基于蛋白质组学的胡椒单萜类化合物对冷鲜肉中关键腐败菌的抑菌机制研究

Pepper monoterpenoids (PMT) have strong antibacterial activity against key spoilage bacteria in chilled fresh Meat. This project is aimed to observe the effect of PMT on cell integrity and elastic coefficient of bacteria by spectral structure analysis. The mechanisms of PMT against the bacterial cell wall and membrane will be explained by monitoring the change of cell permeability with in situ end labeling. Moreover, the mechanism of PMT on genetic materials will be genetically elucidated by conforming the effect of PMT on DNA synthesis by immune coprecipitation technique, conforming the binding site between PMT and DNA by proteomics and bioinformatics method, and monitoring the senior conformational changes of bound DNA by circular dichroism spectrum. Based on proteomics technology and pathway enrichment analysis of differential proteins, the action targets and mechanisms of PMT also will be defined by studying the changes of proteome in key spoilage bacteria affected by PMT. The antibacterial mechanism of PMT will be eventually clarified by verifying the effect of PMT on the differential proteins expression of the key spoilage organisms by bioinformatics technology and revealing the influencing mechanism of the pepper terpenoids on respiration suppression characteristics, action sites and energy metabolism of the key spoilage organisms at the molecular transcription level. The implementation of this project can lay a theoretical foundation for the intensive study and practical application of PMT in the future.

胡椒单萜类化合物(PMT)对冷鲜肉中关键腐败菌有较强的抑菌活性。本项目拟采用光谱结构解析技术观察PMT对菌体细胞完整性和弹性系数的影响，通过原位末端法等监测细胞壁和细胞膜通透性的变化，揭示PMT对细胞壁和细胞膜的作用机制。采用免疫共沉淀技术确定PMT对菌体DNA合成的影响，采用蛋白质组学和生物信息手段确定PMT与DNA的结合位点，采用圆二色谱监测结合后DNA 的高级构象的变化，从基因水平揭示PMT对遗传物质的作用机制。基于蛋白组学技术、差异蛋白的 Pathway 富集分析，研究PMT对关键腐败菌蛋白组变化，明确PMT的作用靶点和作用机制；通过生物信息学技术，验证PMT对关键腐败菌的相关差异蛋白的表达，从分子转录水平上揭示胡椒单萜类化合物对关键腐败菌呼吸作用抑制特性、作用部位以及能量代谢的影响机制，阐明PMT的抑菌机理，以期为后续PMT的深入研究和实际应用奠定理论基础。

胡椒单萜类化合物对冷鲜肉中关键腐败菌有较强的抑菌活性，筛选得到了荧光假单胞菌、单增李斯特菌和热杀索丝菌、大肠杆菌、铜绿假单胞菌、莓实假单胞菌、隆德假单胞菌、金黄色葡萄球菌等指标菌。胡椒单萜化合物能抑制指标菌的正常生长和增殖，破坏了细胞的形态和结构，导致细胞膜的通透性增加，细胞内电解质及大分子物质如蛋白质等的外泄；降低细胞膜电位和代谢活力，抑制呼吸和能量代谢中关键酶活性，阻碍正常能量转化，导致代谢紊乱；代谢组学、蛋白组学和转录组学分析表明胡椒单萜类化合物通过造成细胞内基因差异表达及代谢功能紊乱并发挥其抑菌作用；胡椒单萜化合物与细菌DNA结合并导致DNA构象的改变；通过对单核增生李斯特菌菌体蛋白的DNA进行测序，并将DNA测序结果翻译为氨基酸序列后，共得到4种氨基酸序列：och1（菌体蛋白-15, 17, 20, 25, 27, 31, 34, 35, 39, 43, 44, 47, 48）、och2（菌体蛋白-28）、och3（菌体蛋白-32）和och4（菌体蛋白-36）。将四种菌体蛋白氨基酸序列进行比对，四种菌体蛋白在互补决定区（Complementary Determining Regions, CDRs）则具有较大的序列差异性，其中以CDR1和CDR3的差异更为显著。采用同源建模技术建立了多肽分子的四种结合区域三维模型，并通过分子对接模拟了胡椒单萜化合物与单核增生李斯特菌菌体蛋白的作用模型，菌体蛋白互补决定区CDR1、CDR2和CDR3作为靶蛋白均由胡椒单萜化合物多肽分子簇拥形成，导致菌体呼吸链差异蛋白损伤，因此，单增李斯特菌ATP合成受阻，呼吸功能被抑制，从而导致细菌死亡。. 本研究揭示了胡椒单萜类化合物对关键腐败菌呼吸作用抑制特性、作用靶点以及影响机制，为胡椒单萜化合物的深入研究和实际应用奠定了理论基础。