基于可控酶水解的活性乳酸菌低聚糖制备及其抗肠致病性大肠杆菌黏附活性与机制研究

项目名称： 基于可控酶水解的活性乳酸菌低聚糖制备及其抗肠致病性大肠杆菌黏附活性与机制研究

项目编号： No.31201422

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 食品科学、农学基础与作物学

项目作者： 李伟

作者单位： 南京农业大学

项目金额： 21万元

中文摘要： 肠致病性大肠杆菌（EPEC）黏附于肠黏膜表面是其进一步定植引起婴幼儿腹泻和成人腹泻的的前提条件。近年来研究发现母乳低聚糖（HMO）和一些人工合成的低聚糖可以抑制EPEC对肠道的黏附，但对其抗黏附作用的构效关系和内在机制了解甚微。本研究拟以课题组自主分离鉴定的瑞士乳杆菌所产胞外多糖为研究对象，利用内切聚糖酶对其进行可控酶水解，通过优化酶解条件，控制多糖的水解度，暴露活性低聚糖片段，制备结构上与HMO相似的低聚糖；在对酶解产物进行分离纯化及结构鉴定的基础上，通过体外EPEC竞争性黏附细胞实验以及体内EPEC侵染小鼠等实验，定性、定位、定量分析EPEC肠细胞脱落位点毒力岛基因群中与黏附相关基因和蛋白的表达以及小鼠肠道黏膜免疫球蛋白和特征细胞因子的变化，获得低聚糖抗EPEC黏附作用的构效关系，并揭示二者间的内在机制。本研究对于婴幼儿配方奶粉母乳化以及乳酸菌多糖的深层次开发具有重要的理论和实际意义。

中文关键词： 瑞士乳杆菌 MB2-1；胞外多糖；结构表征；肠致病性大肠杆菌；抗粘附活性

英文摘要： Adherence of enteropathogenic Escherichia coli (EPEC) to intestinal mucosal surface can be considered a prerequisite in the further colonization process cause of diarrhea in infants and adult diarrhea. Recent studies have found that human milk oligosaccharides (HMO) and some synthetic oligosaccharides can inhibit EPEC adhesion on intestinal, but its anti-adhesion effects of structure-activity relationship and mechanism underlying know little. This study is intended to use exopolysaccharides produced by Lactobacillus helveticus as the research object. Through controlled enzymatic hydrolysis using endo-glucanase, optimization of enzymatic hydrolysis conditions, and controlling degree of the polysaccharide hydrolysis, the structural similarity oligosaccharides with HMO were prepared. Base on the purification and structural identification of the hydrolysates, qualitative, positioning, and quantitative analysis were tested using competitive adherent cells experiments in vitro and EPEC infection mice experiments in vivo. The changes of EPEC pathogenicity island gene cluster in locus of entericyte effacement with the adhesion-related gene and protein expression as well as mouse intestinal mucosal immunoglobulin and characteristics of cytokine were also anlalyzed. Furthermore, the relationship of oligosaccharide structu

英文关键词： Lactobacillus helveticus MB2-1；Exopolysaccharide；Structural characterization；Enteropathogenic Escherichia coli；Anti-adhesive activity