.f.
1
1
2
1
2
6
6
ms
3344.84
4789.65
4118.11
382
79.37
162.34
169.8
F pr
< 0.001
< 0.001
< 0.001
0.034
0.389
0.076
0.063
Values are degrees of freedom (df), mean squares (ms), and significance (F pr) from variance analysis.
(P < 0.05) significantly different
There was a significant (P < 0.001) inverse effect of protein concentration on proliferation of
lymphocytes (Table5.1). The highest proliferation was shown at the lowest protein
concentration (0.125 mg/ml) and gradually decreased with increased protein concentration
(Fig. 5.1 and 5.2) for all treatments as can be clearly seen in Figure 2 for deer and cow
unfermented digests. This could be due to cytotoxic effect of milk proteins at high
concentration (Phelan et al., 2009). The viability test confirmed there was a significant (P <
0.05) reduction in the viable cell percentage at the highest protein concentration after 72 hr of
incubation. Phelan et al., (2009) reported exposure to increased concentrations of hydrolysed
milk protein (0 – 50 %, v/v) resulted in a gradual decline in cell viability by affecting cell
membrane integrity.
65
In the second trial the effect of protein concentration in presence and absence of Con A was
considered for unfermented (Fig. 5.3) and Lactobacillus delbrueckiisubsp bulgaricus
fermented digests (Fig. 5.4) on the proliferation of human PBMC. The proliferation activity
with presence of Con A was initially increased with increased concentration of protein and
reached a maximum between 60 - 120 µg/ml for all unfermented and fermented deer and cow
digests. Then the proliferation decreased with increased protein concentration. Sizemore et al.
(1991) studied the effects of cow milk-derived immunomodulatory bioactive peptides YG
(Tyr-Gly) and YGG (Tyr-Gly-Gly) on Con A induced regulatory T cell activity on cell
proliferation. They also observed a biphasic effect as YGG stimulated proliferation at low
concentrations (10-13 – 10-14 mol/l) and inhibited proliferation at higher concentrations.
Kayser and Meisel (1996) also studied the effect of YGG on human peripheral blood
lymphocytes previously activated with Con A and estimated cell proliferation by BrdU
incorporation. The maximum increase in lymphocyte proliferation induced by YGG was
about 20-30% greater than of the control. In addition, the stimulatory effect of YGG on cell
proliferation was abolished at higher peptide concentrations (10-4 - 10-5 mol/l). Laffineur et
al. (1996) showed that unfermented and Lactobacillus helveticus 5089 fermented cow milk
(at protein concentration of 2.5 to 2500 µg/ml) had higher proliferation response at lower
protein concentration and this effect was decreased with the increase in protein concentration.
Digested deer milk at 160 µg/ml protein concentration even without Con A (Fig.5.3.a), which
is a proliferation stimulant, significantly (P < 0.05) enhanced lymphocyte proliferation. None
of the protein concentrations of unfermented cow milk digest enhanced the lymphocyte
proliferation without Con A (P > 0.05).
As shown in figures 5.1, 5.2 and 5.3, the sensitivity of lymphocyte proliferation to protein
concentration was significantly greater (P < 0.001) for cow milk than deer milk. When the
protein concentration increased from 0.125 mg/ml to 0.5 mg/ml, cow milk digests (fermented
and unfermented) showed a more rapid decline in proliferation than cells treated with deer
milk digests. Milk digestions are mixtures of peptides which might have both immune
stimulating and immunosuppressive properties (Gauthier et al., 2006). It could be suggested
that cow milk digests might have more immunosuppressive peptides than deer milk digests.
This may leads to have more immunosupresive peptides than immunostimulative peptides in
cow milk digests when the protein concentration increases. Alternatively deer digest might
have more immunostimulatory peptides than cow digest. So the net balance of peptides
appears to be immunostimulatory in digested deer milk.
66
Deer peptides enhanced lymphocyte proliferation even without mitogen and thereby may
show the same immunostimulating properties in vivo. Immunomodulatory peptides have
potential application in the maintenance of immune health to protect against infections
involving bacterial, viral and parasitic infections (Gauthier et al., 2006; Szwajkowska et al.,
2011) and may suppress tumours (Matsuzaki, 1998; Xu et al., 2011). Therefore,it can be
suggested that there is potential to use deer milk protein for immune health.
Cell Viability
A trypan blue viability test was carried out to determine whether the observed effects could
be due to the protein samples being toxic to the cells. The results of this test are shown in
Table5.2. Only deer and cow digests at 0.5 mg/mL and cow digest at 0.25 mg/ml appeared to
decrease the number of viable lymphocytes after 72 hr of incubation (P < 0.05). This could be
explained by the toxic effect of protein to cells at higher protein concentrations (Phelan et al.,
2009). Jacquot et al. (2010) showed that hydrophobic peptides generated from trypsin and
chymotrypsin digestion of milk proteins were toxic to murine splenocytes. On the other
hand, there was no significant effect on the cell viability for any concentration of both deer
and cow milk digests during the 48 hr incubation for proliferation or cytokine assays.
Table 5.2. Proportion of viable cells compares to control sample after up to three days
incubation with different protein concentrations. Control cells were incubated without milk
protein samples.
Protein
concentration After 24 hr
(mg/mL)
incubation
Deer
digest
Cow digest
Control
% Viable cells
After 48 hr
incubation
After 72 hr
incubation
0.125
0.250
0.500
92 ± 1
90 ± 2
90 ± 2
90 ± 2
88 ± 4
86 ± 2
88 ± 2
84 ± 1
82 ± 3*
0.125
0.250
0.500
91 ± 1
91 ± 2
91 ± 1
90 ± 5
89 ± 2
87 ± 1
85 ± 1
83 ± 1*
80 ± 1*
0
89 ± 1
90 ± 2
86 ± 1
*Significantly different compared to control (P < 0.05). Data are means± S.D. (n = 3)
67
Cytokine production
To develop a better understanding of the immune response, cytokine production by human
PBMC treated with milk proteins was investigated (Barth et al., 2000; Phelan et al., 2009). T
helper -1 (Th1) cells produce IL-2 and IFNγ and TNF-α cytokines resulting in enhanced cellmediated or cytotoxic responses, whereas T helper -2 (Th2) cells produce IL-4, IL-6, IL-5
and IL-10 which generate a humoral or antibody-mediated immune response (Phelan et al.,
2009).
80
IL-2 production (pg/ml)
‫٭٭‬
‫٭٭‬
60
‫٭‬
‫٭‬
40
20
0
Control
D
C
DL
CL
DS
CS
Dsh
Csh
Milk digests & ferment digests
control
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
Figure 5.5. Interleukin-2 (IL-2) production by human peripheral blood mononuclear cells
(PBMC) at different protein concentrations of deer (D) and cow (C) milk digestions and
fermented milk digestions; Lactobacillus delbrueckiisubsp bulgaricus digest of deer (DL) and
cow (CL), Streptococcus salivarius subsp thermophilus digest of deer (DS) and cow (CS),
Lactobacillus casei strain Shirota digest of deer (Dsh) and cow (Csh). PBMCs were
stimulated by Con A. Control cells stimulated by Con A without addition of proteins.
Detection limit 4 pg/ml.
‫ ٭‬Significantly different compared to control P < 0.05
‫ ٭٭‬Significantly different compared to control P < 0.001
68
100
INF Production (pg/ml)
‫٭٭‬
‫٭٭‬
80
‫٭٭‬
‫٭‬
60
‫٭‬
40
20
0
Control
D
C
DL
CL
DS
CS
Dsh
Csh
Milk digests & ferment digests
Control
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
Figure 5.6. IFNγ production by human peripheral blood mononuclear cells (PBMC) at
different protein concentration of deer (D) and cow (C) milk digest and fermented milk
digests; Lactobacillus delbrueckiisubsp bulgaricus digest of deer (DL) and cow (CL),
Streptococcus salivarius subsp thermophilus digest of deer (DS) and cow (CS), Lactobacillus
casei strain Shirota digest of deer (Dsh) and cow (Csh). PBMC, were stimulated by Con A.
Control cells stimulated by Con A without addition of proteins. Detection limit 4 pg/ml.
‫ ٭‬Significantly different compared to control P<0.05
‫ ٭٭‬Significantly different compared to control P<0.001
Production of IL-2 by PBMC (105 cells/mL) supplemented with 10 µg/ml Con A and without
milk digestion acted as a control and is shown in Figure 5.5. All deer milk digests at 0.125
mg/ml protein produce significantly (Figure 5.5.) higher IL-2 levels than control. At the same
time IL-2 production was significantly higher (P < 0.001) with deer milk than cow milk and
showed significantly higher (P < 0.001) production at lower protein concentration (0.125
69
mg/ml) than higher protein concentration (Fig. 5.5). Phelan et al., (2009) also reported that
cow milk protein hydrolysates had generated a Th1 response by enhancing Con A-induced
IL-2 production and Jacquot et al., (2010) showed three peptides from cow milk protein (β
lactoglobulin and α lactalbumin) induced cytokine production (including IL-2 (9.9 pg/ml) and
IFNγ (63.9 to 717.5 pg/ml) ) using murine splenocyte.
At 0.125 mg/ml protein, digested ferments produced significantly higher (P < 0.05) IL-2
levels than unfermented milk digest for both deer and cow milk (Fig. 5.5). This is in
accordance with results of Vinderola et al. (2007), which showed Lactobacillus helveticus
R389 fermented cow milk regulated immune response by IL-2 production. Galdeano et al.,
(2011) observed that fermented cow milk increased the number of IL-2 producing cells in the
small intestine of mice. Digests of deer milk fermented with Lactobacillusdelbrueckiisubsp
bulgaricus,Lactobacillus casei strain Shirota (P < 0.001) and Streptococcus salivarius subsp
thermophilus and unfermented milk digest produced significantly higher IL-2 levels than the
control (P < 0.05). When comparing the three fermented digests of deer milk, Lactobacillus
casei strain Shirota produced significantly higher (P < 0.001) IL-2 level than Streptococcus
salivarius subsp thermophilus, but the IL-2 level was not different (P > 0.05) from ferments
with Lactobacillusdelbrueckiisubsp bulgaricus. Cow milk fermented with Lactobacillus casei
strain Shirota produced significantly higher IL-2 levels than Streptococcus salivarius subsp
thermophilus (P < 0.001). These results showed the same pattern of high IL-2 production at
0.125 mg/ml protein concentration (Fig.5.5) correspondent with high lymphocyte
proliferation at the same protein concentration (Fig. 5.1). This is further evidence to support
the finding that lactobacillus strains were more effective than streptococcus for production of
immunomodulating peptides as shown earlier by the lymphocyte proliferation results. In vivo
study of Vinderola et al., (2007) showed mice fed with fermented milk produce more IL-2
cytokine by increasing IL-2 producing cell numbers.
Deer milk digests produced significantly higher IFNγ in PBMC (P < 0.001) than cow milk
digests and production was higher (P< 0.001) at 0.125 mg/ml protein concentration than the
other two concentrations (Fig. 5.5). Similar to IL-2, deer digest (except Streptococcus
salivarius subsp thermophilus ferment digest) produced significantly higher (P < 0.05) IFNγ
levels compared to control.
At low (0.125mg/ml) protein concentration unfermented deer milk digests significantly
induced IL-2 and IFNγ production (P<0.05). This was further improved (P < 0.001) by
fermentation with Lactobacillus delbrueckiisubsp bulgaricus and Lactobacillus casei strain
Shirota prior to digestion (Fig. 5.4 and 5.5). Similarly Mao et al. (2007) reported that Yak
70
milk casein hydrolysate increased Con A induced IL-2 and IFNγ production in spleen cells.
Our findings clearly indicate that digestion products (fermented or unfermented) act on the
cytokine network, as illustrated by IFNγ and IL-2. Comparison of IFNγ and IL-2 production
with the proliferation response of lymphocytes indicated evidence of parallelism. Cytokine
production (Fig. 5.4 and 5.5) and proliferation response (Fig. 5.1) were maximal at 0.125
mg/mL protein concentration. There was no significant difference (P > 0.05) compared to
control at higher concentration of proteins. Similarly using Con A stimulated human PBMC,
Laffineur et al., (1996) reported Lactobacillus helveticus 5089 fermented cow milk to have
the highest IFNγ and IL-2 productions at protein concentrations of 19 and 9 µg/ml,
respectively, and the production of the cytokines was decreased with increasing protein
concentration. IFNγ is a typical type 1 helper T cytokine, which enhances immune response.
Matsuzaki, (1998) showed that Lactobacillus casei strain Shirota had a potent anti-tumour
effect on transplanTable tumour cells by inducing production of several cytokines including
IFNγ, resulting in the inhibition of tumour growth. IFNγ production has been considered as a
key factor for anti-tumour activity of mushroom bioactives (Xu et al., 2011). Blattman et al.,
(2003) reported that the specific interaction of casein hydrolysates with IL-2 might prove
useful in modulating dysregulated immune responses as well in the treatment of various
immune processes such as chronic viral infections. Our finding suggests deer milk digests
and digests of LAB fermentation can enhance Con A stimulated IL-2 and IFNγ secretion,
which would potentially amplify a lymphocyte (T cell) mediated response and differentiate
helper T cells into Th1 cells. Therefore, these immunomodulatory deer milk digests
(unfermented and fermented) could serve as a potential immunotherapeutic agent by
selectively increasing the pool of activated T lymphocytes.
5.4. Conclusion
The findings from the present study suggest that deer milk digests exhibit immunomodulatory
effects on human PBMC cultures and this effect was further improved by LAB fermentation
prior to digestion. Immunoregulatory activities were measured as the stimulation of
lymphocyte proliferation and the production of IL-2 and IFNγ cytokines. Fermentation with
lactobacillus strains was more effective at producing immunomodulatory peptides than with
streptococcus. To our knowledge, this is the first report of the immunomodulatory activity of
unfermented and fermented deer milk digests although there are historical therapeutic uses of
deer milk.
71
Deer milk is a good source for purification of bioactive peptides as it has twice the protein
content of cow milk (Chapter 3, Opatha Vithana et al., 2012) and the immunomodulatory
activity of deer milk digests were higher than cow digests at the same protein concentration.
New Zealand has 1.2 million farmed deer which is half of world farmed deer population.
Their current use is mainly for venison and velvet (DINZ, 2009). Therefore, there is a good
potential for isolating immunomodulatory peptides from deer milk for pharmaceuticals or
nutraceuticals. This area requires further in vitro and in vivo research to the durability and
sustainability of peptides during digestion and their mechanisms of action. In future work
deer milk digests will be fractionated to isolate specific immunomodulatory peptides.
72
Chapter 6
Comparative Study of Deer Milk with Three Other
Mammalian Milks
6.1. Introduction
The chemical composition of the milk of different species is designed by natural selection to
provide the nutritional needs of the neonate of that species (Park, 2011). Within a given
species, genetic factors and environment conditions such as climate, diet, number and stage
of lactation influence the chemical composition (Blasi et al., 2008). Cow milk has been the
major source of milk and dairy products in developed countries, especially in the Western
world (Haenlein, 2001), although traditional consumption of milk from other mammals such
as goat, sheep, reindeer, buffalo, camel and yak is common many parts of the world.
Information on the milk of some of these species is not readily available, perhaps since the
milk of these mammals is produced for human consumption only in certain regions of the
world (Park, 2011). Milk is a good source of minerals, which are essential nutrients for
human and animals (Osorio et al., 2007). These may be considered as two groups: the major
elements (Ca, K, Mg and Na) and minor or trace elements (Fe, Cu, Mn and Zn) (Rincon et
al., 1994). Milk protein mainly consists of caseins which include αs1-, αs2-, β- and κ-casein,
and whey proteins which include β-lactoglobulin, α-lactalbumin, serum albumin,
immunoglobulins, lactoferrin and lysozyme (Inglingstad et al., 2010).
Milk proteins are natural vehicles for bioactive peptides (Yoav, 2010). There are many
studies and reviews of cow milk bioactives (Minervini et al., 2003; Gobbetti, 2007;
Korhonen, 2009 and Picariello et al., 2010) while only a few study milk from different
species such as sheep and goat (Silva et al., 2006; Papadimitriou et al., 2007; HernandezLedesma et al., 2011). The aim of this chapter is to compare the composition of deer, cow,
sheep and goat milk and the immunomodulatory activity of their milk proteins after in vitro
digestion.
6.2. Material and Methods
6.2.1. Materials
73
Deer milk was obtained from the deer farm at Lincoln University (Canterbury, NZ). Fresh
milk was collected from 6 red deer hinds with an age range from 6 to 11 years. All hinds
were calved between 7/09/2010 to 26/09/2010. The samples were collected as per chapter
3.2.2. Fresh sheep milk samples from individual animals (n = 6) were a kind gift from Blue
River Dairy (Southland, NZ). Fresh cow (n = 10) and goat (n = 6) milks were purchased from
a local farm (Otago, NZ).
A silicabased C-18 RP-HPLC column (250 mm length x 4.6 mm i.d., Microsorb MV C-18,
particle size: 5 µm, pore size: 30 nm), was used for protein separation (Rainin Instruments,
Woburn, MA). Acetonitrile (Fisher Scientific, Pittsburgh, PA) was of HPLC grade, and BisTris buffer, dithiothreitol (DTT), guanidine hydrochloride (GdnHCl), sodium citrate,
trifluoroacetic acid (Sigma, St. Louis, MO) and cow milk protein standards were purchased
from Sigma (St. Louis, MO).
Tetramethylammonium Hydroxide (TMAH) 25% solution in water was purchased from
Sigma-Aldrich, USA. All the milk samples obtained in this research are from mid-lactation
(except analysis of minerals which used milk through lactation). Other materials are as listed
in chapters 3.2.1 and 5.2.1.
6.2.2. Compositional Analysis
Proximate analysis
Protein content was measured by methods described in chapter 3.2.3. Milk fat, ash and
lactose and moisture contents were determined according to standard procedures (AOAC,
2000) and expressed as % of the sample wet weight.
Quantification of milk protein from Deer, Cow, Goat and Sheep Milk
Milk proteins were identified using cow milk protein standards as described by Bobe et al.
(1998). A solution containing 0.1 M Bis-Tris buffer (pH 6.8), 6 M GdnHCl, 5.37 mM sodium
citrate, and 19.5 mM DTT (pH 7) was added directly to 500 µl milk in a 1:1 ratio (v:v) and
incubated for 1 hr at room temperature, then centrifuged for 5 min at 16 000g in a
microcentrifuge. The fat layer was removed with a spatula. The remaining solubilized sample
was diluted 1:3 (v:v) with a solution containing 4.5 M GdnHCl and solvent A, which
consisted of acetonitrile, water, and trifluoroacetic acid in a ratio 100:900:1 (v:v:v; pH 2).
The concentration of milk protein in the final diluted solution was approximately 4 mg/ml.
74
The standard proteins were prepared as per the manufacturer’s instructions. Milk samples of
5-80 µl (usually 20 µl) and standard (20-100 µl) were chromotographed by Reversed-Phase
HPLC (RP-HPLC). Standard curves were developed by measuring the peak areas of various
known amounts of injected milk protein standards. The standard curves were used to
calculate the amount of protein represented by peak areas from milk samples of unknown
composition.
Mineral analysis of Deer, Cow, Goat and Sheep Milk
Mineral analysis was determined by Inductively Coupled Plasma Optical Emission
Spectrophotometer (ICP-OES) as per Ribeiro et al. (2003). A 1 ml sample of milk was mixed
with 1 ml of TMAH and heated for 800C before analysis by ICP-OES (model 720 ICP-OES,
Varian Australia Pty Ltd, Australia). The sample, in an aerosol form, was introduced into
high energy plasma that dissociates the sample into atoms and ions which emit
electromagnetic radiation. The emitted light is spectrally resolved by diffractive optics, and
the intensity of light is measured with a detector (Nolte, 2003). The instrument allows
complete wavelength coverage from 167-785 nm with a resolution of 7 pm and all
wavelengths are captured in one simultaneous reading. The concentration of a specific
element in a sample is related to the intensity of lines in its optical spectrum.
6.2.3. Determination of Buffering Capacity (BC) of Deer, Cow, Goat and Sheep
Milk
Buffering capacity of milk samples were tested using the procedure of Park (1991). Initial pH
values of all milk samples were determined. Aliquots (25 ml) of each sample were placed in
50 ml beakers and titrated slowly with 0.5N hydrochloric acid (HCl) by adding 1 ml each
time with thorough stirring. The pH was measured after completion of each addition and BC
was determined mathematically using the buffering capacity (intensity) formula given below
(Ismail, et al., 1973; Park, 1991).
dB = (ml acid added) (normality of acid)
dpH
(volume of milk) (pH change)
BC values were plotted against pH (end pH) after each addition of HCl.
6.2.4. In vitro Digestion of Deer, Cow, Goat and Sheep Milk
75
In vitro protein digestion of milk was performed in two steps using Pepsin and CPP as
described in chapter 3.2.5.
6.2.5. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDSPAGE) of Deer, Cow, Goat and Sheep Milk
SDS-PAGE was performed for milk before and after digestion to monitor the digestion
according to a standard protocol (Laemmli, 1970) using NuPAGE 4-12% Bis-Trisgels
(Chapter 3.2.5.2). Bio-Rad Precision Plus Protein TM standard was used as a molecular weight
marker.
6.2.6. Immunomodulatory Activityof in vitro Digested Deer, Cow, Goat and
Sheep Milk
6.2.6.1.
In vitroLymphocyte Proliferation Assay
The effect of milk digests on in vitro lymphocyte proliferation was determined in triplicate
using PBMC isolated from human blood by the method described in chapter 5.2.6. Three
different protein concentrations (0.125, 0.25 and 0.5 mg/ml) were tested for each milk digest.
Proliferation was expressed as percentage increase in fluorescence compared to the
fluorescence of the control (no added protein).
% Change in proliferation = Sample fluorescence reading – Control fluorescence reading x 100
Control fluorescence reading
6.2.6.2.
Cytokine Production
IL-2 and IFNγ production of PBMC treated with milk digests at three different protein
concentrations (0.125, 0.25 and 0.5 mg/ml) were determined using ELISA as per chapter
5.2.4.
6.2.7. Statistical Analysis
Data from proximate analysis and protein concentrations were analysed by Minitab 16 using
one way ANOVA to test for species effect. Significant differences among the means were
separated using Tukey’s test.
76
Mineral data from all the species were analysed by one way ANOVA followed by t-test. The
deer mineral data for four different stages of lactation were analysed by canonical variates
analysis (CVA) (Chope and Terry, 2008). In canonical variates analysis all measured
variables (all minerals) were considered jointly to investigate differences between four
milking times. Depending on the dimensionality required, the analysis calculates the linear
composite (that separates different groups in one dimension) called canonical variates 1 and
an orthogonal composite (that separate different group in another dimension) called canonical
variates 2. The analysis generates the percentage of variation due to the linear and orthogonal
components and latent vectors (called loadings) that indicate the contribution level of each
variable to the linear and orthogonal components, thus the importance of each variable to the
overall separation of different stages of lactation.
Data for buffering capacity were fitted to a general linear model (GLM) using end pH as a
covariate for linear and quadratic effects and species as a classificatory variable.
Data for the biological activities were analysed using the REML routine and the significance
of treatment terms and their interactions were determined by Wald statistical tests. In the
REML analysis, species and protein concentration were set as fixed factors, whereas
interaction between species and animals within the same species were set as random factors.
All the sample analyses were performed in triplicates and statistical analyses were carried out
using Genstat (GenStat Release 14.1, Lawes Agricultural Trust, VSN Int. Ltd., Rothamsted,
U.K.).
6.3.
Results
Proximate analysis of Deer, Cow, Goat and Sheep Milk
The chemical composition of deer milk compared with milk from cow, goat and sheep is
shown in Table 6.1. Moisture, dry matter, protein, fat and ash content of deer milk were
significantly different (P<0.05) from cow and goat milk, while there were no significant
differences (P>0.05) between cow and goat milk for the above constituents. Deer milk had
approximately twice the concentration of protein as cow milk. The major protein in deer milk
was β casein and its content was significantly higher (P<0.05) than cow and goat milk β
casein content but not significantly different (P>0.05) from sheep milk. κ-casein-like and αs1casein-like content of deer milk was significantly lower (P<0.05) than the other three species.
77
Of the whey proteins, deer milk had significantly lower (P<0.05) concentration of αlactalbumin-like than all the other species.
Table 6.1. Mean of proximate composition for deer (n = 6), cow (n = 10), sheep (n = 6) and
goat (n = 6) milks and concentration of milk proteins (mg/ml) of deer, cow, sheep and goat
milks (n = 6 each). Values are reported as means± standard deviation.
Proximate
composition
Species
Moisture (%)
Deer
79.4 ± 1.5b
Cow
86.9 ± 0.6
Dry matter (%)
20.6 ± 1.5a
Protein (%)
Sheep
Goat
b
79.6 ± 0.8
87.4 ± 1.2a
13.1 ± 0.6b
20.4 ± 0.8a
12.6 ± 1.2b
7.3 ± 0.4a
3.7 ± 0.3b
6.3 ± 0.7a
3.9 ± 0.3b
Fat (%)
8.1 ± 1.7a
4.7 ± 0.4b
8.1 ± 0.2a
3.8 ± 1.2b
Lactose (%)
3.9 ± 0.3b
3.9 ± 0.2b
5.0 ± 0.4a
4.1 ± 0.2a
Ash (%)
1.2 ± 0.1a
0.7 ± 0.1b
0.9 ± 0.1b
0.9 ± 0.1b
a
Protein profile
κ-casein
4.99 ± 0.61c
9.76 ± 0.61b
12.23 ± 0.08a 9.4 ± 0.10b
c
b
αs2-casein
0.76 ± 0.05
2.56 ± 1.16
13.3 ± 0.07a
0.74 ± 1.15bc
αs1-casein
6.92 ± 0.22c
21.24 ± 0.34a 11.9 ± 1.06b
17.35 ± 2.95ab
β-casein
15.11 ± 0.15d 36.64 ± 0.81a 26.29 ± 1.13c
32.87±1.01b
c
α-Lactalbumin
2.13 ± 0.01
2.99 ± 0.30b
4.02 ± 0.41a
3.49 ± 0.52ab
8.95 ± 0.91b
β-Lactoglobulin A
10.58 ± 0.05a 9.57 ± 0.37a
8.58 ± 0.09b
0.83 ± 0.05b
β-Lactoglobulin B
1.15 ± 0.06b
2.53 ± 0.19a
Not delected
β-Lactoglobulin C
Not detected
0.60 ± 0.06
Not detected Not detected
Mix (Ig~s + Serum
c
b
7.68 ± 0.20
13.55 ± 0.24 29.10 ± 0.78a 2.63 + 0.23d
albumin)
Lactoferrin
2.60 ± 0.60a
2.33 ± 0.54a
1.94 ± 0.32ab 1.38 ± 0.37b
b
Lysozyme
Not detected
9.76 ± 0.61
0.11 ± 0.03b
1.24 ± 0.05a
a-d
Values within each row with different superscripts are significantly different (P < 0.05).
Mineral composition of Deer, Cow, Goat and Sheep Milk
Mineral composition of deer, cow, sheep and goat milks is shown in Table 6.2. The
concentrations of Ca, P and S which are major elements and Zn which is a trace or minor
element were significantly higher (P < 0.05) in deer milk than any of the other three milk
types. Deer milk had significantly higher (P < 0.05) Mg level than cow and goat milk but less
(P < 0.05) than sheep milk. There were no significant differences (P < 0.05) in Fe content
among deer, cow and goat milk, while sheep milk had a significantly higher (P < 0.05) Fe
content than milk from the other three species. Goat milk had significantly higher (P < 0.05)
K, Mn and Cu levels than deer, cow and sheep milk.
78
Table 6.2. Major and minor mineral contents of deer (n=6) of four milking, cow (n=10), sheep (n=6) and goat (n=5) milk. Values are reported
means± standard deviation.
Elements
Deer (µg/ml)
Ca
P
K
Na
Mg
S
Fe
Mn
As
Cr
Cu
Mo
Se
Zn
Al
Pb
2958 ± 101
a
2219 ± 102
b
1148 ± 79
271.6b ± 13
182.7b ± 11
602.3a ± 23
1.23b ± 0.31
0.15b ± 0.023
0.62 ± 0.020
a
0.04 ± 0.003
b
0.11 ± 0.015
0
1.11a ± 0.02
12.09a ± 0.78
6.14b ± 0.28
0.04± 0.03
a
Cow (µg/ml)
c
1601 ± 180
c
1209 ± 104
b
1187 ± 85
391.6a ±45
139c ± 20
354.9c ± 34
1.36b ± 0.71
0.11b ± 0.029
0.60 ± 0.070
b
0.02 ± 0.003
b
0.11 ± 0.04
0.03a ± 0.01
1.00b ± 0.07
6.27c ± 0.65
6.65b ± 0.74
0.06 ± 0.04
Sheep (µg/ml)
b
2427 ± 140
b
1966 ± 86
b
1009 ± 49
366.6a ± 14
229.3a ± 12
533.8b ± 27
2.09a ± 0.14
0.19b ± 0.011
0.62 ± 0.06
a
0.04 ± 0.003
b
0.14 ± 0.01
0
1.10a,b ± 0.14
8.39b ± 0.32
8.02a ± 0.37
0.08 ± 0.10
Goat (µg/ml)
c
1459 ± 206
c
1343 ± 115
a
1626 ± 227
277.8b ± 18
156.8b,c ± 22
349.5c ± 31
1.58a,b ± 0.23
0.54a ± 0.244
0.60 ± 0.02
b
0.03 ± 0.002
a
0.22 ± 0.14
0
1.04a,b ± 0.11
5.32c ± 0.65
6.64b ± 0.46
0.12 ± 0.02
Recovery
(%)
Limits of
detection
98.6
103
105
97
108.1
1.62
0.02
0.14
2.21
0.28
0.19
0.003
0.0003
0.015
0.0002
0.003
0.0006
0.031
0.023
0.246
0.005
105.6
108.2
100
104.8
104.9
106.8
90
106.1
103.6
110
Co, Ni and Cd were not detected in any of the milk
Mo was detected in only one sample of sheep milk and other samples and deer and goat milk were below quantification limit.
a-c
Values within each row with different superscripts are significantly different (P < 0.05).
79
Table 6.3. Mineral composition (µg/ml) of red deer milk (n=6) early, middle and late lactation. Values are reported as means± standard
deviation.
Elements
Ca
Time 1 (µg/ml)
3100 ± 636
Time 2 (µg/ml)
2955 ± 1056
Time 3 (µg/ml)
2942 ± 338
Time 4 (µg/ml)
2833 ± 396
Recovery (%)
98.6
Limits of detection(µg/ml)
1.62
P
2370 ± 389
2182 ± 771
2176 ± 168
2146 ± 273
103
0.02
K
1205 ± 201
1202 ± 424
1150 ± 80
1036 ± 141
105
0.14
Na
289.4 ± 33
272.6 ± 100
258.6 ± 20
266.0 ± 32
97
2.21
Mg
191.8 ± 30
192.4 ± 69
175.7 ± 20
170.9 ± 23
108.1
0.28
S
621.9 ± 109
577.0 ± 194
587.7 ± 41
622.7 ± 85
a
ab
± 0.29
ab
1.06
± 0.17
-
0.19
b
0.921 ± 0.24
105.6
0.003
Fe
1.64 ± 0.66
1.32
Mn
0.176 ± 0.10
0.16 ± 0.04
0.12 ± 0.03
0.13 ± 0.06
108.2
0.0003
As
0.64 ± 0.08
0.64 ± 0.23
0.60 ± 0.06
0.61 ± 0.11
100
0.015
Cr
0.05 ± 0.01
0.04 ± 0.01
0.05 ± 0.01
0.04 ± 0.01
104.8
0.0002
Cu
0.14 ± 0.05
0.11 ± 0.04
0.11 ± 0.03
0.10 ± 0.03
104.9
0.003
Se
1.13 ± 0.07
1.10 ± 0.43
1.08 ± 0.08
1.13 ± 0.10
90
0.031
Zn
13.24 ± 2.09
11.58 ± 3.93
11.64 ± 1.26
11.91 ± 2.09
106.1
0.023
Al
6.47 ± 0.71
6.26 ± 2.27
6.00 ± 0.20
5.83 ± 0.47
103.6
0.246
Pb
0.06 ± 0.01
0.03 ± 0.09
0.01± 0.01
0.07 ± 0.03
110
0.005
Mo, Co, Ni and Cd not detected
Time 1: 17/12/2010
Time 2: 07/01/2011
a-b
Time 3: 21/01/2011
Time 4: 10/02/2011
Values within each row with different superscripts are significantly different (P < 0.05).
80
The changes in mineral composition of red deer milk across lactation were studied using four
collection times and the results are shown in Table 6.3. Only Feshowed significant changes (P <
0.05) during lactation. There was a steady decline in Fe content with the first date tested
(17/12/2010) being significantly higher (P<0.05) than that at the end of lactation (10/02/2011).
Canonical variate analysis is a powerful statistical technique that considers the measured variables
(the concentrations of 15 minerals) jointly to separate milking (stage 1-4) into groups and illustrate
the overall differences in mineral composition among the four groups (Fig. 6.1). Canonical variate 1
reflects the linear component of the analysis and it can be seen that it contributes to the majority of
segregation between stage (time) of lactation. Canonical variate 2 is the orthogonal component that
had much more limited contribution to separating the lactation times (Table 6.4.). Contributions of
each mineral to the differentiate lactations are shown by loading values (Table 6.4.). K, Mg, Al and
Fe seem to be the major contributors in differentiating (P<0.05) the milking times based on
canonical variate 1.
Figure 6.1. Canonical analysis for 15 minerals at four milking times/stages (time 1: 17/10/2010,
time 2: 07/01/2011, time 3: 21/01/2011, time 4: 10/02/2011)of 6 red deer hinds.
81
Table 6.4. Canonical variate analysis for the red deer minerals
Elements
Percentage variation
Ca
K
Mg
Na
Al
Fe
Mn
P
S
As
Cr
Cu
Pb
Se
Zn
Variate 1
79.7
0.205
0.409
0.377
0.359
0.506
0.64
0.307
0.287
0.01
0.198
-0.111
0.341
0.034
0.047
0.286
Variate 2
16.94
-0.044
0.24
0.159
-0.193
0.017
-0.083
-0.077
-0.195
-0.347
0.052
-0.417
-0.136
-0.342
-0.245
-0.367
The linear component of the analysis is represented by variate 1 and orthogonal component of the analysis is
represented by variate 2. The value in the Table represented the contribution of variate 1 and variate 2 in the separation
of the different minerals (percentage variation). The coefficient of mineral content contributes to separation (loadings).
The loadings are treated as vector, which mean that they indicate a direction and have the same weight regardless of the
+/- sign.
Buffering Capacity (BC) of Deer, Cow, Goat and Sheep Milk
Initial pHs of deer, cow, goat and sheep milks were 6.51, 6.56, 6.75 and 6.6 respectively. The
buffering capacity and end pH among the four milks upon titration with 0.05 N HCl are presented in
figure 6.2. Deer milk had significantly greater (P<0.05) BC than any of the other three milks. BC of
sheep milk was significantly higher (P<0.05) than that of cow and goat milk. At the beginning of
titration, buffering index was increased with the decrease of end pH for all four milk types. Then it
reached a plateau at around pH 4 for cow,goat and sheep milk. Deer milk buffering index continued
to increase even after the addition of 5 ml 0.5 N HCl.
82
Buffering Index ( dB/dpH)
0.12
Deer
Cow
Goat
Sheep
Deer (fit)
Cow (fit)
Goat (fit)
Sheep (fit)
0.10
0.08
0.06
0.04
0.02
2
3
4
5
6
7
End pH
Figure 6.2. Buffering index (buffering capacity) of cow (n=6), sheep (n=2) and goat (n=5) milks
compared with deer milk (n=6).
SDS-PAGE of Deer, Cow, Goat and Sheep Milk
Milk from four different species was digested in vitro and the intact proteins remaining in the
digested milk samples were analysed by SDS-PAGE (Figure 6.3). The results revealed that the
casein of all four milks was well digested, while β-lg was still intact. The intensity of β-lg bands of
deer and cow milk digests were less than those of sheep and goat milk digests.
83
MW:
250 kDa
150
100
75
IgHC
LF
SA
50
37
25
20
15
CN
β-lg
α-la
10
M
D C
S
G Dd Cd Sd Gd
Figure 6.3. SDS- PAGE (4-12%) of Deer, Cow, Sheep and Goat milk before and after digestion
with pepsin and CPP. Major bands; immunoglobulins heavy chain (IgHC), lactoferrin (LF), serum
albumin (SA), casein (CN), β-lactoglobulin (β-lg) and α-lactalbumin (α-la). Standard molecular
weight markers are shown on left-hand side of the gel. The lanes: Molecular weight marker (M),
Deer (D), Cow (C), Sheep (S), Goat (G) milk and in vitro digestion of Deer (Dd), Cow(Cd),
Sheep(Sd), Goat(Gd) milk.Each contained had 2 mg/ µl protein.
Immunomodulatory Activityof In vitro Digested Deer, Cow, Goat and Sheep Milk
Lymphocyte proliferation
Lymphocyte proliferation was significantly different (P<0.05) between species (Table 6.5). Positive
values show increased proliferation while negative values demonstrate decrease of proliferation
compared with the positive control. Lymphocyte proliferation was significantly higher at 0.125
mg/ml protein concentration for deer (P<0.001), cow and sheep (P<0.01) than the control (Fig. 6.4).
At the same protein concentration (0.125 mg/ml) deer milk digest showed significantly higher
lymphocyte proliferation compared to cow, sheep (P<0.01) and goat (P<0.001) milk digestswhile
there were no significant differences (P>0.05) among cow, sheep and goat milk digests.
Table 6.5. ANOVA for % change in lymphocyte proliferation.
Fixed term
Species
Protein Concentration
Species. Protein Concentration
Wald statistic
10.66
60.13
11.62
n.d.f.
3
2
6
F statistic
3.55
30.07
1.94
d.d.f.
19.0
176.0
176.0
F pr
0.034
>0.001
0.077
84
Lymphocyte proliferation was significantly different (P<0.001) at different protein concentrations
(Table 6.5). Proliferation decreased with increasing concentration of protein for all four species
(Fig. 6.4).
‫٭‬
‫٭‬
‫٭‬
‫٭٭‬
Figure 6.4.Percentage change of lymphocyte proliferation at a) 0.125mg/ml, b) 0.25mg/ml and c)
0.5mg/ml protein concentration for deer, cow, sheep and goat milk digests.Data are means± S.D. (n =
6 for deer, cow, sheep and n = 5 for goat).
‫ ٭‬Significantly different from control P<0.01
‫ ٭٭‬Significantly different from control P<0.001
Cytokineproduction
IL-2 production (Fig. 6.5) was affected by the type of milk digest from different species (P<0.001)
and the protein concentration (P<0.001). Digested deer milk produced significantly higher IL-2
levels than cow, and goat milk at 0.125 mg/ml protein concentration (P<0.05). At this protein
concentration all milk types showed significantly higher (P<0.001) IL-2 levels than the control. IL2 production was reduced with increased protein concentration. IL-2 production was significantly
lower than control (P<0.001) at 0.5 mg/ml protein concentration, except for lymphocytes treated
with sheep milk digests.
IFNγ production (Fig. 6.6) by PBMC was significantly different when treated with milk digests
from different species and at different protein concentrations (P < 0.001). All samples produced
significantly higher (P < 0.001) IFNγ levels than control. PBMC incubated with deer milk at 0.125
mg/ml protein concentration produced significantly higher (P < 0.001) IFNγ levels than those
treated similarly with cow and sheep milks, while there was no significant difference with goat milk
(P > 0.05) treatment.
85
‫٭٭‬
‫٭٭‬
‫٭٭‬
‫٭‬
‫٭‬
‫٭‬
‫٭٭‬
‫٭٭‬
‫٭‬
‫٭‬
Figure 6.5. Interleukin-2 (IL-2) production by Con A stimulated human peripheral blood
mononuclear cells (PBMCs). Production of IL-2 by PBMCs with different protein concentrations
of deer, cow, sheep (n=6) and goat (n=5) milk in vitro digestion. Control represents cells stimulated
by Con A without addition of proteins. Values are the means± S. D. Detection limit 4 pg/ml.
‫ ٭‬Significantly different from control P<0.01
‫ ٭٭‬Significantly different from control P<0.001
‫٭٭‬
‫٭٭‬
‫٭٭‬
‫٭٭‬
‫٭٭‬
‫٭٭‬
‫٭٭٭٭‬
‫٭٭‬
‫٭٭٭٭‬
‫٭٭‬
Figure 6.6. IFNγ production by Con A stimulated human peripheral blood mononuclear cells
(PBMCs). Production of IFNγ by PBMCs with different protein concentrations of deer, cow, sheep
(n=6) and goat (n=5) milk in vitro digestion. Control represents cells stimulated by Con A without
addition of proteins. Detection limit 4 pg/ml.
‫٭٭‬All samples are significantly different compared to control (P<0.001).
86
6.4.
Discussion
Compositional Analysis
Milk composition varies according to several factors, such as animal, feed and environment
(Mackle et al., 1999). The 20% dry matter content of deer milk was significantly higher (P<0.05)
than cow and goat milks. Thus deer milk has the highest dry matter content among these four milks.
Protein & fat which are constituents of milk dry matter and ash, were all significantly higher
(P<0.05) in deer milk than cow and goat milk. The results for proximate analysis (protein, fat, ash
and lactose) of cow, goat and sheep milks in this study were in accordance with literature (RaynalLjutovac et al., 2008; Ceballos et al., 2009; End et al., 2009). Deer milk had about two times the
protein content of cow milk and these results are consistent with our previous results (Chapter 3,
Opatha Vithana et al., 2010). Similarly deer milk protein of 7.3% in this study is consistent with
Arman et al., (1974) which showed 7.39% protein in red deer milk using six hinds. There was no
significant difference (P<0.05) in lactose content among deer, cow and goat milks. Deer milk had
3.9% lactose in this study versus 5% in sheep milk. It has been reported that sheep milk contains
4.88% lactose (Raynal-Ljutovac et al., 2008).
Milk proteins were identified and quantified by HPLC using cow milk protein standards to get a
general idea of different milk proteins in milk from each species. The β casein-like content of deer
milk was significantly higher (P>0.05) than cow and goat milk and not significantly different
(P>0.05) from sheep milk. Cow milk protein is one of the most common food allergen in
children.Cow milk contains more than 20 proteins (allergens) that can cause allergic reactions (ElAgamy, 2007). Casein and β-lg are the main allergens in cow milk (Natale et al., 2004) and of the
caseins, α-s1 is the major allergen (Cocco et al., 2003). Jarvinen et al., (2002) found that five IgEbinding epitopes (2 on αs1-casein, 1 on αs2-casein, and 2 on κ-casein) were recognized in patients
with persistent allergy. Goat milk is less allergenic than cow milk and β-casein is the major fraction
in goat casein, which is similar to human casein, while αs1-casein is the major fraction of cow casein
(El-Agamy, 2007). The present results showed that the major casein in deer milk is β casein and
that it is low in αs1-casein, which suggests that deer milk might be less allergenic than cow milk.
Further studies should be done to investigate the potential use of deer milk proteins as an alternative
for cow milk for allerginic patients.
In milk, the casein exists as large colloidal particles, 50-600 nm in diameter called “casein micelles”
that are composed of four main caseins: αs1-casein, αs2-casein, β-casein and κ-casein (Semo et al.,
87
2006). Many of the technologically important properties of milk, e.g. its white colour, stability to
heat or ethanol, and coagulation by rennet are due to the properties of the casein micelles (Fox
&Brodkord, 2008). The milk of different species is more or less white due to light scattering for
which the micelles are mainly responsible (Fox &Brodkord, 2008). Therefore our observation of
different colour intensity of different species milk (visual observation) might be due to the
differences of casein micelles based on their compositions. Deer milk was whiter than the milk from
the other three species.
κ-casein-likeprotein content of deer milk was significantly lower (P>0.05) than the other three
species. The size of casein micelle is inversely related to their κ-casein content (Inglingstad et al.,
2010). This suggests that deer milk may form bigger casein micelles than other species. Most
importantly the micelles are coagulated in the stomach of the neonate by chymosin, a proteinase
designed for this function, and coagulation delays the entry of milk into the small intestine, thereby
improving digestibility (Fox &Brodkord, 2008). Inglingstad et al., (2010) studied the comparative
in vitro digestion of cow, goat, horse and human milk proteins and found that horse milk casein had
very rapid gastric digestion compared to the other species. They concluded that lower content of κcasein and larger size of casein micelles in horse milk than other species (cow, goat and human)
may be the reason for the high susceptibility to hydrolysis by gastric enzymes. This suggests deer
milk which showed low content of κ-casein may form bigger casein micelles than other species and
ultimately show higher casein digestibility than other species. This requires further research to be
confirmed.
β-lactoglobulin-like protein, the major whey protein content of deer milk was not significantly
different (P<0.05) to cow milk, when β-lactoglobulin standard of cow milk was used (HPLC), and
also SDS-PAGE results (Fig 6.3.) β-lactoglobulin band of deer milk can be clearly visualized.
McDougall and Stewart, (1975) studied red deer whey proteins by chromatographic separation
(Sephadex G-75 column) using cow milk markers and was able to detect a component which eluted
similarly to cow β-lactoglobulin. Their results also indicate the concentration of β-lactoglobulin
changes with lactation time. Although there are similarities of red deer and cow milk βlactoglobulin, McDougall and Stewart, (1975) reported that differences in the amino acid
composition of β-lactoglobulin were found. Deer β-lactoglobulin contained one more residual of
aspartic acid, alanine and methionine and one less glutamic acid and two less leucine residues than
cow β-lactoglobulin. Another difference was deer β-lactoglobulin A and B genetic variation were
more acidic than those of the corresponding cow β-lactoglobulin.
88
Deer milk had the lowest level of α-lactalbumin-like compared to the other three species. βlactoglobulin and α-lactalbumin are the major whey protein allergens (El-Ghaish et al., 2011; Wal,
1998). Therefore allergenicity caused by whey proteins might be less for deer milk than other milk
types, but this will need to be confirmed in future work.
In this study the mineral composition of deer milk was compared with three other milk types which
are more commonly consumed. The Ca, P and S content of deer milk were higher than cow, goat
and sheep milk. Minerals play an essential role in human health and wellbeing. It is well known that
milk and dairy products are good dietary source of Ca and P (Birghila et al., 2008; Ceballos et al.,
2009). There was 2958 µg/ml Ca in deer milk while cow milk had only 1601 µg/ml. These findings
are in agreement with Arman et al. (1974) who showed 2200 - 2500 µg/g Ca content in deer milk
and Ceballos et al., (2009) who showed 1135.8 µg/g Ca content in cow milk. Sheep milk had
second highest Ca content which was 2427 µg/ml. Similar to our findings, Rincon et al., (1994)
reported sheep milk has significantly (P<0.05) higher Ca level (2056 µg/g) than cow or goat milk.
Mineral results of cow, goat and sheep were in agreement with studies done by Rincon et al.
(1994), Wendorff (2006), Birghila et al. (2008), Raynal-Ljutovac et al. (2008) and Haenlein and
Ceballos et al. (2009). At the same time, red deer milk mineral results of this study generally did
not vary from those of Arman et al. (1974).
Zn was significantly (P<0.05) higher in deer milk than other milk types. Zinc is the trace element in
milk that is present in the largest concentration and found in the milk fat globule. Zn is generally
considered being a stabilizing agent of biological membranes (Linden et al., 2004). Wiking and
Sehested, (2008) reported that the transfer of fat from diet to milk might facilitate transfer of Zn
from diet to milk. This might be the reason that the highest fat containing deer milk has highest Zn
content and was followed by sheep milk which has second highest Zn and fat contents amongthe
four studied milk types. Sheep milk had significantly higher (P<0.05) Mg and Fe contents than any
other species of milk in this study and this was in agreement with Rincon et al. (1994) who showed
that sheep milk has more Mg and Fe contents than cow or goat milk. The level of Mg in red deer
milk was consistent with that reported by Arman et al. (1974) and slightly higher than the results
reported by Vergan et al., (2003). Goat milk had higher K level than deer, cow and sheep milks.
Goat milk is reported to be distinguished by its high K and Cl contents (Raynal-Ljutovac et al.,
2008). Although mineral composition of milk may be affected by the nutrition of the animals, there
are certain aspects that are characteristic for each species which enable the mineral composition of
the milk to be used to identify the species (Rincon et al., 1994; Ceballos et al., 2009).
89
Changes in mineral composition of deer milk during four milking times were studied. Fe
concentration declined during lactation while all other mineral showed no significant differences
(P>0.05) over the four milking times. Fe content at first milking was significantly higher (P>0.05)
than last milking. Feely et al., (1983) showed similar results in human milk where Fe content was
significantly higher (P<0.05) in early lactation and decreased with lactation time.
There was no consistent pattern according to which stage of lactation had highest and lowest
concentration of each element (Table 6.3). However since ANOVA is a unvariable analysis which
allows comparisons to be made between milking based on a single variate, it does not give an
insight into how the milking times are grouped or which is the most important element in defining
group. Canonical variates analysis allows these type of conclusions to be drown, and has previously
been used in discrimination between ultrafiltered milk permeates (Hansen et al., 2010), to
differentiate onion cultivars by mineral content (Chope &Terry, 2009) and jointly separated New
Zealand fish products using five colour parameters (Bekhit et al., 2009). Canonical variates results
differentiate the four milking times considering all the minerals as a whole (Fig. 6.3). Among all the
minerals K, Mg, Al and Fe seem to be major contributors in differentiating (P<0.05) milking times
based on canonical varitae 1 (Table 6.4).
Buffering Capacity of Deer, Cow, Goat and Sheep Milk
The buffering capacity (BC) of milk is an important physico-chemical characteristic that
corresponds to the ability of the milk product to be acidified or alkalinized. This value depends on
several compositional factors such as inorganic phosphate, citrate, organic acids, salts and milk
proteins (Salaun et al., 2005). BC results of this study showed that deer milk had significantly
higher (P<0.001) BC than that of the milk from the other three species. BC of sheep milk was
significantly higher (P<0.05) than that of cow and goat milks.There was no significant difference
(P>0.05) between cow and goat milk BC. Cow and goat milk reached maximum BC between 5.5 –
4.0 pH after addition of 3 ml HCl while sheep milk showed maximum BC after addition of 4 ml
HCl (Fig. 6.2.). Deer milk BC was still increasing even after addition of 5 ml HCl (Fig. 6.2.). The
BC of caseins and whey proteins are maximal at about pH 5.0 –5.5 and pH 3-4, respectively (Salaun
et al., 2005). High protein and mineral contents in deer milk could be the reason for the higher
buffering capacity than other milks. Cations (Ca+2, Mg+2) can modify the acid-base equilibrium of
anions and hence their pKas (negative logarithm of acid dissociation constant) (Salaun et al., 2005).
Deer milk had high P, Ca, Mg and protein levels which may explain its higherBC. Park (1992)
showed that goat milk has higher BC than cow milk and his results supported the idea that goat
90
milk can be utilized in treatment of gastric ulcers. In this experiments deer milk had significantly
higher (P<0.05) BC than cow, goat and sheep milks (Fig.6.2). Therefore deer milk might be a better
source for treatment of gastric ulcers. The high BC may be one reason for the historical use of deer
milk to cure digestive disorders (Park &Haenlein, 2006).
In vitro Digestion
SDS-PAGE showed that casein of all four species was digested completely after in vitro digestion
designed to mimic human digestion in the stomach and intestine (Fig. 6.3). These results were
consistent with the in vitro digestion study of Inglingstad et al. (2010) on cow, goat, horse and
human milks and Almaas et al. (2006) on cow and goat milks. β-lg of all milk types was not
completely digested during the in vitro digestion. The intensity of the β-lg band after in vitro
digestion was lower in deer and cow milk than sheep and goat milk, which suggests β-lg
digestibility of deer and cow milk was greater than for sheep and goat β-lg. Inglingstad et al.,
(2010) reported that cow β-lg was very resistant to in vitro digestion by human gastric and duodenal
enzymes while horse milk β-lg showed better digestibility than cow and goat milk β-lg. Opatha
Vithana et al., (2012) (Chapter 3) reported there was 54% and 55% intact β-lg after in vitro
digestion of deer and cow milks, respectively.
In vitro Lymphocyte Proliferation
All tested milks which had been digested in vitro increased the human lymphocyte proliferation
(positive value) except sheep milk digest at 0.5 mg/ml protein concentration. At 0.125 mg/ml
protein concentration lymphocyte proliferation of deer (P<0.001), cow and sheep milks were
significantly higher (P < 0.01) than the control, while goat milk did not show any significant
difference of proliferation (Fig. 6.4). Cross and Gill (2000) results revealed both suppressive and
stimulating effects on in vitro lymphocyte proliferation, by whole cow casein, α-, β-, κ-casein (and
their derivatives), whole whey protein and lactoferrin. There is sufficient evidence to show digested
cow milk casein and whey proteins stimulate the lymphocyte proliferation (Cross &Gill, 2000;
Silva &Malcata, 2004; Gauither et al., 2006; Korhonen, 2009). The proliferation stimulation
activity may be caused either by whole protein or a specific fraction of protein. Wong et al. (1996)
showed that cow β-casein significantly enhanced the mitogen-induced proliferation of ovine T and
B lymphocytes. Mercier et al. (2004) showed that in vitro proliferation of murine spleen
91
lymphocytes is stimulated by micro filtered cow whey protein isolates (hydrolysed and
unhydrolysed). The immunomodulating potential of peptide fractions isolated from cow β-lg
enzymatic hydrolyzates has also been demonstrated (Prioult et al., 2004). Likewise, peptides
corresponding to cow β-lg fragments f(15–20), f(55–60), f(84–91), f(92–105), f(139–148), and
f(142–148) have been reported to stimulate murine splenocytes proliferation through the
modulation of cytokine secretion (Jacquote et al., 2010). This can suggest immunostimulation
property shown by deer milk digestions in this study may not limited to one protein but majority of
effect may be due to β-casein, since it is the most abundant protein in deer milk.
There was a significant difference (P<0.05) among milks from the four different species in terms of
lymphocyte proliferation (Table 6.4). At 0.125mg/ml protein concentration deer milk digest showed
significantly higher lymphocyte proliferation compared to cow, sheep (P<0.01) and goat (P<0.001)
milks while there are no significant differences (P>0.05) among cow, sheep and goat milk (Fig.
6.4.). While there is sufficient evidence to show cow milk digest stimulate the lymphocyte
proliferation as shown before, very little work has compared the activity to milk from other species.
This is the first reported study on lymphocyte proliferation of deer milk. A few studies have
reported the release of immunostimulating peptides from sheep and goat milk proteins (HernandezLedesma et al., 2011). Peptic hydrolyzates of sheep α-la and β-lg has been demonstrated by ElZahar et al. (2004) to inhibit the growth of Escherichia coli HB101, Bacillus subtilis Cip5262 and
Staphylococcus aureus 9973 in a dose dependent manner, but the peptides responsible for this
activity were not identified. Also Haza et al. (1995) reported immunological characterization of
goat casein by an indirect ELISA procedure.
The results presented in this chapter confirm those reported in chapter 5 which showed higher
lymphocyte proliferation of deer digests than cow digests. Goat milk digestion stimulated the least
lymphocyte proliferation. This could be due to low level of immunstimulating peptides or more
immunosuppressive peptides in goat milk digests than digests of other species since milk protein
hydrolysatesare a mixture of immunostimulating and immunosuppressive peptides.Both inhibition
and stimulation of the immune system could be useful (Cross and Gill, 2000; Gauthier et al., 2006).
Suppressive effect of lymphocyte proliferation might be of great importance when ensuring a state
of tolerance toward non-self-substances such as food molecules entering the body (Eriksen et al.,
2008). By enhancing the proliferation of immune cells, the body may be defended more quickly
from infection (Meisel and Bockelmann, 1999; Gauthier et al., 2006; Szwajkowska et al., 2011) and
suppress tumour growth (Matsuzaki, 1998; Xu et al., 2011). Different milk protein derived
immunomodulating peptides somehow exhibit a regulatory effect on each other to avoid an
92
unfavourable activation or suppression of the neonatal immune system (Cross and Gill, 2000). Both
immunostimulating and immunosuppressive peptides of cow α1-casein have been observed (Gill et
al., 2000). On the other hand, goat milk (which has α1-casein as main protein) is considered to be
less allergenic than cow milk (Eriksen et al., 2008). Results of this study show goat milk protein did
not stimulate (P>0.05) human PBMC at any tested concentrations and exhibited significantly lower
(P<0.001) lymphocyte proliferation at 0.125 mg/ml protein concentration than the other three
species (Fig. 6.3). This could be due to low allergenic protein in goat milk compare to other species.
Further studies should be carried out to explore the mechanisms that regulate these effects.
Protein concentration had a significant (P<0.001) effect on proliferation (Table 6.3). At the lowest
protein concentration (0.125 mg/ml) all milk protein samples showed higher proliferation than at
0.25 mg/ml and 0.5 mg/ml protein concentration (Fig. 6.4). The results in chapter 5 showed
concentration effect on deer and cow milk digest (10 – 360 µg/ml protein) and showed lymphocyte
proliferation increased with increasing protein concentration at first phase and then started to
decrease with the increase in protein concentration. Similar to that all milk digests from four species
showed higher proliferation at lower concentration (0.125 mg/ml) and the proliferation activity
decreased with the increase in protein concentration. This may due to toxic effect of protein to cells
at high concentrations (Phelan et al., 2009). Similar results of lymphocyte proliferation effect with
different concentration was shown by Sizemore et al. (1991), and Kayser and Meisel (1996) using
immunomodulatory di- and tri-peptides. Jacquote et al. (2010) studied seven immunomodulatory
peptides, namely β-lg f15–20 VAGTWY, f55–60 EILLQK, f84–91 IDALNENK, f92–105
VLVLDTDYKKYLLF, f139–148 ALKALPMHIR, f142–148 ALPMHIR and α-la f10–16
RELKDLK, and found they stimulated cell proliferation to varying degrees depending on their
concentration. Some peptides showed decreased lymphocyte proliferation (murine splenocyte) with
increased peptide concentration (10 – 2000 µg/ml) as in the current study. Sheep milk protein at 0.5
mg/ml showed inhibition of Con A stimulation of PBMC (Fig. 6.4) which could be due to a toxic
effect.
Cytokine Production
The present study demonstrated potential immunomodulatory effects of peptides (encrypted in the
milk protein) from milk of four species. Cytokines are involved in the regulation of many biological
systems, including the immune system (Friberg et al., 1994). IL-2, IFNγ and TNF-α are T helper-1
(Th1) related cytokines and IL-4, IL-6, IL-5 and IL-10 are T helper -2 (Th2) related cytokines
(Phelan et al., 2009; Jacquote et al., 2010). At low protein concentration (0.125mg/ml) deer milk
93
peptides treated PBMCs produced significantly higher IL-2 level than cow and goat peptides
(P<0.05). Similarly IFNγ levels were significantly higher for deer milk digest than other milk
digestions except for lymphocytes treated with goat milk digest which showed no significant
difference to deer milk digest at same protein concentration. Jacquote et al., (2010) showed that
cow peptide (β-lg fragment 139–148) exhibited high lymphocyte proliferation of Con A stimulated
murine splenocyte and also showed significantly higher (P<0.01) IFNγ secretion (63.9 to 717.5 pg /
ml) compared to control. The authors reported another immunomodulatory peptide (β-lg fragments
55–60) with higher lymphocyte proliferation and IL-2 production compared to control. The present
study showed deer milk digests at 0.125 mg/ml protein concentration exhibit higher lymphocyte
proliferation and higher IL-2 and IFNγ production compared to control.
Cytokine expression is a complex mechanism and several cytokines could be responsible for the
proliferation response and it could be by increased or decreased level of cytokines (Laffineur et al.,
1996). Laffineur et al. (1996) showed the stimulation of proliferation was modulated by more
cytokines other than IL-2 and IFNγ. Cytokines such as IFNα or IL-12 can stimulate IFNγ
production by natural killer (NK) cells and lymphocytes (Chan et al., 1991). In general IL-2 induces
proliferation and differentiation of T and B cells (Belardelli and Ferrantini, 2002). But in this study
at high protein concentration (0.5 mg/ml) sheep milk digest with less proliferation than the control
value exhibited better production of IL-2, which is unusual compared to other results. IL-2
stimulates the growth of NK cells and enhances the cytolytic function of these cells, producing
lymphokine-activated killer cells (Belardelli &Ferrantini, 2002). Therefore it may suggest that there
might be a cytotoxic effect of sheep digest at the higher concentration and result incell death. In
accordance with our results,a similar effect was shown by Laffineur et al.(1996) for cow β casein
fermented by lactic acid bacteria.
There are cow milk derived immunomodulatory peptides which stimulate lymphocyte proliferations
and at the same time reduce the secretion of IL-4 and IL-2 cytokines (Jacquote et al., 2010). PBMC
of children with cow milk protein allergy produce significantly higher IL-10 (P<0.001) and
significantly lower TNFα (P<0.05) levels with goat milk protein than cow milk proteins. Although
there are many cytokines responsible for immunomodulation, in this study the digested deer milk
significantly increased the production of IL-2 and IFNγ by human PBMC. The specific interaction
of cow casein hydrolysates with IL-2 might prove useful in modulating in vivo immune response
and treatment of chronic viral infections (Blattman et al., 2003). Therefore, there might be a
potential for use of deer milk casein to treat chronic viral infection as well. Further investigation
should be carried out to see in vivo immune responses on deer milk. Cow milk proteins can cause
94
immunological problems for human, such as allergy (El-Agamy, 2007). Mechanism of allergic
reactions to milk protein fall into two categories which are IgE mediated allergy and non IgE
mediated allergy (Crittenden &Bennett, 2005). A pathway of IgE regulation is essentially based on
reciprocal activities of IL-4 and IFNγ (Ebner et al., 1997). In this study deer milk protein at
0.125mg/ml induced lymphocytes to produced significantly (P<0.001) higher IFNγ levels than cow
milk which contain more than 20 allergen proteins (EL-Agamy, 2007) and was not significantly
different (P>0.05) from goat which is less allergenic than cow milk (Alvarez and Lombardero,
2002; Muraro et al., 2002). Human T cells produced more IFNγ after immunotherapy with bee
venom for grass pollen allergy (Jutel et al., 1995). This can suggest high level of IFNγ could exhibit
less allergenic reactions and thereby deer milk might be a good alternative for cow milk
allergicsubjects. But further in vivo studies with animal models should be carried out before coming
to any conclusion.
6.5. Conclusions
The results of this study revealed that red deer milk is a good source of protein, fat and minerals
(especially Ca, P, Zn and Se) compared with cow, sheep and goat milk. The higher buffering
capacity of deer milk than that of milk from the other three species suggests deer milk may be
useful to treat patient with gastric ulcers. Deer milk proteins were digested well during simulated
stomach and duodenum digestion and these hydrolysed products (peptides) exhibited greater
immunostimulating properties compared to cow, sheep and goat milks in terms of proliferation and
IL-2 and IFNγ cytokines production by Con A stimulated human PBMC in vitro.
95
Chapter 7
Fractionation of Immunomodulatory Bioactive Peptides of deer
and Cow Milk Ferments and Digests
7.1. Introduction
One of the primary functions of milk is to protect the health of new born mammal (Korhonen
&Pihlanto, 2006; Szwajkowska et al., 2011). Bioactive peptides including immunomodulatory
peptides are encrypted in milk proteins and can be released by food processing or gastrointestinal
digestion (LeBlanc et al., 2002). Thus the ingestion of milk and its subsequent digestion releases
bioactive peptides from the milk proteins throughout the course of digestion. A spectrum of
opportunities has been created by the formation of bioactive peptides through lactic fermentation
(Szwajkowska et al., 2011). The immunomodulatory action of peptides is related to the stimulation
of proliferation of human lymphocytes and phagocytic activity of macrophages (Clare et al., 2003).
Generally, three strategies are used to identify and characterize biologically active peptides: (i)
isolation from in vitro enzymatic digestion of precursor protein; (ii) isolation from in vivo
gastrointestinal digestion of precursor protein; (iii) chemically synthesised peptides which have a
structure identical to that of those known to be biologically active (Meisel, 2001; Minervini et al.,
2003). Fast protein liquid chromatography (FPLC), a technique developed for the purification of
protein in their native state, has become an increasingly useful tool in the dairy sector and reversed
phase chromatography is used for peptide separation (Gonzalez-Llano et al., 1990).
Jolles et al., (1981) were the first to report that extracts of trypsin hydrolysed human milk possess
immunostimulating activity. In particular, the hexapetide Val-Glu-Pro-Ile-Pro-Tyr, corresponding
to f54-59 of human milk β casein, was isolated from its tryptic hydrolysate (Parker et al., 1984).
Several other immunomodulatory peptides were later isolated, namely f63-68 and f191-193 from
cow milk β casein and f194-199 from cow milk αs1 casein (Parker et al., 1984; Migliore-Samour &
Jolles, 1988) and others from cow whey proteins (Prioult et al., 2004; Gauthier et al., 2006). Most
of the studies isolating bioactive peptides have used cow milk protein as a precursor (Gobbetti et
al., 2002), and only a few have used milk from different species such as goat and sheep (Minervini
et al., 2003; Silva et al., 2006; Papadimitriou et al., 2007; Hernandez-Ledesma et al., 2011).
Chapter 5 showed whole deer milk was more immunostimulatory (increased human lymphocyte
96
proliferation and IL-2 and IFNγ production) than cow milk after in vitro digestion. LAB
fermentation of deer and cow milk prior to in vitro digestion further improved the
immunostimulating activity.
The goal of this study was to fractionate deer and cow milk in vitro digestions (unfermented and
fermented) by FPLC and test their immunomodulatory activity by Con A stimulated human PBMC
proliferation. Then the most active immunostimulating and immunosuppressive fractions were
selected for further studies by human lymphocyte proliferation and IL-2 and IFNγ production after
adjusting the protein concentration to 0.125 mg/ml. Finally one immunostimulating fraction was
tested for lymphocyte proliferation at different protein concentrations with and without Con A
stimulation.
7.2. Material and Methods
7.2.1. Materials
Deer and cow skimmed milk digestions were prepared as described in chapter 3.2.5 and digestions
of Lactobacillusdelbrueckiisubsp bulgaricus,Streptococcus salivarius subsp thermophilus and
Lactobacillus casei strain Shirota fermented deer and cow milks were as described in chapter 4.2.3
(three independent unfermented and fermented digests from each).
ResourceTM RPC 1 ml (ResourceTM reverse phase column, GE Healthcare, Sweden) was purchased
from GE Healthcare Bio-Science AB, Sweden. Trifluoroacetic acid (TFA) and acetonitrile were
purchased from Sigma (St. Louis, MO, USA).
BCA protein assay (bicinchoninic acid assay) kit was from ThermoScience, USA.
7.2.2. Fractionation of Peptides by FPLC
Peptides were separated from deer and cow milk (unfermented and fermented) in vitro digests by
reverse phase FPLC (RP-FPLC) (Biorad) as per Minervini et al. (2003). The absorbance of the
eluate was monitored with a UV detector operating at 280 nm and also the absorbance of fractions
was measured at 220 nm using a FLUOstar Omega (BMG LABTECH) plate reader.
Aliquots (500 µl) of the digests were diluted 1: 4 with 0.05 % (vol/vol) TFA (buffer A) and
centrifuged at 12,000 xg for 10 min, and 1 ml of the supernatant was loaded into the column.
Elution was carried at a flow rate of 1 ml/min with a gradient of acetonitrile (5 to 100 %) in 0.05 %
97
TFA (buffer B). The concentration of acetonitrile was increased linearly from 5 to 46 % between 16
and 62 ml and from 46 to 100 % between 62 and 72 ml. A total of 51 fractions (2 ml each) were
collected and were assayed for immunoactivity using the lymphocyte proliferation assay. Then
selected active fractions were freeze dried and protein concentrations were adjusted to 0.125 µg/ml
by dissolving in water. A lymphocyte proliferation assay was carried out and the cytokine
production was measured with human PBMC.
7.2.3. In vitro Lymphocyte Proliferation Assay
PBMCs were isolated from human blood and in vitro lymphocyte proliferation assay was carried
out as in chapter 5.2.3 for FPLC fractions. Proliferation of each fraction was expressed as
percentage increase of fluorescence compared to the fluorescence of the control (no added proteins).
% Change in proliferation = Sample fluorescence reading – Control fluorescence reading x 100
Control fluorescence reading
7.2.4. Cytokine Production
IL-2 and IFNγ production by PBMC after 24 hr incubation with selected FPLC fractions were
measuredas described in section 5.2.5.
7.2.5. BCA Protein Assay
Protein concentration in each sample was determined using Bicinchoninic Acid reagent (Pierce,
Rockford, Il., USA) according to manufactures instructions. 10 µl samples were assayed in
triplicate and compared to a standard curve generated using dilutions of 2 mg/ml albumin Standard
(Pierce, Rockford, Illinois, USA), to provide concentrations ranging from 0.0625 mg/ml to 1
mg/ml. Samples were diluted before BCA assay where necessary.
7.2. 6. Statistical Analysis
Data are the means and standard deviations of three independent experiments in triplicate. The data
were subjected to factorial analysis of variance (factors were deer and cow, unfermented and
fermented and different protein concentrations), followed by t-test to determine the significant
difference between means using GenStat 14.1. The level of statistical significance was taken as P <
0.05.
98
7.3. Results
Prior to fractionation by RP-FPLC, unfermented and LAB fermented deer milk in vitro digests
showed significantly higher lymphocyte proliferation and cytokine production compared to cow
unfermented and fermented digest (chapter 5).
Deer and cow digestions (unfermented and fermented) were separated using Reverse Phase-FPLC
based on their hydrophobicity. The absorbance at 220 nm of the fractions is shown in Fig. 7.1. The
absorbance profiles from deer milk digests were different to the corresponding cow digests peaks.
The Con A stimulated lymphocyte proliferation caused by fractions of unfermented and fermented
(three species of LAB) deer and cow milk digests is also shown in Fig. 7.1. Positive values
indicated an increase in proliferation compared to controls, while negative values indicated
decreased proliferation. Deer milk digests had more immunostimulating fractions than cow digests.
All digests contained fractions with a considerable lymphocyte proliferation activity, which did not
necessarily correspond to large absorbance peak area. Three fractions with the highest lymphocyte
proliferation for most digests (11, 22 and 30) and three fractions which showed suppressive
lymphocyte proliferation (13, 24 and 36) were selected for further studies.
The protein
concentrations of these fractions were adjusted to 0.125 mg/ml.
Immunostimulating fractions
The proliferation response of human PBMC (ConA stimulated) for positive fractions 11, 22 and 30
for unfermented and fermented deer and cow digests is shown in Fig 7.2. All three peptide fractions
from all the digests (unfermented and fermented) significantly stimulated (P < 0.001) lymphocyte
proliferation compared to the control.
There was significantly higher (P < 0.001) lymphocyte proliferation for all three fractions from
unfermented deer milk than unfermented cow milk (Fig. 7.2 A, B, C). Fractions 11 and 30 of
Lactobacillus casei strain Shirota ferment digests (Fig. 7.2 A and C), and fraction 11 of
Streptococcus salivarius subsp thermophilus ferment digests (Fig. 7.2 A) exhibited significantly
higher (P < 0.05) lymphocyte proliferation for deer than cow. However no fractions from the
Lactobacillus delbrueckiisubsp bulgaricus ferment digests showed significant differences (P > 0.05)
between deer and cow.
99
3.5
40
3.0
30
2.5
20
2.0
10
1.5
0
1.0
-10
40
3
20
2
0
1
-20
0
0.5
-20
-30
0.0
10
20
30
40
50
-40
60
0
10
FPLC Fractions
20
30
40
50
60
FPLC Fractions
Absorbance (220 nm)
% change in proliferation
Absorbance (220 nm)
% change in proliferation
(C)
(D)
3.5
40
3.5
40
2.0
1.5
0
1.0
0.5
-20
Absorbance (220 nm)
2.5
20
% change in proloferation
3.0
3.0
2.5
20
2.0
1.5
0
1.0
0.5
-20
0.0
0
10
20
30
FPLC Fractions
Absorbance (220 nm)
% change in proliferation
40
50
60
Absorbance (220 nm)
0
% change in proliferation
Absorbance (220 nm)
50
Absorbance ( 220 nm)
(B)
% change in proliferation
% change in proliferation
(A)
0.0
0
10
20
30
40
50
60
FPLC Fractions
Absorbance (220 nm)
% change in proliferation
100
(F)
(E)
3.5
3.5
40
40
2.0
0
1.5
-20
1.0
Absorbance (220 nm)
2.5
% change in proliferation
2.5
2.0
0
1.5
-20
1.0
-40
-40
0.5
0.5
-60
-60
0.0
0
10
20
30
40
50
0.0
0
60
10
20
30
40
50
60
FPLC Fractions
FPLC Fractions
Absorbance (220 nm)
% change in proliferation
Absorbance (220 nm)
% change in proliferation
(G)
(H)
3.5
3.5
40
2.5
20
2.0
1.5
0
1.0
0.5
-20
Absorbance (220 nm)
3.0
% change in proliferation
% change in proliferation
40
3.0
2.5
20
2.0
1.5
0
1.0
0.5
-20
0.0
0
10
20
30
FPLC Fractions
Absorbance (220 nm)
% change in proliferation
40
50
60
Absorbance (220 nm)
% change in proliferation
20
20
Absorbance (220 nm)
3.0
3.0
0.0
0
10
20
30
40
50
60
FPLC Fractions
Absorbance (220nm)
% change in proliferation
Figure 7.1. RP-FPLC chromatograms of the unfermented and fermented deer and cow milk after in
vitro digest.Deer milk digests (A); cow milk digests (B); digests of Lactobacillus delbrueckiisubsp
bulgaricus ferment of deer (C) and cow (D); digests of Streptococcus salivarius subsp thermophilus
ferment deer (E) and cow (F); digests of Lactobacillus casei strain Shirota ferment of deer (G) and
cow (H). The dashed line refers to the % change of lymphocyte proliferation compared to positive
control. Data are means of individual samples measured in triplicates, SD not shown for clarity of
graph.
101
Fermentation with Lactobacillusdelbrueckiisubsp bulgaricus had no significant effect (P>0.05) on
lymphocyte proliferation for any of the three fractions (11, 22 and 30) of either deer and cow, while
fermentation with the other two strains significantly lowered (P<0.05) lymphocyte proliferation by
deer and cow digests except for Streptococcus salivarius subsp thermophilus deer fractions11 and
30 and cow 22 and Lactobacillus casei strain Shirota deer fraction 11 (Fig. 7.2.). There was no
significant difference (P>0.05) between fractions (11, 22 and 30) in terms of proliferation.
(A)
(B)
60
60
‫٭‬
‫٭٭‬
‫٭‬
50
% change in proliferation
% change in proliferation
50
‫٭٭‬
40
30
20
10
40
30
20
10
0
0
Milk digest
Ldb digests
Sst digests
LcS digests
Deer
Cow
Milk digest
Ldb digests
Sst digests
LcS digests
Deer
Cow
(C)
60
‫٭‬
% change in proliferation
50
‫٭‬
40
30
20
10
0
Milk digest
Ldb digests
Sst digests
LcS digests
Deer
Cow
Figure 7.2. Percentage (%) change in lymphocyte proliferation in 10 µg/ml Con A stimulated
human PBMC (compared to positive control) of FPLC fraction 11 (A), 22 (B) and 30 (C) of
unfermented and fermented deer ( ) and cow ( ) milk in vitro digests at 0.125mg/ml protein
concentration . Fractions from deer and cow milk digests and digests of Lactobacillus
delbrueckiisubsp bulgaricus (Ldb), Streptococcus salivarius subsp thermophilus (Sst)and
Lactobacillus casei strain Shirota (Lcs) ferment. Data are means± S.D. (n = 3). Samples measured
in triplicates.
‫ ٭‬Deer significantly different than cow P<0.05
‫ ٭٭‬Deer significantly different than cow P<0.001
102
The cytokine production by human PBMC, a metabolic event that preceded cell proliferation
(Hopkins, 2003) was investigated to determine the mechanism of the immunomodulatory activity of
the selected peptide fractions. All three selected fractions (11, 22 and 30) from all digests
(unfermented and fermented) which showed high proliferation, exhibited significantly higher
(P<0.01) IL-2 production by human PBMC compared to the control (Fig. 7.3). At the same time all
three fractions (11, 22 and 30) from both deer and cow produced significantly higher (P<0.05)
IFNγ level when incubated with human PBMC except for fraction 22 of cow unfermented digest,
Lactobacillus delbrueckiisubsp bulgaricus and Streptococcus salivarius subsp thermophilus digests
and fraction 30 of unfermented cow digest (Fig. 7.4).
As shown in figure 7.3, human PBMC with fraction 11 of unfermented deer digest produced
significantly higher (P<0.01) IL-2 level than with the same fraction of cow digest. Similarly IFNγ
production by human PBMC was significantly higher (P<0.05) with all three (11, 22 and 30) deer
fractions (except for fraction 11 of Streptococcus salivarius subsp thermophilus and fraction22 of
Lactobacillus casei strain Shirota) than with same fractions from cow digests (Fig. 7.4).
There was no significant difference (P>0.05) between fractions (11, 22 and 30) in terms of IL-2
production while fractions 11 and 30 showed significantly higher (P<0.001) IFNγ production than
fraction 22.
103
(B)
(A)
120
120
‫٭‬
100
IL2 production (pg/ml)
IL2 production (pg/ml)
100
80
60
40
20
80
60
40
20
0
0
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Deer
Cow
Control
Deer
Cow
(C)
120
IL2 production (pg/ml)
100
80
60
40
20
0
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Deer
Cow
Figure 7.3. Interleukin-2 (IL-2) production by human peripheral blood mononuclear cells (PBMC)
stimulated by Con A treated with FPLC fraction (after 24 hr) 11 (A), 22 (B) and 30 (C) of
unfermented and fermented deer ( ) and cow ( ) milk in vitro digests (0.125mg/ml protein).
Fractions from deer and cow milk digests and digests of Lactobacillus delbrueckiisubsp bulgaricus
(Ldb), Streptococcus salivarius subsp thermophilus (Sst)and Lactobacillus casei strain Shirota
(Lcs) ferment. Control cells were stimulated by Con A without the addition of proteins. Data are
means ± S.D. (n = 3). Samples measured in triplicates. Detection limit 4 pg/ml.
‫ ٭‬Deer significantly different than cow P<0.01
104
(A)
(B)
160
160
‫*٭‬
‫٭‬
120
‫٭‬
100
80
140
INF production (pg/ml)
INF production (pg/ml)
140
‫٭‬
60
120
100
‫٭٭‬
‫٭‬
60
40
40
20
20
0
‫٭‬
80
0
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Deer
Cow
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Deer
Cow
(C)
160
‫٭٭‬
INF production (pg/ml)
140
120
‫٭٭‬
100
‫٭‬
‫٭‬
80
60
40
20
0
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Deer
Cow
Figure 7.4. IFNγ production by human peripheral blood mononuclear cells (PBMC) stimulated by
Con A treated with FPLC fraction (after 24 hr) 11 (A), 22 (B) and 30 (C) of unfermented and
fermented deer ( ) and cow ( ) milk in vitro digests (0.125mg/ml protein).Fractions from deer and
cow milk digests and digests of Lactobacillus delbrueckiisubsp bulgaricus (Ldb), Streptococcus
salivarius subsp thermophilus (Sst)and Lactobacillus casei strain Shirota (Lcs) ferment. Control
cells were stimulated by Con A without the addition of proteins. Data are means ± S.D. (n = 3).
Samples measured in triplicates. Detection limit 4 pg/ml.
‫ ٭‬Deer significantly different than cow P<0.05
‫ ٭٭‬Deer significantly different than cow P<0.001
Another experiment was carried out using fraction 30 of unfermented deer digest to investigate the
proliferation behaviour of human PBMC with and without Con A and to test a wider range of
immunostimulating peptide concentrations (10 to 640 µg/ml). The same experiment was carried out
with whole deer digests in chapter 5. Fraction 30 was selected for further study because there were
105
no significant differences (P>0.05) between tested immunostimulating fractions of deer digests (11,
22 and 30) on lymphocyte proliferation, and it had more protein (peptide) than the other two
fractions (Fig. 7.1). The proliferation response of human PBMC with different concentrations of
peptides with and without Con A stimulation is shown in Figure 7.5. All peptide concentrations
with or without Con A, stimulation significantly induced (P<0.05) lymphocyte proliferation. In the
absence of Con A there was no significant difference (P>0.05) between different peptide
concentrations. When the cells were stimulated with ConA the proliferation was higher at 20 to 80
µg/ml protein concentration range than at other concentrations.
100
a
% increase in proliferation
80
a
ab
bc
cd
60
de
e
40
20
0
0
100
200
300
400
500
600
700
Protein (µg/ml)
With con A
Without Con A
Figure 7.5. % change of lymphocyte proliferation with ConA (
)and without ConA(
)
for different protein concentrations of deer milk digests FPLC fraction 30. Data are means± S.D. (n
= 3).
Different letters within each curve are significantly different (P < 0.05).
106
Immunosuppressing fractions
Although deer and cow milk digests (unfermented and fermented) as a whole exhibited
immunostimulating properties (Chapter 5), there were some immunosuppressing fractions (Fig.
7.1). Three fractions (13, 24 and 36) which suppressed proliferation in common for all deer and
cow digests (fermented and unfermented) were further studied after adjusting the protein (peptides)
concentration to 0.125 mg/ml. Table 7.1 summarises the statistical results from the lymphocyte
proliferation. There was no overall significant difference (P>0.05) between deer and cow milk but
they behaved differently (interaction) in different fractions (P<0.001). In fraction 13 all cow digests
gave lower lymphocyte proliferation than corresponding deer fractions, while fraction 36 was
higher for deer. At the same time there was no significant difference (P>0.05) for human PBMC
proliferation by fermented digests compared to unfermented digests. There were significant
differences (P<0.001) between fractions with fraction 24 inhibiting proliferation significantly more
(P<0.001) than fraction 13 and 36.
Table 7.1. ANOVA analysis for % change of lymphocyte proliferation (compared to positive
control) for FPLC fractions 13, 24 and 36 of unfermented and fermented deer and cow milk digests
at 0.125 mg/ml protein concentration.
Source of variance
d.f.
s.s
m.s.
F
Sig.
Animals (deer and cow)
1
74.8
74.8
0.55
0.46
Unfermented Vs Fermented
Fractions (13, 24, 36)
1
2
107.5
9176.9
107.5
4588.5
0.79
33.6
0.376
<0.001
Animal. Unfermented Vs Fermented
Animal. Fractions
1
2
23.6
20523.8
23.6
10261.9
0.17
75.14
0.678
<0.001
Fractions 13, 24 and 36 of both deer and cow digests (both unfermented and fermented) produced
significantly less (P<0.05) IL-2 with human PBMC compared to the control. Fraction 24 results are
shown in figure 7.6 (A). There was no significant difference (P>0.05) between deer and cow in
terms of IL-2 production for fraction 24 and it is same for the other fractions too (Appendix C).
Similarly there was no significant difference (P>0.05) between deer and cow in fraction 24 for IFNγ
production by human PBMC (Fig 7.6 (B).
In these negative proliferation fractions (13, 24 and 36) IL-2 and IFNγ production by human PBMC
were less than control for both deer and cow digests (Fig. 7.6) while positive proliferation fractions
(11, 22 and 30) produce higher IL-2 and IFNγ than control (Fig. 7.3 & 7.4).
107
(B)
(A)
80
50
INF production (pg/ml)
IL2 production (pg/ml)
40
30
20
60
40
20
10
0
0
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Ldb digests
Sst digests LcS digests
Protein 0.125 mg/ml
Protein 0.125 mg/ml
control
deer
cow
Milk digest
control
deer
cow
Figure 7.6. IL-2 (A) and IFNγ (B) production by human peripheral blood mononuclear cells
(PBMC) stimulated by Con A with FPLC fraction 24 of unfermented and fermented deer ( ) and
cow ( ) milk in vitro digests (0.125mg/ml protein). Fractions from deer and cow milk digests and
digests of Lactobacillus delbrueckiisubsp bulgaricus (Ldb), Streptococcus salivarius subsp
thermophilus (Sst)and Lactobacilluscasei strain Shirota (Lcs) ferment. Control cells were
stimulated by Con A without the addition of proteins. Data are means ± S.D. (n = 3). Samples
measured in triplicates. Detection limit 4 pg/ml.
108
7.4. Discussion
After in vitro digestion unfermented and fermented deer milk showed more immunomodulatory
activity than cow milk (Chapter 5). These digests are a mixture of peptides originating from the
different milk proteins. In order to investigate this further, these digests were separated into
fractions.The fractions with the greatest immunomodulating effects were identified. As a general
rule, the immunomodulatory effects of individual milk components become more clearly defined as
they are progressively purified from the primary milk protein (Cross & Gill, 2000).
RP-FPLC was used in this study to generate fifty one peptide fractions for each sample. Fast protein
liquid chromatography (FPLC), a technique developed for the purification of protein and peptides in
their native state, has become an increasingly useful tool in the dairy sector (Gonzalez-Liano et al.,
1990).
Absorbance detections were done at 280 nm (Appendix D) and 220 nm (Fig. 7.1.).
Separation was not clearly seen from 280 nm absorbance readings as this depends on the content of
tyrosine, tryptophan (aromatic ring) and cysteine (disulphide bond) (Pace et al., 1995). Amarowicz
and Shahidi et al. (1997) reported that protein absorbance reading at 220 nm measured the peptide
bond and therefore 220 nm reading was used in this study. At 280 nm absorbance reading one
fraction had very high reading while other had very low readings. High branched-chain amino acid
to aromatic amino acid ratio (Fischer’s ratio) is important to get good absorbance reading at 280 nm
(Pederoche et al., 2004). The 280 nm measurement suggests that most of the fractions have peptides
with less aromatic ring containing amino acids which could be due to smaller peptides (few amino
acids) than big peptide chains (where the chance of having aromatic ring is high).
As shown in figure 7.1 some high absorbance (peak) fractions did not produce big proliferation
differences (stimulating or inhibiting), while some fractions exhibited the highest proliferation
changes but showed very low absorbance readings (no big peak). This suggests some peptides
present in small quantities contribute to the activity and may have very high immunomodulating
activities.
The lymphocyte proliferation activities of each fraction were shown in figure 7.1 for all digests.
Some fractions stimulated lymphocyte proliferation while others inhibited proliferation activities.
Fractions with a considerable lymphocyte proliferation activity did not necessarily correspond to
peaks with higher absorbance (220 nm). Similarly Minervini et al. (2003) reported large peaks of
RP-FPLC fractions of hydrolysed casein from six species (cow, goat, sheep, buffalo, pig and
109
human) did not necessarily have high ACE inhibitory activity, while fractions with small quantities
of peptides had high ACE inhibitory activity. LeBlanc et al. (2002) showed an HPLC fraction with
lower protein exhibited higher immunomodulating effect than two fractions which had very high
protein content (as shown by high absorbance at 214 nm). There were more fractions exhibiting
lymphocyte proliferation activity in deer than cow milk, while cow milk digests had more
lymphocyte inhibitory fractions than deer milk digests (Fig. 7.1.).
Immunostimulating fractions
All three selected fractions (proliferation stimulating) from deer and cow milk digests (unfermented
and fermented) showed significant (P<0.001) lymphocyte proliferation (Fig. 7.2.) of Con A
stimulated human PBMC in vitro. Tested immunostimulating unfermented and fermented deer
digests fractions showed significantly higher lymphocyte proliferation than cow digests
(unfermented and fermented). There is now a substantial body of evidence to suggest cow milk
hydrolysates as a whole (Laffineur et al., 1996; Vinderola et al., 2006; Eriksen et al., 2008; Phelan
et al., 2009), as well as purified individual constituents (Berthou et al., 1987; Zucht et al., 1995;
Kayser & Meisel, 1996) or fractions (Cross and Gill, 2000; LeBlance et al., 2004; Prioult et al.,
2004), can regulate immune function. Otani et al. (1995) demonstrated different FPLC fractions of
in vitro digests of cow κ-caseinoglycopeptide possess different immunomodulatory capabilities.
Yun et al. (1996) isolated a sub fraction from cow κ-caseinoglycopeptide by size-exclusion
chromatography which promotes proliferation of murine spleen cells. Therefore findings of cow
digests immunostimulating fractions in this study are in accordance with the above studies. To our
knowledge, this study is the first to fractionate the deer milk digests (unfermented and fermented)
and investigate the lymphocyte proliferation activity on human PBMC.
There was a significant difference of proliferation (P<0.05) between unfermented and
Streptococcus salivarius subspthermophilusand Lactobacillus casei strain Shirota fermented digests
of all three fractions (except Streptococcus salivarius subspthermophilusdeer fractions 11 and 30
and cow 22 and Lactobacillus casei strain Shirota deer fractions 11) for both deer and cow (Fig.
7.2.) Although ferment digests showed higher Con A stimulated lymphocyte proliferation than
unfermented digests (chapter 5) as a whole, some purified fermented fractions showed lower
lymphocyte proliferation than the same fraction of unfermented digests. This could be due to
formation of different peptides after fermentation. There is evidence that immunomodulatory
activity was different in purified fractions compared to original milk products (Otani et al., 1993;
Wong et al., 1997;Cross &Gill, 1999).
110
One immunostimulating fraction (fraction 30) of deer (unfermented) was selected to investigate its
proliferation response with and without Con A.
RP-FPLC fraction 30 of unfermented deer digests showed stimulation of human PBMC with or
without Con A (Fig.7.6). Con A is a mitogen that induces cell proliferation (Moller et al., 1986;
Boilard et al., 2001; Zhang et al., 2002). A similar study by Saint-Sauveur et al. (2008) using
fractions isolated (RP-HPLC) from cow whey digests showed enhanced proliferation of both Con A
stimulated and un stimulated splenocytes. Proliferation was significantly higher (P<0.05) at 20 – 80
µg/ml protein concentration than other concentrations for Con A stimulated PBMC, while there
were no proliferation difference (P>0.05) observed between different protein concentrations for
PBMC without Con A stimulation. Since our present results showed that fraction (30) stimulates
human PBMC in vitro even without Con A.This may be due to peptides (or a peptide) present in
this fraction which are responsible for cell proliferation. Therefore we can suggest the same
immunostimuating effect may be exhibitedin vivoin the absence of mitogen.
Stimulation of cytokine production on human PBMC was detected using the ELISA method
(Burrells et al., 1999; Phelan et al., 2009) as a second measure of immunomodulatory activity. Both
IL-2 and IFNγ productions by Con A stimulated human PBMC were increased together with all
three fractions of deer and cow digests (unfermented and fermented). Cytokines are involved in the
regulation of many biological systems including the immune system (Friberg et al., 1994). Cytokine
IL-2 and IFNγ are produced by T helper-1 (Th1) cells and are responsible for enhancing cell
mediated or cytotoxic immune response (together with cytokine TNFα which is produced by Th1)
(Phelan et al., 2009). This suggests increased level of IL-2 and IFNγ cytokine produced by PBMC
with peptide fractions, may be due to changes in immune response. Already there are some studies
showing cow milk has peptides which modulate immune response via cytokine regulation
(Laffineur et al., 1996; LeBlance et al., 2004; Prioult et al., 2004; Vinderola et al., 2006). Prioult et
al. (2004) reported an acidic peptide fraction of cow β lactoglobulin exhibited increased
lymphocyte proliferation and increased IFNγ production in mouse splenocytes.
When comparing the IL-2 cytokine production there was no difference observed between
unfermented and LAB fermented milk digests (for both deer and cow) (Fig. 7.3.). But there were
some differences for IFNγ production as shown in Fig. 7.4. Deer milk fermented with
Lactobacillusdelbrueckiisubsp bulgaricus (except fraction 30)and Lactobacillus casei strain Shirota
ferment digests fractions showed significantly greater (P<0.05) IFNγ production than unfermented
fractions (Fig. 7.4). Similarly all cow LAB ferment fractions showed significantly greater (P<0.05)
111
IFNγ production than the corresponding unfermented fractions (except fraction 22 of
Lactobacillusdelbrueckiisubsp bulgaricus and Streptococcus salivarius subsp thermophilus).
Consistent with our findings Laffineure et al. (1996) reported LAB fermentation of cow milk
significantly improved the IFNγ productionwhile no significant improvement can be observed in
IL-2 levels.
The stimulation of proliferation response (Fig. 7.2) of fractions has been associated with
significantly higher level of IL-2 (Fig. 7.3), the major growth factor activated lymphocyte (Morgan
et al., 1976; Laffineur et al., 1996; Ceyhan et al., 2004) and IFNγ (Laffineur et al., 1996;
Meixner et al.,
2004) (except fraction 22 of cow unfermented digests, Lactobacillus
delbrueckiisubsp bulgaricus and Streptococcus salivarius subsp thermophilus digests and fraction
30 of unfermented cow digests) compared to control (Fig. 7.4). This contrasting result of IFNγ in
some fractions suggests the intervention of cytokines other than IL-2 and IFNγ in lymphocyte
proliferation. Cytokines such as IL-12 (Meixner et al., 2004; Heinzel et al., 1993), IL-4 (Ebner et
al., 1997; Meixner et al., 2004; Jacquot et al., 2010) and IL-10 (Ceyhan et al., 2004; Meixner et al.,
2004; Jacquot et al., 2010) can regulate cell proliferation.
Immunosuppressing fractions
The immunomodulatory potential of peptides can be either enhancement (immunostimualtion) or
suppression (immunosuppression) of immune system (Cross and Gill, 2000; Gauthier et al., 2006).
The main focus of thisstudy was on immunostimulating peptides (since deer milk digestions
showed immunostmulating effect as a whole (chapter 5), but to examine the presence of both
immunostimualtion and immunosuppression type of peptides, three fractions, which suppressed
lymphocyte proliferation for all milk digests (unfermented and fermented) were further studied after
adjusting the protein concentration to 0.125 mg/ml.
Although deer showed significantly higher proliferation than cow in immunostimulating peptide
fractions (11, 22 and 30), there was no significant difference (P>0.05) between deer and cow for
proliferation suppression fractions (Table 7.1). The presence of proliferation suppressive peptides
has been reported in cow milk digests (Prioult et al., 2004; Eriksen et al., Cross &Gill, 2000) which
explains the proliferation suppressive fractions of cow digestions in present study. Also LAB
fermentation did not significantly affect (P>0.05) proliferation for these fractions from both deer
and cow milk compare to unfermented milk (Table 7.1). Similarly Laffieur et al. (1996) reported
112
eight strains of LAB (out of 10 stains tested) did not have significant proliferation effect compared
to unfermented milk.
Similar to lymphocyte proliferation response there were no significant difference (P>0.05) between
deer and cow in cytokine production (IL-2 and IFNγ) by human PBMC.
The proliferation and cytokine production results of immunostimulating and immunosuppressive
peptide fractions suggest it is more worthy to study immunostimulating peptides of deer milk than
cow milk, since deer milk digest as a whole (Chapter 5, fig.5.1) showed immunostimulating effect
than cow milk digest.
7.5. Conclusion
Both deer and cow digests (unfermented and fermented) had immunostimmulating and
immunosuppressive peptide fractions, but deer digest had more proliferation stimulation fractions
than cow digest. Selected high immunostimulating deer digests (unfermented and fermented) FPLC
fractions (11, 22 and 30) showed higher lymphocyte proliferation and higher cytokine IL-2 and
IFNγ production compared to corresponding fractions of cow digests when incubated in vitro with
Con A stimulated human PBMC. Immunostimulating fraction 30 of unfermented deer digests was
able to stimulate the human PBMC even without Con A. The findings of this research suggest that
deer milk digests (and LAB ferment digests) have more immunostimulating activity than cow
digests. Further investigation should be carried out to identify the specific peptide or peptide which
is responsible for immune activity.
113
Chapter 8
Identification of Peptides from Immunostimulating Fractions of
Deer and Cow Milk Digestions
8.1. Introduction
Bioactive peptides of cow and other mammals (sheep, goat, buffalo, pig and human) milk have been
studied previously after separation of proteolysis products by chromatography (FPLC or HPLC)
and the identification of the peptides were carried out by biochemical methods (Minervini et al.,
2003; Jacquot et al., 2010). Mass spectrometry (MS) is commonly used to identify and sequence
purified peptides (Juillard et al., 1995; Rohit et al., 2012; Hou et al., 2012). The use of liquid
chromatography–mass spectrometry (LC-MS) makes it possible to determine the molecular weight
of each peptide, even when testing fractions contain more than one peptide (Juillard et al., 1995).
According to the literature there has been no investigation to identify bioactive peptides from deer
milk protein hydrolysates. At present, deer milk protein sequence information is not available in any
sequencing databases. Therefore in this study, database searching for deer peptides was done on
Bovidae taxonomy; cow, sheep, goat and buffalo.
The aim of this study was to identify the peptide or peptides present in the three most active
immunostimulating fractions (11, 22 and 30) isolated by reverse-phase FPLC(Chapter 7).In vitro
digestions of unfermented and Lactobacillus delbrueckiisubsp bulgaricus fermented deer and cow
milk proteins were used.
8.2. Material and Methods
8.2.1. Materials
Immunostimulating fractions 11, 22 and 30 of deer and cow (unfermented and Lactobacillus
delbrueckiisubsp bulgaricus fermented) digests (chapter 7) were isolated by reverse-phase FPLC.
The fractions were freeze dried and used for analysis.
Formic acid (FA) and acetonitrile were purchased from Sigma (St. Louis, MO, USA). Ultimate
nanoflow HPLC equipment with Famos autosampler and Switchos column switching module were
114
from LC-Packings, The Netherlands. Nanospray needle was purchased from Proxeon, Denmark and
QSTAR Pulsar I mass spectrometer was from AB Sciex, USA.
8.2.2. Liquid Chromatography–Mass Spectrometry (LC-MS/MS)
Freeze dried samples were reconstituted in 25 µl loading solvent (2% acetonitrile, 0.2% FA). LCMS/MS was carried out on an HPLC. A 10 µl of sample was loaded on a C18 precolumn (300 µm
ID, 5 µm particles, 300 Å pore size) at a flow rate of 8 µl/min. The precolumn was then switched in
line with the analytical column (C18, 20 cm, 75 µm ID, 5 µm particles, 300 Å pore size), and eluted
at a flow rate of 150 nl/min, with a linear gradient from 2% to 55% solvent B in 50 min. Solvent A
was HPLC-grade water with 0.2% FA, solvent B was LCMS-grade acetonitrile with 0.2% FA.
Using a stainless steel nanospray needle, the column outlet was directly connected to a QSTAR
Pulsar I mass spectrometer which was programmed to acquire MS/MS traces of the top three 1+,
2+, 3+, 4+ and 5+ peptides (cycle time 6 seconds).
8.2.3. Database searching
Mascot Daemon (v2.2.2) was used to convert the data files to peak lists (Mascot Script for Analyst
v1.6b25). Peak lists were imported in to ProteinScape V2.1 (Bruker) and submitted for searching to
an in-house Mascot server (v2.2.0.6) (Matrix Science, UK). The following search parameters were
used: No enzyme; MS tolerance 75 ppm; MS/MS tolerance 0.3 Da; database NCBInr 14/09/2011.
Bos taurus and Bovidae taxonomy was used for cow and deer milk samples, respectively. Peptides
and proteins with a Mascot score higher than 25 and 50, respectively, were accepted.
8.3. Results and Discussion
Peptides have been identified by coupling high-performance liquid chromatography (HPLC) and
MS. All tested fractions had more than 15 different peptides and one fractions contained more than
100 peptides (fraction 30 of cow milk unfermented and fermented digests) (Table 8.1). Even after
further purification, RP-FPLC fractions of casein hydrolysates of cow, sheep, goat, pig, buffalo and
human milk had mixture of different peptides (Minervini et al., 2003). Peptides present in
immunostimulating RP-FPLC fractions (11, 22 and 30) of deer and cow milk were identified. The
samples in the present work were hydrolysates of casein and whey protein which contribute to the
presence of many different peptides. Mass spectrometric analysis done by Juillard et al. (1995)
identified over 100 different β-casein-derived peptides from cow β casein after fermentation with
Lactobacillus lactis subsp. lactis MG611.
115
Table 8.1. Total number of identified peptides contained and source of protein in FPLC fractions
11, 22 and 30 from unfermented (Cow digests and Deer digests) and Lactobacillus delbrueckiisubsp
bulgaricus fermented cow (CL) and deer (DL) in vitro digestions.
Source
Total number
of peptides
Source of protein
Number of
peptides
Fraction 11
Cow digests
32
CL digests
49
Deer Digests
16
DL digests
36
β Casein
αs1 casein
αs2 casein
κ casein
β Lactoglobulin
β Casein
αs1 casein
αs2 casein
αs1 casein
β Casein
β Lactoglobulin
β Casein
αs1 casein
αs2 casein
β Lactoglobulin
14
10
3
4
1
31
15
3
10a
5a
1a
12a,h
10a
4b
10a
Fraction 22
Cow digests
79
CL digests
73
Deer Digests
51
DL
35
β Casein
αs1 casein
αs2 casein
κ casein
β Lactoglobulin
αs1 casein
β Casein
αs2 casein
κ casein
β Casein
αs1 casein
αs2 casein
κ casein
β Lactoglobulin
β Casein
αs1 casein
αs2 casein
κ casein
β Lactoglobulin
41
22
8
6
2
37
23
9
4
25a,b
12a,b
7a,c
4a,d
3a
14a
13a,b,h
2c
2g
4a
Fraction 30
Cow digests
100
αs1 casein
β Casein
39
35
116
CL
104
Deer Digests
51
αs2 casein
κ casein
β Casein
αs1 casein
αs2 casein
κ casein
β Casein
αs2 casein
κ casein
β Lactoglobulin
DL
50
β Casein
αs1 casein
αs2 casein
κ casein
β Lactoglobulin
Below taxonomy was used for identification of peptides in deer milk samples
a: Bos taurus
e: Ovis vignei bocharensis
b: Capra hircus
g: Ovis orientalis gmelini
c: Ovis aries
h: Bubalus bubalis
d: Ovis vignei blanfordi
17
9
46
40
12
6
21b,h
2c
6e
4a
22a,h
13b,h
8c
2d
5a
In this study, identification of deer milk peptides was carried out by database search of cow, sheep,
goat, buffalo milk proteins since there was no sequencing data available for deer milk proteins.
The main protein source of identified peptides in most tested fractions was β casein. Therefore we
can suggest that immunostimulating activity (chapter 7) in tested fraction was possibly from β
casein originated peptides for both deer and cow milks. The immunomodulatory activities of β
casein derived peptides have been well documented (Coste et al., 1992; Kitazawa et al., 2007;
Phelan et al., 2009).
Some differences in amino acid sequence of deer and cow milk proteins were identified (Table 8.2).
There were four peptides of β casein origin (f 204-222, f 205-222, f 206–222 and f 207-222) present
in fraction 22 of deer milk unfermented digests, which showed sequence differences with cow β
casein where 207th position is leucine in deer as with goat milk versus isoleucine in cow protein
(Table 8.2). Also fraction 11 of digest of Lactobacillus delbrueckiisubsp bulgaricus ferment of deer
milk (11 DL) contained a peptide (β casein f 31–48) which showed a difference with cow β casein
amino acid composition (Table 8.2). In 39th position Arginine (R) was found in deer milk similar to
domestic water buffalo, while cow β casein has Glutamine (Q). A similar study by Masoodi and
117
Shafi (2010) showed the differences in amino acid composition of α1 casein and α2 casein of cow
milk compared to those of sheep and goat milk.
Bovidae (cow, sheep, goat and buffalo) taxonomy was used for identification of peptides in deer
milk protein samples. There are no reported works on amino acid sequencing of deer milk protein
but McDougall (1976) did compare the amino acid composition of red deer milk β lactoglobulin to
that of cow milk. He found that deer milk β lactoglobulin contained one more residue of aspartic
acid, alanine and methionine and one less glutamic acid residue and two less leucine residue than
cow β-lactoglobulin composition. The results of this study show similarities (Fig. 8.1) and
differences (Table 8.2) of deer milk proteins (especially casein) to homologous cow milk proteins
which might lead to altered bioactive properties. As shown in figure 8.1 many of the peptides are
identical in deer and cow fraction 30. This similarity of peptide sequence could be the reason why
both deer and cow fraction showed immunostimulating activity (Chapter 7). At the same time, there
are slight differences observed between deer and cow milk proteins (Table 8.2). This could lead to a
difference of immunostimulating activity and might be the reason why deer milk fractions showed
higher immunostimulating activity than the corresponding cow fraction (Chapter 7).
Table8.2. Comparison of amino acid residues in milk protein of deer with cow giving its specific
position based on results of peptides found in FPLC fractions 11, 22 and 30 of unfermented and
Lactobacillus delbrueckiisubsp bulgaricusfermented cow and deer milkin vitro digestions.
Amino acid
position
β casein 39
β casein 207
α1 casein 163
α2 casein 215
Amino acid in cow milk
Glutamine (Q)
Isoleucine (I)
Glutamic acid (E )
Lysine (K)
Amino acid in deer milk
Arginine ( R) - Similar to goat milk
Leucine (L) - Similar to domestic water buffalo milk
Glutamine (Q) - Similar to goat and sheep milk
Asparagine (N) - Similar to goat and sheep milk
Most abundant peptides identified from each fraction with a mascot value > 70 are shown in Table
8.3. Mascot score is a measure of effectiveness of peptide identification. The fragment β casein 5766 (fraction 22 of cow digests) contained (Table 8.3) the sequence of β casomorphine-7 (β casein
60-66), which has been previously identified as an immunomodulating cow milk peptide (Kayser &
Meisel, 1996). The authors showed β-casomorphin-7 stimulated human lymphocyte proliferation.
β-casomorphin-7 may also affect the human mucosal immune system, possibly via opiate receptors
in lamina propria lymphocytes (Elitsur et al., 1991). The β casein 184-209 fragment present in
fraction 11 of cow digests (Table 8.3) contained another known cow immunostimulating peptide (β
casein 192-209) which enhanced the proliferation of rat lymphocytes (Coste et al., 1992). Similarly
118
the β-cascochemotide (f114-118) an immunomodulating peptide (Kitazawa et al., 2007) exists
within the range of β casein 114-128 peptide (Table 8.3, Fig. 8.1) present in fraction 30 of cow
digest fermented with Lactobacillus delbrueckiisubsp bulgaricus. Cow β casein residues 63-68 and
191-193 have been shown to stimulate phagocytosis of sheep red blood cells by murine peritoneal
macrophages, and to exert a protective effect against Klebsiella pneumoniae infection in mice after
intravenous treatment (Migliore-Samour & Jollès 1988) and these two amino acid sequences can be
seen in peptides detected in cow β casein 57-66 and β casein 184-209, respectively (Table 8.3).
Therefore, it can be suggested that cow milk fractions (11, 22 and 30) which showed high
immunostimulating activity (Chapter 7) may partially have resulted from some of above mentioned
peptide fragments which have been identified in literature. There might be other peptides
contributing to the activity, which have not been identified before.
Although each of the deer milk FPLC fractions exhibited higher lymphocyte proliferation than the
corresponding cow milk fraction (Chapter 7), we did not identify any previously known
immunomodulating peptides in deer samples. Deer fractions had higher activity compared with cow
fractions suggesting new sequences may potentially contributeto this. Also immunostimulating
activity of each fraction may be due to interaction of peptides present in each fraction or few
peptides and their concentration.
Further experiments should be carriedout to identify these peptidesafter purification as well as
testing the immunostimulating activity of each peptide which should be confirmed by synthetic
peptides constructed with the same sequence.
119
Table 8.3. Sequences and corresponding milk protein fragment of peptides contained in FPLC
fractions 11, 22 and 30 from unfermented (Cow digests and Deer digests) and Lactobacillus
delbrueckiisubsp bulgaricus fermented cow (CL) and deer (DL) in vitro digestions.m/z represents
measured mass-to-charge ratio and ∆ m/z represents mass error between the measured mass m/z and
the theoretical m/z for that sequence
Sequence position
Sequencea
m/z
∆ m/z
z
βCN 124-139
βCN 184-209
β lg 92-100
SLTLTDVENLHLPLPL
DMPIQAFLLYQEPVLGPVRGPFPIIV
VLVLDTDYK
888.0039
975.8757
533.2904
3.12
6.10
-8.58
2
3
2
CL
βCN 124-139
βCN 34-48
SLTLTDVENLHLPLPL
QQEEQQQTEDELQDK
887.9974
938.4144
-4.18
0.70
2
2
Deer
Digests
αs1CN 119 - 134
(Bos taurus)
αs1CN 189 - 208
(Bos taurus)
β lg 110-118 (Ovis
aries)
YKVPQLEIVPNSAEER
651.3319
14.66
3
TDAPSFSDIPNPIGSENSEK
1052.9881 0.99
2
VLVLDTDYK
533.2990
7.49
2
SLTLTDVENLHLPLPL
887.9942
-7.77
2
DVENLHLPLPLLQSWM
953.0054
5.10
2
QQEEQQQTEDELQDK
938.4139
0.17
2
βCN 33-52
βCN 57-66
βCN 145-162
βCN 190-202
αs1CN 15- 28
αs1CN 189- 208
αs2CN 116- 128
β lg 10-29
FQSEEQQQTEDELQDKIHPF
SLVYPFPGPI
HQPHQPLPPTVMFPPQSV
FLLYQEPVLGPVR
HQGLPQEVLNENLL
TDAPSFSDIPNPIGSENSEK
QGPIVLNPWDQVK
SLAMAASDISLLDAQSAPLR
852.6958
1089.6059
679.6958
765.9394
802.4236
1052.9829
747.3992
682.6950
-7.72
7.32
17.32
2.33
-2.50
-3.99
-13.29
8.21
3
1
3
2
2
2
2
3
CL
αs1CN 119- 134
αs1CN 181- 200
YKVPQLEIVPNSAEER
TDAPSFSDIPNPIGSENSEK
651.3189
-5.27
1052.9829 -4.04
3
2
Deer
Digests
αs1CN 140- 164
(Bos taurus)
αs1CN 140- 164
EGIHAQQKEPMIGVNQELAYFYPEL
EGIHAQQKEPMIGVNQELAYFYPEL
968.8255
974.1516
3
3
Fraction Source
Cow
11 digests
DL
Cow
22 digests
βCN 124-139 (Bos
taurus,Bubalus
bubalis)
βCN 129-144 (Bos
taurus,Bubalus
bubalis)
βCN 34-48 (Bos
taurus)
13.20
7.41
120
(Bos taurus)
αs1CN 197- 208
(Bos taurus, Capra
hircus)
β lg 92-100 (Bos
taurus)
IPNPIGSENSGK
606.8213
11.12
2
VLVLDTDYK
533.2993
8.06
2
βCN 124-139 (Bos
taurus)
SLTLTDVENLHLPLPL
888.0153
16.00
2
βCN 124-139
αs1CN 50- 65
αs1CN 50- 67
αs1CN 132- 156
SLTLTDVENLHLPLPL
DIGSESTEDQAMEDIK
DIGSESTEDQAMEDIKQM
EGIHAQQKEPMIGVNQELAYFYPEL
888.0093
972.3567
1109.8898
974.1546
9.22
10.06
-3.67
10.46
2
2
2
3
CL
βCN 114-128
βCN 124-139
βCN 144-163
βCN 145-162
βCN 145-163
αs1CN 23- 36
αs1CN 172- 199
αs2CN 125- 138
YPVEPFTESQSLTLT
SLTLTDVENLHLPLPL
MHQPHQPLPPTVMFPPQSVL
HQPHQPLPPTVMFPPQSV
HQPHQPLPPTVMFPPQSVL
HQGLPQEVLNENLL
YVPLGTQYTDAPSFSDIPNPIGSENSEK
TPEVDDEALEKFDK
856.4202
888.0062
761.0555
679.6815
717.3756
802.4202
1009.4803
545.9335
-5.48
5.69
-4.41
-3.72
-4.38
-6.67
-0.02
6.72
2
2
3
3
3
2
3
3
Deer
Digests
βCN 124-139
(Bubalus bubalis)
βCN 126-144
(Bubalus bubalis)
β lg 10-24(Bos
taurus)
β lg 10-29 (Bos
taurus)
β lg 92-100 (Bos
taurus)
DL
Cow
30 digests
DL
SLTLTDVENLHLPLPL
888.02 20.9
2
TLTDVENLHLPLPLLQSWM
1118.5817 -5.42
2
SLAMAASDISLLDAQ
761.3813
5.76
2
SLAMAASDISLLDAQSAPLR
682.7030
19.96
3
VLVLDTDYK
533.3198
46.56
2
βCN 124-139
(Bubalus bubalis)
βCN 129-138 (Bos
SLTLTDVENLHLPLPL
888.0030
2.16
2
taurus)
DVENLHLPLP
573.8124
1.89
2
SLAMAASDISLLDAQSAPL
937.4762
-6.67
2
β lg 10-28 (Bos
taurus)
a Single letter amino acid code is used
Peptides with > 70 mascot score were shown
121
10
20
30
40
50
60
70
80
90
100
RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ TEDELQDKIH PFAQTQSLVY PFPGPIPNSL PQNIPPLTQT PVVVPPFLQP EVMGVSKVKE AMAPKQKEMP
120
FPKYPVEPFT
130
ESQSLTLTDV ENLHLPLPLL
140
150
160
QSWMHQPHQP LPPTVMFPPQ
170
180
SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF
190
200
LLYQEPVLGP
VRGPFPIIV
110
209
C
CL
D
DL
Figure 8.1. Localization of identified peptides in FPLC fraction 30 of unfermented cow (C) and deer (D) milk digests and digests of
Lactobacillus delbrueckiisubsp bulgaricus ferment of cow (CL) and deer (DL) on cow β casein.
Peptides with > 70 mascot score were shown
122
β casein 124–139 peptide was found in all tested fractions of both deer and cow milks (Table
8.3). This unexpected result is fully unknown since separation is based on hydrophobicity of
peptide. The mascot score for the 124–139 peptides is different in different fractions.
Therefore it could be suggested that due to high abundance of this peptide, it diffused to more
than one fraction. These results are similar to those reported by Minervini et al. (2003) which
reported the appearance of the same peptides in different FPLC fractions which use
hydrophobicity as separation principle.
This study used three fractions (11, 22 and 30) out of fifty FPLC fractions, where a high
immunostimulating activity was shown (Chapter 7) and β casein 124 – 139 peptide was
found in most of these fractions (both deer and cow) abundantly. This suggests there might be
immunostimulating property of this peptide and there could be really interesting findings of
this particular peptide and further investigation is strongly recommended. Di- and tripeptides
are too small to be reliably identified with this method and therefore another method should
be used in future work.
8.4. Conclusion
Mixtures of different peptides with different protein origins have been identified in
immunostimulating FPLC fractions from both deer and cow milk digests. The main protein
source was found to be β casein. The results obtained in this study showed that there are
differences in the amino acid sequence of deer and cow milk proteins. Some previously
identified immunostimulating peptide sequences were found in tested FPLC fractions in cow
digests whereas there were no previously identified immunostimulating peptides in deer
digests. Further studies should be carried out to identify these unknown immunostimulating
peptides after further purification and the activity confirmed by synthesis.
123
Chapter 9
General Discussion, Conclusion and Future Work
Digestion and fermentation of milk protein produces bioactive peptides and currently milk
proteins are considered the most important commercial source of bioactive peptides
(Korhonen, 2009). Using deer and cow milks obtained from Lincoln University deer and
dairy farm, respectively, the present study has shown that deer milk has twice protein content
of cow milk. Also deer milk produced more peptides than cow milk after in vitro digestion
(Chapter 3). LAB fermentation (Lactobacillus delbrueckiisubsp bulgaricus, Streptococcus
salivarius subsp thermophilus and Lactobacillus casei strain Shirota) prior to in vitro
digestion improved the peptide production compared to unfermented milk digestion. There
were more peptides generated after an in vitro digestion of deer milk ferment than cow milk
after similar treatment (Chapter 4).
Immunomodulating activities of the above unfermented and fermented milk digestions were
tested using human PBMC and deer milk showed more immunostimulating properties than
cow milk after in vitro digestion and this was further improved by LAB fermentation prior to
digestion based on lymphocyte proliferation and cytokine production (IL-2 and INFγ).
Immunostimulating activities were higher at lower protein concentration (0.125 mg/ml) than
higher concentrations for both milk types which could be explained by cytotoxic effect at
higher concentrations of protein (Phelan et al., 2009). Similar results of higher lymphocyte
proliferation at lower protein concentration were shown by several authors (Sizemore et al.,
1991, Kayser & Meisel, 1996; Jacquote et al., 2010).
In vitro assays,as used in this work, are useful for identification of potential bioactive
peptides in food and the results of in vitro assays may indicate what may happen when
potentially immunomodulating peptides are consumed (Eriksen et al., 2008; Agyei &
Danquah, 2012). The action of immunomodulatory peptides is relatively nonspecific and the
exact mechanism of action as well as the in vivo fate of these peptides is largely unknown
(Pihlanto, 2011; Agyei & Danquah, 2012). It is known some small peptides are easily
absorbed in the intestine while little is known about the absorption of larger bioactive
peptides (Pihlanto, 2011). When the bioactive peptides are not absorbed in the intestinal tract,
their activity may result either directly in the intestinal tract or via receptors and cell
124
signalling in the gut (Pihlanto, 2011). Immunomodulatory peptides which are absorbed from
the gut can either suppress or stimulate certain immune responses (Agyei & Danquah, 2012).
Therefore it is of great importance to establish if a protein (or peptide/peptides) has the same
biological function in vivo as it has in vitro before using for human.
Variation in milks between animal species was considered in chapter 6 and
immunomodulating activity of deer milk was compared with three other domestic species
(cow, goat and sheep). The immunostimulatoryactivity of deer milk digests was not only
higher than cow digestions, but also goat and sheep milk digestions (two other commonly
used milk types).
The peptides in unfermented and fermented digests of deer and cow milk were fractionated
using FPLC technique and three high immunostimulating peptide fractions (11, 22 and 30)
were identified (Chapter 7). Each of these deer milk digests FPLC fractions showed more
immnostimulating activity than corresponding cow milk digest fractions.
Identification of peptides present in above immunostimulating fractions was done using LCMS/MS. Mixtures of different peptides were found in each fraction and most of them were
from β casein (Chapter 8). Some previously characterised immunostimulating peptides
sequences were found within peptides present in unfermented and fermented cow digest
fractions (β casomorphine-7, β-cascochemotide, β casein f 192-209, f 63-68 and f 191-193).
There were no previously identified immunostimulating peptides found in deer fractions
suggesting deer milk might have novel (unidentified) immunostimulating peptides which
have very high activity.
Peptide identification was done only in three high
immunostimulating fractions (11, 22 and 30) which we selected previously from 51 FPLC
fractions. There was a common peptide (β casein fraction 124 – 139) observed in most of the
above immunostimulating fractions for both deer and cow. Also some differences of amino
acid sequence of deer milk protein (β casein , α1 casein, α2 casein) to cow milk protein were
identified, which could be another reason to have different level of immunostimulating
activities by deer and cow milk proteins.
As it is discussed and pointed out throughout this thesis there were so many previous studies
on immunomodulating activity of cow milk and some on other domestic species like sheep,
goat and camel and still the interest of milk from these species can be seen as there are
sufficient amount of very recent studies and reviewed on bioactive peptides on above milk
species (Tanabe, 2012; Nagpal et al., 2012; Choi et al., 2012 and Ebaid et al., 2012). There is
125
no reported work on bioactive peptides on red deer milk which could have potential
immunobioactivity based on historical use of deer milk to cure some medical problems
(Fondahl, 1989; Park &Haenlein, 2006). The present work was able to fulfil that gap by
identifying immunostimulating activity of deer milk and showing higher immunostimulating
activity than cow, goat and sheep milk.
Although the main research area of this study is immunostimulating peptides of deer milk,
some other observations during this research were very interesting and led to studying other
properties of deer milk such as buffering capacity and mineral content. During the first
experiment (Chapter 1) there was the observation of high tolerance of deer milk to pH
changes with addition of acid (1M HCl) or base (1M NaOH) during in vitro digestion pH
adjustment prior to enzyme addition. Also pH of deer milk did not drop below 4.6 after LAB
fermentation while cow milk pH dropped below 4.3 where casein is precipitated (Chapter 4).
These observations led to the measurement ofthe buffering capacity of deer milk compared to
cow milk. In this study higher buffering capacity of deer milk than cow, sheep and goat milk
was identified, which may have potential to treat patient with gastric ulcers (Chapter 6). But
further studies should be carried out to confirm the suggested property. At the same time,it
was found that deer milk is a better source of minerals (especially Ca, P and Zn) compared
with cow, sheep and goat milk.
New information generated through this research were that red deer milk is a better source of
immunomodulating bioactive peptides than cow milk protein and LAB fermentation prior to
digestion improved the formation of bioactive peptides in both deer and cow milks. This
potentially could open commercial opportunities for the deer industry in New Zealand. New
Zealand has 1.2 million farmed deer which is half of world farmed deer population. They are
farmed mainly for meat and velvet (DINZ, 2009). This present research is an initial indication
of the potential of utilising milk as another deer product. Based on present results there is
considerable potential to isolate immunomodulatory peptides from deer milk for
pharmaceuticals and furtherresearch should be carried out. Also this work indicates deer milk
is a usefulsource of novel bioactive peptides.
Future work
•
Many different peptides have been identified in this study from three
immunostimulating FPLC fractions from unfermented and fermented deer milk
digestions. It will be important to determine which, if any, of these
126
areimmunostimulating
peptides
by
further
purification
by
appropriate
chromatography techniques together with biological assays (lymphocyte proliferation
and cytokine production). It will also be important to develop genetic resources for
deer either using genomic sequencing or expressed sequence tags as this will allow
the identification of unique deer peptides. This would be helpful to identify one or
several most active immunostimulating peptides in each active fraction. Then the
activity of each peptide should be tested to confirm the immunostimulating property
after synthesis and those peptides may have potential commercial value as cow milk
derived commercial bioactive products (Ricci-Cabello et al., 2012).
•
It is also interesting to investigate the cytokine production with different time
intervals (6hrs interval) for longer time (72 hr).
•
Once any particular immunomodulating peptide/ peptides were identified from deer
milk protein, in final stages animal trials should be beneficial to identify optimum
plasmatic concentration of these substances and identify the therapeutic potential of
immunostimulating bioactive peptides.
•
Some observations in this research led to the interest to study in areas which not
directly relevant to present topic, but could be potential use of deer milk. Low growth
of LAB strains in deer milk than cow milk (Chapter 4) created a really interesting area
to study on antibacterial properties of deer milk.
•
It is also interesting to investigate other bioactivities in deer milk like opiate-like,
mineral
binding,
antioxidant,
antithrombotic,
hypocholesterolemic,
and
antihypertensive actions since many bioactive peptides are multifunctional and can
exhibit more than one of the effects (Korhonen, 2009).
•
Also further studies of the buffering capacity of deer milk is beneficial to confirm the
observed higher buffering capacity, find out what cause the high buffering capacity
and finally to investigate potential use of deer milk to treat human gastric problems.
•
LAB fermentation of present work was carried out in aerobic condition without pH
control. It is interesting to find behaviour of tested lab strains in different fermentative
conditions such as anaerobic and with pH control for both deer and cow milks and test
the differences in peptide formation and immunomodulating activity.
127
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Appendix A
Appendix A
Identification of deer milk proteins compare to cow milk
proteins& selection of SDS-PAGE concentration
Identification of deer milk proteins compare to cow milk proteins
MW:
250 KD
150
100
75
50
37
Ig HC
LF
SA
CN
25
20
15
10
β-lg
α-la
M C1 C2
D1
C3
C4
D2
D3
Figure 1.SDS- PAGE (4-12%) of skimmed cow and deer milk and milk digested with pepsin
and CPP. High molecular weight marker is shown on left-hand side of the gel. The lanes are:
M: High molecular marker,C1: Cow milk (10 µl sample),C2: Cow milk (20 µl sample),D1:
Deer milk (10 µl sample),C3: Cow milk hydrolysed (10 µl sample),C4: Cow milk
hydrolysed (20 µl sample),D2: Deer milk hydrolysed (10 µl sample),D3: Deer milk
hydrolysed (20 µl sample). protein.Immunoglobulins heavy chain (IgHC), lactoferrin (LF),
serum albumin (SA), casein (CN), β-lactoglobulin (β-lg) and α-lactalbumin (α-la).Each
contained had 2 mg/ µl
152
The shown cow protein bands were identified accordance with Almaas et al., 2006 (20%
SDS-PAGE) and Inglingstad et al., 2010 (12.5 % and 15 % SDS-PAGE) gel results. Deer
protein bands were identified based on similarity to cow milk protein bands.
Selection of SDS – PAGE concentration
MW:
250 kDa
150
100
75
50
37
25
20
15
10
C C30 C50 C60
D D30 D50 D60
Figure 2.SDS- PAGE (8-16%) of skimmed cow & deer milk, cow & deer milk digested, with
pepsin and CPP. The lanes are: High molecular marker (M), Cow milk(C), Digest after30min
(C30), 50min (C50), 60min (C60), Deer milk (D), Digest after30min (D30), 50min (D50),
60min (D60). Each contained had 2 mg/ µl
MW:
250 kDa
150
100
75
50
37
25
20
15
10
CL DL
CS DS
Csh Dsh
C D
153
Figure 3.SDS-PAGE (8-16%) of fermented cow and deer milks by Lactobacillus
delbrueckiisubsp bulgaricus, Streptococcus salivarius subsp thermophilus and Lactobacillus
casei strain Shirota at 37°C for 24 hr. The lanes are Lactobacillus delbrueckiisubsp bulgaricus
fermented cow (CL) and deer (DL) milk, Streptococcus salivarius subspthermophilusfermented
cow (CS) and deer (DS) milk, Lactobacillus casei strain Shirota fermented cow (Csh) and
deer (Dsh) milk, unfermented cow (C) and deer (D) milk. Each contained had 2 mg/ µl
C
C60
D60
D D
M
Figure 4.SDS-PAGE (20%) of skimmed cow & deer milk, cow & deer milk digested, with
pepsin and CPP. The lanes are: High molecular marker (M), Cow milk(C), Cow milk digest
after 60min (C60), Deer milk (D), Deer milk digest after 60min (D60). Each contained had 2
mg/ µl
As preliminary studies different concentration of gels were tested base on literature.
Vinderola et al., 2006 used 12 % SDS – PAGE for LAB fermented cow milk and Matar et
al., 1995 used 12.5% seperating gel and 4% stacking gel. Almass et al., 2005 used 20% SDSPAGE for cow and goat milk and their digests. Inglingstad et al., 2010 was used 12.5 % and
15 % gel to analyse cow, goat, horse, human milk and their digest.
Milk, milk ferment and milk digests were run with 4-12% precast gel which used in
experiment chapters (fig. 3.3, 3.4, 4.7, 6.3), 8-16% gel (Fig 2 and 3) and 20 % gel (fig. 4).
As shown in figure 2 and 3 protein bands of milk and digests were not clear in 8-16% gel and protein
did not run well in 20 % gel (fig. 4) while 4-14% gel (experiemental chapters) showed cleare band.
Since this study aimed to analyse milk protein bands and digested protein bands together 4-12
% gels were selected which gave clear bands in both case.
154
Appendix B
Paper accepted forProceedings of the Nutrition Society of New Zealand (2011), Volume
35.
The release of peptides by in-vitro digestion of
fermented red deer (Cervus elphus) and cow (Bos
taurus) milk.
N. L. O PATHA V ITHANA1 , S. L. MASON1 , A. E. A. BEKHIT2 , J. D. MORTON1
1
Lincoln University, New Zealand; 2Otago University, New Zealand
ABSTRACT
Fermentation and digestion release peptides from milk as a source of amino acid and energy, as well as a source
of biofunctional peptides that may have health benefits. The aim of this study was to ferment deer and cow milk
using three strains of Lactic acid bacteria (LAB) and to compare the release of peptides following in-vitro
digestion of the fermented milks. The three strains were Lactobacillus belburueckii subspbulgaricus,
Streptococcus salivarius subsp thermophilus and Lactobacillus casei strain Shirota and the fermentations were
carried at 37 oC for 24 hours. The in-vitrodigestion was performed in two steps; imitating both the human
stomach (Pepsin, pH 2.5) and the duodenum (Corolase PP, pH 7.5). Release of peptides during milk
fermentation and subsequent digestion was quantified using OPA (o-phthaldialdehyde) assay. Changes in
protein and peptide profiles were evaluated using sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) and quantified using ImageJ software. After 24 hours of fermentation, the pH of cow milk
dropped below 4.6 and precipitated caseins while pH of deer milk remained above pH 4.6 for all three strains.
Deer milk fermentations gave higher peptide production than cow milk fermentations. Following in-vitro
digestion peptide production was significantly greater in deer milk than cow milk (P≤ 0.05). Lactobacillus casei
strain Shirota showed the highest release of peptides. The main milk proteins were degraded during
fermentation of both milks. Lactoferrin (LF) in cow milk was more resistant to fermentation than that in deer
milk for all 3 strains. After 24 hours of fermentation by Lactobacillus casei strain Shirota, cow LF was still
intact while 47% of the deer LF had degraded. Digestibility of β-lactoglobulin, α-lactalbumin of deer milk and
immunoglobulin of cow milk was improved by fermentation.This study showed LAB fermentation prior to invitro digestion increased the digestibility and release of peptides from both milks. This effect was greater in deer
milk than cow milk.
INTRODUCTION
Fermented milk products, in addition to providing a source of energy and nutrients, are also a source of peptides
with biological functions that may improve health when ingested. The health benefits of fermented milk have
been recognized and documented (Gobbetti et al., 2006). Over the last decade a great number of peptide
155
sequences with different bioactivities (eg. immunomodulatory, antihypertensive, antithrombotic, antibacterial,
antioxidative and opioid activity) have been identified in various milk proteins (Korhonen, 2009). Many
commercial lactic acid bacteria (LAB) based starter cultures are highly proteolytic (Korhonen, 2009). The in
vitro study by Pihlanto-Leppala et al. (1998) demonstrated that fermentation of milk with starter cultures or
enzymes derived from such cultures prior to treatment with digestive enzymes can enhance the release and alter
the profile of bioactive peptides produced. Further, Matar et al. (1996) showed that fermentation of milk by
LAB has a major impact on the release of novel low molecular mass peptides during in vitro digestion. It is
likely that peptides are also released during digestion in the gastrointestinal tract. Deer milk has 8.8 % protein
which is twice as concentrated as cow milk (Opatha Vithana et al., 2010). This could be a good substrate for
proteolytic activity of LAB stains and may produce more peptides which might have biological functions
beneficial to health.
The aim of this study was to compare deer and cow milk during fermentation using three LAB cultures in terms
of peptide production before and after in vitro digestion and compare deer and cow milk protein digestibility.
This is the first report studying the fermentation of deer milk followed by in vitro digestion and the information
from the present work is potentially useful in understanding the release of peptides in animal nutrition and to
evaluate the potential use of deer milk products for human use.
METHODS
Milk Fermentation
Skim deer and cow milk were inoculated withLactobacillus delbrueckiisubsp bulgaricus, Streptococcus
salivarius subsp thermophilus and Lactobacillus casei strain Shirota with 2% (vol/vol) inoculums separately and
incubated at 37°C for 24 hr (Matar et al., 1996).
In vitro Digestion
In vitro digestion was performed for milk and fermented milk in two steps using Pepsin and Corolase PP
(mixture of trypsin, chymotrypsin and several amino and carboxypeptidases) according to Eriksen et al. (2008).
The release of peptides during milk fermentation and digestion was quantified using OPA (o-phthaldialdehyde)
assay(Church et al., 1983). The changes in protein and peptide profiles were compared by sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as per Lammli (1970) and quantified using ImageJ
software (rsbweb.nih.gov/ij/).
Statistical analysis
All experiments were carried out in triplicate and the reported values are mean ± standard deviation. The data
were subjected to one way analysis of variance (ANOVA), followed by Tukey’s test to determine the significant
difference between means at p˂0.05 level using Minitab (16 version).
RESULTS
The pH of deer milk was 6.1 prior to fermentation whereas it was 6.5 for cow milk. After 24 hr of fermentation;
the pH of cow milk was significantly lower than deer milk ferments for all three stains of LAB;Lactobacillus
casei strain Shirota, Lactobacillus delbrueckiisubsp bulgaricus and Streptococcus salivarius subsp
thermophilus. The pH fell below 4.6 for cow milk at which pH the casein is precipitated. Deer milk pH did not
fall below 4.6 for any of the three LAB fermentations over 24 hr (Fig 1).
(b)
(c)
7.0
7.0
6.5
6.5
6.5
6.0
6.0
6.0
5.5
pH
7.0
pH
pH
(a)
5.5
5.5
5.0
5.0
5.0
4.5
4.5
4.5
4.0
4.0
0hr
6hr
12hr
24hr
44hr
Fermentation time
72hr
4.0
0hr
6hr
12hr
17hr
Fermentation time
24hr
0hr
6hr
12hr
17hr
24hr
Fermentation time
Figure 1. pH during fermentation of deer ( ◦ ) and cow ( • ) milk at 37°C. (a) Lactobacillus casei strain
Shirota (Lcs), (b) Lactobacillus delbrueckiisubsp bulgaricus (Ldb), (c) Streptococcus salivarius subsp
thermophilus (Sst). Data are mean of three independent fermentations ± S.D.
The proteolytic activities of the studied strains (Fig.2) were significantly higher in deer milk than cow milk
(p<0.05). The highest production of peptides was generated by Lactobacillus casei strain Shirota (Fig.2) after
24 hours of fermentation at 37°C. Once ferments were digested, significantly ( p<0.05) higher peptide
production was observed in deer milk compared to cow milk for all three strains and the highest production was
156
found in digest of Lactobacillus casei strain Shirota fermented deer milk. Fermented milk digests always have
slightly higher peptide concentration than raw milk digest. When comparing the three stains in terms of peptide
production there was no significant difference after in vitro digestion of both deer and cow milk ferment.
Figure 2. Peptide production following in vitro digest of raw milk and milk ferment for 24 hr as well as
undigested ferment. Cow( ) and deer ( ) milk fermented with (a) Lactobacillus casei strain Shirota (b)
Lactobacillus delbrueckiisubspbulgaricus (c) Streptococcus salivarius subsp thermophilus. Data are means± SD
(n = 3).
The degradation of milk proteins was visualized using SDS gel electrophoresis. The main milk proteins started
to degrade during fermentation of both milks (Fig 3). After in vitrodigestion, most of the milk protein bands
completely disappeared and appearance of new lower molecular mass bands on the gel could be seen.
Quantification of protein bands using ImageJ software is shown in Table 1.
(i)
(ii)
(iii)
IgHC
LF
SA
CN
βLG
αLA
M
C Cf Cfd
D Df Dfd C Cf Cfd
D Df Dfd C Cf Cfd
D Df Dfd
Figure 3. SDS-PAGE (4-12%) of deer and cow milk, ferment and digested ferment using(i) Lactobacillus casei
strain Shirota, (ii) Lactobacillus delbrueckiisubsp bulgaricus and (iii) Streptococcus salivarius subsp
thermophilus. The wells contains (C) cow milk, (Cf) cow milk ferment, (Cfd) cow milk ferment digest, (D)
deer milk, (Df) deer milk ferment, (Dfd) deer milk ferment digest, (M) molecular weight marker. Each
contained had 2 mg/ µl protein.
Table 1. Remaining protein content (%) in digest of milk, Lactobacillus casei strain Shirota, Lactobacillus
delbrueckiisubspbulgaricus and Streptococcus salivarius subsp thermophilusfermented (24 hr at37°C) milk
before and after in vitro digested (30min simulated stomach + 30min simulated duodenum). Values are obtained
by ImageJ.
Lactobacillus delburueckii
Streptococcus salivarius subs
Lactobacillus casei strain Shirota subsp bulgaricus
thermophilus
Ferment
Ferment
Ferment
Milk Digest
Ferment
Digest
Ferment
Digest
Ferments
Digest
Deer
Cow
Deer
Cow
Deer
Cow
Deer
Cow
Deer
Cow
Deer Cow
Deer
Cow
IgHC
LF
1± 2
0±1
21±5
1±1
62±1
47±3
64±2
101±1
0±0
1±1
0±0
2±1
71±4
42±5
74±3
63±4
0±0
0±0
8±1
2±1
73±3
48±2
77±3
69±5
0±0
0±0
0±0
0±0
SA
0±0
1±1
77±1
68±1
0±1
1±0
62±7
63±6
0±0
1±1
70±1
67±6
0±0
0±0
157
CN
β-lg
0±0
54±5
1±1
55±8
83±2
87±1
85±1
86±1
0±0
52±1
0±1
57±1
76±8
85±2
82±4
91±7
1±0
51±1
1±1
57±4
85±2
83±4
84±1
78±2
2±0
51±1
2±0
68±1
α-la
11±2
27±5
92±2
90±1
8±4
17±1
87±2
90±3
1±1
13±4
85±3
91±2
15±3
23±4
Data are mean ± S.D. (n=3)
Lactoferrin (LF) of cow milk is more resistant to degradation during fermentation by all three LAB strains than
deer milk (Table 1). After 24 hours of fermentation by Lactobacillus casei strain Shirota, LF was still intact
(100%) in cow milk while 47% LF remained intact after deer milk fermentation. Digestibility of α –lactalbumin
(α –la) of deer milk was significantly (p< 0.05) improved by Lactobacillus delbrueckiisubsp
bulgaricusfermentation. About 11% intact α –la was found after in vitro digestion of raw deer milk
whileLactobacillus delbrueckiisubsp bulgaricus ferment digest only contained 1% intact α –la (Table 1). The
digestibility of β lactoglobulin in Streptococcus salivarius subsp thermophilus fermened deer milk was also
significantly (p< 0.05) greater than β-lactoglobulin in cow milk ferment by the same culture. Deer milk
fermented by the other two stains and digested showed slightly higher digestibility than cow ferment digest for
β-lactoglobulin (β-lg). There was a significant difference observed in immunoglobulin (IG) of cow milk. Intact
protein level was 21% after in vitro digestion of raw milk and this decreased to 0 (no intact protein) after in vitro
digestion of milk fermented by all three strains. These differences in digestibility of milk protein may lead to
formation of novel peptides.
DISCUSSION
Deer milk was slightly more acidic than cow milk. Differences in pH and buffering capacity of fresh milk
reflect compositional variation (McCarthy, 2002). After 24 hour of fermentation, the deer milk ferment showed
significantly higher pH (p<0.05) than cow milk which went below the casein iso electric point resulting in
precipitation of casein for all three strains (Fig.1). The smaller pH drop of deer milk may be due to several
factors such as low bacterial growth resulting in low lactic acid formation or a higher buffering capacity of deer
milk than cow milk.The ultimate pH of cheese results from the lactic acid produced (from starter culture lactose
metabolism) and moderated by the buffering capacity of the milk (Pendey et al., 2003). Principal buffer
components in milk are soluble phosphate, colloidal calcium phosphate, citrate, bicarbonate, casein, salt and a
number of minor constituents (McCarthy, 2002). Goat milk has high buffering capacity due to its high content
of major buffering components such as minerals and protein (Park, 1992). The mineral and casein content of
red deer milk is about twice that of cow milk (Opatha Vithana et al., 2010). This high casein and minerals can
lead to high buffering capacity of deer milk and could reduce the pH drop during fermentation when compared
to cow milk. Further studies should be done to confirm the nature and the components contributing to the
buffering capacity of deer milk.
Lactobacillus delbrueckiisubsp bulgaricus and Lactobacillus casei strain Shirota fermentation of deer milk
produced significantly (p<0.05) higher peptide concentration than the fermentation of cow milk (Fig 2). When
all three strains ferments were digested, deer milk ferment produced significantly higher peptide concentration
than cow milk ferment. Gobbetti et al. (2000) showed that during milk fermentation, probiotic strains may
produce several oligopeptides which generate bioactive peptides only after subsequent digestion by pepsin and
trypsin. Peptide bonds that are protected within the native protein structure might be exposed by the action of
LAB enzymes, thereby allowing the release of new peptides. According to Korhonen and Pilanto (2006), the
use of both fermentation and digestion has been more effectiv in generating biofunctional peptides than
fermentation or digestion alone. Mata et al. (1996) reported that proteolysis during fermentation enhanced the
release of peptides and may lead to the formation of novel peptides during gastrointestinal digestion. Overall
deer milk digest produce more peptides than cow milk and this was further increased by fermentation prior to
digestion.
The ability to produce extracellular proteinases is a very important feature of LAB. They catalyse proteolysis of
milk proteins, providing the amino acids essential for growth of LAB (Fira et al., 2001). LAB fermentation in
yogurt is a desirable process improving milk digestibility and enhancing nutritional quality by protein
degradation and hence, changes the texture, the taste and the aroma of fermented products (El-Ghaish et al.,
2011). In this study LAB fermentation improved the digestibility of β-lg of deer milk more than for cow milk
(Table 1). β-lg is the main cause of 80% of milk allergies in children and infants. β-lg is the major whey protein
in milk and in dairy products and it is of particular interest since it is the only whey protein of cow milk absent
in human milk (El-Ghaish et al., 2011). Kleber et al., 2006 studied the ability of some LAB strains to reduce the
antigenicity of β-lg using skim milk and sweet whey by indirect competitive ELISA, using polyclonal
antibodies. They observed the reduction of antigenicity of skim milk by 90% and sweet whey by 70% compared
to the initial value. Our results suggest that less β-lg remained in the ferment digest of deer milk compared to
cow ferment digest which may leads to less allergenicity of LAB fermented digest in deer milk than cow milk.
α-Lactalbumin is another whey protein which is less allergenic than to β-lg (Wal, 1998). 11% intact α –la of
deer milk was reduced to 1% by digestion with Lactobacillus delbrueckiisubsp bulgaricus and to 8% by
158
digestion with Lactobacillus casei strain Shirota ferments (Table 1). There was significantly lower concentration
of intact α –la in digest of deer ferment than cow ferment for all three strains. Bu et al. (2010) found that LAB
fermentation reduced the antigenicity of cow α –la and β-lg by hydrolysis. Since our results showed less α –la
and β-lg in deer ferment digest than cow ferment digest, fermented deer milk digest may be less allergen than
fermented cow milk digest. Digestibility of cow IG was significantly improved after fermentation by all tested
LAB strains (Table 3). Cow milk IGs are relatively resistant to proteolytic digestive enzymes in the
gastrointestinal tract, but can be sensitive to microbial enzymes (Marnila and Korhone, 2011). In this study
lactoferrrin (LF) in cow milk was significantly more resistant (P<0.05) to fermentation than that in deer milk for
all three strains. Lactoferrin is an 80 kDa iron-binding glycoprotein of the transferrin family that is expressed in
most biological fluids and is a major component of the mammalian innate immune system (Gonzalez-Chavez et
al., 2009). In characterising the various peptides generated by LF hydrolysis, it was found that minimal
variations in the amino acid sequence change the antimicrobial activity of the peptide. For example, LFampin
268–284 and LFampin 265–284, chemically synthesised fragments from the N-terminal sequence of bLF, differ
in only three amino acids (265Asp-Leu-267Ile) but exhibit different antibacterial activities (Kraan van der et
al., 2006). Therefore different levels of hydrolysis in deer and cow milk during fermentation may form different
peptides which may have different strength of biological activity. Similar to our cow milk fermentation results,
results of Franco et al. (2010) indicate that LF structure does not seem to be altered much by the activity of
commercial LAB bacteria (yogurt). In contrast digestibility of deer milk is higher than cow milk and was further
improved by LAB fermentation prior to digestion.
CONCLUSION
Fermentation prior to digestion increased the production of peptides from and the digestibility of major milk
proteins from both deer and cow. Deer ferment digest produced more peptides than cow ferment digest. There
was no significant difference in terms of peptide level produced after digestion among three LAB stains used in
this experiment. Digestibility of major milk proteins was higher in deer ferment than cow ferment. This study is
a novel approach which might have a great impact in diversifying income stream for deer farmers and generate
new information which will help in understanding fawn nutrition. In future work deer and cow milk peptides
will be fractionated using FPLC and immunomodulatory activity will be studied using human peripheral blood
mononuclear cells (PBMC).
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Appendix C
IL-2 production by human peripheral blood
mononuclear cells (PBMC) stimulated by Con A with
FPLC fraction 13 and 36 of unfermented and fermented
deer and cow milk in vitro digests
Fraction 36
50
50
40
40
IL2 production (pg/ml)
IL2 Production (pg/ml)
Fraction 13
30
20
10
30
20
10
0
0
Control
Milk digest
Ldb digests
Sst digests LcS digests
Control
Protein o.125 mg/ml
control
D
C
Milk digest
Ldb digests
Sst digests LcS digests
Protein 0.125 mg/ml
control
deer
cow
Figure 1. IL-2 production by human peripheral blood mononuclear cells (PBMC) stimulated
by Con A with FPLC fraction 13 and 36 of unfermented and fermented deer ( ) and cow ( )
milk in vitrodigests (0.125mg/ml protein). Fractions from deer and cow milk digests and
digests of Lactobacillus delburueckii subsp bulgaricus (Ldb), Streptococcus salivarius subs
thermophilus (Sst) and Lactobacillus casi strain Shirota (Lcs) ferment. Control ( ) cells
were stimulated by Con A without the addition of proteins. Data are mean ± S.D. (n = 3).
Samples measured in triplicates.
161
Appendix D
Deer and cow milk digests FPLC fractions absorbance at
280 nm
Figure 1.Absorbance at 280 nm of RP-FPLC chromatograms of the deer milk digests
Figure 2. Absorbance at 280 nm of RP-FPLC chromatograms of the cow milk digests
162