食用海葡萄硫酸多糖CLGP调节肠道菌群的构效关系研究

The structures of sulfate polysaccharides are special and complex, therefore their known structure-activity relationships need to be further studied due to the limitations of qualitative and quantitative analytical measurements on sulfate polysaccharides. Recently, our group have separated and purified two novel sulfate polysaccharide fractions (CLGP3 and CLGP4) with homogeneous weight containing large number of sulfate groups and branches from Caulerpa lentillifera, which is an edible green seaweed. The previous study on gut microbiota fermentation of CLGP3 and CLGP4 indicated that CLGP3 and CLGP4 could modulate the composition of the gut microbiota, but their exact relationships have not systematical study. Fortunately, we have established new methods that could be used to qualitative and quantitative structural analysis of sulfate polysaccharides and sulfate oligosaccharides. Therefore, we can systematical study on the structure-activity relationships of CLGP3 and CLGP4 by using these new methods. Firstly, according to the structural characteristics of CLGP3 and CLGP4, their sulfate groups and branches will be selectively removed to produce various sulfate polysaccharides with different sulfation pattern and branches; moreover, CLGP3 and CLGP4 will be degraded by mild acid hydrolysis and enzymatic digestion to produce various sulfate oligosaccharides with different structures. Subsequently, the main structures and sulfation patterns of above products will be fine analysis to determine the accurate structure of CLGP3 and CLGP4. Furthermore, the regulatory effects of CLGP3, CLGP4 and above products on the gut microbiota will be evaluated and compared by QPCR and 16S high-throughput sequencing techniques in vitro and in vivo to fully reveal the exact relationships between accurate structure and regulatory effect on the gut microbiota of CLGP3 and CLGP4. This project will provide scientific basis for the nutritional evaluation and development of new kinds of edible seaweeds.

硫酸多糖结构特殊复杂，由于分析手段和方法的限制，其构效关系有待深入研究。课题组前期从食用海葡萄中分离纯化出两种结构新颖、富含硫酸基团、多分支的均一性海葡萄硫酸多糖（CLGP3和CLGP4），体外酵解发现其可有效改善肠道菌群组成，但其调节肠道菌群的构效关系还没有系统研究。本项目拟在课题组已建立的硫酸多糖和寡糖结构分析新方法的基础上，分别对海葡萄硫酸多糖进行可控性脱除和降解，获得具有不同硫酸基团取代模式和分支结构的脱除产物以及不同结构类型的硫酸寡糖片段；通过分析CLGP3、CLGP4及其脱除产物的重复单元结构和硫酸基团取代模式，解析硫酸寡糖的序列结构，明确海葡萄硫酸多糖的精细结构；利用荧光定量PCR和16S高通量测序技术从体内外系统研究和对比海葡萄硫酸多糖结构对肠道菌群组成和多样性的影响，全面揭示海葡萄硫酸多糖调节肠道菌群的确切构效关系。本项目将为新型食用海藻的营养评价和开发利用提供科学依据。

海藻是海洋生物资源的重要组成部分，各种食用海藻已成为居民膳食的重要组成部分。海藻多糖与陆生植物多糖最大的不同是海藻多糖主要是硫酸多糖，独特的结构赋予了海藻多糖具有多种生物活性，是健康食品的新型基料和优良的膳食补充剂。海葡萄学名长茎葡萄蕨藻（Caulerpa lentillifera），属于绿藻门（Chlorophyta）蕨藻科（Caulerpaceae）蕨藻属（Caulerpa），其营养丰富，是一种宝贵的可食用绿藻。海葡萄富含多糖，尤其是硫酸多糖，但国内外有关海葡萄硫酸多糖的研究起步较晚，其构效关系研究尚不充分，仍有待深入研究。据此，本项目以海葡萄为原料，通过建立的海葡萄硫酸多糖硫酸基团、支链的可控性脱除方法，结合NMR、甲基化、GC-MS等技术，成功解析了海葡萄硫酸多糖组分CLGP4的精细结构，推测CLGP4为多分支的木糖半乳甘露聚糖，主要以→4)Manp(1→重复单元为主链结构，并且在→4)Manp(1→残基的C-2位存在分支；分支糖链由→2)Manp(1→、→3)Galp(1→和→4)Xylp(1→以及末端Gal和Xyl组成；有20%的硫酸基团处于→4)Xylp(1→残基的C-3位上，有40%的硫酸基团处于→3)Galp(1→残基的C-6 位上，有40%的硫酸基团处于→2)Manp(1→残基的C-3位上；继而，从体外和体内两方面研究了海葡萄多糖对肠道菌群的调节作用，发现海葡萄多糖不仅可提高SCFAs的产量，还能够改变肠道菌群的结构，促进能够降解和代谢多糖的肠道有益菌生长，抑制有害菌生长；此外，通过与硫酸半乳聚糖（卡拉胶寡糖）做对照，对比了不同种类硫酸多糖对肠道菌群的作用效果，发现不同结构的硫酸多糖对肠道菌群的影响存在差异性。该项目为阐明功能海藻硫酸多糖的构效关系奠定了理论基础，也为海葡萄资源的综合开发、利用提供了科学依据。