Study On The Functional Characteristics Of Lactobacillus Reuteri SH 23 Upon Stimulated Gastrointestinal Fluid Stress

Lactobacillus is one of the main genus of Lactic acid bacteria(LAB)in human and animal gastrointestinal tract,which has the physiological functions of maintaining the balance of intestinal flora and regulating immunity.The prerequisite for these functions is that LAB can survive in the harsh gastrointestinal environment and colonize in epithelial cells.Therefore,systematically understanding the stress response and adhesion mechanism of LAB in the gastrointestinal environment is the basis for exploring the potential functions of LAB.In this study,the correlation of bacteria surface protein changes and adhesion ability were analyzed when Lactobacillus reuteri SH 23(L.reuteri SH 23)exposing to the stimulated gastrointestinal fluid.Furthermore,transcriptomics and proteomics were used to investigate the mechanism of adaptation and adhesion of L.reuteri SH 23 in the presence of simulated gastrointestinal fluid.LPx TG-motif cell wall anchor domain protein of L.reuteri SH 23 was cloned and expressed which used to study its potential probiotic characteristics.This provided basic information for further exploration of the adhesion mechanism of L.reuteri SH 23 to the intestinal mucosal barrier.The main experimental results were as follows:(1)L.reuteri SH 23 was screened with the potential resistant to gastrointestinal fluids compared with other strains,though the adhesion related activities characteristics were reduced under gastrointestinal fluids.Through scanning electron microscopy and transmission electron microscope,the appearance of cells showed a bit of incompleteness after intestinal fluid treatment.These data suggested that L.reuteri SH 23 was affected by the simulated gastrointestinal fluids in vitro.(2)According to the comparative transcriptome analysis,gene expression involved in the cell envelope,metabolic processes,common stress response,regulatory systems and transporters were affected,while gene related to the mucus-binding proteins(Mub)like LPx TG-motif cell wall anchor domain protein(Lreu23DRAFT?5180 and Lreu23DRAFT?4529)of L.reuteri SH 23 showed significant increase in this environment.(3)With the approach of proteomics analysis,the surface proteomes of L.reuteri SH 23 were identifed and analyzed in response to simulated gastrointestinal fluid.Moonlight protein,involved in glycolysis(Enolase and Phosphoglycerate mutase),chaperone activity,protein synthesis and nucleic acid stability(30S and 50S ribosomal proteins),Amino acid ABC transporter,LPx TG anchor domain surface protein and Mub protein were used to analyze the adhesion mechanism of L.reuteri SH 23 in adapting to the intestinal mucosal barrier.The study found that LPx TG anchor domain surface protein was more abundant,which was consistent with transcriptomics data.That would improve the colonization ability of L.reuteri SH 23 to the host.(4)In order to further study the probiotic characteristics of the surface proteins of the LPx TG anchor domain,A4V07?06340(from proteomics)and Lreu23DRAFT?5180(from transcriptomics)were cloned and over-expressed in E.coli.Furthermore,recombinant protein(A4V07?06340)could increase the adhesion rate of L.reuteri SH 23 to Caco-2 cells,and reached to the highest rate at 50 ?g/m L.At the same time,it could also increase the survival rate of L.reuteri SH 23 in stimulated gastrointestinal fluids.