Isolation And Screening Of Probiotic Lactic Acid Bacteria In Liquor Pit And Study On The Exopolysaccharides Biological Activity

Lactic acid bacteria?LAB?are the important member of the probiotics,and are widely distributed.The exopolysaccharides of LAB have excellent physicochemical properties and biological activities.LAB and LAB EPS have gradually become a hot spot in recent years.In this study,lactic acid bacteria were isolated from three categories,white wine pit mud,fermented grains and Daqu and then identified by16S rDNA sequencing and API50 CH.Lactobacillus were screened the probiotic properties in vitro.L.buchneri WXL017 strain expressed better comprehensitive probiotic properties in in vitro experiment and high production of EPS.The isolation,purification and immunological activities of the EPS produced by WXL017 were carried out systemically.The main results are as follows:Through streaking in the plate,gram staining,H₂O₂ enzyme testing,PCR sequencing,establishing phylogenetic tree,and API50 CH identification,147 LAB strains were isolated from white wine pit mud,fermented grains and Daqu,which were identified as 19 species namely:L.buchneri,L.harbinensis,L.brevis,L.sunkii,L.parabuchneri,L.coryniformis,L.acidipiscis,L.fermentum,L.diolivorans,L.paracasei,L.paraplantarum,L.plantarum,L.lactis,E.xiangfangensis,E.faecium,E.thailandicus,P.pentosaceus,P.parvulus,P.lolii.There were 12 Lactobacillus species and 7 Lactococcus species.94 Lactobacillus strains were resistant to artificial gastric juice and bile salts,adhered to Caco-2 cells,and evaluated EPS production.WXL017,WXL051 and WXL054 were screened out with good probiotic potential.L.buchneri WXL017 was fermented.After bacteria removal,protein removal,and alcohol precipitation process,WXL017 crude EPS was obtained.A single component was obtained by DEAE-Sepharose Fast Flow and Sepharose CL-6B columns,which was acidic polysaccharide.UV and FTIR spectroscopy detection showed WXL017EPS had characteristic absorption peaks of polysaccharides.There were no nucleic acid and protein impurities.Macrophage RAW264.7 was stimulated by purified WXL017 EPS,and the proliferation and NO release ability were significantly increased.The mice were immunized with purified WXL017 EPS mixed with 146S antigen.The results showed that the level of 146S-specific Ig G antibodies in the serum and the proliferation of splenic lymphocytes were significantly increased by WXL017 EPS.It demonstrated that WXL017 EPS enhanced mice 146S specific immune response.This study reveals that there are abundant lactic acid bacteria in liquor pits.Different LAB strains exhibit good biological characteristics and probiotic potential,providing a wealth of raw materials for the study of the performance of lactic acid bacteria.The purified exopolysaccharides-WXL017 EPS has strong immunological activity and provides theoretical and experimental basis for the selection and application of lactic acid bacteria exopolysaccharides immunopotentiator.