Effects Of Different Bile Salt Hydrolase Of Lactobacillus Plantarum AR113 On Its Physiological Activities

Bile salt hydrolase(BSH)is a metabolite produced during the growth and reproduction of microorganisms.The enzyme has the ability to hydrolyze conjugated bile salts.A large number of studies have shown that BSH can improve the survival rate and stability of probiotics in gastrointestinal tract,and can also participate in the regulation of bile acids to help probiotics to achieve some of their probiotic functions,such as lowering cholesterol.Therefore,the BSH of probiotics has always been a research hotspot.However,the relationship between BSH and the performance and application of probiotics has not been established.Therefore,we aim to investigate the effect of BSH on the basic physiological performance of L.plantarum AR113 from the perspective of bsh gene,taking L.plantarum AR113 of which containing 4 bsh genes as the research object.Based on the existing research in our laboratory,and further applying the bsh knockout strains to the treatment of enteritis mice,by comparing with L.plantarum AR113,and analyze the mechanism of he mechanism of bsh gene in relieving enteritis preliminarily.The main findings are as follows:(1)Seven bsh knockout strains were constructed,including L.plantarum AR113?bsh 1,L.plantarum AR113?bsh 2,L.plantarum AR113?bsh 3,L.plantarum AR113?bsh 4,L.plantarum AR113?bsh 13,L.plantarum AR113?bsh 132 and L.plantarum AR113?bsh 134.By measuring the BSH activity,it was found that the bsh knockout strains prefer to hydrolyze glycine-conjuncted bile salts in the order of GC>GDC> GCDC,and also found that the bsh 1 and bsh 3 genes contributed significantly to the BSH activity,and the bsh 1 gene's contribution is greater than the bsh 3 gene.(2)By measuring the growth curves,it was found that the knockout of bsh gene had no significant effect on the normal growth of L.plantarum.The bile salt tolerance experiment showed that the bsh 1 gene was related to the bile salt stress ability of L.plantarum,and in the face of bile salt stress,the bsh expression level of the bsh knockout strain was up-regulated,which was conducive to the growth of the strain.By simulating the survival rate of bsh knockout strains by simulating the gastrointestinal tract,it is concluded that the bsh 1 and bsh 3 genes play an important role in the resistance of the strain to the stress of the physiological factors of the gastrointestinal tract.(3)The therapeutic effect on enteritis shows that knocking out the bsh 1 or bsh 3gene may reduce the BSH activity of the strain,so it has little effect on regulating the weight loss,the increase of the active disease index,the shortening of the colon,and the increase of activity of MPO.There is also no significant improvement on the up-regulated expression of pro-inflammatory cytokines TNF-?,IL-6,IL-1? and down-regulated expression of inflammatory cytokine IL-10 induced by DSS.In addition,knocking out the bsh 1 or bsh 3 havs no significant effect on expression levels of mucin MUC2 and tight junction proteins ZO-1,Occuldin and Claudin.All of these indicates that bsh 1 and bsh 3 genes can adjust the expression of inflammatory factors and intestinal barrier factors,to reduce the level of inflammation in the intestinal colon tissue and relieve the damage of intestinal permeability and epithelial structure effectively.(4)Analysis on the composition of intestinal flora in mice showed that both L.plantarum AR113 and ?bsh 134 can effectively adjust the diversity and richness of intestinal flora caused by DSS,and promote the increase of abundance of intestinal probiotics includings Lactobacillus,Rikenellaceac?RC9?gut?group and Turicibact,which are beneficial to restore the intestinal microecological balance and alleviate intestinal inflammation.However,AR113 and ?bsh 134 had no significant difference in the regulation of intestinal microorganisms,indicating that the knockout of bsh 1,bsh 3and bsh 4 genes had little effect on the regulation of intestinal flora by L.plantarum AR113.