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Background and Objectives: Human epithelial cells have been widely used to study the interaction between intestinal cells and pathogens, in vitro. In this study, the effect of probiotic bacteria Bacillus coagulans and its supernatant on the growth inhibition, cytotoxicity and induction of apoptosis caused by Salmonella Typhimurium and its adhesion to HT-29 cells were investigated. Materials and Methods: B. coagulans supernatant was used to obtain the minimum inhibitory concentration. To evaluate the cytotoxicity and percent of apoptotic cells, B. coagulans and its supernatant (2, 4, 6 and 8% concentrations) with S. Typhimurium was added to HT-29 cells. The MTT assay was used in order to evaluate the cytotoxicity. Percent of apoptotic cells was reported using a fluorescence staining method. Additionally, the adhesion of S. Typhimurium to HT-29 cells was investigated. The effect of B. coagulans on the level of adhesion was also studied. Results: The most inhibitory effect was shown at the concentration of 80000 µg/ml supernatant of B. coagulans (54.77% ± 1.43). The simultaneous culture of S. Typhimurium with B. coagulans had the lowest amount of cytotoxicity and induced apoptosis among the all co-culture groups of S. Typhimurium with B. coagulans or its supernatant. The determined cytotoxicity and induced apoptosis were 26.06 % ± 3.79 and 17.63 % ± 2.14 respectively. In the adhesion test, it was observed that B. coagulans can significantly prevent adhesion of S. Typhimurium to HT-29 cell. Conclusion: B. coagulans can reduce the adhesion, cytotoxicity and induction of apoptosis caused by S. Typhimurium in HT-29 cells in vitro.

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Aims This study aimed to evaluate the effects of three Bacillus probiotics on Salmonella Typhimurium, and interleukin‐8 (IL‐8) gene expression in the co‐culture of the Bacillus and the pathogen in vitro. Methods and Results Bacillus subtilis, Bacillus indicus and Bacillus coagulans were initially turned to spore and heat‐inactivated forms. The cellular damages of the probiotics on the HT‐29 cells were investigated individually and in combination with S. Typhimurium using 3‐(4,5 dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and fluorescence assays. To extract cell free supernatants (CFS) of the probiotics, they were cultured in selective media. The inhibitory activity of CFSs were then assayed against the pathogen. The gene expression of IL‐8 of the HT‐29 cells was evaluated by real‐time PCR in all the groups. The results showed that the CFSs of three probiotics could inhibit the growth of S. Typhimurium by more than 50%. Inhibitory effects of B. indicus and B. subtilis CFSs were related to the production of pepsin‐sensitive compounds, except B. coagulans in which the high inhibitory effect was due to organic acids. The spores of the three probiotics and the heat‐inactivated forms of B. subtilis and B. coagulans could reduce the cytotoxicity of S. Typhimurium. The cell viability also increased applying both forms probiotics against the pathogen. In all co‐culture groups, the IL‐8 gene expression induced by S. Typhimurium was reduced. Conclusions The three Bacillus probiotics can be considered as proper candidates for the prevention and treatment of S. Typhimurium food poisoning. Significance and Impact of the Study Applying probiotics as live bacteria is universally noted in foods. This study tried to discover the effects of Bacillus probiotics in the form of spore or even heat‐killed bacteria against S. Typhimurium and evaluate ratio of IL‐8 gene expression in cell culture. The most effective Bacillus probiotic will be recommended. This approach will help to use probiotics as nonvegetative cells in foods to fight gastrointestinal pathogens.