Sobre la concepción de plano, su polisemia y su identificación

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

125 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 LC- MS/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

126 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

127 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.

128

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