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

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 structure and anti-EPEC adhesion effect and internal mechanism were obtained. This study has important theoretical and practical for infant formula as well as deep development of lactic acid bacteria polysaccharide.

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。

肠致病性大肠杆菌（Enteropathogenic Escherichia coli，EPEC）是引起全球婴幼儿急、慢性腹泻和成人散发性腹泻的一类重要病原菌。传统的抗生素疗法存在产生抗药性和易复发等缺点，因此微生态等替代疗法成为目前研究热点。瑞士乳杆菌MB2-1分离自药食兼用的传统新疆赛里木酸奶，胞外多糖（EPS）产量很高。最新研究发现乳酸菌EPS可对EPEC自身及其毒素起直接抑制和封闭作用，但对其抗黏附机制了解甚少。本项目以乳清为基质，接种瑞士乳杆菌MB2-1，通过改变外源碳源种类、浓度和控制发酵条件，获得多种含特征活性结构单元的EPS组份，通过体外抗粘附抑制实验和一维和二维核磁共振等分析手段对高活性EPS组份进行高效筛选和结构表征，确定了纯高活性EPS组份的精细结构和高级结构，结果表明三种EPS纯组分抗黏附活性均能达到60%以上，在2h时LHEPS-1的抗黏附活性最高，可达到83%，而且该EPS样品对于对三种食源性致病菌的生物膜也具有较强的抑制作用，最高可达到70%以上。该研究结果可为开发功能性抗EPEC乳制品提供理论依据，同时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也具有重要的理论和实际意义。

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