马乳酒样乳杆菌胞外多糖的结构解析及免疫调节活性机理研究

The exopolysaccharides (EPS), excreted by lactic acid bacterial (LAB), have attracted extensive attention due to the physiological activities of improving yoghurt quality and immune regulation. However, it’s difficult to purify and determined the senior structure of the EPS. Thus, there is deficiency in advanced structure and physiological activity mechanism of EPS, which produced by lactic acid bacteria.. Our group had isolated the strain Lactobacillus kefiranofaciens ZW3 from Tibet Kefir grain. The EPS, produced by L. kefiranofaciens ZW3, showed good characteristics of sticky and antioxidant in our previously study. Moreover, the fermentation conditions and purification process also had been optimized. The techniques of HPGPC, FT-IR, GC-MS and 1D/2D NMR will be used to analysis the structure of uniform molecular size EPS. The effects of EPS on the proliferation of muring macrophage cell line RAW 264.7 will be determined by MTT assay. The concentration and mRNA expression of cytokines TNF-α, INF-γ, IL-10 and IL-12 will be determined by ELISA and fluorescent quantitative reverse transcription PCR technique. Furthermore, the signal pathways of NF-κB and MAPK were detected with the methods of Western-blot. Thereby the immunomodulatory mechanism of EPS will be clarified through molecule level and protein expression. Moreover, the functional groups of EPS will be modified by chemical modification methods. The differences of immunomodualtory activity were analysis, which preliminarily reveals the relationship between structure and physiological activity.

乳酸菌胞外多糖由于具有多种生理活性，近年来引起了广泛的关注。由于胞外多糖分离纯化及结构分析较困难，影响了胞外多糖高级结构与生理活性之间构效关系的分析。课题组前期从藏灵菇中分离得到胞外多糖高产菌株——马乳酒样乳杆菌ZW3(1518.4mg/L)，优化了发酵条件及分离纯化工艺，初步分析了理化性质，并完成了该菌株的全基因组测序。本课题在此基础之上进一步研究该胞外多糖的一级结构，包括分子量、官能团、单糖组成、糖苷键的连接方式等；采用小鼠巨噬细胞株RAW 264.7细胞作为体外模型，分析该多糖的免疫调节活性，从分子水平和蛋白表达水平阐述其免疫调节活性的作用机理；利用化学修饰法对该胞外多糖的官能团进行修饰，通过比较修饰前后免疫调节活性的差别，初步地揭示其结构与生理活性之间的构效关系。通过本课题研究，可以进一步丰富马乳酒样乳杆菌ZW3胞外多糖的研究成果，为将其作为新型食品添加剂及保健食品奠定基定基础

乳酸菌胞外多糖是乳酸菌在生长代谢过程中分泌到细胞壁外的一种天然高分子化合物。由于其能够改善发酵乳制品风味与质地，并具有多种生理活性，近年来受到国内外学者的普遍关注。然而，由于乳酸菌胞外多糖的产量普遍偏低、分离纯化及结构分析较困难，限制了胞外多糖高级结构与生理活性之间构效关系的研究，影响了其进一步开发利用。. 本项目以课题组前期从我国西藏传统乳制品发酵剂藏灵菇（Kefir粒）中分离得到胞外多糖高产菌株——马乳酒样乳杆菌ZW3合成的胞外多糖为研究对象，进一步优化了菌株在发酵罐中合成胞外多糖的培养条件。发酵液经分离纯化后，采用理化方法及现代仪器分析技术，探究了该胞外多糖的一级结构，包括分子量、官能团、单糖组成、糖苷键的连接方式等；采用小鼠巨噬细胞株RAW 264.7细胞作为体外模型，分析该多糖的免疫调节活性，并从分子水平和蛋白表达水平阐述其免疫调节活性的作用机理。此外，利用化学修饰法对该胞外多糖的进行了乙酰化修饰，通过比较修饰前后免疫调节活性的差别，初步地揭示其结构与生理活性之间的构效关系。. 本项目的研究成果不仅可为多糖的结构特性、生理活性、作用机制及结构-效应之间关系研究提供可借鉴的研究方法，而且可为乳酸菌胞外多糖的进一步开发利用提供理论指导与技术支持，为新型食品添加剂及保健食品原料提供新的选择。