茯茶中冠突散囊菌作用下的代谢物变化及形成机理研究

Eurotium cristatum is the dominating microorganism during the processing of Fuzhuan tea. It is also the source of catalyzing enzyme for the quality characteristic of Fuzhuan tea. However, there are few studies on the change and formation of metabolites in Fuzhuan tea during fermentation by Eurotium cristatum, which significantly restrict the development of research on the mechanism for the formation of the quality characteristic and bioactive components of Fuzhuan tea. In this project, Eurotium cristatum are used as single strain culture and sterile raw dark green tea are used as substrate for the solid state fermentation. HPLC-MS/MS and HPLC-NMR are used for analyzing the difference of chemical constitution between the samples before and after fermentation. According to the molecular weight and spectrographic information of metabolites, standards, database and reference, the difference metabolites will be identified and the "characteristic metabolites", which derived from catechin, theanine and alkaloid, are selected. The origin of substrate and the biosynthetic pathway of "characteristic metabolites" are clarified by liquid state fermentation, which used various combinations of catechin, theanine and alkaloid as substrate. The enzyme catalyzing the change of metabolites and the biosynthesis of "characteristic metabolites" are studied by the proteomic technology. This objective of this project is revealing the mechanism for the change and formation of metabolites in Fuzhuan tea during fermentation by Eurotium cristatum and is providing a theoretical basis for the researchs on the mechanism for the formation of the quality characteristic and comprehensive exploitation of Fuzhuan tea.

冠突散囊菌是茯茶加工过程中的优势微生物，是茯茶品质形成的主要生化动力来源，但关于茯茶中冠突散囊菌作用下的代谢物变化及形成的研究较少，严重阻碍了茯茶品质风味形成机理及功能活性成分的深入研究。本项目拟采用项目组前期分离的茯茶优势微生物-冠突散囊菌为单一发酵菌株，以无菌黑毛茶为底物进行固态发酵。采用HPLC-MS/MS和HPLC-NMR联用技术分析发酵前后茶叶化学物质组成差异；根据分子质量信息和光谱信息结合标准品、数据库、文献等对差异代谢物进行鉴定，并筛选出由儿茶素/茶氨酸/生物碱衍生形成的"特征代谢物"；通过儿茶素/茶氨酸/生物碱不同组合为底物进行液态发酵，明确"特征代谢物"的底物来源及生物合成途径；通过蛋白质组学技术研究催化茯茶中代谢物变化与"特征代谢物"形成的微生物酶；以期探明茯茶中冠突散囊菌作用下的代谢物变化及形成机理，为揭示茯茶品质风味形成机理及茯茶深度开发利用奠定理论基础。

本项目采用冠突散囊菌为单一菌种进行固态发酵，研究固态发酵前后差异代谢产物及其形成机制。结果表明，单菌固态发酵前后共鉴定49种差异代谢物。另有部分单菌固态发酵后差异代谢物含量较低且不稳定，未能成功鉴定。我们推测，茯茶加工过程中微生物代谢的衍生产物可能不仅仅是由冠突散囊菌单一菌催化产生，而是在多种核心功能微生物共同催化下形成。因此，对传统茯茶加工过程中代谢产物的变化也进行分析，鉴定茯茶中94种非挥发性成分，其中56种为差异代谢物。同时，筛选到16种与冠突散囊菌密切相关的代谢产物（冠突散囊菌大量生长后其含量显著增加直至加工完成），检索所有公开数据库及文献报道均未能鉴定，推测可能是冠突散囊菌催化形成的新型化合物，需进一步纯化制备后进行核磁和红外光谱鉴定。.由于核心功能微生物组对茯茶物质转化的重要性，我们也系统研究了从茶鲜叶至成品茯茶整个加工过程中微生物群落的动态变化。结果表明，黑毛茶“渥堆”过程中前期真菌群落以Cyberlindnera属、Aspergillus属、Uwebraunia属、未知分类的Pleosporales目真菌属为优势菌属，后期以Cyberlindnera属为优势菌属；而细菌群落前期以Klebsiella属、Lactobacillus属细菌群落为优势菌属，后期以Klebsiella属为优势菌属。茯砖茶压制加工过程中，前期真菌群落以Aspergillus属、Cyberlindnera属和Candida属为优势菌属，加工后期以Aspergillus属为优势菌属。真菌群落在茯茶加工过程中起主导作用。.冠突散囊菌对茶叶主要生化成分的生物转化研究结果表明，冠突散囊菌可分泌胞外酶水解EGCG为EGC和没食子酸。利用茶氨酸作为维持自身生长繁殖的营养基础，促进菌体的生长并能延缓菌体的自溶，其含量在发酵液中迅速下降可能是被由于冠突散囊菌分泌的胞外酶水解所致。咖啡碱、可可碱和茶碱均能在一定程度上刺激冠突散囊菌的生长，但不能被冠突散囊菌代谢分解。同时，冠突散囊菌可催化可可碱和茶碱合成咖啡碱。