高产胞外多糖嗜热链球菌的筛选及胞外多糖的结构分析

Selection of Streptococcus thermophilus for High Extracellular Polysaccharide Production and Structural Analysis of Extracellular Polysaccharide

目的:嗜热链球菌是一种对人体有明显食疗作用的有益菌。以从传统发酵乳制品中分离得到的442株嗜热链球菌为研究对象,从中筛选出1株高产胞外多糖嗜热链球菌MGD1-4,对该菌株发酵液所产胞外多糖进行分离、纯化与结构分析。方法:采用DEAE-Cellulose 52离子交换柱及Sepharose CL-6B凝胶过滤层析方法从嗜热链球菌MGD1-4液体培养基中获得的胞外多糖中分离得到单一组分,利用凝胶渗透色谱仪(GPC)测定各组分分子质量,采用高效液相色谱(HPLC)、红外光谱(IR)及核磁共振(NMR)等对各EPS纯化组分的单糖的组成及结构进行解析。结果:EPS-1a为中性多糖,分子质量为1.572×10^(5)u;EPS-3a为酸性多糖,分子质量为3.825×10^(5)u,且二者均为单一多糖。单糖组成测定结果表明:EPS-1a主要由葡萄糖、甘露糖和半乳糖构成,三者的物质的量比为3.62∶1∶2.99,占总糖量90%以上;EPS-3a主要由甘露糖、半乳糖和阿拉伯糖构成,占总糖量80.6%,其物质的量比为1.19∶1∶1.08。对1D NMR的研究表明,EPS-1a和EPS-3a的糖环形式均为吡喃糖环,糖苷键构型均为α型。结论:通过分析该菌株所产胞外多糖结构,初步确定其分子结构及理化性质,对进一步研究EPS的构效关系具有重要意义。

Objective:Streptococcus thermophilus is a kind of beneficial bacteria with obvious therapeutic effect on human body.In this experiment,442 strains of S.thermophilus isolated from traditional fermented dairy products as research objects to screen out a high-yield extracellular polysaccharide strain MGD1-4 and the extracellular polysaccharide was isolated,purified and analyzed structurally.Methods:A few single fractions were purified from the extracellular polysaccharide produced by the fermentation broth of the strain,using the method of anionic chromatography of DEAE-Cellulose 52 and Sepharose CL-6B.And the structure was analyzed by gel permeation chromatography(GPC),high performance liquid chromatography(HPLC),infrared spectroscopy(IR)and nuclear magnetic resonance(NMR).Results:It found that EPS-1a was a neutral polysaccharide with a molecular mass of 1.572×10^(5) u,EPS-3a was an acidic polysaccharide with a molecular mass of 3.825×10^(5) u,and both of them were single polysaccharide.The results of monosaccharide composition indicated that EPS-1a was mainly composed of glucose,mannose and galactose.The molar ratio of three was 3.62∶1∶2.99 and accounting for more than 90%,but EPS-3a was mainly composed of mannose,galactose and arabinose,which accounts for 80.6%of total,its molar ratio was 1.19∶1∶1.08.Combined with the results of 1D NMR,it was showing that the sugar ring of EPS-1a and EPS-3a were all pyranose rings,containingαglycosidic bond.Conclusion:By analyzing the structure of extracellular polysaccharide produced by the strain,the physical and chemical properties were initially determined,which is of great significance for further study of the structure-activity relationship of extracellular polysaccharide.