泡菜工业化发酵生产过程中重要风味物质的形成机理及其代谢通路的解析研究

With the extension of fermentation time or the changing of vegetable varieties, the microbial community structure of the traditional Chinese Paocai changes constantly and becomes complex and diverse. Meanwhile, flavor compounds in Paocai are also gradually forming, changing and becoming complex; having a significant influence on the quality and the style stability of the Paocai. In order to find out the related enzyme genes about synthesizing metabolites during Paocai fermentation, metabonomics is used to investigate the change rules (including variety and quantity) of the products of microbial metabolism; metatranscriptomics is applied to reveal the dynamic changes of active microbes and the distribution of differentially expressed genes. Combining the active microbial composition with correlation analysis between functional gene and environmental factors, it would be helpful to reveal the response of microorganism in the gene expression and the metabolic regulation to different fermentation environmental conditions, interpreting the formation and change mechanism of the flavor, analysising the metabolic pathways of important flavors compounds in Paocai. Furthermore, the comparative analysis of functional genes during different fermentation stages of industrial Paocai, the screening out and the heterologous expression of key genes that could regulate the flavor compounds forming may contribute to shorten fermentation time and improve sensory quality of Paocai with the application of genetically engineered strains. This research may lay the theoretical and technical foundation for the industrialization production of Paocai.

中国传统泡菜在发酵生产过程中，随着发酵时间的延伸和投入蔬菜种类的改变，泡菜微生物群落结构组成不断发生演替，形成复杂多样的微生物多样性。同时，泡菜中的风味物质也逐渐形成和变化，决定传统泡菜生产的质量和风格的稳定性。基于代谢组学技术分析泡菜中微生物代谢产物种类及含量的变化规律，并进行发酵微生物宏转录组测序，研究泡菜发酵过程中活性微生物演替规律与差异表达基因的分布，能够找寻到代谢产物合成的关联酶基因。结合活性微生物及其功能基因与环境因子的相关性分析，有利于全局反映泡菜微生物在基因表达和代谢通路调控水平上对不同发酵环境条件的响应规律，揭示泡菜风味物质的形成和变化机制，解析重要风味物质的代谢通路；结合比较分析泡菜工业化生产不同发酵阶段的功能基因，筛选出调控风味物质的显著关键基因并进行异源表达，有利于借助基因工程菌缩短泡菜发酵时间，改善感官品质，为泡菜工业化生产发酵理论的提升与发酵技术的进步奠定基础。

泡菜在发酵生产过程中微生物群落结构组成不断发生演替，形成复杂多样的微生物多样性，微生物菌群的演变关系到泡菜品质。采用多组学技术研究泡菜发酵过程中活性微生物演替规律与差异表达基因的分布，能够找寻到代谢产物合成的关联酶基因。结合活性微生物及其功能基因与环境因子的相关性分析，有利于全局反映泡菜微生物在基因表达和代谢通路调控水平上对不同发酵环境条件的响应规律，揭示泡菜风味物质的形成和变化机制，解析重要风味物质的代谢通路；结合比较分析泡菜工业化生产不同发酵阶段的功能基因，筛选出调控风味物质的显著关键基因并进行异源表达。本项目采用宏转录组测序技术、qRT-PCR、PCA、RDA、PLS-DA分析、克隆及异源表达等技术分析不同工厂、不同原料泡菜理化性质检测、风味分析与宏转录组测序。初步揭示了工业规模泡菜关键风味形成与功能微生物之间相互关系，为泡菜工业化生产发酵理论的提升与发酵技术的进步奠定基础。采用代谢组学、宏转录组学方法分析工业化生产泡豇豆、泡萝卜、泡青菜不同发酵阶段关键风味物质、活性微生物演替规律与差异表达基因分布。用RT-qPCR和代谢组学检测进行差异表达基因的表达量和生理验证，并结合活性微生物/功能基因与环境因子的相关性分析。挖掘调控挥发酚类、苯乙醇等香气成分形成的关键酶，筛选出了催化羟基肉桂酸类物质脱羧的3个酶基因（DhFDC1、LvPAD和LvUbiD）并进行异源表达，提高了对传统发酵泡菜中微生物的群落演替和风味物质形成机制的认识，为改善泡菜的感官及品质奠定理论和工业生产技术基础。