Phenotypic Characteristics And Biosynthesis Mechanism Of Exopolysaccharide From Streptococcus Thermophilus IMAU20246

Exopolysaccharides(EPS)of lactic acid bacteria are biopolymers secreted into extracellular matrix during the growth of lactic acid bacteria,which have good physical properties and physiological activities.In this study,S.thermophilus IMAU20246 with good fermentation characteristics and high EPS production was taken as the research object,to explore the effects of different carbon sources on the yield,molecular weight and phenotypic characteristics of EPS.On this basis,combined with the results of genome re-sequencing,the eps gene cluster of the strain was analyzed by bioinformatics method,the EPS synthesis pathway was predicted,and the expression levels of EPS synthesis related genes in different growth stages of the strain were studied.The main conclusions are as follows:1.Lactose,glucose,galactose and sucrose were used as the main carbon sources in the M17 medium,respectively,and the EPS production of S.thermophilus IMAU20246 was determined using the phenol-sulphuric acid method.The results showed that the highest EPS production was achieved when sucrose was used as the main carbon source,reaching 617±5.9 mg/L,which was significantly higher than the other three carbon sources(P<0.05).2.The crude polysaccharide produced by S.thermophilus IMAU20246 in liquid medium with different carbon sources was separated and purified,and four components were obtained,namely EPS 1-1,EPS 2-1,EPS 3-2 and EPS 4-1.The neutral polysaccharides EPS 1-1,EPS 2-1 and EPS 4-1 were mainly composed of glucose,galactose,mannose and rhamnose in different molar ratios,while the acidic polysaccharide EPS 3-2 was mainly composed of glucuronic acid,galactose,mannose and ribose.Analysis of the infrared spectra revealed that EPS 1-1,EPS 3-2 and EPS 4-1were all mannopyranosyl heteropolysaccharides with co-existence of α and βconformations,while EPS 2-1 was an α-configuration mannopyranosyl heteropolysaccharide.These results indicate that the addition of different carbon sources has an effect on the glycosidic bond configuration and monosaccharide composition of EPS.3.The S.thermophilus IMAU20246 genome was resequenced and bioinformatics analysis revealed that the S.thermophilus IMAU20246 genome contains a complete eps gene cluster,which is 18.1 kb in length and encodes 15 genes related to EPS biosynthesis.Based on genomic information,it is hypothesized that S.thermophilus IMAU20246 synthesizes 7 sugar nucleotides by transporting glucose,mannose,fructose and galactose,lactose,alginose,cellobiose and sucrose,which are involved in the biosynthesis of EPS.4.The RT-q PCR technique was used to analyze the expression of the eps gene cluster at different growth stages.The results showed that eps gene clusters were expressed at all cell growth stages,especially the glycosyltransferase genes eps E,eps F,eps H and eps J were expressed at the highest level when EPS production reached its maximum,indicating that glycosyltransferases are key genes in determining EPS production.In this study,the phenotypic characteristics of EPS synthesized by S.thermophilus IMAU20246 under different carbon sources were studied,and the eps gene cluster and its expression were analyzed,and the mechanism of EPS biosynthesis was explored,which provided theoretical basis for further study of lactic acid bacteria EPS.