In Vitro Screening Of Lactobacillus Strains Potential To Stimulating Immunity And Their Action Mechnism

The study of probiotic bacteria, especially the probiotic bacteria having the immunostimulating ability is promising and has gained most interesting. Some Lactobacillus strains could exerted their immunity enhancing effects by augmenting both non-specific and specific host immune responses.The purpose of this study was to isolate Lactobacillus strains from traditional fermented foods made by locals in Gansu, Qinghai, Xinjiang and Tibet and the feces of healthy infants, and to screen potential probiotics having immunostimulating activity from the isolates.The total of 168 bacilli strains and 39 coccus strains were isolated from the traditional foods and infant feces. The bacilli and coccus strains were gram-positive, catalase-negative and F6PPK-negative, so they were presumedly considered as lactic acid bacteria. Yeasts were also isolated from the traditional foods.The isolated bacilli strains were co-cultured with human peripheral blood mononuclear cells (PBMCs) and the interleukin 12 (IL-12, p70) concentration of the culture supernate were determined by ELISA method. The total of 34 bacilli strains, which stimulated PBMCs to produce IL-12(p70) at higher concentration, were screened from 168 strains of isolated bacilli. The concentrations of interferon gamma (IFN-γ)and tumor necrosis factor alpha (TNF-α), which were produced by PBMCs co-cultured with the 34 strains, were determined by ELISA method. Among the 34 bacilli strains, eight bacilli strains stimulated PBMCs to produce IL-12(p70), IFN-γand TNF-αat higher concentrations compared to rest strains. The eight bacilli strains, J23ANL, J5ANL, IN1ANL, SB5AL, SB31AL, M5AL, G15AL and T3AL were screened as potential probiotics having immunostimulating activity. Using Biolog MicroLog2 system, the strains J23ANL, M5AL and G15AL were identified as Lactobacillus paracasei supsp. paracasei, J5ANL, IN1ANL, SB5AL and SB31AL as Lactobacillus rhamnosus, T3AL as Lactobacillus coryniformis supsp. torquens.The eight Lactobacillus strains were examined in vitro for resistance to simulated gastro and intestinal juice, adhesion to HT-29 cells, and antagonistic activity against pathogens. All eight strains retained their viability after 1 h of exposure to simulated stomach juice with some reduction of the log of viable colony counts (1.94~3.48); after 3 h of exposure to simulated stomach juice, the viability of the strains was lost markedly and the strain G15AL, T3AL and SB31AL didn't maintain viability. All eight strains retained their viability even after 4 h of exposure to simulated small intestinal juice just with slightly reduction of the log of viable cology counts (0.18~1.64). The strains IN1ANL, M5AL and G15AL had adhesive ability with more than 40 bacteria per 100 HT-29 cells; the rest strains didn't have adhesive ability with fewer 40 bacteria per 100 HT-29 cells. All the eight strains had antagonistic activity against E.coli, S.typhimurium and S.sonnei.The effects of live bacteria, heat-killed bacteria, cell wall and genomic DNA of the eight Lactobacillus strains on PBMCs proliferation, cytokine secretion and NK cell activity were determined to study the immunostimulating activities of the bacteria cellular components. The live bacteria, heat-killed bacteria and cell walls of the eight strains exerted proliferative effect on PBMCs and the stimulate index of PBMCs declined with the increase of concentrations of the bacteria and cell walls. The stimulate index of PBMCs exerted by genomic DNA of the strains were little than those of PBMCs exerted by live bacteria, heat-killed bacteria and cell walls. The live bacteria of all the eight strains could significantly augment natural killer cell (NK) activity and the effect varied with different strain and dose. The cell wall of the strains could significantly augment NK cell activity the effect varied with different strain and dose. Except the DNA of strain SB31AL, other strains'DNA could enhance NK cell activity significantly. The enhancement of NK cell activity by the strains was related with the PBMCs cytokine secretion induced by the strains. The live bacteria, heat-killed bacteria, cell wall and genomic DNA of the eight strains stimulated PBMCs to produce IL-12(p70), IFN-γand TNF-α.The concentrations of IL-12(p70) and TNF-αstimulated by bacterial cell wall and DNA of the eight strains was lower than those stimulated by live bacteria of the strains.The antiproliferative effects of the heat-killed bacteria, bacterial cell wall and genomic DNA of the eight strains on HT-29 and K562 cancer cells were examined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The heat-killed bacteria, bacterial cell wall and genomic DNA of the eight strains all inhibited the growth of HT-29 cells significantly. The heat-killed bacteria of the eight strains inhibited the growth of K562 cells; except J23ANL and J5ANL, the cell wall of the other strains inhibited the growth of K562 cells; the DAN of the strain G15AL, M5AL and SB31AL inhibited the growth of K562 cells. Among of the eight stains, the inhibition rates of HT-29 by Lactobacillus coryniformis ss torquens T3AL were significantly higher than those by the rest strains. The results of Single cell gel electropherosis assay showed that t he strain T3AL exerted markedly antiproliferative effect on HT-29 cell through the induction of cell apoptosis.The results of in vitro assays showed that the eight Lactobacillus strains had immunostimulating activities, could be tolerant to gastrointestinal conditions, exerted antagonistic activity against pathogens and inhibited the growth of cancer cells. The probiotic potential of the eight Lactobacillus strains will be further studied in vivo.