Cytotoxicity in Vero cells and cytokines analyses in Balb/c mice as safety assessments of the probiotic mixture Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007 for use as a feed additive.

The objective was to carry out cytotoxicity assays in Vero cells and cytokines analyses in Balb/c mice as safety assessments to evaluate the probiotic mixture (M) Saccharomyces cerevisiae RC016 (Sc) and Lactobacillus rhamnosus RC007 (Lr) for use as feed additive. Vero cells (104 cells per well) were exposed to Sc (2·08 × 107 , 2·08 × 106 ; 2·08 × 105 cells per ml), Lr (8·33 × 107 ; 8·33 × 106 ; 8·33 × 105 cells per ml) and their M (1 : 1). Sc concentrations did not affect the Vero cells viability; in contrast, they were lower when exposed to Lr (P Ë‚ 0·0001). Vero cells showed increasing viability with M decreasing concentrations (91% viability with M2). Control BALB/c mice received only phosphate buffer saline and the others received the M. The IL-10, IL-6 and TNFα concentrations from intestinal fluid were analysed and no significant differences were observed among treatments. The same occurred with the ratio between IL-10/TNF-α. Beneficial effects of probiotics are associated with the regulation of the excessive inflammatory response; it is desirable they can modulate the cytokines production only under pathological conditions. Here, M administration to healthy mice did not induce negative side effects and expands the knowledge about beneficial effects of using probiotic microorganisms in mixture for feed additives development.

Beneficial effects of Saccharomyces cerevisiae RC016 in weaned piglets: in vivo and ex vivo analysis.

Probiotics represents an alternative to replace antibiotics as growth promoters in animal feed and are able to control enteric bacterial diseases and to improve gut immunity. Saccharomyces cerevisiae RC016 showed previously inhibition/coagregation of pathogens) and mycotoxins adsorbent ability (aflatoxin B1, ochratoxin A and zearalenone). The aim of this work was to evaluate beneficial properties of S. cerevisiae RC016 in a non-inflammatory in vivo model in weaned piglets and in an intestinal inflammation ex vivo model induced by the mycotoxin deoxynivalenol (DON). Secretory immunoglobulin A (s-IgA) levels, intestinal cytokines, goblet cells and production parameters were evaluated in a pig model. For the in vivo assays, twelve pigs were weaned at 21 days and assigned to two groups: Control (n=6) and Yeast (n=6). Animals received yeast strain for three weeks. After 22 days the small intestine was recovered for determination of goblet cells and s-IgA. For the ex vivo assay, jejunal explants were obtained from 5 weeks old crossbred piglets and treated as follow: (1) control; (2) treated for 3 h with 10 µM DON used as an inflammatory stressor; (3) incubated with 107 cfu/ml yeast strain; (4) pre-incubated 1 h with 107 cfu/ml yeast strain and then treated for 3 h with 10 µM DON. CCL20, interleukin (IL)-1ß, IL-8 and IL-22 gene expression was determined by qPCR. Oral administration of S. cerevisiae RC016 increased s-IgA, the number of goblet cells in small intestine and all the growth parameters measured. In the ex vivo model, the cytokine profile studied showed a potential anti-inflammatory effect of the administration of the yeast. In conclusion, S. cerevisiae RC016 is a promising candidate for feed additives formulation to improve animal growth and gut immune system. This yeast strain could be able to improve the gut health through counteracting the weaning-associated intestinal inflammation in piglets.

Aflatoxin B1 adsorption/desorption dynamics in the presence of Lactobacillus rhamnosus RC007 in a gastrointestinal tract-simulated model.

AIMS: (i) To determine the aflatoxin B1 (AFB1 ) adsorption and desorption dynamics in the presence of Lactobacillus rhamnosus RC007 under simulated transit of AFB1 at each gastrointestinal tract (GIT-saliva, stomach and intestine) stage consecutively and then, separately, (ii) to study the ability of L. rhamnosus RC007 to biotransform AFB1 as a strategy that complements the adsorption process. METHODS AND RESULTS: The AFB1 adsorption and desorption assay simulating the GIT passage of AFB1 (93·89 ng g-1 ) in the presence of L. rhamnosus RC007 (108 CFU per ml) was conducted. Moreover, lactic acid production was determined. Results demonstrated that predominant environmental conditions in salivary solution induced a low AFB1 adsorption, while the transit through the gastric solution and intestinal solution allowed high percentages of adsorption and did not generate significant AFB1 desorption. CONCLUSIONS: The AFB1 adsorption and desorption dynamics in the presence of L. rhamnosus RC007 was favoured by gastric and intestinal environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of the adsorption dynamics of AFB1 with a micro-organism of interest will allow predicting its behaviour at each stage of the GIT.

Gut-borne Saccharomyces cerevisiae, a promising candidate for the formulation of feed additives, modulates immune system and gut microbiota.

The aim was to evaluate the effect of Saccharomyces cerevisiae RC016 on immune parameters and gut microbiota in healthy mice. Animals received S. cerevisiae RC016 for 10 days. Microbial translocation to liver and changes in some bacterial populations in caecum were determined. Immune stimulation was assessed at gut level (measure ofimmunoglobulin A (IgA)+ cells and luminal cytokine profile) and by evaluating the activity of peritoneal macrophages. Oral administration of S. cerevisiae RC016 did not induce microbial translocation to liver. Mice that received yeast increased the number of IgA+ cells in their intestines, the phagocytic activity of peritoneal macrophages and decreased tumour necrosis factor alpha (TNF-α) levels in the small intestine with increases of interleukin-10/TNF-αratio. Administration of S. cerevisiae RC016 caused the decline of a logarithmic unit for Enterobacteriaceae counts compared to the control. The immune and gut microbiota modulation observed demonstrates that S. cerevisiae RC016 is a promising candidate for the formulation of feed additives to improve animal productivity. The beneficial in vivo effects observed for the potential probiotic S. cerevisiae RC016 with previously reported mycotoxin-bindingproperties, demonstrated that this strain could be suitable to be included in a novel product to improve animalproductivity, with both probiotic and mycotoxin-binding properties. However, studies in the specific host will be necessary to confirm this potential.

Lactobacillus rhamnosus RC007 intended for feed additive: immune-stimulatory properties and ameliorating effects on TNBS-induced colitis.

Lactobacillus rhamnosus RC007 is a potential probiotic bacterium that can exert beneficial effects as supplement for animal feed, by improving the immune status in healthy host, and by providing therapeutic benefits to infected/inflamed animals. The aim of the present work was to evaluate in vivo the beneficial properties of L. rhamnosus RC007, intended for animal feed, when administered to healthy and trinitro-benzene-sulfonic-acid (TNBS) colitis induced BALB/c mice. The administration of L. rhamnosus RC007 to healthy mice during 10 days increased the phagocytic activity of peritoneal macrophages and the number of immunoglobulin A+ cells in the lamina proper of the small intestine. Significant increases of monocyte chemotactic protein 1, interleukin (IL)-10 and tumour necrosis factor alpha (TNF-α) concentrations, and in the ratio between anti- and pro-inflammatory cytokines (IL-10/TNF-α) were observed in intestinal fluids after administration of bacteria. In the inflammation model, less body weight loss, macroscopic and histological damages in the large intestine were accompanied by increased IL-10/TNF-α ratio in the intestinal fluids of mice from the L. rhamnosus-TNBS group when compared to the TNBS group. In a healthy host, the oral administration of L. rhamnosus RC007 kept the gut immune system stimulated allowing a faster response to noxious stimulus. Mice that received L. rhamnosus RC007 also decreased the severity of the intestinal inflammation.

Effect of breast feeding time on physiological, immunological and microbial parameters of weaned piglets in an intensive breeding farm.

The aim of this work was to study the long-lasting consequences of different weaning age on physiological, immunological and microbiological parameters of weaned piglets. Piglets were weaned at 14 days (14W) or 21 days (21W). Blood samples were taken for IgG and cortisol determination on preweaning day and at 4; 20 and 40 post-weaning days. Three animals of each group were sacrificed. Small intestines for morphometric studies and secretory-IgA determination in fluid were taken. The cecum was obtained for enterobacteria, lactobacilli and total anaerobes enumeration. A significant decrease in piglet's plasma IgG concentrations was observed immediately after weaning and no differences were found between 14W and 21W. An increase in intestinal S-IgA was observed according to piglet's age. This increase was significantly higher in piglets 14W compared to piglets 21W. Animals from 14W group showed a decrease in villus length and in the number of goblet cells and intraepithelial lymphocytes. Other parameters were not affected by the weaning age. A short-term increase in cortisol was observed after weaning in both experimental groups. Enterobacteria decreased significantly after weaning in both groups, reaching values of weaning after 40 days. Lactobacilli counts decreased in both groups after weaning; however their counts were always higher than those obtained for enterobacteria. No differences were observed between 14W and 21W with regards to counts of anaerobes. The shortening of breast feeding time would favor an early synthesis of intestinal S-IgA after weaning. The changes observed in the microbiota could decrease postweaning enteric infections. However, early weaning induced negative effects on the cells of gut innate immunity and villi atrophy. This work provides knowledge about advantages and disadvantages at different weaning and long-lasting consequences on pig health. It is critical that swine producers become aware of the biological impacts of weaning age, so as to be able to decide the appropriate management strategies according to their facilities and rearing environment.

Comparison of toxicogenic and immunosuppressive capacity of Aspergillus fumigatus strains isolated from clinical and corn silage samples.

AIMS: To compare clinical and environmental Aspergillus fumigatus strains on their toxicogenic and immunosuppressive capacity. METHODS AND RESULTS: A total of 51 strains of A. fumigatus isolated from clinical and corn silage samples were assayed. All A. fumigatus strains were assayed for gliotoxin production, therefore strains with different gliotoxin capacities and isolated from different sources were selected and assayed for their effects on bovine macrophages and lymphocytes. Spore diffusates (SDs) obtained from all A. fumigatus strains were able to inhibit macrophage phagocytosys, regardless of their gliotoxin production capacity. However, most but not all strains were able to inhibit bactericidal activity. SDs from all A. fumigatus strains reduced lymphocytes viability. The heat treatment was not always able to inhibit the negative effect on immune cells. CONCLUSIONS: There was no difference between clinical and environmental isolates in their toxicogenic and immunosuppressive capacity. Gliotoxin would not be responsible for the immunosuppressive activity observed by the assayed A. fumigatus strains. However, gliotoxin could be present in the SD, together with some other substances. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained suggest that any environmental strain of A. fumigatus is a putative infectious strain. Prevention measures should be applied to control environmental Aspergillus conidia.

Genotoxicity and cytotoxicity evaluation of probiotic Saccharomyces cerevisiae RC016: a 60-day subchronic oral toxicity study in rats.

AIMS: To acquire data on the safety-in-use of the probiotic Saccharomyces cerevisiae RC016 and test its ability to reduce genotoxicity caused by dietary aflatoxins (AFs). METHODS AND RESULTS: The probiotic was orally administered to Wistar rats. Six groups (n = 6) were arranged: feed and probiotic controls, two levels of AFs-contaminated feed and two treatments including both the probiotic and the toxin. Genotoxiciy and cytotoxicity were evaluated with the bone marrow micronuclei assay and the comet assay and internal organs were macroscopically and microscopically examined. The tested S. cerevisiae strain did not cause genotoxicity or cytotoxicity in vivo, and it was able to attenuate AFs-caused genotoxicity. Saccharomyces cerevisiae RC016 did not cause any impairment on the rats' health and it showed no negative impact on the weight gain. Moreover, RC016 improved zootechnical parameters in AFs-treated animals. The beneficial effects were likely to be caused by adsorption of AFs to the yeast cell wall in the intestine and the consequent reduction in the toxin's bioavailability. CONCLUSIONS: The dietary administration of RC016 does not induce genotoxicity or cytotoxicity to rats. SIGNIFICANCE AND IMPACT OF THE STUDY: Incorporation of RC016 in the formulation of feed additives increases animal productivity. Similar effects may even occur in human food applications.

Selection of lactic acid bacteria to promote an efficient silage fermentation capable of inhibiting the activity of Aspergillus parasiticus and Fusarium gramineraum and mycotoxin production.

AIMS: To select lactic acid bacteria with potential silage inoculant properties. The bio-control activity against mycotoxicogenic fungi and the presence of antibiotics resistance gene were also evaluated. METHODS AND RESULTS: Lactobacillus rhamnosus RC007 and Lactobacillus plantarum RC009 were selected on the basis of growth rate and efficacy in reducing the pH of maize extract medium; therefore, they were evaluated for their bio-control ability against Fusarium graminearum and Aspergillus parasiticus. Studies on lag phase, growth rate and aflatoxin B1 (AFB1) and zearalenone (ZEA) production were carried out in vitro under different regimes of aw (0·95 and 0·99); pH (4 and 6); temperature (25 and 37°C); and oxygen availability (normal and reduced). Lactobacillus rhamnosus RC007 was able to completely inhibit the F. graminearum growth at all assayed conditions, while Lact. plantarum RC009 only did it at pH 4. Both Lactobacillus strains were able to significantly reduce the A. parasiticus growth rate mainly at 0·99 aw . A decrease in ZEA production was observed as result of Lactobacillus strains -F. graminearum interaction; however, the A. parasiticus- Lact. plantarum interaction resulted in an increased AFB1 production. Lactobacillus rhamnosus RC007 proved to have no genes for resistance to the tested antibiotics. CONCLUSIONS: The ability of Lact. rhamnosus RC007 to rapidly drop the pH and to inhibit fungal growth and mycotoxin production and the absence of antibiotic resistance genes shows the potential of its application as inoculant and bio-control agent in animal feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of selecting bacteria for silage inoculants not only for the improvement of silage fermentation but also for their effects on mycotoxicogenic fungi and the resulting mycotoxin production due to the risk that they may involve.

In vitro study on the effect of Saccharomyces cerevisiae strains on growth and mycotoxin production by Aspergillus carbonarius and Fusarium graminearum.

The effect of Saccharomyces cerevisiae RC008 and RC016 strains, previously selected based on their aflatoxin B1 mycotoxin binding ability and beneficial properties, against Aspergillus carbonarius and Fusarium graminearum under different interacting environmental conditions was evaluated. In vitro studies on the lag phase, growth rate and ochratoxin A/zearalenone and DON production were carried out under different regimens of a(w) (0.95 and 0.99); pH (4 and 6); temperature (25 and 37 °C) and oxygen availability (normal and reduced). Both yeast strains showed antagonistic activity and decreasing growth rate compared to the control. In general, the RC016 strain showed the greatest inhibitory activity. Except at the interacting condition 0.95 a(W), normal oxygen availability and 37 °C, at both pH values, A. carbonarius and F. graminearum were able to produce large amounts of mycotoxins in vitro. In general, a significant decrease in levels of mycotoxins in comparison with the control was observed. S. cerevisiae RC008 and RC016 could be considered as effective agents to reduce growth and OTA, ZEA and DON production at different interacting environmental conditions, related to those found in stored feedstuff. The beneficial and biocontrol properties of these strains are important in their use as novel additives for the control of mycotoxigenic fungi in stored feedstuffs.

Statistical optimization of culture conditions for biomass production of probiotic gut-borne Saccharomyces cerevisiae strain able to reduce fumonisin B1.

AIM: To evaluate the ability of probiotic Saccharomyces cerevisiae RC016 strain to reduce fumonisin B(1) (FB(1)) in vitro and to optimize the culture conditions for the growth of the yeast employing surface response methodology. METHODS AND RESULTS: Using Plackett-Burman screening designs (PBSD) and central composite designs (CCD), an optimized culture medium containing (g l(-1)) fermentable sugars provided by sugar cane molasses (CMs), yeast extract (YE) and (NH(4))(2) HPO(4) (DAP) was formulated. The S. cerevisiae RC016 strain showed the greatest binding at all assayed FB1 concentration. The CMs, YE, DAP concentrations and incubation time influenced significantly the biomass of S. cerevisiae RC016. CONCLUSION: A combination of CMs 17%; YE 4·61 g l(-1) and incubation time 60 h was optimum for maximum biomass of S. cerevisiae RC016. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of this work lies in the search for live strains with both probiotic and fumonisin B1 decontamination properties that could be sustainably produced in a medium just containing cheap carbon, nitrogen and phosphorus sources and would be included in a novel product to animal feed.

Saccharomyces cerevisiae strains and the reduction of Aspergillus parasiticus growth and aflatoxin B1 production at different interacting environmental conditions, in vitro.

The effect of Saccharomyces cerevisiae RC008 and RC016, previously selected based on their aflatoxin B(1) binding ability and beneficial properties, against Aspergillus parasiticus under different interacting environmental conditions was evaluated. Studies concerning the lag phase, growth rate and aflatoxin B(1) production were carried out in vitro under different regimes of a (w) (0.95 and 0.99), pH (4 and 6), temperature (25 and 37°C), and oxygen availability (normal and reduced). Both yeast strains showed great antagonistic activity at pH 4, decreasing growth rate compared with the control. The RC008 strain showed the greatest inhibitory activity at all assayed conditions. A. parasiticus produced large amounts of AFB(1) in vitro. A significant decrease of AFB(1) levels in comparison with the control were observed with yeast interaction. Differences between control and treatment values ranged from 130 to 5400 ng ml(-1). S. cerevisiae RC008 and RC016 could be considered as effective agents in reducing growth and AFB(1) production at different interacting environmental conditions, related to that found in stored feedstuff. The importance of the present work lies in the search for live strains with both probiotic and biocontrol properties able to prolong the safe storage of feedstuff and exert beneficial properties after animal consumption and which could be included in a novel product for animal feed.

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness.

AIMS: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B1 binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. METHODS AND RESULTS: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. CONCLUSIONS: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin-adsorbent interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.

Saccharomyces cerevisiae strains retain their viability and aflatoxin B1 binding ability under gastrointestinal conditions and improve ruminal fermentation.

The aim was to evaluate both the ability of yeast strains to survive and bind AFB(1) under ruminant gastrointestinal conditions and the effect of these yeast strains on ruminal fermentation. Yeast viability was studied under simulated gastrointestinal conditions. AFB(1) binding ability was evaluated at different pH values as present in the ruminant gastrointestinal tract. The effect of yeast strains on cellulose digestion and volatile fatty acids production by ruminal bacteria was also evaluated. All yeast strains were able to survive under gastrointestinal conditions and to adsorb AFB(1) at the different pH assayed. The strain RC016 showed the highest binding percentage at the three tested pH. The number of cellulolytic bacteria in ruminal fluid increased in the presence of RC008 and RC016 yeast strains. The concentration of acetate and propionate after ruminal fermentation increased with the addition of RC008 and RC016 strains; this effect was less significant with RC009 strain. Strains RC008 and RC016 are potential probiotic to be included in animal feed: they help to increase fibber digestibility and could reduce AFB(1 )bioavailability in the gastrointestinal tract. Viable S. cerevisiae RC008 and RC016 strains with both probiotic and mycotoxins adsorption properties could be used as feed additives in ruminant feedstuff.

Adjuvant effect of a probiotic fermented milk in the protection against Salmonella enteritidis serovar typhimurium infection: mechanisms involved.

Probiotics may offer protection against Salmonella enteritidis serovar Typhimurium infection via different mechanisms. The aim of this study is to investigate, using mouse models, the effect of the administration of fermented milk containing the probiotic bacteria L. casei DN-114 001 in the protection against Salmonella enteritidis serovar Typhimurium when this product is administered continuously before and after infection or only post-infection. The adjuvant effect of this probiotic fermented milk (PFM) against S. Typhimurium was also evaluated in newborn mice, whose mothers received the PFM during the suckling period or their offspring after weaning. The results obtained showed that PFM administration after salmonella infection was useful to decrease the severity of the infection. The best effect was obtained with continuous PFM administration. In the newborn mice model, PFM administration to the newborn mice after weaning showed the best effect against the pathogen. PFM administration to the mother during the suckling period was beneficial against this enterophatogen when their offspring did not receive probiotics after weaning. Continuous PFM administration to adult mice (before and after infection) was important to maintain the intestinal barrier and the immune surveillance in optimal conditions to diminish the pathway of entrance of salmonella and the spread of this pathogen to deeper tissues. In the newborn mice model, it was observed that PFM administration to the offspring after weaning or their mother during the suckling period had a protective effect against salmonella infection, however, in the mice from mothers that received PFM during nursing which were fed with PFM after weaning, we found a down regulated immune maturity that was not protective against this infection.

Immune response of non-pathogenic gram(+) and gram(-) bacteria in inductive sites of the intestinal mucosa study of the pathway of signaling involved.

The gut associated lymphoid tissue (GALT) is anatomical and functionally divided in inductive and effectors sites. In previous works we demonstrated that non-pathogenic bacteria with probiotic characteristics can improve the gut mucosal immune system, with an increase in the number of IgA and cytokines producing cells in the effector site of the intestine. In the present work we studied the effect of non-pathogenic Gram(+), Gram(-) bacteria and a Gram(+) probiotic strain on the inductor site (PP) after the oral administration to BALB/c mice. We also studied some signals induced by the assayed strain in the effectors site, such as the enzyme calcineurin and TLR-9 as a way to understand the mechanisms induced in such bacterial stimulation. The implicance of the lipoteichoic acid (LTA) in the immunostimulation was analyzed. All strains increased the number of IFN-gamma and TNF-alpha(+) cells, but not of IL-10(+) cells in the total population of PP. The release of IFN-gamma and TNF-alpha was only induced by LPS stimulation. All assayed strains increased the number of calcineurin(+) cells, while only Gram(+) strains increased the number of TLR-9(+) cells. The immunostimulatory properties of the purified LTA from Gram(+) strains was evaluated on a monocyte-macrophage U937 cell line. These cells showed capacity to release TNF-alpha and IL-10 in response to all LTA assayed in a dose-dependent way. Gram(+) strains induced signals through the calcineurin enzyme able to activate the transcriptional factor NFAT and through TLR-9. The LTA molecule from Gram(+) strains would not be the only structure involved in the immunostimulatory properties observed, specially for the probiotic strain.

Gut immune stimulation by non pathogenic Gram(+) and Gram(-) bacteria. Comparison with a probiotic strain.

We analyzed the gut immune stimulation induced by Gram-positive bacteria: non probiotic Lactobacillus acidophilus CRL 1462 and Lactobacillus acidophilus A9; two potentially probiotic strains: L. acidophilus CRL 924 and Lactobacillusdelbrueckii subsp. bulgaricus CRL 423; comparatively with a probiotic strain: Lactobacillus casei CRL 431. We also studied Gram-negative bacteria: Escherichia coli 129 and E. coli 13-7 in BALB/c mice. All the strains increased the number of IgA+ cells. We analyzed the cytokines IFNgamma, TNFalpha, IL-17, IL-12, IL-6 and MIP-1alpha. The Gram(+) strains increased the number of IL-10+ cells. Gram(-) strains did not increase IL-10+ cells, but they increased the number of IL-12+ cells. The probiotic strain increased mainly IFNgamma and TNFalpha. In the study of the receptors TLR-2, TLR-4 and CD-206, we demonstrated that only the probiotic strain increased the number of CD-206+ cells. All the Gram(+) strains increased the number of TLR-2+ cells and the Gram(-) strains of the TLR-4+ cells. The probiotic strain induced the release of IL-6 by a preparation enriched in intestinal epithelial cells (IEC). Gram(+) and Gram(-) bacteria activated different immune receptors and induced a different cytokine profile. The probiotic strain showed a great activity on the immune cells and the enriched population in IEC, activating mainly cells of the innate immune system.