植物乳杆菌生物胺分解酶的表征及其在果酒中的作用机制研究

Biogenic amines are toxic metabolites that are formed during the wine-making process. They can cause serious health problems to human beings, and therefore are regarded as one of the most harmful toxic compounds in fruit wines, which will definitely lower the food safety level of the product. Among the current methods employed to control the content of biogenic amines in fruit wines, the use of biogenic amine-degrading enzymes seems the most attractive, due to the advantage of high effectiveness and low costs such method possesses. Lactobacillus plantarum has been proved to be an effective lactic acid bacterium that can be used for the degradation of biogenic amine in fruit wines. However, concerning the identity of the biogenic amine-degrading enzyme produced by L. plantarum, disputes have arisen, because different researchers viewed this enzyme as different enzymes, but until now no solid evidence has been provided. In order to solve the problem stated above, we aim to purify and characterize these amine-degrading enzymes from L. plantarum F4 which was isolated in our preliminary study and exhibited remarkable biogenic amine-degrading activity, and reveal the identity of these enzymes by comparing their nucleotide/amino sequences with the data from NCBI. In addition, the catalytic mechanism of the degradation of biogenic amines by the biogenic amine-degrading enzymes, and the related degradation kinetics will also be elucidated. The current study will provide evidence for the safe and wide application of L. plantarum related biogenic amine-degrading enzymes in fruit wine industry.

生物胺是果酒（葡萄酒）酿造过程中产生的一类不良代谢产物，是果酒中潜在的主要食品安全问题之一。利用胺分解酶消除果酒中的生物胺兼具高效性和经济性，是现有控制生物胺含量的策略中效果最佳的一种。植物乳杆菌的降胺活性已获得广泛认可，但有关其所产的胺分解酶的化学本质却引起了学术分歧，且有关该酶的催化机制尚不明了。本项目针对以上问题开展研究，以自主筛选的具有高降胺活性的L. plantarum F4为研究对象，拟通过分离纯化从受试菌株中获得多种胺分解酶纯品，解析其生化特征和酶学性质，并确认其中的主体酶。进而针对各种胺分解酶，克隆其全基因序列并进行序列比对，揭示其酶化学本质。此外，本项目还致力于阐明L. plantarum胺分解酶的催化机制，构建生物胺分解动力学模型，这对于确保胺分解酶在果酒中的安全性，进而推广应用具有重要的理论和实践意义。

生物胺是果酒酿造过程中产生的一类不良代谢产物，是果酒中的主要食品安全问题之一。利用植物乳杆菌胺分解酶来分解果酒中的生物胺是现有策略中效果最佳的一种，但是胺分解酶的化学本质存在学术分歧。本项目以自主筛选的具有高降胺活性的L. plantarum SGJ-24为研究对象，通过细胞破碎、硫酸铵沉淀、DEAE阴离子交换层析和Superdex 75凝胶过滤层析，分离获得了胺分解酶纯酶，SDS-PAGE显示为电泳纯，亚基分子量为36 kDa。该酶的N末端序列为SVKIGINGFGRIGRL，经Protein BLAST对比，揭示该酶的化学本质是3-磷酸甘油醛脱氢酶。该酶分解组胺的最适反应温度为40℃，在55℃以下较为稳定；最适pH是7.5，在pH 6.5 ~ pH 8.5条件下比较稳定；DTT、β-巯基乙醇、EDTA和Mg2+抑制了酶的活力；SDS、Fe2+、Fe3+、K+、Na+、Pb2+和Ca2+能够促进酶活力。项目在研期间共接收或发表期刊论文8篇，其中SCI收录3篇，EI收录1篇；获得授权国家发明专利2项；参加国际和国内学术会议5次；培养硕士研究生3名。