Antivirulence activities of Bioactive Peptides produced by Lactobacillus helveticus and Lactobacillus acidophilus against Salmonella enterica serovar Typhimurium
During fermentation, Lactobacillus acidophilus and Lactobacillus helveticus release many small peptides as secondary metabolites. Previous studies have showed the protective effects of these biomolecules against enteric pathogens in vitro and in vivo. The purpose of the present study is to observe the effects of the bioactive peptides from Lactobacillus helveticus and Lactobacillus acidophilus on the virulence factors of Salmonella Typhimurium and to observe the effects of fermentation conditions on the antivirulence activities of the bioactive peptides from Lactobacillus acidophilus. Cell-free spent media (CFSMs) were prepared from Lactobacillus helveticus (LH-2) fermented skim milk and Lactobacillus acidophilus (La-5) fermented whey protein based media. Lactate dehydrogenase (LDH) production, which is used as an indicator to cytotoxicity, was assayed in Salmonella infected RAW 264.7 cells co-incubated with CFSMs. The effects of the CFSMs on the gene expression of Salmonella were analyzed using a two-step RT-qPCR assay. Additionally, the antivirulence effects of the La-5 CFSMs produced under different fermentation conditions were compared and correlated with specific peptides in the La-5 CFSMs. The LH-2 and La-5 CFSMs significantly decreased (p< 0.05) the cytotoxicity caused by Salmonella infection in RAW 264.7 cells. In terms of gene expression, both LH-2 and La-5 CFSMs showed two-fold down-regulation in sipB and more than six-fold down-regulation in hilA, ssrB, sopD and invA genes expression. The La-5 CFSM produced under strict anaerobic condition with medium rate of agitation and fresh culture inoculation showed highest down-regulation of all the genes tested. The down-regulation of the virulence genes when correlated with reverse phase- HPLC profiles, significant correlation (p<0.01 or p<0.05) was found for few specific peaks. These results suggest that the bioactive peptides obtained from Lactobacillus helveticus and Lactobacillus acidophilus can protect against cell damage and apoptosis caused by Salmonella Typhimurium infection in vitro. Bioactive peptides obtained from both Lactobacillus species exhibited antivirulence effects against Salmonella Typhimurium. The production method can be optimized to increase the efficacy of antivirulence effect of the peptides.