Streptococcus Thermophilus Producing High Yield Of Exopolysaccharides(EPSs)—Screening,Characteristion And Application

Extracellular Polysaccharide(EPS)is secreted during the process of growth and metabolism to extracellular mucus or capsular polysaccharide,which is one of important secondary metabolites.EPS has not only excellent biological activities,such as anti-tumor and immunomoregulation but also can improve the viscosity and stability of the fermented milk product Now days,the structure and multiple functions of EPS were widely studied.In this study was mainly on isolate and screening a strain of lactic acid bacteria one producing high-yield EPS from traditional yoghurt of the south of Gansu province,which was identified S.thermophilus,and was named as MN-BM-A01 after multiple classified study,systematic classified study and whole genome sequencing and analysis.Extracellular polysaccharide fermentation of the strain was optimized;extracellular polysaccharide separation and purification,and structure identification were conducted;influence of polysaccharide on structure of fermented yoghurt texture was researched.(1)451 strains screened from fermented yoghurt in the south of Gansu province via determination of cell morphological characteristics,physiological and biochemical characteristics,microscopic and biological.Based on results of multiple comparative studies of the speed of producing acids,the production of viscosity and flavor and the production of extracellular polysaccharides,MN-BM-A01 was determined as a strain of S.thermophilus,the maximum yield of extracellular polysaccharide S.thermophilus strains could reach(38.5±1.15 mg/L after 6h,and167.23±11.46 mg/L after 24h).The cultivating temperature was 43? for S.thermophilus MN-BM-A01.Based on results of single factor tests and response surface test,glucose and WPC80 were determined as the optimum carbon source and nitrogen source.The addition amount of glucose was 45.29 g/L,sucrose was 61.26 g/L,WPC 80 was 20.00g/L.Under the optimal fermentation condition,the EPS yield reached 320±2.45mg/L after 6h,which was roughly 8 times higher than original condition.(2)Crude polysaccharide was obtained from fermentation broth of extracellular polysaccharide of S.thermophilus MN-BM-A01 via TCA deproteinization and ethanol precipitation.Purified polysaccharide was then obtained via dialysis,ultrafiltation,and chromatography.Infrared spectroscopic analysis indicated that extracellular polysaccharide contain acetamido,acidophobe,and?-pyranose ring structure.The results of HPLC-tested molecular molecular distribution indicated that main molecular weight of polysaccharide was 2.7×104 Da.Monosaccharide composition analyzed via GC-MS indicated that polysaccharide was mainly composed by mannose,glucose,and galactose.Glucose occupied the biggest composition,which was 55.23%.(3)S.thermophilus MN-BM-A01 whole genome was 1.88 M,without plasmids.Contains 1704 proteins,6 rRNA operon and 67 tRNA genes were found.Carbohydrate metabolism system analysis showed that MN-BM-A01 can met lactose,glucose,galactose,sucrose,starch and other sugars.Protein metabolism system analysis showed that MN-BM-A01 has 157 genes involved in protein metabolism,including the key enzyme of casein hydrolysate.No resistance gene was found from ResFinder database.CRISPRfinder predicted 2 CRISPR system in MN-BM-A01.MN-BM-A01 had a 36.0 kb EPS gene cluster,containing 39 genes.There was a high changeability of EPS gene cluster with homology comparison.The bioinformation analysis suggested that MN-BM-A01 can grow rapidly in the milk environment,and it has the ability to defend the phage infection.(4)The characters of yoghurt by MN-BM-A01 were dertermined.Rheology analysis indicated that the viscosity of yoghurt which was ferment by MN-BM-A01 were higher than other gelatin systems.According to stability analysis,the gel formed by MN-BM-A01 was the most stable.It is detected by confocal laser scanning microscope that either yogurt fermented with Streptococcus thermophilus MN-BM-A01 is able to form even-distributed casein micelle network.The sensory modeling showed that with comparison of MN-BM-A01 fermented yoghurt and commercially available bacteria fermented yoghurt,the results indicated that the viscosity,suck strength,shape,thickness and surface brightness of the yoghurt ferment by MN-BM-A01 were significantly higher than that fermented by commercially available bacteria.In summary,Streptococcus thermophilus MN-BM-A01 selected from built fermentation system of screening strains high in EPS has characteristic of both excellent fermentation and affluence in EPS.MN-BM-A01 has a proteitial prospects of commercial starter culture.