Na+/H+胁迫影响粪肠球菌酪胺产生的分子机制

The excessive intake of biogenic amines can cause adverse physiological effects to humans, sometimes even life-threatening. In view of the high tyramine detection rate and content in meat products, study on its generation mechanism will help to ensure the quality and safety of the meat products. Based on our previous research, the applicants determined Enterococcus faecalis is the main tyramine producing bacteria in water-boiled salted duck and isolated one E. faecalis strain designated as E. faecalis R612-Z1. Its tyramine generating capacity is influenced by pH and NaCl concentration. This project focuses on this strain, genome sequencing and revers-transcript PCR will be used first to determine the encoding genes of the tyrosine decarboxylation pathway and their transcription patterns, after that heterologous expression will be used to study the activity of the main functional areas within the pathway under Na+/H+ stress; And then Real-time PCR and HPLC will be employed to detect the expression differences of the tyrosine decarboxylase gene, the tyramine production and the strain biomass under Na+/H+ stress as well as their correlation, combined transcriptome analysis with the energy level analysis within the cell determined by fluorescence detection to determine the relationship between the intracellular environment and the tyramine production. Comprehensive consideration of the effects of Na+/H+ stress on both the tyrosine decarboxylase pathway itself and the intracellular environment, the molecular mechanisms of how Na+/H+ stress affects the tyramine production in E. faecalis will be clarified. The results of this research will provide theoretical basis to the tyramine regulation in meat products.

过量摄入生物胺会引起人体的不良生理反应，甚至危及生命。鉴于酪胺在肉制品中较高的检出率和含量，对其产生机理的研究将有助于保障肉品的质量安全。前期研究发现E. faecalis是盐水鸭中的主要产酪胺菌，且分离到一株E. faecalis R612-Z1，其酪胺产生受pH和NaCl浓度的影响。本课题以该菌为研究对象，首先利用基因组测序和RT-PCR方法确定酪氨酸脱羧通路的主要编码基因及其转录方式，通过外源重组表达研究该通路各功能区在Na+/H+胁迫下的活性；然后利用Real-time PCR和HPLC测定胁迫时酪氨酸脱羧酶基因表达量、酪胺产量和菌株生物量的变化及相关性，结合转录组学分析，配合荧光检测判定胞内的能量水平，确定胞内大环境与酪胺产生的关系。综合通路本身和胞内环境两方面结果，阐明Na+/H+胁迫影响E. faecalis酪胺产生的分子机制。研究成果将为肉制品中酪胺产生的调控提供理论依据。

过量摄入生物胺会引起人体的不良生理反应，甚至危及生命。鉴于酪胺在肉制品中较高的检出率和含量，对其产生机理的研究将有助于保障肉品的质量安全。前期研究发现E. faecalis是盐水鸭中的主要产酪胺菌，且分离到一株E. faecalis R612-Z1，其酪胺产生受pH 和NaCl 浓度的影响。本课题以该菌为研究对象，首先利用基因组测序和RT-PCR方法确定酪氨酸脱羧通路的主要编码基因及其转录方式，发现E. faecalis酪氨酸脱羧通路中的主要基因酪氨酸脱羧酶基因tyrDC、转运蛋白基因tyrP及Na+/H+反向转运蛋白基因nhaC-2位于同一操纵子上，并且酪氨酸脱羧酶在蛋白结构上存在有磷酸吡哆醛结合位点。外源表达了E. faecalis和E. faecium的TyrDC蛋白，发现两者均对酪氨酸和苯丙氨酸具有催化活性，最适底物均为酪氨酸，最适温度为25 ℃，pH为5.5~6.0。NaCl浓度对酶催化酪氨酸的活性影响不大，但是对两个酶催化苯丙氨酸的活性均有很强的抑制作用。高浓度NaCl和强酸条件均能刺激单位菌体E. faecalis酪氨酸脱羧通路活性，从而增强其产酪胺能力。通过转录组测序发现，Na+/H+胁迫能够引起与氨基酸代谢、核酸代谢与能量代谢相关的通路的表达量变化，这些代谢通路的改变都可能影响了菌体的整体应答与酪胺的产生。根据转录组的数据，进一步研究发现菌体在Na+/H+胁迫下代谢精氨酸的能力提高，说明精氨酸脱亚胺酶途径对粪肠球菌R-612-Z1适应胁迫环境起着重要的作用。由于E. faecalis代谢产生酪胺会引起食品的质量安全，本项目实施过程中研究了nisin与ε-pL抑制菌体生长和酪胺产生的协同作用，研究发现两者主要通过影响细胞膜电势来破坏细胞膜结构达到协同抗菌作用，能够有效的抑制肉制品中的E. faecalis的生长及酪胺的产生。研究成果将为肉制品中酪胺产生的调控提供理论依据。