Galactooligosaccharides reduce infection caused by Listeria monocytogenes and modulate IgG and IgA levels in mice

International Dairy Journal 41 (2015) 58e63

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Galactooligosaccharides reduce infection caused by Listeria monocytogenes and modulate IgG and IgA levels in mice Vikas Sangwan a, Sudhir K. Tomar a, *, Babar Ali a, Ram R.B. Singh b, Ashish K. Singh b a b

Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001, India Dairy Technology Division, National Dairy Research Institute, Karnal 132001, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 May 2014 Received in revised form 29 September 2014 Accepted 30 September 2014 Available online 12 October 2014

The effect of galactooligosaccharides (GOS) on infection caused by Listeria monocytogenes and levels of immunoglobulins (IgG and IgA) was studied in mice. b-Galactosidase extracted from native strains of Streptococcus thermophilus was used for GOS production. Seventy two mice were divided in to four groups; normal control group (NCG), pathogenic control group (PCG), and test groups 1 and 2. NCG and PCG were fed with standard rodent diet and the remaining groups were fed with standard diet supplemented with GOS (6% of diet). Six mice from each group were sacrificed on days 2, 5 and 8 postchallenge. Lower counts of L. monocytogenes were observed in the intestine, liver and spleen of groups fed with GOS. Feeding of GOS augmented the IgA (small intestine) and IgG (blood) concentration in mice. Findings of the study indicate that GOS have potential to modulate the immune system along with a protective effect against L. monocytogenes. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Gastrointestinal (GI) infections induced by food borne pathogens are a major clinical problem worldwide. Listeria monocytogenes, a facultative intracellular pathogen, causes a lifethreatening food-borne disease in humans and other mammals with a very high mortality rate of about 20%, which is more significant than any of the other more notorious food-borne pathogens such as Salmonella and Campylobacter (Melton-Witt, Rafelski, Portnoy, & Bakardjiev, 2012). The growing resistance of pathogenic bacteria to antibiotics has led to the requirement for alternative therapies/strategies aiming at prevention and treatment of diseases caused by food borne pathogens. The use of prebiotics is emerging as a strategy for the reduction and prevention of GI infections. Prebiotics are described as functional components of food that are metabolised by particular commensal bacteria conferring benefits upon health and wellbeing of the host. An increase in health beneficial microbes lead to the inhibition of pathogenic adherence and invasion in the colonic epithelia by alteration in the colonic pH, production of short chain fatty acids (SCFA), improvement in mucus production, induction of

* Corresponding author. Tel.: þ91 184 2259196. E-mail addresses: [email protected], [email protected] (S.K. Tomar). http://dx.doi.org/10.1016/j.idairyj.2014.09.010 0958-6946/© 2014 Elsevier Ltd. All rights reserved.

cytokine production, and competition for the same glycoconjugates present on the surface of epithelial cells (Delgado, Tamashiro, Marostica, Moreno, & Pastore, 2011). GOS are well documented to be effective prebiotic ingredients that modulate intestinal microbiota, barrier functions, and provide other beneficial health effects such as stool improvement, mineral absorption, weight management, carcinogenesis, and allergy alleviation (Figueroa-Gonzalez, Quijano, Ramirez, & Cruz-Guerrero, 2011; Ohr, 2010). Reports are available showing the potential of GOS in reducing infection caused by Salmonella enterica serovar Typhimurium (Searle et al., 2009, 2010). Only a few reports are available showing the ability of GOS to reduce the infection caused by L. monocytogenes (Ebersbach et al., 2010). In addition to their ability to reduce pathogenic infection, the administration of prebiotics has been associated with immunomodulatory effects encompassing innate, adaptive immunity as a result of the interaction with the microbiota (Vulevic, Drakoularakou, Yaqoob, Tzortzis, & Gibson, 2008). The largest component of the immune system, gut-associated lymphoid tissue (GALT), constitutes about 60% of all lymphocytes in the body. GALT is perpetually in contact with the microbiota and their metabolic products; therefore, dietary substrates reaching the large intestine that are able to influence the microbiota are eventually liable to affect it (Tzortzis & Vulevic, 2009). Prebiotics exert their influence on the immune system both directly by interacting with

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carbohydrate receptors on immune cells and indirectly by enhancing the number of health promoting bacteria, specifically lactobacilli and bifidobacteria (Neyrinck et al., 2011). A higher number of these bacteria alters the levels of various immunomodulating molecules such as endotoxins, lipopolysaccharides and SCFA (Watzl, Girrbach, & Roller, 2005). This leads to beneficial changes in the immune system such as modulation of immune cells in Peyer's patches (Hosono et al., 2003), increased cytotoxicity of natural killer cells (Kelly-Quagliana, Nelson, & Buddington, 2003) and increased production of IgA and various interleukins (Hosono et al., 2003). Scientific evidence from in vivo infection studies reporting that dietary non-digestible carbohydrates increase host resistance to intestinal bacterial infections is lacking. Only a few in vivo studies have so far directly investigated the effect of GOS against bacterial infections. This study was designed to assess the effect of GOS on resistance of mice to intestinal colonisation and translocation of L. monocytogenes and on the concentration of IgA and IgG.

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Fig. 1. HPLC chromatogram of GOS produced by the action of b-galactosidase extracted from Streptococcus thermophilus. The peaks of glucose, galactose, lactose and galactooligosaccharides (GOS) are identified and retention times defined (n ¼ 5).

2. Materials and methods 2.1. Chemicals All the components of the animal diet, except casein, were procured from Hi Media Laboratories Pvt. Ltd, (Mumbai, India). Casein was locally procured from Modern Dairy plant, Karnal, Haryana, India. Alloxan was procured from SigmaeAldrich (CA, USA). GOS used as a standard in this study was supplied by FrieslandCampina Domo, Great Ocean Ingredients Plant, Allansford, Australia on a complementary basis.

composition as detected by high performance liquid chromatography (HPLC). The HPLC system (all from Shimadzu, Kyoto, Japan) consisted of a manual injector (20 mL), a CTO-20A pump, a RID-10A refractive index detector and a Phenomenax Luna Amino (NH2) carbohydrate column (250 mm  5 mm i.d.). The column temperature was maintained at 40  C using a CTO-20A column heater (Shimadzu). The mobile phase was acetonitrile (75%, v/v) and distilled water (25%, v/v) filtered through a sterile micro-filter (0.45 mL) and deaerated for 20 min in ultrasonic equipment before use. Flow rate was kept at 1 mL min1 (Neri et al., 2009).

2.2. Bacterial strain 2.4. Animals L. monocytogenes ATCC 15313 was used to challenge the experimental mice. This non-lethal strain has been reported as an enteric pathogen and has been used in a number of studies reporting the effect of dietary oligosaccharides in immune modulation in mice and efficiency of probiotic organisms to provide protection against pathogens (Kelly-Quagliana et al., 2003; Martins et al., 2010). The culture was inoculated into brain heart infusion (BHI) broth and thereafter streaked on polymyxinacriflavin-lithium chloride-ceftazidime-aesculin-mannitol (PALCAM) agar supplemented with Listeria selective supplement (Hi Media Laboratories Pvt. Ltd). Typical grey-green colonies surrounded by dark brown to black halos were picked up and inoculated in BHI broth. The culture was incubated 18e24 h aerobically at 37  C. Culture was stored as glycerol stock at 20  C until use. The day prior to infection, the culture was thawed, inoculated in BHI broth, and grown overnight at 37  C without agitation. The following day, the culture was washed once in phosphate buffered saline (PBS) and again resuspended in PBS to a final concentration of 2  108 cfu mL1. 2.3. Test substance Two different GOS preparations were used for the study. Laboratory GOS (Test substance 1) was produced from b-galactosidase extracted from native strains of Streptococcus thermophilus using lactose supplemented whey (30% lactose concentration) as a substrate (Fig. 1). Vivinal GOS syrup (Test substance 2, consisting of around 60% GOS by weight along with lactose, glucose and a small amount of galactose) was procured from FrieslandCampina Domo, was used as a standard. Both of the GOS samples had the same

Seventy two male BALB/c mice (six weeks old) were obtained from Small Animal House, National Dairy Research Institute, Karnal, Haryana, India and housed in ventilated cages under controlled temperature and humidity conditions with a 12 h light/dark cycle. The experimental protocol was approved by the Institute Ethical Committee for Animal Experiments (ECAE). 2.5. Grouping of animals and feeding schedule Mice were divided into 4 groups, normal control group (NCG), pathogenic control group (PCG), test group 1 (TG1) and test group 2 (TG2); the grouping and feeding schedule are given in Table 1. All the animals were allowed a 7 day adaptation period to remove the effect of stress that may be experienced by the animals due to separation from the main stock. All groups were fed ad libitum on

Table 1 Grouping and diets of mice.a Group

Code

Diet

Normal control group Pathogenic control group Test group 1

NCG PCG

Normal diet Normal diet þ pathogenic challenge

TG1

Test group 2

TG2

Normal diet supplemented with laboratory-produced GOS (60 g kg1 of diet) þ pathogenic challenge Normal diet supplemented with Vivinal GOS (60 g kg1 of diet) þ pathogenic challenge

a

Eighteen mice were in each group (n ¼ 18).

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standard rodent diet (AIN93) and water. After the adaptation period was over, TG1 and TG2 were fed with a diet containing GOS, whereas NCG and PCG were fed with the normal diet for 7 days. Thereafter, groups PCG, TG1, and TG2 were challenged with 500 mL of a bacterial suspension containing 109 cfu (de Waard, Garssen, Bokken, & Vos, 2002) of L. monocytogenes given orally through gavage while NCG was kept unchallenged. The day on which mice were given a challenge was considered as day 0. From the day of challenge, all the groups were fed on their respective diets as mentioned in Table 1.

at room temperature for 1 h. After washing, 100 mL of colour development solution was added to each well and incubated for 9 min for proper development of colour; 100 mL of stop solution was added to each well to stop the reaction. A Victor X3 plate reader (Perkin Elmer, Singapore) was used to read the plate at 450 nm wavelength and the readings were compared with the standard curve prepared by plotting absorbance against mouse IgA concentrations. 2.9. Detection of IgG concentration in serum by enzyme linked immunosorbent assay

2.6. Collection of samples Six mice from each group were euthanised by cervical dislocation on day 2, 5 and 8 post-challenge for the removal of blood, small intestine, large intestine, liver and spleen. Before sacrificing, the animals were fasted overnight. Liver, spleen and large intestine were collected in 0.1% peptone water. Small intestine was carefully removed from gastro-duodenal to ileocaceal junctions and collected in 5 mL PBS (pH 7.2). 2.7. Colonisation assays To determine the counts of L. monocytogenes in large intestine, liver and spleen, these organs were weighed and homogenised using an overhead stirrer-homogeniser in 5 mL of 0.1% peptone water. Large intestine was homogenised along with the content. Cell suspensions were serially diluted in peptone water and aliquots were plated on PALCAM agar supplemented with Listeria spp. selective supplement and incubated for 48e72 h at 37  C. 2.8. Detection of IgA concentration in intestinal fluid by enzyme linked immunosorbent assay The procedure used for collection of the intestinal fluid was a modification of the procedure described by Lim and Watson (1981). Small intestine was washed with PBS and scraped with a sterile scalpel. Thereafter, the contents were centrifuged at 3000  g for 30 min at 4  C and the supernatant obtained was stored at 20  C for enzyme linked immunosorbent assay (ELISA). All the steps from removal of intestine to collection of intestinal fluid were performed at lower temperature (in ice) only. ELISA detection of IgA was performed using a Mouse IgA ELISA kit (Komabiotech Seoul, Korea). ELISA was performed as per the guidelines given by Komabiotech. Briefly, the wells of the microplate were washed with water solution provided in the kit. Diluted sample (supernatant of intestinal fluid diluted 1:10,000; 100 mL) was added to each well in duplicate and incubated for 1 h at room temperature and washed thereafter. Diluted (1:40,000) detection antibody (100 mL) was added to each well and incubated

ELISA detection of IgG was performed using Mouse IgG ELISA kit (Komabiotech). The same protocol was followed as described above, except the sample dilution was 1:15,000 in this case. The standard curve was prepared by plotting absorbance against mouse IgG concentrations (ng mL1). 2.10. Statistical analyses Values were expressed as means and standard error of six mice. Statistical analysis was carried out using Statistical Analysis System (SAS, V. 6, SAS Institute Inc., Cary, NC, USA). The statistical model used was one way and repeated measurement analysis of variance (ANOVA). Differences among means were compared by the Duncan multiple range test. 3. Results 3.1. Effect of GOS on colonisation of L. monocytogenes Mice were challenged with L. monocytogenes by oral administration. The challenged animals were found to be less active compared with the unchallenged ones: healthy mice were very active in the cage, the challenged mice were very inactive. The infected mice were observed to be weak compared with the healthy mice. The severity of infection was monitored in the animals by determining the pathogenic count in the intestine, liver and spleen. As anticipated, no infection was found in organs of NCG throughout the experiment. Infection was developed in all the animals of PCG, TG1 and TG2. Intensity of infection was less pronounced in groups fed with GOS supplemented diet (TG1 and TG2) compared with PCG that was fed on basal diet alone. L. monocytogenes count in different organs of all the groups on day 2, 5 and 8 are shown in Table 2. The highest listeria count was observed in intestine followed by liver and spleen. In the intestine, the highest count was observed in the mice sacrificed on day 2 post infection and a reduction was observed thereafter. While in liver and spleen, the lowest counts were observed in mice sacrificed on day 2 and highest on day 8 after the pathogenic challenge.

Table 2 L. monocytogenes counts in intestines, liver and spleen of different groups of mice on day 2, 5 and 8.a Group

L. monocytogenes counts (log cfu g1) Intestines

NCG PCG TG1 TG2

Liver

Spleen

Day 2

Day 5

Day 8

Day 2

Day 5

Day 8

Day 2

Day 5

Day 8

e 5.57 ± 0.03a 5.27 ± 0.02b 5.06 ± 0.01c

e 4.75 ± 0.02a 4.39 ± 0.02b 4.28 ± 0.01c

e 4.51 ± 0.02a 4.13 ± 0.02b 3.98 ± 0.02c

e 2.95 ± 0.01a 2.72 ± 0.01b 2.46 ± 0.01c

e 3.37 ± 0.01a 3.05 ± 0.01b 2.93 ± 0.02c

e 4.35 ± 0.01a 3.53 ± 0.03b 3.39 ± 0.04c

e 1.95 ± 0.02a 1.78 ± 0.02b 1.76 ± 0.03b

e 2.66 ± 0.02a 2.38 ± 0.06b 2.12 ± 0.03c

e 3.96 ± 0.01a 2.94 ± 0.02b 2.75 ± 0.03c

a Groups are: NCG, normal control group; PCG, pathogenic control group; TG1, test group 1; TG2, test group 2. Values are means ± standard error of the mean (n ¼ 6); values with different superscript letters in a column are significantly different at the level of <0.05. A dash represents no growth.

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Table 3 IgA and IgG concentrations in different groups of mice fed with basal and GOS supplemented diets.a Group

Concentration of IgA (mg mL1)

Concentration of IgG (mg mL1)

Day 2 NCG PCG TG1 TG2

1.45 3.15 4.10 4.38

± ± ± ±

Day 8 a

0.04 0.03b 0.63c 0.06d

1.39 3.60 4.94 4.71

± ± ± ±

a

0.02 0.03b 0.07c 0.06c

1.33 3.99 4.24 3.42

± ± ± ±

Day 2 a

0.02 0.03b 0.08c 0.10d

2.27 4.37 5.70 6.01

± ± ± ±

Day 5 a

0.06 0.07b 0.08c 0.10c

2.32 4.59 6.30 6.70

± ± ± ±

Day 8 a

0.05 0.05b 0.11c 0.09c

2.26 4.59 5.47 4.03

± ± ± ±

0.03a 0.05b 0.10c 0.12d

a IgA concentration was determined in the intestine; IgG concentration was determined in blood collected from the heart. Groups are: NCG, normal control group; PCG, pathogenic control group; TG1, test group 1; TG2, test group 2. Values are means ± standard error of the mean (n ¼ 6); values with different superscript letters in a column are significantly different at the level of <0.05. A dash represents no growth.

3.2. Effect of GOS on IgA level in mice Supernatant obtained after centrifugation of homogenate of small intestine was used for the estimation of IgA using ELISA kit. TG1 and TG2 had a significantly higher (P < 0.05) concentration of IgA than that in NCG and PCG. The lowest concentration of IgA was found in NCG. Concentration of IgA in PCG was significantly higher (P < 0.05) than in NCG. Diet supplemented with GOS significantly increased the IgA concentration in mice compared with the normal diet. IgA concentration in different groups at different time intervals during the trial is shown in Table 3. 3.3. Effect of GOS on IgG level in mice Due to the importance of IgG in pathogenic infections, their concentration was measured in blood. IgG levels were recorded in serum after day 2, 5 and 8 of pathogenic challenge. Feeding with GOS was also found to be associated with an increase in the IgG concentration in mice (Table 3). The lowest concentration of IgG was observed in NCG compared with PCG, TG1 and TG2. A significantly higher concentration of IgG was observed in TG1 and TG2 compared with PCG. 4. Discussion GOS was used to reduce the infection caused by L. monocytogenes. GOS was prepared in our laboratory using bgalactosidase extracted from native strain of S. thermophilus (Sangwan, Tomar, Ali, Singh, & Singh, 2014). Whey supplemented with lactose was used as a cost effective substrate for the production. We optimised various experimental conditions to increase the product yield. The GOS thus produced was found to be similar in composition to the commercial sample as analysed by HPLC (Fig. 1). The prepared GOS mixture was quantified using HPLC and it was found to consist of 94 g L1 GOS. The commercial sample of GOS (Vivinal GOS) was used as a standard, both for comparison in detection by HPLC and in the animal trial. L. monocytogenes, acquired by eating contaminated food products, is a potentially deadly pathogen that colonises the GI tract and cause acute gastroenteritis and the more severe invasive disease listeriosis. In the small intestine it enters cells of the intestinal epithelium passing through the intestinal barrier and carried via the blood to the liver that is the main site of replication (Drevets & Bronze, 2008; Sleator, Watson, Hill, & Gahan, 2009). Consumption of food products contaminated with Listeria is the most common cause of infection in naturally occurring listeriosis, therefore in this study, pathogen was given by an oral route and effect of GOS on their colonisation was studied in the intestine, liver and spleen. We found that infection caused by Listeria reached the liver and spleen after passing through the intestine. Samples obtained from all the challenged animals were found to be infected with L. monocytogenes. The listeria strain used in this study is not lethal,

and therefore no mortality was observed in any of the challenged group. At a dose of greater than 1.2  1011 cfu, no mortality was observed with this strain after 6 days post infection (Liu, 2004); the dose used in this study was considerably lower, and none of the mice died. After 2 days post infection L. monocytogenes was found to be present in all the three tested organs, i.e., intestine, liver and spleen, showing the translocation of Listeria from intestine to other organs. Consumption of GOS reduced the counts of Listeria in all the organs (Table 2). Groups TG1 and TG2 had significantly lower (p < 0.05) Listeria counts compared with PCG throughout the experimental period. This may be due to the presence of prebiotics in diet protecting the gut from infections and inflammation by inhibiting attachment and/or invasion of pathogenic bacteria or their toxins to colonic epithelium. This attachment is mediated by glycoconjugates on glycoproteins and lipids present on the microvillus membrane (Tzortzis & Vulevic, 2009). Prebiotic GOS contain structures similar to those found on microvillus membrane that interfere with the bacterial receptor by binding to them and thus preventing bacterial attachment to colonic epithelium (Sangwan, Tomar, Singh, Singh, & Ali, 2011). Consumption of different prebiotics (XOS, GOS and inulin) was found to be associated with a significant reduction in the number of intestinal pathogens including L. monocytogenes and S. enterica serovar typhimurium, which may be due to an enhancement of the immune response caused by selective stimulation of beneficial microorganisms in the intestine (Buddington, Donahoo, & Buddington, 2002; Ebersbach et al., 2010; Orrhage & Nord, 2000; Searle et al., 2009, 2010). The intestine is protected from pathogens by the mucosal immune system which produces IgA. Animal studies related to the immune modulating effect of prebiotics reported an increase in IgA (Ouwehand, Derrien, de Vos, Tiihonen, & Rautonen, 2005). The main function of faecal IgA is to agglutinate microorganisms and to prevent the adherence of pathogenic bacteria and viruses to the mucosal surface (Hanson et al., 2003; Van de Perre, 2003). Another important function of IgA is the maintenance of intestinal microbial homoeostasis; this is illustrated by animal studies reporting a shift from unbalanced to normal composition of microflora when IgA concentration becomes normal from their low levels (Suzuki et al., 2004). We also examined the influence of GOS feeding on intestinal IgA secretion (small intestine was used because protective IgA responses to pathogens are predominantly initiated in Peyer's patches that are present in the small intestine) and observed that its oral administration augmented the IgA secretion in mice (Table 3). The IgA concentration in TG1 and TG2 was higher compared with that in PCG on day 2 and day 5. On day 8, IgA levels in TG2 were found to be decreased significantly (p < 0.05) compared with those in TG1 and PCG, which may be due to the reduction in number of L. monocytogenes in mice of TG2. The concentration of IgA in PCG on day 2, 5 and 8 showed a constant rise because of increased pathogenic count in the mice of this group. On

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a given day, IgA level was higher in mice of TG1 and TG2 compared with PCG, because of the effect of prebiotic feeding. The level of IgA in groups TG1 and TG2 showed an initial increase from day 2 to day 5 and decreased thereafter, which may be attributable to decreased pathogenic count in these groups. Mice of PCG were found to have higher IgA titre compared with NCG on all the days. The response of mice to the pathogenic challenge also seemed to contribute to higher IgA concentration in PCG compared with NCG. A few studies have been undertaken on immune-modulating effects of GOS, which have been studied more extensively in combination with fructooligosaccharides (FOS). Several studies report an increase in intestinal or faecal IgA levels upon supplementation with prebiotic preparations (Bakker-Zierikzee et al., 2006; Benyacoub et al., 2008; Hosono et al., 2003; Nakamura et al., 2004; Roller, Rechkemmer, & Watzl, 2004; Scholtens et al., 2008; Swanson et al., 2002). Stimulation of IgA synthesis following prebiotic supplementation may also contribute by an increase in the number of probiotic organisms. As IgA antibodies present at the mucosal surface of the gut prevent adherence of pathogens to the gut mucosa, increased IgA concentrations suggest an enhanced local immune capacity and greater protection against pathogenic invasion. IgG is the main antibody isotype found in blood and extracellular fluid. It binds to most of the pathogenic organisms and controls the infections by using several immune mechanisms including agglutination and immobilisation, complement activation, opsonisation for phagocytosis, and neutralisation of toxins. People with IgG deficiency are more prone to infections and are known to be deficient in other immunoglobulins such as IgA and IgM. Therefore, the concentration of serum IgG was also measured in this study (Table 3). A similar trend as for IgA, was observed in the case of IgG. We found that the concentration of IgG in NCG remained almost similar throughout the experiment, whereas the concentration of IgG in animals of group PCG remained higher throughout the experiment with a small increase from day 2 to day 8. NCG was found to have the lowest IgG levels compared with PCG, TG1 and TG2. Significantly (p < 0.05) higher concentrations of IgG were observed in TG1 and TG2 compared with PCG and after an initial increase in IgG levels of TG1 and TG2 from day 2 to day 5, a reduction was observed on day 8. Only a few studies reported the effect of prebiotics on IgG concentration. The IgG levels have been reported to be increased by mannan-oligosaccharide (MOS) supplementation in turkeys (Cetin, Guclu, & Cetin, 2005) and layers (Woo, Kim, & Paik, 2007). Yin et al. (2008) reported that dietary supplementation with galacto- MOS or chitosan oligosaccharide enhanced (p < 0.05) IL-1b gene expression in jejunal mucosa and lymph nodes, as well as serum levels of IL-1b, IL-2, IL-6, IgA, IgG and IgM compared with supplementation with antibiotic in earlyweaned pigs. Janardhana et al. (2009) demonstrated that FOS treatment significantly enhanced the IgM and IgG antibody titres in plasma. 5. Conclusion Although more research is required, prebiotics are providing a potential option for modulation of gastrointestinal complications and the immune system. The mechanisms involved in imparting immune modulation to the host remain to be determined; however, this study provides encouraging data to suggest that consumption of GOS can improve immune parameters and reduce or delay pathogenic infections. The findings of the study presented here indicate the immune modulating effect of GOS along with its protective effect against pathogenic L. monocytogenes. These GOS mediated effects could be due to its action as a receptor mimic or may be due to direct interaction between GOS and gut immune

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