传统黄酒酿造中生物胺形成的分子基础及调控机制

Biogenic amines (BAs), which have potential toxicity and the indicator of the hygeian conditions，are frequently found in fermented foods. Relatively high levels of BAs have been reported in Chinese rice wines. There is a synergic effect between BAs and alcohol, which enhances the toxic effects of BAs. However, there are few studies on the identification and regulation of biogenic amines in Chinese rice wines. It is necessary to further study the formation and regulation mechanism of biogenic amines in Chinese rice wines. In view of this, the project takes the typical traditional shaoxing rice wines as the research objects. To illustrate the change rule of biogenic amines during the brewing of Chinese rice wines, the amino acid decarboxylase and amine oxidase gene expression, corresponding enzyme activities, the content of biogenic amines and their precursor amino acids were determinated by fluorescence quantitative PCR, modern enzymology and chromatography/mass spectrometry methods, respectively. To resolve the relationships between microbial communities structure and the content of biogenic amines, the microbial communities during the brewing of shaoxing rice wines were monitored by using culture-dependent and culture-independent methods. To analyze the metabolic pathways and key regulatory genes closely related to biogenic amine formation, the metabolic networks of the key culturable microorganisms were constructed. Based on these knowledge, the effect of the raw material composition, process conditions and the microbial community structure on the synthesis and degradation of biogenic amines during the brewing of shaoxing rice wine were clarified. The project aims to clarify the molecular and regulatory mechanism of biogenic amines formation during the brewing of traditional Chinese rice wines, to improve the safety of Chinese rice wines, and also to provide research ideas for the control of other metabolic hazards in traditional fermented foods.

生物胺普遍存在于发酵食品中，具有潜在的毒性和对卫生条件的指示作用。黄酒中生物胺含量较其它酒高，酒中的乙醇对生物胺的毒性有协同增效作用，而国内外对其中生物胺的研究较少，有必要深入研究黄酒中生物胺的形成和调控机制。为此，本课题选用代表性的传统绍兴黄酒为对象，采用荧光定量PCR、现代酶学和色谱/质谱等技术，检测氨基酸脱羧酶/胺氧化酶基因的表达、相应酶的活性和生物胺及其前体氨基酸的含量，揭示黄酒酿造中生物胺的变化规律；采用免培养分子生态学技术、结合经典微生物分离培养方法探究黄酒酿造中的菌群演替，解析菌群结构与生物胺含量之间的关联。选取可培养的关键生物胺产生菌，构建代谢网络，分析与生物胺形成密切相关的代谢途径及调控基因，研究原料组分、工艺条件和菌群结构对生物胺合成与降解的影响。课题旨在阐明黄酒酿造中生物胺形成的分子基础和调控机制，为提升黄酒的安全性奠定基础，同时也为其它代谢危害物的调控提供研究思路。

黄酒中生物胺含量较其它酒高，酒中的乙醇对生物胺的毒性有协同增效作用，而国内外对其中生物胺的研究较少，有必要深入研究黄酒中生物胺的形成和调控机制。本项目所做工作主要包括以下方面：①建立了黄酒中多种生物胺检测的SPE-HPLC-UV方法。该方法针对复杂的黄酒基质，采用固相萃取有效降低了基质效应对生物胺检测的影响，而且方法简单，设备普适性强，可用于黄酒中7种生物胺的常规监测；②调研了不同产区黄酒中的生物胺和氨基酸含量，并结合多元统计分析方法对黄酒产地进行区分判别，所建立的模型可以为黄酒的鉴伪和原产地保护提供参考方法；③建立了能够同时检测酪胺、腐胺和组胺产生菌的多重PCR方法，该方法特异性强，可应用于酒曲安全预警及生物胺产生菌的检测。④建立了定量检测组胺、腐胺和酪胺产生菌的荧光实时定量PCR检测方法，该方法灵敏度高，特异性强，可实时监测黄酒酿造过程中生物胺产生菌的变化情况。⑤利用聚合酶链反应-梯度凝胶电泳结合第一代测序以及高通量测序两种方法研究黄酒曲中细菌和真菌多样性。发现不同酒曲微生物多样性差异很大，麦曲中细菌和真菌的多样性均最为丰富；⑥从表型到基因型系统系统研究了环境因素对屎肠球菌产酪胺的影响，该结果对黄酒和其它发酵制品中酪胺的控制提供理论参考；⑦采用脱羧酶培养基从黄酒酒曲中分离组胺产生菌，并采用HPLC和PCR方法进行确认。采用随机扩增的多态性DNA方法对组胺产生菌分型并采用16S rRNA序列，rpoB或dnaJ基因对单型组胺产生菌分析鉴定，发现所有组胺产生菌均属于肠杆菌科。这些菌株在含8%乙醇的TS培养基中均无法生长。小规模黄酒发酵表明，无论采用何种酒曲，在发酵初期，发酵酒样中的组胺含量与肠杆菌科的相关性比其与乳酸菌的相关性更强。选择肠杆菌科细菌含量较低的酒曲酿造，并且抑制发酵早期的肠杆菌科细菌的生长是降低黄酒中组胺含量的有效方法。