Isolation And Identification Of Weissella Confusa From Giant Panda Feces And Its Effect On Intestinal Mucosal Barrier Function Of GFA Mice

Giant panda is a rare species in china,various measures have been taken to protect the giant pandas.Weissella confusa,a lactic acid bacteria species,was supposed to be probiotics recently,however,the beneficial effect on animal intestinal health of Weissella confusa was still unknown.The present study have isolated a probiotic Weissella confusa strain from giant panda feces and the beneficial effect to animal intestinal barrier function of this Weissella confusa strain was measured.Lactic acid bacteria(LAB)strains were firstly isolated from giant panda feces by plate separation.In order to screen an probiotics from all isolated LAB strains,in vitro probiotic performance was measured,including antimicrobial effect,antioxidant activity,auto-aggregation and cell surface hydrophobicity.After probiotics potential was confirmed,biochemical identification and 16 S rRNA sequencing were used for strain indentification.Besides,safety of this strain was also measured by antibiotic susceptibility and hemolysis test.After isolation and identification,the selected probiotics strain was administrated to the animal test.72 four-weeks old mice were divided into 6 groups: Control group(C1),Giant-panda flora associated mouse group(C2),ETEC control group(C3),Low-dose Weissella confusa group(W1),Middle-dose Weissella confusa group(W2),High-dose Weissella confusa group(W3).Mice were first treated by mixed-antibiotics for build giant panda flora associated mouse model.Then mice except group C1 and C2 were infected by ETEC for 4days and Weissella confusa treating was continued for 5days.After experiment finished,all mice were executed by cervical dislocation,mice ileum jejunum and serum samples were collected for intestinal barrier function test.qPCR technique was used for tight-junction protein mRNA expression test and intestinal flora quantity measure of some certain species.Mice serum D-lactic concentration and intestine mucosal SIgA concentration was measured by enzyme-linked immuno sorbent assay(ELISA).The richness and diversity of mice intestinal flora was measured by PCR-DGGE.The results shows: one LAB strain that own probiotics potential was selected,it can inhibit variety pathogen growth including ETEC,as well as scavenge different kinds of free radical,own pretty good surface hydrophobicity and self-aggregation rate.This strain was then identified as Weissella confusa by 16 S rRNA sequencing and biochemical test.Named Weissella confusa BSP201703.The intestinal flora evenness and similarity of the giant-panda flora associated mouse group and the giant panda feces was detected by PCR-DGGE and result shows the multiply antibiotics treatment could remove 90 percent mouse autochthonous flora,make the mice intestinal more liable to foreign flora colonization.After treat with selected Weissella confusa strain,group W1,W2,and W3 mice shows alleviate of inflammation caused by ETEC infection according to the result of pathological section,compare with control group,group W2,which treated by middle-dose Weissella confusa treat can significantly improve mRNA expression of tight junction proteins ZO-1,Claudin-1 and Occludin-1 in jejunum and ileum.The result of mouse gut flora composition analysis by quantitive real-time PCR(qPCR)and PCR-DGGE shows that Weissella confusa BSP201703 treatment can regulate evenness and diversity of mouse intestinal,but not significant.Besides,Weissella confusa BSP201703 treatment can reduce serum D-lactic concentration,increase jejunum mucous and ileum mucous SIgA concentration.In conclusion,one probiotic Weissella confusa strain BSP201703 was isolated from giant panda feces and it shows beneficial effect on adjust intestinal barrier function.This Weissella confusa strain could be a new probiotics for animal and human but more researches about other function of this strain was needed.