Screening And Potential Evaluation Of Probiotic Lactobacillus From Calves

Lactic acid bacteria is a common probiotic.Screening and developing lactic acid bacteria with probiotic effects can reduce the use of antibiotics,which is of great significance to the sustainable development of animal husbandry.This study aims to screen out new strains of lactic acid bacteria with potential probiotic function from healthy calf feces,and further evaluate their probiotic effects in mouse inflammation models,providing a theoretical basis for the development and application of probiotic lactic acid bacteria.Through streaking,picking,Gram stain identification and 16SrDNA sequencing analysis,a total of a new strain of lactic acid bacteria was obtained,named Lactobacillus reuteri RGW1,and sent to the Chinese Culture Collection Center for preservation,with the culture number CGMCC No.18626.Through in vitro tests,the inhibitory ability of L.RGW1 against pathogenic bacteria was analyzed.It was found that L.RGW1 could effectively inhibit the growth of Escherichia coli K99 and Salmonella dublin,and its antibacterial effect on Salmonella dublin was significantly greater than the widely used probiotic L paralimentarius.Increasing the pH of the L.RGW1 culture medium to 7.0,maked it lose its ability to inhibit E.coli K99 and Salmonella dublin,but neither heat treatment nor proteinase K treatment affected its antibacterial properties.Antibiotic sensitivity analysis showed that L.RGW1 was sensitive to ampicillin,amoxicillin,chloramphenicol,cefazolin,erythromycin,tetracycline and penicillin.L.RGW1's sensitivity to erythromycin was significantly greater than L.paralimentarius,while its sensitivity to other antibiotics were less than that of L.paralimentarius.L.RGW1 does not contain the virulence genes including Gelatinase,Protein for activation of cytolysin,Enterococcal surface protein,Cell wall adhesins,Hyaluronidase,Aggregation substance,Histidine decarboxylase,Adhesion of collagen,Tyrosine decarboxylase and Ornithine decarboxylase,Adhesion of collagen,Tyrosine decarboxylase and Ornithine decarboxylase.L.RGW1 can be survived in a low pH environment of 3.In an environment with a pH=4,the number of survived L.RGW1 was significantly greater than L.paralimentarius(P=0.007).The L.RGW1 has ability to tolerate bile salt.In the environment containing 0.1%bile salt,the survived L.RGW1 was significantly greater than L.paralimentarius(P<0.01),and in the environment containing 0.5%bile salt,the survived L.RGW1 was significantly less than L.paralimentarius(P=0.006).It can tolerate simulated gastric juice and intestinal fluid,and its tolerance to simulated gastric juice was significantly lower than L.paralimentarius(P<0.01),while its tolerance to simulated intestinal fluid was significantly higher than L.paralimentarius(P<0.01).At the same time,the hydrophobicity of L.RGW1 was significantly higher than that of L.paralimentarius(P=0.001),and it had good adhesion to Caco-2 intestinal epithelial cells.Therefore,in vitro test results showed that L.RGW1 had the ability to inhibit E.coli K99 and S.dublin,and was sensitive to commonly used antibiotics,with no virulence genes.It can also resiste the gastrointestinal environment,and had a strong adhesive ability to intestinal epithelial cells.Thus,the L.RGW1 has the potential to be used as a probiotic.The probiotic effect of L.RGW1 was further explored in vivo in a mouse model.Totally,36 five-week-old C57/BL female mice were usded in the present study,and were randomly divided into four groups.The mice were given sterilized PBS(control group),L.RGW1 cell-removed culture fluid(supernatant group),and radiation-inactivated L.RGW1 culture fluid(irradiation group)and L.RGWl culture fluid(bacteria group)for 7 days.Mice were injected intraperitoneally with LPS to induce acute inflammation at day 8.Changes in the feed intake and body weight were analyzed during the feeding trial.Mice were slaughtered after the feeding trial,and the differences in serum inflammatory factor concentration and intestinal tissue morphology in each group were analyzed.Compared to the control group,the body weight of the irradiation group mice was significantly reduced on day 2 and 4,and there was no significant change in the body weight of the supernatant group and bacteria group.No significant change was observed in the feed intake among every group,except for the feed intake in irradiation group was significantly lower than that in control group on day 1.The serum TNF-? concentration in supernatant group was significantly lower than that in control group,while the serum IL10 and TGF-?concentration in bacteria group was significantly higher than that in control group.Compared to control group,the proliferation of lymphocytes in the colon and cecum tissues of mice in supernatant group,irradiation group and bacteria group were decreased,while the height of colon villi and the ratio of villi/crypts in bacteria group increased significantly(P=0.022),and the depth of colon crypts in supernatant group decreased significantly.Therefore,intragastric administration of L.RGW1 to mice can inhibit acute inflammation induced by LPS and promote colonic villi growth.In summary,this experiment successfully obtained a new strain of lactic acid bacteria L.RGW1 from the feces healthy calf.After in vitro probiotic potential analysis and preliminary evaluation of in vivo probiotic effects,this lactic acid bacteria had the potential to be used as a probiotic.Subsequent experiments should further be carried for exploring the mechanism of L.RGW1,and finding the bioactive substances of L.RGW1 for its probiotic effects,and thus provide a theoretical basis for its subsequent commercial development and wide application in the aquaculture industry.