发酵香肠中多酚类群体感应抑制剂对产气肠杆菌生物胺代谢的调控机制

Fermented sausage is a kind of traditional Chinese fermented meat products, but it contents high concentration of biogenic amines, which is a potential carcinogen. Enterobacter aerogenes is the main biogenic amine-producing bacteria in fermented sausage. The amine-producing capacity of E. aerogenes relates to acyl-homoserine lactones (AHLs) quorum sensing (QS). Rose polyphenols have high quorum-sensing inhibition activities and can decrease the formation of biogenic amines. Although biogenic amine reduction activity of the rose polyphenols have been confirmed, the activity of each type of polyphenol is still unclear. The aim of this project is to study the regulation mechanism of polyphenolic quorum sensing inhibitor on the biogenic amine metabolites of E. aerogenes during sausages fermentation. According to the rose polyphenols types, the activity of these polyphenolic quorum sensing inhibitors are evaluated to screen a kind of polyphenol with a high capacity of biogenic amine reduction. A strain of E. aerogenes with AHL-degrading activity is obtained by aiiA gene transduction. Finally, quench the quorum sensing system by degradation or interference of quorum sensing signal. After quorum quenching, the differences of bacterial activity, metabolite, quorum sensing, and protein expression are determined. The regulation mechanism of polyphenolic quorum sensing inhibitor on the biogenic amine metabolites of E. aerogenes in fermented sausages is studied by proteomics to develop a novel food safety control by quorum-sensing inhibition.

发酵香肠是我国传统发酵肉制品的典型代表，但其生物胺含量偏高，存在潜在的致癌性。现有研究已证实产气肠杆菌是发酵香肠中最主要的一种产生物胺细菌，其产生物胺特性受酰基高丝氨酸内酯（acyl-homoserine lactones，AHL）型群体感应（quorum sensing，QS）调控。来源于玫瑰花的多酚类QS抑制剂能降低产气肠杆菌的产生物胺能力，但前期研究对象为多酚混合体，多酚单体的降生物胺活性及其调控机制尚未确证，本项目针对此立项研究。首先对玫瑰多酚中各种多酚单体的活性进行比较，筛选出降生物胺活性较高的多酚单体；然后将aiiA基因导入产气肠杆菌，构建可降解信号分子的重组菌；最后通过降解信号分子和干扰信号分子传导两个途径，分析菌体活性、代谢产物、群体感应及蛋白质表达的差异。目的是从蛋白质组学角度阐明多酚类QS抑制剂对产气肠杆菌生物胺代谢的调控机制，为开发新型食品微生物控制技术提供理论依据。

近年来研究证实食源性细菌可通过群体感应系统调控与食品质量安全相关的菌群特性。本项目以发酵香肠为研究对象，根据其菌相分布情况，从降解信号分子和干扰信号分子传递两个角度进行全面的研究，以探究AHLs在细菌产生物胺中的作用，阐释多酚单体构象与其降生物胺活性之间的构效关系，并揭示多酚类群体感应抑制剂在发酵香肠生物胺调控中的应用价值。研究内容主要分六个部分：发酵香肠微生物多样性分析、发酵香肠中肠杆菌群体感应系统检测、多酚类群体感应抑制剂特性研究、多酚结构与群体感应抑制活性之间的构效关系、群体感应抑制剂对发酵香肠食用安全性的影响、群体感应抑制剂对发酵香肠产胺菌代谢的调控。研究发现玫瑰多酚群体感应抑制剂在酸性条件下性质稳定，在中性条件下其生物活性下降。儿茶素是玫瑰多酚中含量较高的酚类单体，其对产气肠杆菌产生物胺具有强抑制作用，有效浓度可低至0.078mg/L。群体感应抑制剂可使发酵体系内依赖于群体感应系统的肠杆菌、假单胞菌等细菌的数量及种类降低，而有助于乳酸菌的富集。通过外源加强AHLs信号、AiiA酶降解信号分子、抑制剂干扰AHLs三方面研究发现，干扰AHL信号分子的传导能有效抑制产气肠杆菌产腐胺、尸胺、组胺和酪胺的能力。其中，儿茶素通过降低产气肠杆菌的氨基酸代谢能力及蛋白酶活性，抑制产气肠杆菌的产胺能力。研究结果证实利用群体感应抑制剂，可抑制生物胺的积累，优化微生态体系，提高产品安全性的目的。群体感应调控可作为一种新型食品微生物安全防控技术，群体感应抑制剂具有重要的开发意义和广阔的应用前景。

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