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Goat Milk Yoghurt by Using Lacto-B Culture Modulates the Production of Tumor Necrosis Factor-Alpha and Interleukin-10 in Malnourished Rats
Goat Milk Yoghurt by Using Lacto-B Culture Modulates the Production of Tumor Necrosis Factor-Alpha and Interleukin-10 in Malnourished Rats
Korean Journal for Food Science of Animal Resources. 2014. Feb, 34(1): 88-98
Copyright © 2014, Korean Society for Food Science of Animal Resources
  • Received : October 01, 2013
  • Accepted : January 23, 2014
  • Published : February 28, 2014
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About the Authors
Nurliyani
nurliyani@yahoo.com
BJ. Istiti Kandarina
Department of Public Health, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia
Sari Kusuma
Department of Nutrition and Health, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia
Yunita Dewi Trisnasari
Department of Nutrition and Health, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

Abstract
Total spleen lymphocytes, lymphocyte proliferation, tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in spleen lymphocyte culture were studied in malnourished Wistar rats fed with goat milk yoghurt. Malnourished rats were created by using standard feed restriction as much as 50% of normal rats for 21 d. Goat milk yoghurt containing three types of micro-organism e.g., Lactobacillus acidophilus, Sterptococcus thermophilus and Bifidobacterium longum derived from Lacto-B culture in powder form. After 21 d, the rats continued to receive restricted feeding and supplemented with goat milk yoghurt for 7 d. Total splenocytes were counted by hemocytometer. Splenocytes proliferation was expressed as stimulation index, whereas the TNF-α and IL-10 of spleen lymphocyte culture were measured by ELISA technique. The total number of sple-nocytes and stimulation index of splenocytes in moderate malnourished and normal rats supplemented with goat milk yoghurt was not significantly different. The level of TNF-α in the rat supplemented with goat milk yoghurt was lower ( p <0.05) than the control group, whereas the level of IL-10 in the rat supplemented with goat milk yoghurt was higher ( p <0.05) than the control group. In conclusion, goat milk yoghurt supplementation in malnourished rats could decrease TNF-α as a represen-tation of the pro-inflammatory cytokine, while it increases IL-10 as a representation of the anti-inflammatory cytokine.
Keywords
Introduction
Goat milk has many advantages in terms of percentage of an over-sized fat droplets smaller than cow's milk (Sil-anikove ., 2010) . Because the goat milk fat globule has a greater surface area, and lipases in the gut are sup-posedly able to attack the lipids more rapidly. Almost 20% of the fatty acids of goat milk fall into the short-chain fatty acids category (C4:O to C12:O) compared with l0- 20% for cow milk. Lipases attack the ester link-ages of the shorter-chain fatty acids more readily, so these differences may contribute to more rapid digestion and absorption of goat milk fat (Arora ., 2013) . Some physicochemical properties of goat milk such as smaller fat globules, higher percentage of short and medium chain fatty acids, and softer curd formation of its proteins are advantageous for higher digestibility and healthier lipid metabolism relative to cow milk (Park, 1994) . Thus goat milk has been recommended for either infants, old, convalescent people (Kompan and Komprej, 2012) , and goat milk-based diet (animal fat) has a benefecial effect and thus its consumption may be recommended espe-cially in cases of malabsorption syndromes (Alferez ., 2001) . Goat milk has a nutritional value similar to that of cow milk and could be used as an alternative to cow milk for rehabilitating undernourished children (Razafindra-koto ., 1993) .
The prevalence of malnutrition in Indonesia is still rel-atively high despite a declining trend from 2007 to 2010. The proportion of malnourished children under five in Indonesia decreased from 18.4% in 2007 to 17.9% in 2010 (Riskesdas, 2010) . Protein malnutrition disrupts the normal ecology of the microflora affecting strictly anaer-obes, impairs host immune response and antibacterial de-fenses, enhances the susceptibility to infection, and leads to mucosal atrophy (Dock ., 2004) . The direct rela-tionship between malnutrition and death is mainly due to the immunodeficiency status and consequently, greater susceptibility to infectious agents (Franca et al ., 2009). The severity of the malnutrition depends on the degree of nutrient imbalance and the interaction between nutrients and the age of the host (Nunez ., 2013) . Severe pro-tein malnutrition, mainly in newborns and small children, also provokes thymus atrophy that, in turn, reduces thy-mus cell number and also severely affects the develop-ment of peripheral lymphoid organs. The immediate con-sequence of this atrophy is leucopenia, decreased CD4/ CD8 ratio and increased number of immature T cells in the periphery (Savino, 2002) .
The spleen has a very high blood flow, that it filters off cell-free fluid from the blood into the lymphatic system and that the rate of filtration is modulated by neuronal and humoral factors (Semaeva ., 2010) . The spleen was chosen as a representative of systemic lymphoid organ to evaluate the systemic immune system status (Ramiro-Puig ., 2007) .
The induction of proinflammatory and anti-inflamma-tory cytokines is important in determining whether the im-mune system is successful in providing protection against specific pathogenic organisms (Al-Bana ., 2008) . Pro-duction TNF-α by bone marrow cells is also significantly lower in malnourished animals (França ., 2009) .
Probiotic yoghurt intake was associated with significant anti-inflammatory effects that paralleled the expansion of peripheral pool of putative T regulatory (Treg) cells in in-flammatory bowel disease (IBD) patients (Baroja ., 2007) . The concept that probiotics act via the induction of regulatory cells is inherently attractive, because it seems unlikely that they could act by adequately replacing all bacteria in the intestinal microflora that are capable of causing inflammation in experimental mice/individuals susceptible to IBD-like inflammation or IBD (Giacinto ., 2005) . Several different mechanism of probiotics are: 1) involves altering the composition of the intestinal microbiota by producing bacteriocins, which are products that eliminate certain bacteria, or by altering pH, which will alter the growth characteristics of certain bacteria, 2) involves altering the epithelial barrier function of the intestine, and 3) have important immunoregulatory activ-ity, so certain probiotics and their products can activate regulatory T cells and regulatory pathways, leading to downregulation of inflammation (Sartor, 2011) . The pre-vious study by Giacinto . (2005) showed that probi-otic administration was associated with an early increase in the production of IL-10 and an increased number of regulatory CD4 + T cells. The increase in CD4 + CD25 high T cells correlated with the decrease in the percentage of TNF-α- or IL-12-producing monocytes and dendritic cell (DC) (Baroja., 2007) .
Recently, yoghurt are often added by probiotics to im-prove health effects, and originally yoghurt is made using a starter Lactobacillus bulgaricus and Streptococcus ther-mophilus cultures that can be derived from the powder or liquid culture. Probiotic such as Bifidobacterium longum reported by the previous study (Pavlovic ., 2006) grew better in goat milk than in cow milk. According to Dock . (2004) , S. thermophilus and L. helveticus enhance the recovery of gut atrophy induced by malnutrition.
In this study, yoghurt was made directly using Lacto-B powder which inoculated into the milk. Lacto-B is a non dairy powder-containing probiotic consists of B. longum , L. acidophilus , and S. thermophilus known as treatment for diarrhea. Probiotic strains such as Lactobacillus spe-cies, Bifidobacterium species and Streptococcus species have long history of safe use and are Generally Recognized As Safe (GRAS). Multi-strain and multi-species probiot-ics have improved functionality as compared to single strain (Dash, 2009). This study aimed to determine the effect of goat milk yoghurt which is made with culture from Lacto-B powder on total of rat splenocytes, splenocyte proliferation, levels of TNF-α and IL-10 in spleno-cyte culture supernatant of malnourished rats.
Materials and Methods
- Goat milk yoghurt preparation
Goat milk yoghurt was prepared from goat milk of Eta-wah Crossedbred from Indonesia and culture of Lacto-B powder. Goat milk was pasteurized at 85°C for 30 min and cooled to 43-45°C. After cooled, pasteurized goat milk was inoculated with 3% Lacto-B powder (Novell Phar-maceutical Laboratories) containing S. thermophilus, L. acidophilus and B. longum and incubated at 45°C for 6 h (Dave, 1998 ; Sunarlim, 2009 with slight modification).
- Experimental animals
Three week old male Wistar rats weighing 25.5-41.0 g, were individually caged and housed. During 7 d the rats fed unrestricted amounts of a standard laboratory diet AIN-93G (Reeves , 1993) and then randomly assigned to two groups: one maintained in the same conditions (con-trol) and another group fed 50% of the intake of the con-trol's (restricted or malnourished) (Rosso ., 1981) for 21 d. Both groups were allowed to drink water ad libitum. After 21 d, the rat were assigned to four groups: 1) Mal-nourished rats (MC), 2) Malnourished rats with goat milk yoghurt (MY), 3) Normal rats (NC), and 4) Normal rats with goat milk yoghurt (NY). MC and NC groups (con-trol without yoghurt) were given aquadest with a volume equal to the volume of treated yoghurt. Aquadest as con-trol or yoghurt were given orally with force feeding. The dose of goat milk yoghurt was 2 mL/100 g body weight/day, whose consumption is equivalent to the children con-sumption of 100mL yoghurt/day (WHO, 2006) . Each group of rat using 7 replications (n=7 rats). Rat body weight was measured every 3 d. All of groups were treated for 7 d, and then were killed. Lymphocytes are isolated from rat spleen for cultured. All procedures related to animal expe-riment were conducted following the recommendation of Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine Universitas Gadjah Mada, Indonesia (KE/FK/753/EC).
- Lympocyte proliferation assay
Lymphocyte proliferation was assayed according to Jain et al . (2008) with slight modifications. Spleen was remo-ved and placed in 10 mL of RPMI 1640 (Sigma) media containing 10% fetal bovine serum (FBS) (Gibco) and 2% penicillin-streptomycin (Gibco). The spleen was washed with RPMI media. Aseptically collected spleen tissues were gently teased with sterile needles and forceps to release splenocytes into the RPMI 1640 (Sigma) media containing 10% FBS and 2% penicillin-streptomycin. Ti-ssue suspensions were allowed to stand for 2 min to sedi-ment large tissue clumps. The upper portion containing splenocytes was collected and centrifuged at 1000×g for 5 min at 40°C. Suspension was incubated with erythrocyte lysis buffer (0.17 M Tris HCl and 0.16 M NH 4 Cl, pH 7.2) for 1 min and washed twice by centrifuging as above with RPMI 1640 medium. Cell viability was checked by try-pan blue (0.4% solution) and counted by haemacytome-ter. Viable splenocytes (1.5×10 6 cells/mL) were finally cul-tured in heat inactivated 10% FBS enriched RPMI 1640 medium supplemented with or without mitogen 5 μg/mL phytohaemagglutinine (PHA) (Murex) and incubated at 37°C in a humidified atmosphere of 5% CO 2 incubator for 72 h. Then, 10 μL of methylthiazoletetrazolium (MTT) (5 mg/mL) was added in each well and incubated for 4 h at 37°C. Acidified isopropanol (100 μL of 0.1 N HCl in anhydrous isopropanol) was added and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed using an Enzyme-linked immunosorbent assay (ELISA) plate reader with 550 nm. The lymphocyte proliferation was expressed as stimula-tion index (SI) which was calculated as the corrected ab-sorbance of mitogen-stimulated cells divided by the cor-rected absorbance of unstimulated cells (Keller ., 2005) .
- Measurement of cytokine levels (TNF-α and IL-10)
Splenocytes were cultured as described above, and su-pernatant was collected after 72 h to analyze cytokine lev-els. Rat specific ELISA kits for measurement of TNF-α and IL-10 (eBioscience, Bender MedSystem, Vienna, Aus-tria) were used. Assays were performed according to ins-truction provided by the manufacturers. Briefly, microw-ell plate was washed twice with Wash Buffer. The Stan-dard was reconstituted with 250 μL aquabidest for Stand-ard of TNF-α, and 380 μL of aquabidest for IL-10, and then prepared standard with 1:2 dilution in small tube. For TNF-α: 225 μL of reconstituted standard TNF-α (concen-tration = 5 ng/mL) was pipetted into S1 tube (standard 1) containing 225 μL Sample Diluent (concentration = 2500 pg/mL). This procedure was done for the next tubes until the concentration of the final tube (S7) was 39.1 pg/mL. The procedure for analysis of IL-10 was the same as TNF-α analysis. For IL-10: 225 μL of reconstituted standard (concentration = 2000 pg/mL) was pipetted into S1 tube (standard 1) containing 225 μL Sample Diluent (concen-tration = 1000 pg/mL). The S1 standard (225 μL) was trans-ferred into S2 standard tube (concentration = 500 pg/mL), and continue this procedure was done for the next tubes until the concentration of the final standard (S7) was 15.6 pg/mL. Each standard (100 μL) was pipetted into well for standard, and 100 μL of Sample Diluent was pipetted into well for blank. Each well for sample was filled with 50 μL of Sample Diluent and added 50 μL sample. Biotin conju-gate solution was added into microwell plate, and covered with adeshive film. The plate was incubated at room tem-perature (18-25°C) for 2 h. Adhesive film was removed, and washed microwell plate 4 times with 400 μL Wash Buffer for each well. Streptavidin-HRP solution (100 μL) was added into all well, and covered with adhesive film. The plate was incubated at room temperature for 1 h. Micro plate was washed 4 times, whereas washing for plate in IL-10 assay as much as 3 times, and added 100 μL tetram-ethyl- benzidine (TMB) substrate into wells. The plate was incubated at room temperature for 10 min, and avoided from direct exposure to light. The enzyme reaction was stopped by pipetting 100 μL of Stop Solution into each well. The absorbance of each microwell was read on ELISA plate reader 405 nm. Washing for plate in IL-10 assay as much as 3 times.
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Body weight (g) of malnourished and normal rats. (day 0-6: adaptation; day 6-27: restriction feeding; day 27-33: yogurt supplementation and continued restricted feeding. MC: Malnourished-control; MY: Malnourished-yoghurt; NC: Normal-control; NY: Normal-yoghurt)
- Statistical analysis
The data of total splenocytes, lymphocyte proliferation, TNF-α and IL-10 levels of supernatant lymphocyte cul-ture in malnourished and normal rats supplemented with goat milk yoghurt, were analyzed by ANOVA using SPSS 12.0 (2003).
Results
To study the effects of goat milk yoghurt containing S. thermophilus, L. acidophilus and B. longum on improve-ment the immunity of malnourished rats, the goat milk yoghurt has been supplemented for 7 d after 21 d recei-ved restricted feeding and continued restricted feeding together with goat milk yoghurt supplemented.
- Effects on body weight
The rat body weight that received 50% restricted feed-ing and normal rats for 21 d and after supplemented with goat milk yoghurt is showed on Fig. 1 .
A 50% restricted feeding resulted in moderate malnutri-tion, because the weight gain of malnourished rats in this study after malnutrition period for 21 d only 30.56% of normal rats. After restriction feeding period, the malnour-ished rats have lower ( p <0.05) body weight than the nor-mal rats ( Table 1 ).
Body weight of rats after 7 d received goat milk yoghurt and continue restricted feeding given in Table 2 .
Based on Table 1 and 2 , increasing of body weight of malnourished rat supplemented with goat milk yoghurt was higher (near twice: 89.40˗73.93=15.47 g) than the control (88.16˗78.77=9.39 g) rats. However, the body weight of malnourished rats significantly lower ( p <0.05) than the normal rats ( Table 2 ).
- Effect on total splenocytes
To study the immunity of malnourished rats, splenocytes counts were quantified. The results showed that the aver-age of splenocytes counts of malnourished and normal rats treated with goat milk yoghurt was not significantly different with the control rats ( Table 3 ).
Body weight (g) of rats after 21 d of restriction feeding and normal rats before supplemented with goat milk yoghurt
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Superscript with the different letters indicates significantly different (p<0.05).
Body weight (g) of malnourished and normal rats treated with goat milk yoghurt
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Superscript with the different letters indicates significantly different (p<0.05).
Splenocytes count (×106) of malnourished and normal rats treated with goat milk yoghurt
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Superscript with the same letter indicates not significantly different.
- Effects on lympocyte proliferation
Measurement of the proliferative response of lympho-cytes is the most commonly used technique for evaluating cell-mediated immune response. Quantitative analysis of proliferative response involves measuring the number of cells in culture in the presence and absence of a stimula-tory agent such as an antigen or a mitogen. Concanavalin A (Con A) and PHA stimulate T cells (Meydani and Ha, 2000) .
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Stimulation index (SI) of splenocytes of malnourished and and normal rat treated with goat milk yoghurt. (superscript with the same letter indicates not significantly different)
Lymphocyte proliferation that was expressed as an sti-mulation index of lymphocytes in malnourished and nor-mal rat was not significantly different. Furthermore, no significant changes were also observed in stimulation in-dex of lymphocytes of rat treated with goat milk yoghurt for 7 d compared to the control in normal and malnour-ished rat ( Fig. 2 ).
The average of lymphocyte stimulation index in the mal-nourished and normal rats supplemented with goat milk yoghurt was 1.62±0.68 and 1.60±0.72, respectively.
- Effect on TNF-α level in splenocyte culture super-natant
Levels of TNF-α was significantly decreased ( p <0.05) in supernatant of cutured splenocytes from rat received goat milk yoghurt for 7 d compared to the control rat ( Fig. 3 ), whereas TNF-α levels in malnourished and normal rat were not significantly different. The average of TNF-α in rats supplemented with goat milk yoghurt was 19.90±5.87 pg/mL, whereas in the control rat was 25.01±4.08 pg/mL.
- Effect on IL-10 level in splenocyte culture superna-tant
Goat milk yoghurt that supplemented in malnourished and normal rats increased level of IL-10 in spleen lympho-cyte culture significantly ( p <0.05) ( Fig. 4 ). The average of IL-10 rats supplemented with goat milk yoghurt was 78.05±23.72 pg/mL, whereas in the control rat was 53.80±18.79 pg/mL.
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Levels of TNF-α (pg/mL) in supernatant splenocytes culture of malnourished and normal rat treated with goat milk yoghurt. (superscript with the different letter indicates significantly different)
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Levels of IL-10 (pg/mL) in splenocytes culture superna-tant culture of malnourished and and normal rat trea-ted with goat milk yoghurt. (superscript with the differ-ent letter indicates significantly different)
Discussion
Malnutrition causes decreasing the weight depending on degree of malnutrition. According to Cortés-Barber-ena . (2008) , the mild malnutrition or first degree when the weight deficit reached 10-25%; moderate or second degree when the weight deficit reached 25-40%; and severe or third degree when the weight deficit was greater than 40% of that of age-matched control rats. In-creasing of rat body weight in this study after receiving goat milk yoghurt was nearly twice when compared to the control rat 7 d before. This is accordance with the previ-ous study (Prada ., 2007) , in the rats treated with low protein diets for 60 d, 30 d of recovery feeding showed that body weight was twice when compared to that obser-ved in the group one month before and no significant dif-ference in proteinemia, albuminemia, circulating free fatty acid (FFA) levels and liver lipid content between the groups of low protein diets and normal protein. The low of body weight in malnourished rats in this study also similar to the other previous study using rats 5 wk old with 55% dietary restricted (Hubert ., 2000) and pro-tein restricted (Prada ., 2007) . Decreasing this body weight gain through by reducing the intake of calories or other nutrients per unit body mass (Hubert ., 2000) . The chil-dren on goat milk outgained the cow milk children in bodyweight over the 2-wk trial period and fat absorption tended to be better in the goat milk children (Razafindrakoto et al ., 1993).
Splenocytes counts of moderate malnourished and nor-mal Wistar rats in this study similar to the previous study ( Cortés-Barberena ., 2008) , total splenic lymphocytes from normal Wistar rats was not significantly different with the moderate malnourished rats, but was significantly different between normal or moderate and severe malnou-rished rats. Maybe moderate malnutrition are not causing spleen damage that produces lymphocytes, but in the case of severe malnutrition occurs spleen damage. This possi-bility supported by the study effect of severe malnutrition in Wistar rats showed that malnutrition plays an impor-tant role in thymus and spleen atrophy by increasing the rate of spontaneous apoptosis, and indicate that severe malnutrition is associated with a significant increase of spontaneously apoptotic cells in the thymus (9.8-fold) and spleen (2.4-fold). These alterations may contribute to the immune suppression found in malnourished organ-isms (Ortiz ., 2008) .
The total number of lymphocyte from normal rats was higher than the diabetic rat (260±23 vs. 123±25) × 10 6 / spleen, because that lymphocytes are more prone to injury in the diabetic state (Chi ., 1982) . In this study, the number of lymphocytes in normal or malnourished rat was lower ( Table 3 ) than the lymphocytes in the previous study by Chi et al . (1982). This differences may be due to differences of age and species of rats, because lymphocy-tes number may also decrease with age, whereas splenic morphology is affected by species, age and genetic (Cesta, 2006) , that also effects on lymphocytes number. Accord-ing to Cheung and Nadakavukaren (1983), that age related to cellularity changes (cells/g of tissue) of the spleen of rats and the spleen increase in weight with age. In this study, source of lymphocytes using Wistar rats and previ-ous study by Chi . (1982) using mixed of Wistar and Sprague Dawley rat lymphocytes.
Lactic acid bacteria (LAB) of goat milk yoghurt in this study was 9.1×10 8 CFU/mL and Bifidobacteria was 2.0×10 6 CFU/mL. Supplementation this goat milk yoghurt for 7 d did not effect on lymphocyte number in spleen of nor-mal and malnourished rats. This results similar to the pre-vious study by Galdeano and Perdigon (2006) , L. casei with dose of 10 8 CFU/mouse/day, that fed for 7 d did not influence the T-cell population of lamina propria in the mice small intestine.
There was no different between splenocyte proliferation response in moderate malnourished and normal rats fed goat milk yoghurt, because of the possibility of moderate malnutrition in rats no significant damage of the spleen as occurs in severe malnutrition. Giving yoghurt with a dose of 2 mL/day that containing LAB 9.1×10 8 CFU/mL and bifidobacteria 2.0×10 6 CFU/mL have no effect on prolif-erative response in normal and malnourished rats. First, this is because in normal (healthy) rats, yogurt supple-mentation is only used for the maintenance, and there is a homeostasis in the immune system and also there was no significant damage to the spleen in moderate malnutri-tion. Tolerance and homeostasis are maintained by spe-cialized subsets of lymphocytes. Subsets of CD4 + T cells have drawn most of the attention so far, and there are sev-eral phenotypes, depending on the type of cytokines or sur-face molecules they express (Corthesy ., 2007) . Se-cond, this possibility to occure spleen lymphocyte prolif-eration response is required a certain dose and duration of administration of probiotics and optimal mitogen concen-tration as well. According to Kirjavainen . (1999) , L. rhamnosus GG (LGG) and Propionibacterium freudenre-ichii subsp. shermanii JS (PJS) have specific dose and duration-dependent immunomodulatory effects on the pro-liferative activity of B and T lymphocytes and may also reduce lymphocyte sensitivity to the cytotoxic effects of lectin mitogens. A significant decrease in basal lymphop-roliferation (by 32 to 42%) was observed with PJS treat-ment after the 3- and 7-d periods; however, this activity returned to control levels after 14 d of treatment, which also resulted in significantly enhanced T-cell proliferation at optimal and supraoptimal ConA concentrations (by 24 to 80%). The 14 d LGG treatment also enhanced the latter activity (by 119%). In this study using 5 μg/mL of PHA mitogen-specific T cell proliferation alone, so it is not stim-ulated B cells, and the PHA dose may also not optimal to induce lymphocyte proliferation response. Study by Pala-cios . (2007) using 60 μg/mL T-cell mitogen PHA, showed that in vitro lymphocyte proliferation stimulated by T-cell mitogens decreased with age in free-living ver-tebrate.
After supplemented with goat milk yoghurt, the average of IL-10 was higher ( Fig. 4 ) than TNF level ( Fig. 3 ). The results showed that goat milk yoghurt containing L. aci-dophilus, S. thermophilus and B. longum tend to induce anti-inflammatory cytokine in malnourished and normal rat. This results similar to the previous study using probi-otic E. coli , that the probiotic strain was able to downreg-ulate the increased levels of TNF-α both in the intestine from colitis rats, and in plasma and lungs in mice treated with LPS (Arribas ., 2009) . In addition, the ability of this probiotic bacterium E. coli to reduce TNF-α produc-tion in inflammatory conditions was also demonstrated in the trinitrobenzene sulfonic acid (TNBS) model of rat colitis or when it was evaluated in the dextran sodium sulfate (DSS)-induced colitis in mice (Grabig ., 2006) . TNF-α producing cells in the thymus increased signifi-cantly in the malnourished control group compared with the welnourished control group, and no significant differ-ences were observed among the test groups, milk, probi-otic fermented milk, and bacterial-free supernatant, where the values obtained were similar to the welnourished con-trol group (Nunez ., 2013) . According to study by Galdeano . (2011) , the administration of probiotic fer-mented milk as a dietary supplement during the re-nutri-tion process in a murine immunodeficiency model by malnutrition showed could be a good adjuvant diet to im-prove the gut and systemic immune response for the pro-tection against Salmonella infection (Galdeano ., 2011) . This probiotics interact with immunocompetent cells using the mucosal interface and modulate locally the pro-duction of proinflammatory cytokines (Borruel ., 2002) . These proinflammatory cytokines are responsible for the presence of a chronic systemic inflammatory state, which shares numerous characteristics with the ‘‘acute-phase response’’ found in critically ill patients (Mantovani ., 2004) . Practically, measurement of circulating levels of TNF-α would enable not only to identify malnourished patients in a more rapid and cost-effective way, but would also help to select those who might benefit from some type of nutritional intervention. Usually, malnourished patients had higher values of IL-1 and TNF-α (16.7 and 28.0 pg/mL) (Correia ., 2007) .
Goat milk yoghurt containing L. acidophils, S. thermo-philus and B. longum that supplemented in malnourished rats could increase the level of IL-10 in splenocytes cul-ture supernatant and also in normal rats. This result simi-lar to the previous study (Nunez ., 2013) using mice that received probiotics fermented milk containing L. del-brueckii subsp. bulgaricus (10 8 CFU/mL) and S. thermo-philus (10 8 CFU/mL) and the probiotic bacterium L. casei DN-114-001 (10 8 CFU/mL) during re-nutrition period showed significantly increased number of IL-10 produc-ing cells in the thymus. The increases of IL-10 can con-tribute to the decrease in cellular apoptosis observed after re-nutrition.
Probiotics in fermented milk was the most effective re-nutrition supplement to improve the histology of the thy-mus, decreasing cellular apoptosis in this organ (Nunez ., 2013) . Other previous study reported the effects of E. coli Nissle 1917 on the secretion of the cytokine IL-10 in Con A-activated splenocytes obtained from mice after LPS-induced septic shock (Arribas ., 2009) . That ef-fects are dose dependent, which was shown after per-forming additional experiments with different doses of the probiotic E. coli coli strain Nissle 1917 (10 6 -10 9 CFU per mice) (Hockertz, 1997) .
IL-10 is secreted by T lymphocytes, and monocytes play a central role in downregulating inflammatory cascades by suppressing the secretion of proinflammatory cytokines such as TNF-α (Kuhn ., 1993 ; Van Furth ., 1996) . IL-10 also inhibits the production of chemokines by leu-kocytes and down regulates the expression of intercellular adhesion molecule 1 and chemoattractant proteins by en-dothelial cells, thus inhibiting leukocyte migration to the site of infection. In turn, TNF-α can induce the production of IL-10 upon stimulation with endotoxin (Van Furth ., 1996) .
In this study, goat milk yoghurt using Lacto-B powder directly, which consists of mixture three types of bacteria as much as 1×10 9 CFU/mL. The total of LAB in the goat milk yoghurt was 9.1×10 8 CFU/mL and bifidobacteria was 2.0×10 6 CFU/mL. This number of lactic acid bacteria still in the range according to Nunez . (2013) . Anti-inflam-matory effects such as stimulation of IL-10-producing cells, are strain-dependent traits, and their effectiveness also depends on the concentrations used and the method of administration. Oral administration of a probiotic mix-ture that consisted of B. longum Bar 33 and L. acidophi-lus Bar 13 prevented inflammation and mucosal ulcerations in a trinitrobenzene sulfonic acid- (TNBS-) in-duced colitis mouse model (Roselli ., 2009) . This pro-tection was associated with an upregulation of IL-10 that caused an inhibition of the TNBS-induced increase of the CD4 + population, downregulation of IL-12, and a differ-ent pattern of Foxp3 + CD4 + CD25 + Treg cells in the intra-epithelial and lamina propria lymphocytes (Mengheri, 2008) . In other probiotic mixture containing L. acidophi-lus, L. casei, L. reuteri, B. bifidium , and S. thermophilus induced both Tcell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction in mice. It also induced gener-ation of CD4 + Foxp3 + Tregs from the CD4+CD25 popu-lation and increased the suppressor activity of naturally occurring. Conversion of T cells into Foxp3 + Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-β, COX-2, and indole-amine 2,3-dioxygenase. The therapeutical effect of the probiotics is associated with enrichment of CD4 + Foxp3 + Tregs in the inflamed regions (Kwon ., 2010) . The forkhead family protein Foxp3 (forkhead box P3) is a transcription factor highly expressed in CD4 + Tregs. It is a regulator of T-cell tolerance and is necessary for the de-velopment and function of Tregs (Ziegler, 2006) .
The other previous study showed that a yoghurt pro-duced with a pool of potentially probiotic LAB strains was effective in inhibiting the propagation of a dimethyl-hydrazine- (DMH-) induced colon cancer in mice by inc-reasing the number of IL-10-secreting cells, increasing cellular apoptosis and decreasing procarcinogenic enzy-mes (de LeBlanc and Perdigon, 2010) . By increasing IL-10 levels and in consequently decreasing inflammatory cytokines such as TNF-α and IFN-γ, some LAB can pre-vent the appearance of local inflammatory diseases and could be used as an adjunct therapy with conventional treatments (de LeBlanc ., 2011) .
Components of food that is consumed, naturally have the potential to interact with a variety of lymphoid cells throughout the gastrointestinal tract. Protein administered orally, has been known to interact with specific secondary lymphoid organs such as tonsils and Peyer's patches. Complex interactions that occur on the side (lymphoid or-gans) is crucial for the development of the specific im-mune response. Several factors can influence the interac-tions, including the form of solids or liquids from food component and oral exposure methods (Sfeir ., 2004) . Immunomodulating requires intact or active peptides that are able to achieve immunocompetent cells. Most of the proteins are absorbed through the apical membrane of epithelial cells, which do not bind to the receptor will undergo lysosomal degradation (Perdue, 1999) .
For the occurrence of the immune responses depends on the interaction with the active component with the im-munocompetent cells, and immunogenicity of LAB in in-testinal depends on degree of interaction with lymphoid tissue (Meydani and Ha, 2000) . According to Sfeir . (2004) , complex interactions between protein and immu-nocompetent cells is crucial for occurrence the specific immune response. Active component in yoghurt consists of bacterial and non-bacterial components that can role as immunomodulators (Meydani and Ha, 2000) . Cell surface components in lactic acid bacteria, such as peptidoglycan, lipoproteins, and diacylated lipopeptide, act as ligands for immune cells that bind to Toll-like receptor (TLR) 2 and TLR1 or 6 heterodimers on the macrophage cell surface and then stimulate nitrite oxide (NO) production (Itoh ., 2012) .
The LAB are Gram-positive bacteria and their cell walls comprise a complex mixture of glycolipids, lipoproteins and phosphorylated polysaccharides embedded in a thick layer of peptidoglycan (PGN), a polymer of β-(1,4)-linked N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides. TLR2 has been shown to recog-nize lipoteichoic acid (LTA), lipoarabinomannan, lipopro-tein (LP) and PGN, and is likely to be involved in the recognition of LAB. Lactic acid bacteria act as immuno-regulators through interaction of lipoprotein with TLR2 and as immunostimulators through interaction of pepti-doglycan with nucleotide-binding oligomerization domain-2 (NOD2). As dendritic cell (DC) are potent stimulators of naive T cells and express several pattern recognition receptors (PRRs), they serve as an important link between the microbiota and polarization towards T helper type 1 (Th1), Th2- or regulatory T-cell-dominated environments. Cell surface-expressed and intracellularly expressed PRRs collectively recognize lipid, carbohydrate, protein and nu-cleic acid structures that are broadly expressed by differ-ent groups of microorganisms (Zeuthen ., 2008) .
Recognition of microbial components by TLRs initiates signal transduction pathways, which triggers expression of genes. These gene products control innate immune res-ponses and further instruct development of antigen-spe-cific acquired immunity. Individual TLRs play important roles in recognizing specific microbial components (Takeda and Akira, 2005) .
The presence of LAB is thought to be essential for yo-ghurt to exert immunostimulatory effects but components of nonbacterial yogurt, such as whey protein, short pep-tides, and conjugated linoleic acid (CLA), are believed to contribute to yoghurt’s beneficial effects as well. It is pro-posed that the LAB that survive through the gastrointesti-nal (GI) tract, whether intact or modified, can bind to the luminal surface of M cells. LAB-bound M cells reaching to the dome region of Peyer’s patch (PP) cells stimulate local immune response, which can further activate the local immune system, resulting in stimulation of the local and the systemic immune response (Meydani and Ha, 2000) . Whey protein had activity as an anti-allergy, anti-inflam-matory, immunomodulatory and antioxidant. One of the proteins in the milk whey fraction which has the broadest range of biological activities related to the body's defense system, is lactoferrin, which among other acts as immu-nomodulator (Shimazaki ., 1998 ; Zimecki and Kruzel, 2000) . Study by Ward . (2002) , showed that lactofer-rin had been known inhibit allergen-induced skin inflam-mation in both mice and humans, most likely secondary to TNF-α production. The potency of probiotic bacteria to regulate immune responses is a complex interaction bet-ween the host immune system and different bacterial com-pounds, including chromosomal DNA and cell wall com-ponents as well as soluble metabolites (Corthesy ., 2007) .
The other non-bacterial component in goat milk yoghurt was prebiotics oligosaccharides. Acording to Wu . (2006) , goat milk showed rich in sources of prebiotic oli-gosaccharides that resemble milk oligosaccharides (breast milk) (Bode, 2006) , and in contrast to other prebiotics such as inulin or frukto short-chain oligosaccharides (Lara-Vil-loslada ., 2006) . The similarities found between human and goats’ milk oligosaccharides in terms of the presence of neutral (galactosyl-lactose and lacto-N-hexaose) and sialylated (3-, 6-sialyl-lactose and disialyl-lactose) struc-tures suggest that the latter may, to some extend, carry out or resemble physiological activities described for the for-mer (Martinez-Ferez ., 2006) . Goat milk oligosaccha-rides also have anti-inflammatory effects in rats with ex-perimental colitis and may be useful in the management of inflammatory bowel disease (Daddaoua ., 2006) .
In conclusions, supplementation of goat milk yoghurt in malnourished rats could decrease the pro-inflammatory cyto-kine, while it increases the anti-inflammatory cytokine, represented by TNF-α and IL-10 levels in the sple-nocyte culture supernatant, respectively. However, that goat milk yoghurt feeding does not give any effect on total splenocyte numbers and splenocyte proliferation.
Acknowledgements
The authors thank Prof. dr. Marsetyawan HNE Soesatyo, M.Sc., Ph.D for reading the manuscript. This research was supported partially by Anggaran Dana Masyarakat Univer-sitas Gadjah Mada (Nomor LPPM-UGM/2175/BID.I/2012 TANGGAL 11 JULI 2012).
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