中国白酒中异味萜类化合物产生微生物的代谢机制及其对酿造微生物群落结构调控的研究

Chinese light-style liquor is one of the most popular traditional alcoholic beverages all over the word, and is famous for natural and refreshing flavor. It has been demonstrated that earthy off-flavor in light-aroma type Chinese liquor is highly detrimental to liquor aromatic quality in the early research on Fen-flavor liquor. However, it is still unknown about the source and the metabolic mechanism of the off-flavor. Therefore, it would be significant to scientifically investigate into the biosynthesis mechanism of earthy off-flavor, and the role of earthy off-flavor producers on redistribution of microbial structure and fermentation performance..With the knowledge of the source of earthy off-flavor (GM), the project is going to obtain the GM microbial producers with the flavor-oriented technology. It will simulate the solid-state fermentation of liquor fermentation and then disturb the fermentation system, and then reveal the metabolic pathway and the regulation mechanism of GM biosynthesis. In addition, it will also investigate the role and the mechanism of GM producer on microbial structure and performance in liquor fermentation, by using the uncultured and quantitative analysis, systems biology technology. The project would not only explain the basic theory issues of off-flavor in liquor making process, but also improve the quality and safety of Chinese light-style liquor, and promote the development of the traditional liquor making process.

清香型白酒以其"清新爽净"的口感而闻名于世。尽管国家早在"汾酒试点"的研究表明：土霉异味是造成清香型白酒品质下降的主要原因，但对引起其产生的原因和机理并不清楚，严重影响清香型白酒品质和产业水平的提升。因此，科学解析清香型白酒中土霉味微生物的代谢机制及其对酿造微生物群落结构与功能的影响，对于丰富中国白酒独特酿造的理论基础与实践具有重要价值。.本研究拟在前期明确土霉味萜类物质化学本质(GM)的基础上，采用风味定向筛选技术，获得GM产生主要微生物菌群；通过白酒酿造模拟体系的构建及干预，认识"多菌种"固态发酵体系中GM合成代谢途径及调控机制；同时，基于未培养定量技术以及系统生物学技术的应用，解析GM产生微生物对酿造微生物群落结构及功能的影响及其机制。本研究不但能够解决清香型白酒生产过程中的关键基础理论问题，而且对于清香型白酒品质与安全的提升，以及传统白酒酿造产业的技术升级都具有重要的理论与实践作用

本研究运用风味导向学术思想，首次明确了白酒中的土霉味物质的来源为微生物代谢合成。并采用现代分子生物学手段建立基于土味素关键合成酶编码基因实时荧光定量PCR方法对产土味素的链霉菌进行快速检测和跟踪。发现并证实产土味素链霉菌的重要代谢物对固态发酵微生物群落结构的调节规律和机制，建立了揭示中国白酒复杂体系中异味物质微生物产生机理的研究方法。运用风味导向技术，从复杂的白酒酿造菌群结构中成功筛选得到五株产生明显土霉气味的土味素代谢菌株。根据其菌落、细胞形态和生理生化特征，以及放线菌/链霉菌特异分类鉴定引物鉴定，确定这五株菌株均为链霉菌（Streptomyces）。系统研究白酒异常酿造微生态中各菌种间生长规律，发现产土味素链霉菌与白酒酿造中的功能微生物有明显的竞争关系。经过进一步超高压液相色谱-四级杆飞行时间串联质谱（UPLC-Q-TOF-MS/MS）检测，分子模拟，添加标准品等方式，确定抗真菌活性物质为一系列七烯大环内酯类同系物的抗生素。从而明确了产土霉异味链霉菌对白酒酿造群落结构的影响机制。通过考察不同微生物中的土味素合成酶编码基因的同源性，建立了基于土味素合成酶编码基因保守序列的实时荧光定量PCR方法。可实现在白酒酿造复杂基质中与土霉异味直接相关的灵敏、快速的生物定量分析，为可疑风味污染源预测、传播环境调查、产生菌溯源等工作奠定基础。