传统食醋中四甲基吡嗪形成机制及其代谢调控

Tetramethylpyrazine is an important flavor substance in many kinds of traditional fermented food, and it is also the functional component of brewed vinegar, whose process feature is solid-state fermentation process participated with multi-strains. It is thought that tetramethylpyrazine is the product of maillard reaction, while its microbial synthesis pathway and the source of precursors are still unclear, and this lead to a big defect of production control technology. The purpose of the present study is to reveal the accumulation regularity of tetramethylpyrazine systematically using HS-SPME-GC-MS and HPLC methodology in the entire process of Shanxi aged vinegar, a geographical indication product in China. The composition of microbial community will be investigated with high-throughput sequencing and DGGE, and the correlation between microbial community and biosynthesis of tetramethylpyrazine will be further studied. The production strains of tetramethylpyrazine will be screened and the biosynthesis pathway will be verified with methodology of isotope labeling and metabolic flux. The fermentation of tetramethylpyrazine and precursors of maillard reaction will be analyzed using isotope labelling technique in order to clarify the formation mechanism and metabolic control strategy of tetramethylpyrazine. Research results will provide theoretical basis and technical support for vinegar industry to establish the production and control technology of tetramethylpyrazine in urgent need. It is advantageous to control the indicators of functional components in vinegar effectively and to ensure the product quality. The results will be helpful for improving the product value and market position of traditional vinegar.

四甲基吡嗪是多种传统发酵食品的重要风味物质，也是以多菌种参与、固态发酵为工艺特征的酿造型食醋的功效组分。四甲基吡嗪的形成多认为是美拉德反应产物，而对微生物合成途径以及前提物的来源缺乏认识，导致生产控制技术存在较大缺陷。本课题以我国地理标志产品山西老陈醋为研究对象，采用HS-SPME-GC-MS、HPLC等分析技术，系统揭示四甲基吡嗪在食醋生产全过程的演替规律；采用宏基因组学高通量测序与DGGE等分子生物学技术解析微生物群落组成以及与四甲基吡嗪合成量之间的关联性；分离并验证四甲基吡嗪产生菌，采用同位素代谢流分析技术解析其合成代谢途径；采用同位素标记技术，分析四甲基吡嗪发酵与美拉德反应的前体物，以期阐明四甲基吡嗪的形成机制和代谢调控策略。研究成果将为建立食醋行业急需的四甲基吡嗪生产调控技术提供理论依据和技术支撑，有利于有效控制食醋的功效组分指标并保障产品质量，提升传统食醋产品价值和市场地位。

四甲基吡嗪（Tetramethylpyrazine，TMP）是山西老陈醋等传统食醋中重要的功能和风味物质，本项目以山西老陈醋为例，重点围绕传统食醋发酵过程TMP及其相关前体物代谢途径展开研究。对山西老陈醋酿造过程化合物组成进行了系统分析，从中筛选并验证了24种TMP相关前体物，其中含C类化合物对TMP形成贡献较大的主要为乙偶姻、葡萄糖，含N类化合物贡献较大的主要为NH4+、谷氨酰胺。醋酸发酵阶段是TMP前体物积累的主要阶段，熏醅阶段是TMP生成的主要阶段。.利用宏基因组学方法构建了山西老陈醋发酵过程TMP及其相关前体物的代谢网络，确定了乙偶姻途径是TMP形成相关的关键代谢节点，关键酶主要有乙酰乳酸合成酶、乙酰乳酸脱羧酶、双乙酰还原酶、丁二醇脱氢酶，氨甲酰磷酸激酶等。相关微生物主要有Acetobacter pasteurianus、Streptococcus infantarius、Lactobacillus hilgardii、Lactobacillus delbrueckii、Komagataeibacter europaeus。利用宏转录组学方法对代谢网络的动态变化进行了分析，结果表明，总酸、乙醇、还原糖等条件对谷氨酰胺合成酶、氨甲酰磷酸激酶、双乙酰还原酶、丁二醇脱氢酶的表达有重要影响，其中还原糖和总酸能够促进上述4种酶的表达，乙醇对上述4种酶的表达有抑制作用。.分离纯化9株具有生成乙偶姻能力的微生物。分析了pH、有机酸等条件对乙偶姻代谢的影响，发现有机酸通过抑制微生物的生长，从而抑制了乙偶姻的生成。选育获得具有较好耐酸能力的菌株Lactobacillus plantarum AF1-9，利用该菌株进行了原位强化发酵实验，在醋酸发酵初期阶段添加L. plantarum AF1-9至109 CFU/100 g醋醅，发酵结束醋醅中乙偶姻浓度提高了24.1%，产品中TMP达到14.5 mg/100g醋醅，与对照相比提高了13.3%。.相关研究成果获天津市科技进步一等奖1项，发表论文14篇，其中SCI论文7篇，EI论文2篇；申请发明专利3项，其中授权1项。