己酸二元发酵体系中甲烷菌促进己酸生成的机制研究

In the traditional fermentation of Luzhou-flavor liquor, caproic acid bacteria is one of the most important acid producing microbes, as it produced caproic acid to react with ethanol, thereafter caused the formation of ethyl caproate, which is the dominant aromatic ingredient in Luzhou-flavor liquor. It was reported that caproic acid was mainly formed by the mutual interactions of caproic acid-producing bacteria with methanogenic bacteria in the pit. However, the mechanism was rarely reported. Therefore, based on our provous study, we would attempt to explore the mutual interaction mechanisms of the two selected bacteries during the caproic acid fermentation process and the plan of research are as follows: (1) Using the molecular biological technology such as 16S rDNA method, in combination with the traditional morphological, physiological and biochemical characteristics to identify the two obtained microorganisms; (2) Real-time PCR will be applied to study the quantitation of the two groups in different time during caproic acid fermentation; (3) The specific methanogenic inhibitors will be used to explore the mechanism of the methanogenic bacteria involed in the caproic acid acculumation; (4) Using the metabolomics technology and in combination with metabolic pathways analysis to explore the relationship between the key metabolites of the the two selected bacteries in binary system of caproic acid fermentation.

己酸菌是传统浓香型白酒酿造过程中最重要的功能菌，其所产生的己酸与乙醇发生酯化反应，生成浓香型白酒的主体香成分己酸乙酯。研究表明，在发酵窖池内，甲烷菌的存在促进了己酸的生成。但关于其作用的机制，目前尚少见报导。鉴于此，申请人利用前期工作中获得的由己酸菌和甲烷菌组成的二元发酵体系为研究对象，探索甲烷菌促进己酸生成的机制。研究内容包括：（1）通过16S rDNA 技术等分子生物学技术，并结合形态学、生理学等常规鉴定方法，将获得的两种微生物进行鉴定至种；（2）Real-time PCR研究二元体系发酵的不同时期两种微生物的动态分布；（3）利用产甲烷抑制剂研究甲烷菌对促进二元体系发酵中己酸累积的贡献；（4）利用代谢组学技术，结合代谢途径分析来探索二元体系发酵中两菌间关键代谢物关系。

己酸菌是传统浓香型白酒酿造过程中最重要的功能菌，其所产生的己酸与乙醇发生酯化反应，生成浓香型白酒的主体香成分己酸乙酯。研究表明，在发酵窖池内，甲烷菌的存在促进了己酸的生成。但关于其作用的机制，目前尚少见报导。鉴于此，申请人利用前期工作中获得的由己酸菌和甲烷菌组成的二元发酵体系为研究对象，解析甲烷促进己酸生成的机制。本项目开发一种生产己酸的新工艺，并将其应用于中国浓香型白酒的酿造。结果表明产甲烷菌166可以将C.Kluyveri H068产生的积累的H2转化为甲烷，从而消除了氢对C.Kluyveri H068的产物抑制，提高了己酸的产量。此外，通过实验室规模实验，对二元发酵体系进行了优化，结果表明，以10%的混合接种量和2:1（v/v）、20 g/l的醋酸钠浓度、4%的乙醇含量为最佳条件，确定了己酸生产的最佳条件。%（v/v）和酵母提取物浓度为10g/L。此外，将优化条件扩大到1000 L发酵罐，并对所得己酸发酵液进行回窖发酵。结果表明，该方法能有效地提高我国浓香型白酒的品质。