含硫香料对常见食源性致病菌毒力消减作用的分子机制研究

Foodborne pathogens are critical in food safety risk, and usage of food additive is common control method. Sulfur-containing flavor compounds have become a very important class of flavors, in which sulfides have good antibacterial activities against common pathogens. However, how about antibacterial properties of other sulfur-containing flavors? How to attenuate the virulence of pathogens by sulfur-containing flavors with strong antibacterial effect? The answers of these questions remain uncertain. Therefore, concerning these questions the objective of this project is to elucidate the molecular mechanism involved in virulence attenuation of sulfur-containing flavors from the points of virulence factor expression and cellullar immunologic response. The tested foodborne pathogens are Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella typhimurium in this project. Firstly, a comprehensive and systematic screening of sulfur-containing flavors with strong antibacterial activities is conducted. The proper conditions for virulence attenuation of sulfur-containing flavors are determined by investigation of bacterium metabolic kinetics. Secondly, in order to determine the target virulence molecular, the effects of sulfur containing flavors on gene and protein expressions of virulence factors are investigated by the methods of RT-qPCR and Western blotting. Lastly, the effects of sulfur-containing flavors on cellullar immunologic response induced by pathogens and target toxin are analyzed by molecular biological methods, such as flow cytometry, gene chip, etc. Consequently, the studies of this project will provide foundational information for enriching food preservatives.

食源性致病菌是食品安全的重要风险隐患，运用食品添加剂是常用的控制手段。含硫香料是一类重要的食用香料，其中硫醚类香料对常见致病菌具有良好的抑菌效果。然而，其他种类的含硫香料的抑菌效果如何？具有强抑菌功效的含硫香料如何消减致病菌的毒力？目前仍未明确。针对上述问题，本项目拟以金黄色葡萄球菌、单增李斯特菌、大肠埃希氏菌O157:H7及鼠伤寒沙门氏菌为供试菌，从毒力因子表达和细胞免疫应答两方面研究含硫香料对致病菌的毒力消减作用的分子机制。首先，全面系统筛选具有高抑菌活性的含硫香料，通过细菌代谢动力学，明确含硫香料毒力消减作用的适宜条件。其次，采用RT-qPCR、Western blotting的方法，研究含硫香料对毒力因子基因和蛋白质表达的影响，明确靶点毒力分子。最后，采用流式细胞仪、基因芯片等分子生物学手段，研究含硫香料对致病菌及靶点毒素引起的细胞免疫应答的影响，为丰富食品防腐因子奠定研究基础。

食源性致病菌是食品安全的重要风险隐患，运用食品添加剂是常用的控制手段。本项目针对金黄色葡萄球菌、单增李斯特菌、大肠杆菌、沙门氏菌及副溶血性弧菌，采用多种含硫香料开展致病菌毒力消减作用研究。在进行抗食源性致病菌含硫香料的筛选及构效关系解析的基础上，进一步明确含硫香料对常见食源性致病菌代谢动力学的调节作用，并依据毒力基因表达和氧化应激明确含硫香料对常见食源性致病菌毒力消减作用的分子机制。通过筛选9种异硫氰酸类含硫香料，发现3-(甲硫基)丙基异硫氰酸酯、异硫氰酸苄酯和异硫氰酸苯乙酯对金黄色葡萄球菌、单增李斯特菌、副溶血性弧菌及沙门氏菌四种供试菌均有抑制作用，其中异硫氰酸苄酯作用效果最为突出，说明苄基的存在，对含硫香料的抑菌能力有重要贡献。针对食源性致病菌的代谢动力学研究，以产志贺毒素的大肠杆菌和产肠毒素的大肠杆菌作为供试菌株，选取异硫氰酸苄酯和异硫氰酸苯乙酯进行研究。扫描电镜结果显示经含硫香料处理的细菌形态出现凹陷，菌体总ATP水平下降，细胞外ATP水平上升。亚抑菌浓度的含硫香料可显著下调多种毒力基因的表达水平，包括志贺毒性大肠杆菌中的eaeA、stx2、ehxA，肠毒素性大肠杆中的estA，沙门氏菌中的hilA、hilC、hilD，副溶血性弧菌中tdh、 fliI、VPA0318、VP1057、VPA0243、VPA0202和金黄色葡萄球菌中cp5D、cp8F、nuc、spa、clf。利用转录组技术，进一步分析异硫氰酸苄酯对金黄色葡萄球菌和副溶血性弧菌的代谢调控作用，发现异硫氰酸苄酯处理金黄色葡萄球菌和副溶血性弧菌后，氧化还原过程是显著富集的生物过程。鉴于此，进一步以β-胡萝卜素降解为模型，对部分含硫香料的促氧化作用进行了研究。以上研究成果发表SCI/EI收录论文6篇、中文核心期刊论文1篇，培养硕士研究生4名，为丰富食品防腐因子奠定研究基础。