脉冲电场介导碳纳米管灭活副溶血性弧菌机理研究

Food-borne pathogens are important factors affecting food safety and threatening human health. Developing efficient sterilization technology is of great significance to food safety. As an emerging nanomaterial, carbon nanotubes (CNTs) have attracted much attention because of their unique physical antimicrobial activities. CNTs have been applied to sterilization of pathogenic microbes but cannot meet the requirement of high efficiency sterilization due to cytotoxicity. External energy regulation is an important way to enhance the interaction of carbon nanotubes with cells. In this study, a typical food-borne pathogenic bacterium, Vibrio parahaemolyticus, was used as the bacteria model. The effect of pulsed electric field (PEF) on carbon nanotubes inactivation effect and microbial mechanism was investigated in detail. To achieve a precise targeted regulation of carbon nanotubes under the effects of the PEF, surface functionalization of CNTs and establishment of a mathematical model for directional regulation were studied. On the other hand, the enhancement of carbon nanotubes under the action of electric field was explored from the cell level. The morphological structure of the cells and the possible changes of the intracellular matter were studied. The microbial mechanism of CNTs under PEF mediation was obtained based on the above investigations. This topic is the basic research work of the development of green and efficient sterilization technology. The successful development of the project will further promote the wide application of nanomaterials and pulsed electric fields in the field of food biochemistry.

食源性致病菌是影响食品安全和人类健康的重要因素，开发高效灭菌技术对保障食品安全具有重要的意义。碳纳米管作为一种新兴的纳米材料，其特有的物理抗菌作用备受关注，并已应用于微生物的灭菌。因受细胞毒性限制，低浓度碳纳米管不能满足高效灭菌要求。外界能量的调控是增强碳纳米管对细胞作用功效的重要方法。本项目以典型的食源性致病菌-副溶血性弧菌为研究对象，初步探索脉冲电场介导碳纳米管对食源性治病菌的灭活效果及作用机制。课题一方面利用碳纳米管功能化修饰和定向调控过程仿真模型的建立，实现脉冲电场作用下碳纳米管定向精准调控。另一方面从细胞层面探索脉冲电场介导下碳纳米管的增强作用使菌体细胞形态结构和胞内物质可能发生的改变，进而揭示脉冲电场介导碳纳米管灭菌机理。本课题是开发绿色高效灭菌技术的基础研究，课题的成功开展将进一步推动纳米材料和脉冲电场在食品生化领域的广泛应用。

食品安全一直是全球普遍关注的一个重要问题。全球70%食源性疾病病例与各种致病性微生物污染的食品有关。灭菌成为食品加工过程的一个重要环节。高温、化学药剂和生物法是最常用的灭菌方式。这些方法不同程度存在耗时、程序复杂或易产生污染等问题。碳纳米管（CNTs）由于其特殊的形状及其特有的物理抗菌作用为食源性致病菌灭菌技术提供了新的视野。碳纳米管抗菌效果及机理研究虽取得了一些进展，但目前这项技术的应用还存在一些局限性，碳纳米管在微生物抑制和杀灭过程受到浓度的制约，低浓度碳纳米管没有毒副作用，但杀菌效果有限。.基于此，本项目首先从碳纳米管表面结构设计出发，通过强酸氧化对原始多壁碳纳米管进行纯化和短切，制备出分散性较好的酸化多壁碳纳米管，对多壁碳纳米管的抑菌性能研究发现主要来源于多壁碳纳米管对副溶血性弧菌细胞的物理吸附作用。大量的较长的多壁碳纳米管缠绕包裹在细菌表面，增大了与细菌接触的相互作用，从而引起菌体细胞表面结构的破坏，使细胞生长受到抑制甚至死亡，少量的短切的多壁碳纳米管对细菌的细胞膜损伤（物理穿刺作用）也会致死菌体；在此基础上通过非共价作用（π-π堆积、疏水作用等）制备了一种二氢杨梅素（DMY）功能化多壁碳纳米管复合材料（f-MWNTs-PEG-DMY）。f-MWNTs-PEG-DMY的抑菌性能主要来源于f-MWNTs-PEG-DMY对菌体细胞的物理吸附作用以及负载在碳管表面的DMY对副溶血性弧菌强的抑制作用。另一方面从细胞膜通透性、细胞形态观察、氧化应激三方面探索超声物理场介导下碳纳米管的增强作用使菌体细胞形态结构和胞内物质可能发生的改变，发现超声增加了f-MWNTs的分散性，破坏细菌细胞膜表面结构，使f-MWNTs进入细菌内部造成菌体死亡。本课题促进了绿色高效灭菌技术发展，推动了纳米材料和物理场在食品生化领域的广泛应用。