旧金山乳杆菌合成乙酯类香气物质的关键基因和代谢途径解析

Ethyl esters produced by lactic acid bacteria (LAB) during sourdough fermentation are important flavor compounds in flour-based fermented foods. However, the metabolic mechanism on the synthesis of ethyl esters by sourdough LAB is still unclear, which restricts the application of LAB in the flour-based fermented food industry. On the basis of the preliminary work, this project takes the most representative LAB in traditional sourdough, i.e., Lactobacillus sanfranciscensis, as the research object, and uses the specific simulated catalytic system containing cell-free extract to determine the way of synthesis of ethyl ester and the type of the key enzyme needed; separates and purifies the key enzyme by ion exchange chromatography, hydrophobic interaction, gel filtration, etc., and characterizes its enzymatic properties; based on the annotation proteins of the whole genome of L. sanfranciscensis, combines N-terminal sequencing with MALDI-TOF/TOF sequencing of the purified enzyme to locate key genes required for the synthesis of ethyl esters; constructs key gene-deleted strains by gene knockout to verify the key role of the gene in ethyl ester synthesis, and finally confirms the key gene (key enzyme) for ethyl ester synthesis; illuminates the complete metabolic pathway of the synthesis of ethyl esters in L. sanfranciscensis with the help of KEGG analysis and Geno ontology analysis. The project aims to provide a theoretical basis for the metabolic regulation of ethyl esters synthesis during sourdough fermentation and the development of dough fermenting agents with excellent flavor properties.

乳酸菌在酸面团发酵过程中代谢产生的乙酯类物质是发酵面食中重要的风味成分，然而目前对酸面团乳酸菌产乙酯类物质的相关代谢机理仍不清晰，制约了乳酸菌在发酵面制品行业的应用。本项目在前期工作的基础上，以传统酸面团中最具代表性的乳酸菌旧金山乳杆菌为研究对象，采用含无细胞提取物的特定模拟催化体系，确定乙酯合成的方式和关键酶的种类；利用离子交换色谱、疏水相互作用、凝胶过滤等技术手段分离纯化关键酶，并表征其酶学性质；结合N端测序和MALDI-TOF/TOF测序，基于旧金山乳杆菌全基因组注释蛋白，获得关键酶序列信息，并锁定关键基因；通过基因敲除构建关键基因缺失菌株，验证其在乙酯合成中的关键作用，最终确定关键基因（关键酶）；借助KEGG和GO分析，阐明旧金山乳杆菌产乙酯类物质的完整代谢通路。本项目旨在为实现面团发酵过程中乙酯类物质的代谢调控及开发风味性能优良的面食发酵剂提供理论基础。

乳酸菌在酸面团发酵过程中产生乙酯类物质的相关代谢机理不清晰，制约了乳酸菌在发酵面制品行业的应用。本项目以传统酸面团中最具代表性的乳酸菌旧金山乳杆菌为研究对象，对其代谢产生乙酸乙酯的关键基因和途径进行了解析。本研究并筛选出了两株高产乙酸乙酯的旧金山乳杆菌，并确定了乙酸乙酯合成的关键时间点，通过添加不同乙酸乙酯合成的前体物质，结果显示加入低浓度（2mM）乙醇明显可以提高旧金山乳杆菌对乙酸乙酯的产量。基于转录组学研究和KEGG和GO分析，分析挖掘了旧金山乳杆菌合成乙酸乙酯的关键基因，其中参与丙酮酸代谢的乙醇脱氢酶基因（LS451_RS06270）发生了显著变化，表明乙醇刺激了乙醇脱氢酶(ADH)的活性，促进了乙醇、乙醛和丙酮酸的转化，进一步合成乙酸乙酯。进一步在发酵酸面团和馒头中，也发现低浓度的乙醇促进了其中乙酸乙酯的生成和风味的提升。本项目的完成将为实现面团发酵过程中乙酯类物质的代谢调控及开发风味性能优良的面食发酵剂提供理论基础。