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Comparison of the bioactivity of whole and skimmed digested sheep milk with that of digested goat and cow milk in functional cell culture assays
Accepted Manuscript Title: Comparison of the bioactivity of whole and skimmed digested sheep milk with that of digested goat and cow milk in functional cell culture assays Authors: Sonya Mros, Alan Carne, Minh Ha, Alaa El-Din Bekhit, Wayne Young, Michelle McConnell PII: DOI: Reference:
S0921-4488(17)30054-8 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.02.018 RUMIN 5434
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
21-12-2016 16-2-2017 19-2-2017
Please cite this article as: Mros, Sonya, Carne, Alan, Ha, Minh, Bekhit, Alaa El-Din, Young, Wayne, McConnell, Michelle, Comparison of the bioactivity of whole and skimmed digested sheep milk with that of digested goat and cow milk in functional cell culture assays.Small Ruminant Research http://dx.doi.org/10.1016/j.smallrumres.2017.02.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparison of the bioactivity of whole and skimmed digested sheep milk with that of digested goat and cow milk in functional cell culture assays
Sonya Mrosa, Alan Carneb, Minh Hab Alaa El-Din Bekhitc Wayne Youngd and Michelle McConnella*
a
Department of Microbiology and Immunology
b
Department of Biochemistry
c
Department of Food Science
University of Otago, PO Box 56, Dunedin d
AgResearch Ltd Grasslands, Palmerston North
*Corresponding author Email address [email protected]
1
Highlights
Sheep milk simulated gastric digests increase TNF- α production by THP-1 cells
Sheep milk simulated gastric digests modulate mitogenic stimulation of splenocytes
Cow milk simulated gastric digests increase IL-10 production by THP-1 cells
Abstract Milks from sheep, goat and cow were subjected to simulated gastric digestion prior to being analysed in cell culture assays to compare the bioactivity of the digests. All milks displayed a similar profile on 1D SDS-PAGE following digestion. Overall, the effects seen from the digested milks (whether whole or skimmed) in cell culture assays from the three animal species were similar with the exception of digested sheep milk which increased the amount of TNF-α produced by THP-1 cells in the presence of lipopolysaccharide (LPS) and digested cow milk which increased the amount of IL-10 produced by THP-1 cells in the presence of LPS. While sheep milk did not stimulate splenocyte proliferation directly, digested sheep milk had a greater effect on the response of splenocytes to mitogenic stimulation than cow and goat milk digests. Keywords Milk, sheep, goat, cow, bioactivity, cell culture assays
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1. Introduction Although milk production in New Zealand is dominated by cow dairying there is a rapidly expanding industry producing sheep milk that is primarily used for the production of cheese, as well as milk powder, yoghurt and ice cream (Peterson and Prichard, 2015). Milk has long been regarded as a functional food in addition to its primary purpose of nutrition (Gobbetti et al., 2007, Mills et al., 2011, Severin & Wenshui, 2005). Bioactivity in milk is reported to be associated with proteins, peptides, lipids and carbohydrates (Korhonen and Pihlanto, 2006, Krissansen, 2007). In their native state many of the milk proteins are not bioactive (Korhonen and Pihlanto, 2003). However, bioactive peptides can be released as a result of hydrolysis of proteins or by fermentation with lactic acid bacteria (Mills et al., 2011). Proteolytic hydrolysis of proteins can release peptides exhibiting a number of biological activities, including antimicrobial and immunomodulatory (Korhonen and Pihlanto, 2006). Considerable research has been undertaken investigating peptides of bovine origin (Korhonen and Pihlanto, 2006) but less is known about peptides from milk proteins of other species. Since there are known homologies between sequences of bovine, ovine and caprine milk proteins the likelihood of similar bioactive peptides being released is high (Recio and Visser, 2000). However, we have recently reported in a proteomic study that although the whey proteome of sheep showed some similarities to that of cow, a considerable number of differences were identified (Ha et al., 2015), suggesting that sheep milk may contain some different bioactivities.
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The use of cell culture based systems to investigate the immunomodulatory properties of products is well established (Gonzales et al., 2015, Haverson et al., 2002; Kanwar and Kanwar, 2009; Park, 2009) as it enables investigation of the effect of products on different types of cellular function. To learn more about milk from sheep in New Zealand, both whole and skimmed milks from sheep, goat and cow were subjected to simulated gastric proteolytic digestion using enzymes involved in human digestion and the resulting hydrolysates were analysed using a number of functional cell culture assays (including assays designed to measure immunomodulatory effects of the milk hydrolysates in the presence and absence of mitogenic stimulation of the neutalisation effects of the milk hydrolysates in the presence of toxin) to compare the bioactivity profiles obtained from peptides generated by the simulated digestion in the three milk types. 2. Materials and methods All chemicals were obtained from Sigma Aldrich, Auckland, New Zealand, unless otherwise stated. Raw milks were obtained from local commercial farms. 2.1. Milk sample collection and preparation Ethical approval was not required for this study. Individual milk samples were collected from animals that had been tested for mastitis and were shown to be healthy. Milk from each animal was expressed by hand into pre-chilled autoclaved glass bottles and kept on ice during transportation to the laboratory. On arrival at the laboratory one half of each milk sample was centrifuged at 4000 x g for 30 min
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at 4°C to float the cream fraction. The cream was removed and any pelleted casein fraction was resuspended into the skim milk prior to it being used for proteolytic digestion. 2.2. In vitro digestion with pepsin and pancreatin Bicinchoninic acid (BCA) protein assays were performed to determine the total protein levels (mg/ml) of each of the milk samples. Digestion of the milks with pepsin (Sigma, EC3441, 2650 U.mg-1) and pancreatin (Sigma, P1750, 1750 U.mg-1) were carried out according to the method of Hernandez-Ledsema et al. (2007). The milks were first adjusted to pH 3.5 and then hydrolysed with pepsin, made up at 20 mg.ml-1, for each milk sample based on the total protein level, then incubated, at 37°C for 30 min, while stirring the solution at 150 rpm. The digests were placed in ice water to stop hydrolysis and then the pH was adjusted to 7.0 with 0.1M NaOH. Pancreatin was added at a concentration of 50 mg.ml-1 based on the total protein level. Samples were then incubated for 60 min at 37°C with stirring at 150 rpm . The proteases were inactivated by heating at 95°C for 15 min followed by cooling to room temperature. Digested and undigested milk samples were displayed on 1D-SDS-PAGE, conducted on Bolt 4-12% gradient Bis-Tris gels (Life Technologies, Auckland, New Zealand) as reported previously (Ryder et al., 2015). 2.3. Splenocyte proliferation assay Spleens were harvested from adult C57Bl/6 mice (UoO AEC ET16/11), macerated and then passed through a cell strainer (70 μm; Falcon, Becton Dickinson, NJ, USA) before being treated with 5 ml of red blood cell ACK lysis buffer for 3 min, with shaking at 1 min intervals. 5
Aliquots of the splenocyte preparation (100 µl) (2 x 106.ml-1) were dispensed into 96 well flat bottom tissue culture plates (Falcon™) and with either 50 μl complete media alone (Dulbecco’s Modified Eagle Media (Gibco®) supplemented with 10% Foetal Calf Serum (Gibco®) and 1% Penicillin/Streptomycin (10,000 U.ml-1 Gibco®)) as a cells-only control, or with 50 μl complete media plus sub-optimal concentrations determined previously of (i) pokeweed mitogen (PWM) (Sigma, L8777) to give a final concentration of 2.5 µg.ml-1, or E.coli lipopolysaccharide LPS (Sigma, L2880) to give a final concentration of 1.25 µg.ml-1, or (ii) concanavalin A (Con A) (Sigma, C-2010) to give a final concentration of 1.25 µg.ml-1. Plates were incubated in 5% CO2 for 72 h at 37°C, with, or without the milk hydrolysates (at 1/1000 dilution) in six wells per animal milk sample. Milk products (1/1000 dilution) alone were also incubated with splenocytes in six wells per animal milk sample. Further controls of mitogens alone at final concentrations in the well of 1.25 µg.ml-1 of LPS, 10 µg.ml-1 of PWM and 5 µg.ml-1 of Con A were also used. Following incubation, tritiated thymidine (Perkin Elmer, 6-3H 185MBq) a lowenergy beta particle emitter, was added (50 µCi per well), and the plates were reincubated for a further 18 h. The contents of the 96 well plate were transferred onto a Printed Filtermat A (Perkin Elmer, USA) for use with a 1450 MicroBeta™ (Perkin Elmer) scintillation counter using a 96 Cell Harvester (TomTec, CT, USA). The Filtermat was dried in a microwave oven for 2.5 min and then placed into a sample bag (MicroBeta™, Perkin Elmer) with 4.5 ml of Ultima Gold™ liquid scintillation cocktail (Perkin Elmer) and heat sealed closed. The prepared Filtermats were then placed into a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence
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Counter (Wallac, Finland) to count the photons produced in the reaction of the beta particles with the scintillator, and quantify the beta emitting isotopes. 2.4. THP-1 assay THP-1 cells (ATCC # TIB-202™) were dispensed into wells of a 96 well flat bottom tissue culture plate (Falcon™) at a concentration of 3x105 cells.ml-1, with the addition of phorbol myristate acetate (PMA) at 20 ng.ml-1 to induce cell differentiation into macrophage-like cells, and incubated at 37°C in 5% CO2 until the formation of a confluent monolayer (24-48 h). Cells were then stimulated with either 1/1000 (final dilution in cell culture medium) dilutions of the digested and undigested milks alone, or 100 ng.ml-1 E. coli LPS alone, or LPS + milks. The supernatants were harvested after 24 h incubation and were used to determine the levels of the cytokines TNF-α and IL-10 using OptEIA™ ELISA kits (BD Biosciences) as per the manufacturer’s instructions. 2.5. Anti-toxin assay A. Toxin preparation and titration of activity E. coli 026 (ATCC9026), a known Shiga-like toxin producing strain of enterotoxigenic E. coli, was grown overnight in tryptic soy broth at 37°C. The culture was centrifuged at 4000 x g for 10 min to pellet the cells. The supernatant was retained and filtered through a 0.45 m filter to remove any remaining bacterial cells. Serial dilutions of the toxin in 100 l volumes were added to wells of a 96 well flat bottom tissue culture plate (Falcon™) containing confluent monolayered Vero cells (ATCC CCL-81™) and incubated for 96 h. After incubation 10 µl of 7
phosphate buffered saline (PBS) at pH 7.2, containing MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) final concentration: 5 mg.mL-1 (Molecular Probes™ ThermoFisher Scientific, USA) was added to each well. After 4 h incubation at 37°C, the supernatant was removed and 100 µl dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. After vigorous shaking, the absorbance was measured in a micro-plate reader (Varioskan Flash, Thermo Scientific, USA) at 570 nm. Dilutions of Shiga-like toxin showing a cytopathic effect of >50% compared to untreated cells were used in a toxin neutralization assay based on that of Paton and Paton (1998) as described below. B. Toxin neutralisation Serial dilutions of the digested sheep milk samples were made and assayed in triplicate in 100 l amounts on Vero cells to check cytotoxicity. The 1/10 and 1/100 dilutions caused some toxicity in the cells, so the 1/1000 dilution that did not cause toxicity towards the Vero cells, was mixed with Shiga-like toxin (10 l milk digest + 90 l toxin) and the mixture was incubated for 60 min at 37°C. A 100 l volume of the mixture was then added to a confluent monolayer of Vero cells, and plates were incubated for 72 h at 37°C in a 5% CO2 incubator. Controls were prepared using Vero cells only, and cells plus 10 l of PBS + 90 l Shiga-like toxin. At the end of 72 h incubation the MTT method described above was used to determine cell viability and the results were expressed as % of viable cells when compared to the Vero cells only control. A 1/1000 dilution of each of the digested milks was also added as controls.
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2.6. Statistical Analysis All analyses were carried out using results from three independent experiments. Sheep milk simulated gastric digests Statistical analysis was undertaken by ANOVA using GraphPad Prism Version 6.05 3. Results 3.1. In vitro digestion Analysis of protein profiles from undigested skimmed sheep, cow and goat milk by SDS-PAGE showed the proportion of casein isoforms in sheep and goat milk were different to that of cow milk, and goat milk had a higher proportion of lower molecular weight proteins than the other two milks (Figure 1). The immunoglobulins (Ig) band appeared less intense in skimmed milk compared with whole milk whereas the lactoferrin (Lf) band was more intense in goat milk compared with cow and sheep milk. Following digestion all milk samples appeared similar on SDS-PAGE. In all of the milk samples the caseins were fully digested but lactoglobulin (-Lg) was only partially digested (Figure 1). 3.2. Splenocyte proliferation assay None of the whole milks and skim milks stimulated proliferation of mouse splenocytes. A similar result was obtained for all of the digested milk samples (data not shown). However, whole sheep milk and skimmed goat milk in the presence of Con A reduced the stimulatory effect on cell proliferation observed with Con A alone (p<0.05; Figure 2). None of the other milks had any effect. In the presence of
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pokeweed mitogen (PWM) a number of the milks reduced the effect of the PWM induced cell proliferation on the splenocytes. Undigested whole and skimmed sheep milk both reduced the PWM effect (p<0.05; Figure 3), as did both undigested and digested whole cow milk (p<0.05; Figure 3), whereas goat milk had no effect. Digested skim milks from all three animal types augmented the PWM effect. Undigested whole and skim milk from sheep reduced the LPS effect on splenocyte proliferation (p<0.05; Figure 4), whereas milks from the other animals had no effect. Digested sheep milk and the milks from all other animals had no effect on the LPS stimulation (Figure 4). 3.3. THP-1 assay Whole digested sheep milk reduced the amount of TNF-α produced by the unstimulated THP-1 cells (P<0.05; Figure 5).
However, both undigested and
digested whole and skim milk in the presence of LPS stimulated the production of more TNF-α than the LPS alone (p <0.05; Figure 5). In contrast, the effects on IL-10 production were markedly different. All the milks alone stimulated the production of IL-10 by differentiated THP-1 cells. For whole and digested sheep and goat milks this was less than the amount produced following LPS stimulation of the cells (p<0.05; Figure 6). In the presence of LPS goat and sheep milk did not have any effect on the IL-10 produced compared to LPS alone but cow milk augmented IL-10 production compared to LPS alone (p<0.05; Figure 6). 3.4. Toxin neutralisation
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Some, but not all, milk from the three animal types was able to partially neutralize the toxins (Figure 7, but none were able to neutralize sufficient toxin to prevent cell damage at some level. While the overall trend appeared to be that goat milk was more effective at neutralizing toxin than sheep milk, and that sheep milk was more effective than cow milk (Figure 7) there was no statistical difference between the groups. 4. Discussion The whole milk protein profiles were similar to those obtained in a previous study (Ha et al, 2014). However, the peptide profiles of all the milks were similar following simulated gastric digestion, suggesting that digested milks may have similar effects in functional assays. A review by Park and Nam (2015) suggests that similar bioactive compounds are derived from digestion of bovine, caprine and ovine milks with some bioactive peptides from ovine and caprine milks having identical sequences. In the study reported here, the prediction was that the milks would have similar activities, which proved true for the splenocyte assays where none of the milks stimulated splenocyte proliferation. However, some differences were observed with the effects on mitogen-induced proliferation, particularly with sheep and goat milk, but not with cow milk. This may be because peptides generated from these may be slightly different despite having similar primary protein structures (Ha et al., 2015). This ability to down-regulate mitogenic stimulation may be related to the ability of some of the milks to stimulate IL-10 cytokine production but not TNF-α. The main role for TNF-α is in the regulation of immune cells. It is a pro-
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inflammatory cytokine and is released in response to lipopolysaccharide, other bacterial products and IL-1. TNF-α attracts neutrophils to the site of infection, causes inflammation and has been associated with a number of conditions such as rheumatoid arthritis and ankylosing spondylitis where disease treatment often includes a TNF inhibitor (Scott et al., 2015). In the study reported here the addition of milks increased the effects of LPS stimulation of the differentiated macrophages although the milks alone had no effect on TNF production. On the other hand, IL-10 is an anti-inflammatory cytokine that has multiple effects in immune-regulation and inflammation and down-regulates production of some of the inflammatory cytokines. From the results obtained it seems that while none of the milks produce an inflammatory stimulus on differentiated macrophages as measured by TNF-α production they do stimulate the production of IL-10, which is an antiinflammatory cytokine that dampens the immune response. Interestingly, when in association with an inflammatory substance such as LPS, the milks induced an increased TNF-α inflammatory response but did not have as much effect on the IL10 pro-inflammatory response except for cow milk, which caused an increased IL10 response in the presence of LPS. Taken in conjunction with the splenocyte results this suggests that digested milks could play a role in vivo in cases of inflammation. Previous studies have shown that trypsin and chymotrypsin digested whey protein from cow milk causes less stimulation of splenocytes than undigested peptides (Mercier et al., 2004) and that more stimulation was observed with digested whey protein peptides than undigested (Saint-Sauveur et al., 2008). These differences may be related to the
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amount of protein added to the cells. Saint-Sauveur et al. (2008) also reported a reduction in Con A stimulation of the splenocytes with the proteolytic digest having a similar effect to what we observed for both undigested whole sheep milk and skimmed goat milk. Similarly, we observed reduced effects on pokeweed mitogen stimulation of splenocytes with whole and skimmed sheep milk and whole and digested cow milk. Further work needs to be undertaken to find the active component of the milks causing this effect. Strains of enterotoxigenic E.coli are responsible for childhood diarrhoea particularly in developing countries and are also often associated with cases of Traveller’s Diarrhoea (Bourgeois et al., 2016; Iseri et al., 2011). Yoghurt is often recommended as a prophylactic for Traveller’s Diarrhoea, so it was of interest to determine whether digested milk samples could play a role in neutralization of the toxin. However, the ability of milk digests to neutralize toxin from enterotoxigenic E.coli was limited and although the trend appeared to be that goat milk was more effective than sheep milk which in turn was more effective than cow milk, there was no statistical difference between the milks from the different species in their ability to neutralize toxin. The effects seen with yoghurt in vivo may be as a result of stimulation of the gastrointestinal tract to produce more mucus, thus preventing the toxin from causing damage (Plaisancie et al., 2013). 5. Conclusion The immunomodulatory activity of the resultant digested milks from the three different animals was similar in all of the assays used with the exception of sheep milk, which reduced splenocyte proliferation in response to LPS and cow milk,
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which increased IL-10 production in response to LPS stimulation. Some undigested milks from all species were able to reduce the effects of suboptimal concentrations of mitogens on splenocyte proliferation. Whole digested sheep milk reduced the amount of TNF-α produced by the unstimulated THP-1 cells whereas the milks from the other species did not. This work shows that digested sheep milk may have some different bioactive effects than milk from cow and goat, but more bioactive assays and animal feeding trials need to be undertaken to confirm this. Conflict of interest None declared. Acknowledgments This research was supported by a grant (C10X1305) from the New Zealand Ministry of Business, Innovation and Employment.
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Gonzales, G. B., Van Camp, J., Vissenaekens, H., Raes, K., Smagghe, G., Grootaert, C. 2015. Review on the use of cell cultures to study metabolism, transport, and accumulation of Flavonoids: from mono-cultures to co-culture systems. Compr. Rev. Food Sci. Food Saf., 14, 741-754. Ha, M., Bekhit A.E.D., McConnell M., Mason S., Carne A. 2014. Fractionation of whey proteins from red deer (Cervus elaphus) milk and comparison with whey proteins from cow, sheep and goat milks Small Rumin. Res. 120, 125-134 Ha, M., Sabherwal, M., Duncan, E., Stevens, S., Stockwell, P., McConnell, M. ,Bekhit, A.E. D., Carne A. 2015 In-depth characterization of sheep (Ovis aries) milk whey proteome and comparison with cow (Bos taurus). PLoS One. 10, e0139774. Haverson, L.H., Ohlsson, B.B., Hahn-Zoric, M., Hanson, L.A.A., Mattsby-Baltzer, I. 2002. Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-κB Cell Immunol. 220, 83-95
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inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal. BMC Immunol.10:7 doi:10.1186/1471-2172-10-7 Korhonen, H., Pihlanto-Leppala, A., 2003. Food-derived bioactive peptides— opportunities for designing future foods. Curr. Pharm. Des. 9, 1297–1308. Korhonen, H., Pihlanto A. 2006. Bioactive peptides: production and functionality. Int. Dairy J. 16, 945-960. Krissansen, G.W. 2007. Emerging health properties of whey proteins and their clinical implications. J. Am. Coll. Nutr. 26, 713 S – 723 S. Mercier, A., Gauthier, S.F., Fliss, I. 2004. Immunomodulating effects of whey proteins and their enzymatic digests Int. Dairy J. 14, 175-183 Mills, S., Ross, R.P., Hill, C., Fitzgerald, G.F., Stanton, C. 2011. Milk intelligence: mining milk for bioactive substances associated with human health. Int. Dairy J. 21, 377401
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Paton, J.C., Paton, A.W. 1998 Pathogenesis and diagnosis of Shiga toxin--producing Escherichia coli infections Clin. Microbiol. Rev. 11,450—79 Park, Y.W. 2009. Bioactive components in milk and dairy products edited by Young W. Park., Wiley Blackwell pp3-10 Park, Y.W., Nam, M.S. 2015. Bioactive peptides in milk and dairy products. Korean Soc. Food Sci. Anim. Rec. 35(6), 831-840. Peterson, S.W., Prichard, C. 2015. The sheep dairy industry in New Zealand: a review. Proc. New Zeal. Soc. An. Prod. 75, 119-126. Plaisancie, P., Claustre, J., Estienne, M., Henry, G., Boutrou, R., Paquet, A., Leonil, J. 2013. A novel bioactive peptide from yoghurt modulates expression of the gelforming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine. J. Nutr. Biochem. 24, 213- 221 Recio, I., Visser, S. 2000. Antibacterial and binding characteristics of bovine, ovine and caprine lactoferrins: a comparative study. Int. Dairy J. 10, 597-605 Saint-Sauveur, D., Gauthier, S.F., Bouti, Y., Montoni, A. 2008, Immunomodulating properties if a whey protein isolate, its enzymatic digest and peptide fractions. Int. Dairy J. 18, 260-270
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Severin, S., Wenshui, A. 2005. Milk Biologically Active Compounds as Nutraceuticals : Review. Crit Rev Food Sci. 45, 645-656 Scott D.L., Ibrahim F., Farewell V., O’Keefe A.G., Walker D., Kelly C., Birrell, F., Chakravaty K., Maddison P., Heslin M., Patel A., Kingsley G.H. 2015 Tumour necrosis factor inhibitors versus combination intensive therapy with conventional disease modifying anti-rheumatic drugs in established rheumatoid arthritis: TACIT noninferiority randomised controlled trial BMJ 2015;350:h1046 Silva, S.V., Malcata, F.X., 2005. Caseins as source of bioactive peptides. Int. Dairy J. 15, 1–15. Timm, M., Saaby, L., Moesby, L.Wind Hansen E 2013 Considerations regarding use of solvents in in vitro based assays Cytotechnology 65(5), 887–894.
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Figure Legends Figure 1. Digested samples of cow, goat and sheep milk using pepsin and pancreatin. Lane 1 ladder; lane 2 cow whole; Lane 3 cow whole digest; Lane 4 cow skim; Lane 5 cow skim digest; Lane 6 sheep whole; Lane 7 sheep whole digest; Lane 8 sheep skim; Lane 9 sheep skim digest; Lane 10 goat whole; Lane 11 goat whole digest; Lane 12 goat skim; Lane 13 goat skim digest. Igs,immunoglobulins; Lf, lactoferrin;Lp, lactoperoxidase; Sa, serum albumin; α-CN, alpha-casein; κ-CN, kappacasein; β-CN, beta-casein; β-Lg, beta-lactoglobulin; Lz, lysozyme; -La, alphalactalbumin.
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Figure 2. Splenocyte stimulation by sheep, goat and cow undigested and digested whole milk. The milks were used at 1/100 dilution from ten different animals of each species, with and without 1.25 µg.ml-1 Con A (a stimulator of T cell compartment). Average of three independent experiments (mean ± standard error). Solid lines above the bars indicate statistical differences p<0.05.
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Figure 3. Splenocyte stimulation by sheep, goat and cow undigested and digested whole milk. The milks were used at 1/100 dilution from ten different animals of each species, with and without 2.5 µg.ml-1 Pokeweed mitogen (stimulates B cell compartment). Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate statistical differences p<0.05.
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Figure 4. Splenocyte stimulation by sheep, goat and cow undigested and digested whole milk. The milks were used at 1/100 dilution from ten different animals of each species, with and without 1.25 µg.ml-1 lipopolysaccharide (stimulates monocyte compartment). Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate statistical differences p<0.05.
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Figure 5. TNF-α produced by differentiated THP-1 cells after 24 h in the presence of sheep, goat and cow undigested and digested whole and skimmed milk. with and without the addition of 100ng.ml-1 of LPS. Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate p<0.05 Cells with LPS only vs milk samples with and without LPS (2 way ANOVA multi comparisons) 3000 2500
TNF -α Pg ml-1
2000 1500 Undigeste d Digested
1000 500 0 Cells Cells milk milk milk milk milk milk milk milk milk milk milk milk only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS whole Sheep
skim
whole
skim Goat
whole
skim Cow
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Figure 6. IL10 produced by differentiated THP-1 cells after 24 h in the presence of by sheep, goat and cow undigested and digested whole and skimmed milk with and without the addition of 100ng.ml -1of LPS Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate p<0.05 Cells with LPS only vs milk samples with and without LPS (2 way ANOVA multi comparisons) 450 400 350
IL-10 pg.ml-1
300 250 200
Undigested
150
Digested
100 50 0 Cells Cells milk milk milk milk milk milk milk milk milk milk milk milk only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS whole Sheep
skim
whole
skim Goat
whole
skim Cow
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Figure 7. Neturalisation of shiga-like toxin. Percentage of Vero cells remaining viable as measured by MTT assay after exposure to sheep, goat and cow undigested and digested whole milk incubated with Shiga-like toxin from E. coli 0126 for one hour prior to application to cells. Average of three independent experiments using
120 100 80 60 40 20 0
Cells only Cells + toxin 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
% of viable cells compared to cells only
the same set of milks (mean ± standard error).
Cow milk + toxin
Sheep milk + toxin
Goat milk + toxin
1:1000 dilution of digested whole milk
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S0921-4488(17)30054-8 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.02.018 RUMIN 5434
To appear in:
Small Ruminant Research
Received date: Revised date: Accepted date:
21-12-2016 16-2-2017 19-2-2017
Please cite this article as: Mros, Sonya, Carne, Alan, Ha, Minh, Bekhit, Alaa El-Din, Young, Wayne, McConnell, Michelle, Comparison of the bioactivity of whole and skimmed digested sheep milk with that of digested goat and cow milk in functional cell culture assays.Small Ruminant Research http://dx.doi.org/10.1016/j.smallrumres.2017.02.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparison of the bioactivity of whole and skimmed digested sheep milk with that of digested goat and cow milk in functional cell culture assays
Sonya Mrosa, Alan Carneb, Minh Hab Alaa El-Din Bekhitc Wayne Youngd and Michelle McConnella*
a
Department of Microbiology and Immunology
b
Department of Biochemistry
c
Department of Food Science
University of Otago, PO Box 56, Dunedin d
AgResearch Ltd Grasslands, Palmerston North
*Corresponding author Email address [email protected]
1
Highlights
Sheep milk simulated gastric digests increase TNF- α production by THP-1 cells
Sheep milk simulated gastric digests modulate mitogenic stimulation of splenocytes
Cow milk simulated gastric digests increase IL-10 production by THP-1 cells
Abstract Milks from sheep, goat and cow were subjected to simulated gastric digestion prior to being analysed in cell culture assays to compare the bioactivity of the digests. All milks displayed a similar profile on 1D SDS-PAGE following digestion. Overall, the effects seen from the digested milks (whether whole or skimmed) in cell culture assays from the three animal species were similar with the exception of digested sheep milk which increased the amount of TNF-α produced by THP-1 cells in the presence of lipopolysaccharide (LPS) and digested cow milk which increased the amount of IL-10 produced by THP-1 cells in the presence of LPS. While sheep milk did not stimulate splenocyte proliferation directly, digested sheep milk had a greater effect on the response of splenocytes to mitogenic stimulation than cow and goat milk digests. Keywords Milk, sheep, goat, cow, bioactivity, cell culture assays
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1. Introduction Although milk production in New Zealand is dominated by cow dairying there is a rapidly expanding industry producing sheep milk that is primarily used for the production of cheese, as well as milk powder, yoghurt and ice cream (Peterson and Prichard, 2015). Milk has long been regarded as a functional food in addition to its primary purpose of nutrition (Gobbetti et al., 2007, Mills et al., 2011, Severin & Wenshui, 2005). Bioactivity in milk is reported to be associated with proteins, peptides, lipids and carbohydrates (Korhonen and Pihlanto, 2006, Krissansen, 2007). In their native state many of the milk proteins are not bioactive (Korhonen and Pihlanto, 2003). However, bioactive peptides can be released as a result of hydrolysis of proteins or by fermentation with lactic acid bacteria (Mills et al., 2011). Proteolytic hydrolysis of proteins can release peptides exhibiting a number of biological activities, including antimicrobial and immunomodulatory (Korhonen and Pihlanto, 2006). Considerable research has been undertaken investigating peptides of bovine origin (Korhonen and Pihlanto, 2006) but less is known about peptides from milk proteins of other species. Since there are known homologies between sequences of bovine, ovine and caprine milk proteins the likelihood of similar bioactive peptides being released is high (Recio and Visser, 2000). However, we have recently reported in a proteomic study that although the whey proteome of sheep showed some similarities to that of cow, a considerable number of differences were identified (Ha et al., 2015), suggesting that sheep milk may contain some different bioactivities.
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The use of cell culture based systems to investigate the immunomodulatory properties of products is well established (Gonzales et al., 2015, Haverson et al., 2002; Kanwar and Kanwar, 2009; Park, 2009) as it enables investigation of the effect of products on different types of cellular function. To learn more about milk from sheep in New Zealand, both whole and skimmed milks from sheep, goat and cow were subjected to simulated gastric proteolytic digestion using enzymes involved in human digestion and the resulting hydrolysates were analysed using a number of functional cell culture assays (including assays designed to measure immunomodulatory effects of the milk hydrolysates in the presence and absence of mitogenic stimulation of the neutalisation effects of the milk hydrolysates in the presence of toxin) to compare the bioactivity profiles obtained from peptides generated by the simulated digestion in the three milk types. 2. Materials and methods All chemicals were obtained from Sigma Aldrich, Auckland, New Zealand, unless otherwise stated. Raw milks were obtained from local commercial farms. 2.1. Milk sample collection and preparation Ethical approval was not required for this study. Individual milk samples were collected from animals that had been tested for mastitis and were shown to be healthy. Milk from each animal was expressed by hand into pre-chilled autoclaved glass bottles and kept on ice during transportation to the laboratory. On arrival at the laboratory one half of each milk sample was centrifuged at 4000 x g for 30 min
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at 4°C to float the cream fraction. The cream was removed and any pelleted casein fraction was resuspended into the skim milk prior to it being used for proteolytic digestion. 2.2. In vitro digestion with pepsin and pancreatin Bicinchoninic acid (BCA) protein assays were performed to determine the total protein levels (mg/ml) of each of the milk samples. Digestion of the milks with pepsin (Sigma, EC3441, 2650 U.mg-1) and pancreatin (Sigma, P1750, 1750 U.mg-1) were carried out according to the method of Hernandez-Ledsema et al. (2007). The milks were first adjusted to pH 3.5 and then hydrolysed with pepsin, made up at 20 mg.ml-1, for each milk sample based on the total protein level, then incubated, at 37°C for 30 min, while stirring the solution at 150 rpm. The digests were placed in ice water to stop hydrolysis and then the pH was adjusted to 7.0 with 0.1M NaOH. Pancreatin was added at a concentration of 50 mg.ml-1 based on the total protein level. Samples were then incubated for 60 min at 37°C with stirring at 150 rpm . The proteases were inactivated by heating at 95°C for 15 min followed by cooling to room temperature. Digested and undigested milk samples were displayed on 1D-SDS-PAGE, conducted on Bolt 4-12% gradient Bis-Tris gels (Life Technologies, Auckland, New Zealand) as reported previously (Ryder et al., 2015). 2.3. Splenocyte proliferation assay Spleens were harvested from adult C57Bl/6 mice (UoO AEC ET16/11), macerated and then passed through a cell strainer (70 μm; Falcon, Becton Dickinson, NJ, USA) before being treated with 5 ml of red blood cell ACK lysis buffer for 3 min, with shaking at 1 min intervals. 5
Aliquots of the splenocyte preparation (100 µl) (2 x 106.ml-1) were dispensed into 96 well flat bottom tissue culture plates (Falcon™) and with either 50 μl complete media alone (Dulbecco’s Modified Eagle Media (Gibco®) supplemented with 10% Foetal Calf Serum (Gibco®) and 1% Penicillin/Streptomycin (10,000 U.ml-1 Gibco®)) as a cells-only control, or with 50 μl complete media plus sub-optimal concentrations determined previously of (i) pokeweed mitogen (PWM) (Sigma, L8777) to give a final concentration of 2.5 µg.ml-1, or E.coli lipopolysaccharide LPS (Sigma, L2880) to give a final concentration of 1.25 µg.ml-1, or (ii) concanavalin A (Con A) (Sigma, C-2010) to give a final concentration of 1.25 µg.ml-1. Plates were incubated in 5% CO2 for 72 h at 37°C, with, or without the milk hydrolysates (at 1/1000 dilution) in six wells per animal milk sample. Milk products (1/1000 dilution) alone were also incubated with splenocytes in six wells per animal milk sample. Further controls of mitogens alone at final concentrations in the well of 1.25 µg.ml-1 of LPS, 10 µg.ml-1 of PWM and 5 µg.ml-1 of Con A were also used. Following incubation, tritiated thymidine (Perkin Elmer, 6-3H 185MBq) a lowenergy beta particle emitter, was added (50 µCi per well), and the plates were reincubated for a further 18 h. The contents of the 96 well plate were transferred onto a Printed Filtermat A (Perkin Elmer, USA) for use with a 1450 MicroBeta™ (Perkin Elmer) scintillation counter using a 96 Cell Harvester (TomTec, CT, USA). The Filtermat was dried in a microwave oven for 2.5 min and then placed into a sample bag (MicroBeta™, Perkin Elmer) with 4.5 ml of Ultima Gold™ liquid scintillation cocktail (Perkin Elmer) and heat sealed closed. The prepared Filtermats were then placed into a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence
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Counter (Wallac, Finland) to count the photons produced in the reaction of the beta particles with the scintillator, and quantify the beta emitting isotopes. 2.4. THP-1 assay THP-1 cells (ATCC # TIB-202™) were dispensed into wells of a 96 well flat bottom tissue culture plate (Falcon™) at a concentration of 3x105 cells.ml-1, with the addition of phorbol myristate acetate (PMA) at 20 ng.ml-1 to induce cell differentiation into macrophage-like cells, and incubated at 37°C in 5% CO2 until the formation of a confluent monolayer (24-48 h). Cells were then stimulated with either 1/1000 (final dilution in cell culture medium) dilutions of the digested and undigested milks alone, or 100 ng.ml-1 E. coli LPS alone, or LPS + milks. The supernatants were harvested after 24 h incubation and were used to determine the levels of the cytokines TNF-α and IL-10 using OptEIA™ ELISA kits (BD Biosciences) as per the manufacturer’s instructions. 2.5. Anti-toxin assay A. Toxin preparation and titration of activity E. coli 026 (ATCC9026), a known Shiga-like toxin producing strain of enterotoxigenic E. coli, was grown overnight in tryptic soy broth at 37°C. The culture was centrifuged at 4000 x g for 10 min to pellet the cells. The supernatant was retained and filtered through a 0.45 m filter to remove any remaining bacterial cells. Serial dilutions of the toxin in 100 l volumes were added to wells of a 96 well flat bottom tissue culture plate (Falcon™) containing confluent monolayered Vero cells (ATCC CCL-81™) and incubated for 96 h. After incubation 10 µl of 7
phosphate buffered saline (PBS) at pH 7.2, containing MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) final concentration: 5 mg.mL-1 (Molecular Probes™ ThermoFisher Scientific, USA) was added to each well. After 4 h incubation at 37°C, the supernatant was removed and 100 µl dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. After vigorous shaking, the absorbance was measured in a micro-plate reader (Varioskan Flash, Thermo Scientific, USA) at 570 nm. Dilutions of Shiga-like toxin showing a cytopathic effect of >50% compared to untreated cells were used in a toxin neutralization assay based on that of Paton and Paton (1998) as described below. B. Toxin neutralisation Serial dilutions of the digested sheep milk samples were made and assayed in triplicate in 100 l amounts on Vero cells to check cytotoxicity. The 1/10 and 1/100 dilutions caused some toxicity in the cells, so the 1/1000 dilution that did not cause toxicity towards the Vero cells, was mixed with Shiga-like toxin (10 l milk digest + 90 l toxin) and the mixture was incubated for 60 min at 37°C. A 100 l volume of the mixture was then added to a confluent monolayer of Vero cells, and plates were incubated for 72 h at 37°C in a 5% CO2 incubator. Controls were prepared using Vero cells only, and cells plus 10 l of PBS + 90 l Shiga-like toxin. At the end of 72 h incubation the MTT method described above was used to determine cell viability and the results were expressed as % of viable cells when compared to the Vero cells only control. A 1/1000 dilution of each of the digested milks was also added as controls.
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2.6. Statistical Analysis All analyses were carried out using results from three independent experiments. Sheep milk simulated gastric digests Statistical analysis was undertaken by ANOVA using GraphPad Prism Version 6.05 3. Results 3.1. In vitro digestion Analysis of protein profiles from undigested skimmed sheep, cow and goat milk by SDS-PAGE showed the proportion of casein isoforms in sheep and goat milk were different to that of cow milk, and goat milk had a higher proportion of lower molecular weight proteins than the other two milks (Figure 1). The immunoglobulins (Ig) band appeared less intense in skimmed milk compared with whole milk whereas the lactoferrin (Lf) band was more intense in goat milk compared with cow and sheep milk. Following digestion all milk samples appeared similar on SDS-PAGE. In all of the milk samples the caseins were fully digested but lactoglobulin (-Lg) was only partially digested (Figure 1). 3.2. Splenocyte proliferation assay None of the whole milks and skim milks stimulated proliferation of mouse splenocytes. A similar result was obtained for all of the digested milk samples (data not shown). However, whole sheep milk and skimmed goat milk in the presence of Con A reduced the stimulatory effect on cell proliferation observed with Con A alone (p<0.05; Figure 2). None of the other milks had any effect. In the presence of
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pokeweed mitogen (PWM) a number of the milks reduced the effect of the PWM induced cell proliferation on the splenocytes. Undigested whole and skimmed sheep milk both reduced the PWM effect (p<0.05; Figure 3), as did both undigested and digested whole cow milk (p<0.05; Figure 3), whereas goat milk had no effect. Digested skim milks from all three animal types augmented the PWM effect. Undigested whole and skim milk from sheep reduced the LPS effect on splenocyte proliferation (p<0.05; Figure 4), whereas milks from the other animals had no effect. Digested sheep milk and the milks from all other animals had no effect on the LPS stimulation (Figure 4). 3.3. THP-1 assay Whole digested sheep milk reduced the amount of TNF-α produced by the unstimulated THP-1 cells (P<0.05; Figure 5).
However, both undigested and
digested whole and skim milk in the presence of LPS stimulated the production of more TNF-α than the LPS alone (p <0.05; Figure 5). In contrast, the effects on IL-10 production were markedly different. All the milks alone stimulated the production of IL-10 by differentiated THP-1 cells. For whole and digested sheep and goat milks this was less than the amount produced following LPS stimulation of the cells (p<0.05; Figure 6). In the presence of LPS goat and sheep milk did not have any effect on the IL-10 produced compared to LPS alone but cow milk augmented IL-10 production compared to LPS alone (p<0.05; Figure 6). 3.4. Toxin neutralisation
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Some, but not all, milk from the three animal types was able to partially neutralize the toxins (Figure 7, but none were able to neutralize sufficient toxin to prevent cell damage at some level. While the overall trend appeared to be that goat milk was more effective at neutralizing toxin than sheep milk, and that sheep milk was more effective than cow milk (Figure 7) there was no statistical difference between the groups. 4. Discussion The whole milk protein profiles were similar to those obtained in a previous study (Ha et al, 2014). However, the peptide profiles of all the milks were similar following simulated gastric digestion, suggesting that digested milks may have similar effects in functional assays. A review by Park and Nam (2015) suggests that similar bioactive compounds are derived from digestion of bovine, caprine and ovine milks with some bioactive peptides from ovine and caprine milks having identical sequences. In the study reported here, the prediction was that the milks would have similar activities, which proved true for the splenocyte assays where none of the milks stimulated splenocyte proliferation. However, some differences were observed with the effects on mitogen-induced proliferation, particularly with sheep and goat milk, but not with cow milk. This may be because peptides generated from these may be slightly different despite having similar primary protein structures (Ha et al., 2015). This ability to down-regulate mitogenic stimulation may be related to the ability of some of the milks to stimulate IL-10 cytokine production but not TNF-α. The main role for TNF-α is in the regulation of immune cells. It is a pro-
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inflammatory cytokine and is released in response to lipopolysaccharide, other bacterial products and IL-1. TNF-α attracts neutrophils to the site of infection, causes inflammation and has been associated with a number of conditions such as rheumatoid arthritis and ankylosing spondylitis where disease treatment often includes a TNF inhibitor (Scott et al., 2015). In the study reported here the addition of milks increased the effects of LPS stimulation of the differentiated macrophages although the milks alone had no effect on TNF production. On the other hand, IL-10 is an anti-inflammatory cytokine that has multiple effects in immune-regulation and inflammation and down-regulates production of some of the inflammatory cytokines. From the results obtained it seems that while none of the milks produce an inflammatory stimulus on differentiated macrophages as measured by TNF-α production they do stimulate the production of IL-10, which is an antiinflammatory cytokine that dampens the immune response. Interestingly, when in association with an inflammatory substance such as LPS, the milks induced an increased TNF-α inflammatory response but did not have as much effect on the IL10 pro-inflammatory response except for cow milk, which caused an increased IL10 response in the presence of LPS. Taken in conjunction with the splenocyte results this suggests that digested milks could play a role in vivo in cases of inflammation. Previous studies have shown that trypsin and chymotrypsin digested whey protein from cow milk causes less stimulation of splenocytes than undigested peptides (Mercier et al., 2004) and that more stimulation was observed with digested whey protein peptides than undigested (Saint-Sauveur et al., 2008). These differences may be related to the
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amount of protein added to the cells. Saint-Sauveur et al. (2008) also reported a reduction in Con A stimulation of the splenocytes with the proteolytic digest having a similar effect to what we observed for both undigested whole sheep milk and skimmed goat milk. Similarly, we observed reduced effects on pokeweed mitogen stimulation of splenocytes with whole and skimmed sheep milk and whole and digested cow milk. Further work needs to be undertaken to find the active component of the milks causing this effect. Strains of enterotoxigenic E.coli are responsible for childhood diarrhoea particularly in developing countries and are also often associated with cases of Traveller’s Diarrhoea (Bourgeois et al., 2016; Iseri et al., 2011). Yoghurt is often recommended as a prophylactic for Traveller’s Diarrhoea, so it was of interest to determine whether digested milk samples could play a role in neutralization of the toxin. However, the ability of milk digests to neutralize toxin from enterotoxigenic E.coli was limited and although the trend appeared to be that goat milk was more effective than sheep milk which in turn was more effective than cow milk, there was no statistical difference between the milks from the different species in their ability to neutralize toxin. The effects seen with yoghurt in vivo may be as a result of stimulation of the gastrointestinal tract to produce more mucus, thus preventing the toxin from causing damage (Plaisancie et al., 2013). 5. Conclusion The immunomodulatory activity of the resultant digested milks from the three different animals was similar in all of the assays used with the exception of sheep milk, which reduced splenocyte proliferation in response to LPS and cow milk,
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which increased IL-10 production in response to LPS stimulation. Some undigested milks from all species were able to reduce the effects of suboptimal concentrations of mitogens on splenocyte proliferation. Whole digested sheep milk reduced the amount of TNF-α produced by the unstimulated THP-1 cells whereas the milks from the other species did not. This work shows that digested sheep milk may have some different bioactive effects than milk from cow and goat, but more bioactive assays and animal feeding trials need to be undertaken to confirm this. Conflict of interest None declared. Acknowledgments This research was supported by a grant (C10X1305) from the New Zealand Ministry of Business, Innovation and Employment.
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Paton, J.C., Paton, A.W. 1998 Pathogenesis and diagnosis of Shiga toxin--producing Escherichia coli infections Clin. Microbiol. Rev. 11,450—79 Park, Y.W. 2009. Bioactive components in milk and dairy products edited by Young W. Park., Wiley Blackwell pp3-10 Park, Y.W., Nam, M.S. 2015. Bioactive peptides in milk and dairy products. Korean Soc. Food Sci. Anim. Rec. 35(6), 831-840. Peterson, S.W., Prichard, C. 2015. The sheep dairy industry in New Zealand: a review. Proc. New Zeal. Soc. An. Prod. 75, 119-126. Plaisancie, P., Claustre, J., Estienne, M., Henry, G., Boutrou, R., Paquet, A., Leonil, J. 2013. A novel bioactive peptide from yoghurt modulates expression of the gelforming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine. J. Nutr. Biochem. 24, 213- 221 Recio, I., Visser, S. 2000. Antibacterial and binding characteristics of bovine, ovine and caprine lactoferrins: a comparative study. Int. Dairy J. 10, 597-605 Saint-Sauveur, D., Gauthier, S.F., Bouti, Y., Montoni, A. 2008, Immunomodulating properties if a whey protein isolate, its enzymatic digest and peptide fractions. Int. Dairy J. 18, 260-270
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Severin, S., Wenshui, A. 2005. Milk Biologically Active Compounds as Nutraceuticals : Review. Crit Rev Food Sci. 45, 645-656 Scott D.L., Ibrahim F., Farewell V., O’Keefe A.G., Walker D., Kelly C., Birrell, F., Chakravaty K., Maddison P., Heslin M., Patel A., Kingsley G.H. 2015 Tumour necrosis factor inhibitors versus combination intensive therapy with conventional disease modifying anti-rheumatic drugs in established rheumatoid arthritis: TACIT noninferiority randomised controlled trial BMJ 2015;350:h1046 Silva, S.V., Malcata, F.X., 2005. Caseins as source of bioactive peptides. Int. Dairy J. 15, 1–15. Timm, M., Saaby, L., Moesby, L.Wind Hansen E 2013 Considerations regarding use of solvents in in vitro based assays Cytotechnology 65(5), 887–894.
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Figure Legends Figure 1. Digested samples of cow, goat and sheep milk using pepsin and pancreatin. Lane 1 ladder; lane 2 cow whole; Lane 3 cow whole digest; Lane 4 cow skim; Lane 5 cow skim digest; Lane 6 sheep whole; Lane 7 sheep whole digest; Lane 8 sheep skim; Lane 9 sheep skim digest; Lane 10 goat whole; Lane 11 goat whole digest; Lane 12 goat skim; Lane 13 goat skim digest. Igs,immunoglobulins; Lf, lactoferrin;Lp, lactoperoxidase; Sa, serum albumin; α-CN, alpha-casein; κ-CN, kappacasein; β-CN, beta-casein; β-Lg, beta-lactoglobulin; Lz, lysozyme; -La, alphalactalbumin.
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Figure 2. Splenocyte stimulation by sheep, goat and cow undigested and digested whole milk. The milks were used at 1/100 dilution from ten different animals of each species, with and without 1.25 µg.ml-1 Con A (a stimulator of T cell compartment). Average of three independent experiments (mean ± standard error). Solid lines above the bars indicate statistical differences p<0.05.
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Figure 3. Splenocyte stimulation by sheep, goat and cow undigested and digested whole milk. The milks were used at 1/100 dilution from ten different animals of each species, with and without 2.5 µg.ml-1 Pokeweed mitogen (stimulates B cell compartment). Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate statistical differences p<0.05.
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Figure 4. Splenocyte stimulation by sheep, goat and cow undigested and digested whole milk. The milks were used at 1/100 dilution from ten different animals of each species, with and without 1.25 µg.ml-1 lipopolysaccharide (stimulates monocyte compartment). Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate statistical differences p<0.05.
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Figure 5. TNF-α produced by differentiated THP-1 cells after 24 h in the presence of sheep, goat and cow undigested and digested whole and skimmed milk. with and without the addition of 100ng.ml-1 of LPS. Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate p<0.05 Cells with LPS only vs milk samples with and without LPS (2 way ANOVA multi comparisons) 3000 2500
TNF -α Pg ml-1
2000 1500 Undigeste d Digested
1000 500 0 Cells Cells milk milk milk milk milk milk milk milk milk milk milk milk only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS whole Sheep
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skim Goat
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Figure 6. IL10 produced by differentiated THP-1 cells after 24 h in the presence of by sheep, goat and cow undigested and digested whole and skimmed milk with and without the addition of 100ng.ml -1of LPS Average of three independent experiments using the same set of milks (mean ± standard error). Solid lines above the bars indicate p<0.05 Cells with LPS only vs milk samples with and without LPS (2 way ANOVA multi comparisons) 450 400 350
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100 50 0 Cells Cells milk milk milk milk milk milk milk milk milk milk milk milk only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS only + LPS whole Sheep
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Figure 7. Neturalisation of shiga-like toxin. Percentage of Vero cells remaining viable as measured by MTT assay after exposure to sheep, goat and cow undigested and digested whole milk incubated with Shiga-like toxin from E. coli 0126 for one hour prior to application to cells. Average of three independent experiments using
120 100 80 60 40 20 0
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% of viable cells compared to cells only
the same set of milks (mean ± standard error).
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1:1000 dilution of digested whole milk
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