基于黏附效应的益生瑞士乳杆菌胞外多糖（EPS）肠道锚定、定殖及菌群调节机制研究

Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. Non-humanized probiotics, including lactic acid bacteria, play a prebiotic effects role as a prerequisite that they must adhere to the host surface and colonize the human intestine. Recent studies showed that some extracellular macromolecules secreted on the surface of lactic acid bacteria, such as exopolysaccharides (EPS), are involved in the related anchoring and adhesion processes. However, very few studied have investigated the structure-activity relationship and intrinsic mechanism of adhesion effect. In our previous studies, Lactobacillus helveticus MB2-1, a strain isolated from traditional Sayram yogurt from Xinjiang province showed a high yield of exopolysaccharides (EPS), which has a strong intestinal adhesion activity. This project intends to establish a method for selective oriented structure of EPS based on bioinformatics and molecular biology, to construct different types of EPS deletion mutants. In addition, the different kinds and types of EPSs in response to gastrointestinal pressure, intestinal adhesion, microflora and immune regulation will be determined. Furthermore, the relationship between the structure of EPS and the intestinal anchoring, adhesion, colonization and biologic activity of L. helveticus MB2-1 will be revealed in qualitative, positioning and quantitative manners. Its mechanism of action will also be clarified. This project is considered to provide solid theoretical and practical basis for development of non-humanized probiotics' functional dairy products .

益生菌是一类摄入一定数量后对宿主有益的活性微生物的总称。乳酸菌在内的非人源益生菌发挥益生作用的前提，是其必须能黏附于宿主细胞表面，并定殖于人体肠道。近年来研究表明乳酸菌表面分泌的一些胞外大分子物质，如胞外多糖（EPS）参与了相关的锚定、黏附过程，但对其构效关系和内在作用机制了解甚微。在前期研究中，本课题组从新疆传统药食兼用型赛里木酸奶中分离到一株高产胞外多糖（EPS）的瑞士乳杆菌MB2-1，该菌株具有很强的肠道黏附活性。本课题拟建立基于生物信息学和分子生物学的瑞士乳杆菌MB2-1 EPS缺失突变株选择性定向构造方法，构建不同类型EPS缺失突变株；明确不同类型和种类EPS在应对消化道压力及在肠道黏附、菌群和免疫调节中所起的作用；定性、定位、定量揭示EPS结构与瑞士乳杆菌MB2-1肠道锚定、黏附、定殖及活性间的关系，并阐明其作用机制；为非人源益生菌功能性乳制品的开发提供坚实的理论和应用基础。

益生菌通过菌体表面特性或分泌胞外粘性大分子物质等方式黏附于宿主细胞表面，并定殖于人体肠道，进而发挥多种益生功能。这些生物大分子如胞外多糖（EPS）参与了相关的锚定、黏附过程，但目前对其构效关系和内在作用机制还不明晰。本项目前期于新疆传统药食兼用型赛里木酸奶中分离到一株高产胞外多糖（EPS）的瑞士乳杆菌MB2-1，该菌具有很强的肠道粘附活性，因此对其胞外肽聚糖和胞外粘液多糖的结构与益生活性进行了系统研究。衍生化修饰L. helveticus MB2-1 EPS组分的多光谱与核磁结构特征及其体外抗氧化活性得到分析，A-EPS-2、C-EPS-2、S-EPS-2和P-EPS-2的得率分别为46.0±2.3%、46.4±3.5%、50.4±4.2%和51.3±2.8%，取代度分别为0.439、0.526、0.625和0.432。在益生活性研究中，瑞士乳杆菌MB2-1的EPS-1和EPS-2均可以显著促进多种益生菌的生长，其中EPS-1发酵12 h时选择性指数（SI）达1.7，较同时间菊糖组高1.4倍，且丁酸的累积量达到5.35 mM，表明EPS-1具有优良的益生特性。同时分析了瑞士乳杆菌LZ-R-5胞外多糖的一级和高级结构，以及其在肠道黏附、菌群和免疫调节中所起的作用，模拟发酵结果表明R-5-EPS对有害菌Bacteroides-Prevotella group和Clostridium histolyticum group在 48 h内保持着抑制作用，而体外实验提示R-5-EPS可以刺激RAW264.7细胞中细胞因子TNF-α，IL-6和IL-1β的产生，从而增强免疫活性。基于同源重组与loxP系统建立了瑞士乳杆菌缺失突变株选择性定向构造方法，完成瑞士乳杆菌多糖合成关键基因YveK敲除与表型变化分析，结果表明YveK基因的缺失导致自凝聚能力下降，疏水性增加，生物膜成膜能力降低。YveK基因通过调控c-EPS和s-EPS的生物合成，改变了菌体荚膜结构和菌株表面特性，进而影响瑞士乳杆菌胞外多糖的相关益生活性。本项目揭示了EPS结构与瑞士乳杆菌肠道锚定、黏附、定殖及生物活性间的关系，并通过多糖合成关键基因敲除引起的菌株特性变化，建立了瑞士乳杆菌突变株构造方法并阐明EPS生物合成机制，为高产胞外多糖菌株筛选与乳酸菌代谢物生物合成提供思路。