基于Cpx信号通路的EGCG抗菌机理及其水凝胶复合物的功效研究

As an antibacterial agent derived from plant, EGCG has been widely used in many fields including food preservation and chemistry engineering. However, since its antibacterial mechanism still remains unclear and it is very easy to be oxidized, the application of EGCG is limited. Based on our preliminary work that the antibacterial activity of EGCG is triggered by ROS induced via Cpx system（published on Feb 2018）,in this project, to further study the antibacterial mechanism related to the Cpx pathway, we plan to construct DegP and Ftsh gene deletion and overexpression mutant strains and determine the CpxP transcription level, ROS level and the survival rate in these mutants. Besides, to overcome its instable chemical property, we propose to obtain a novel hydrogel complex containing EGCG and cellulose ploymer, which has the advantages of low cost, environmentally friendly, high activity utilization. This study will provide theoretical basis and application guidance not only for expanding the application of traditional agricultural products (tea), but also for the use of natural abundant fibrous source. Moreover, it will have great significance on ensuring food safety and protect national health.

针对植物源抗菌剂EGCG（表没食子儿茶素没食子酸酯）在食品、化工等领域应用中存在的抗菌机理不明晰、化学性质不稳定（易氧化）等突出问题，基于申请人2018年2月发表论文中对“EGCG抗菌效应与Cpx诱导产生的ROS相关”的前期研究，项目拟对蛋白水解酶Ftsh、DegP的基因进行敲除和过表达，通过检测CpxP的转录水平、ROS的含量以及菌株存活率，从Cpx信号通路水平深入阐释EGCG的抗菌机理。同时为克服EGCG易氧化的问题，合成低廉、环保、活性利用度高的EGCG/纤维素聚合物新型水凝胶复合物。这将为开发中国传统特色农产品（茶叶）资源、环境友好地利用自然界丰富的纤维素原料提供理论依据和应用指导；并对保证食品安全，保障国民健康具有重要意义。

项目针对植物源抗菌剂EGCG(表没食子儿茶素没食子酸酯)在食品、化工等领域应用中存在的 抗菌机理不明晰、化学性质不稳定(易氧化)等突出问题，首先明晰了EGCG抗菌功效: 实验使用二倍稀释法测定了EGCG的最小抑菌浓度（minimum inhibitory concentration，MIC）；采用稀释平板法测定了细菌在EGCG处理后的存活率；通过探针法检测了EGCG对细胞膜电位的影响；利用PI荧光染色检测了EGCG对细胞膜的损伤；通过考马斯亮蓝染色法测定了EGCG处理后细菌胞内蛋白的渗漏情况；最后使用扫描电子显微镜（scanning electron microscope，SEM）观察了EGCG对细菌形态的影响。以上实验结果显示， EGCG对W3110的MIC为1.2mg/mL；当浓度低于1.6mg/mL时，其具有抑菌功效，而当其浓度为1.6和1.8mg/mL时，细菌被处理3h后的存活率分别约为40%和15%；不同浓度EGCG均能导致细菌膜电位在1h内迅速下降50%左右；1.8mg/mL EGCG处理3h后，细菌的PI荧光上升约4倍，胞内蛋白渗漏水平显著上升（大于0.4mg/mL），且细菌在SEM下的形态出现明显的改变。其次，揭示了EGCG对细菌转录组的影响，从细菌分裂、蛋白合成以及Cpx双组份系统等分子水平和信号通路方面揭示了EGCG的抗菌机制。最后合成了低廉、环保、活性利用度高的EGCG/纤维素聚合物新型水凝胶复合物。这些成果丰富了EGCG的抗菌理论，也为其作为抗菌剂和食品保鲜剂的应用提供依据。

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