DNaseI修饰型壳聚糖-乳酸链球菌素纳米粒对单增李斯特菌生物被膜的清除作用及机制研究

单增李斯特菌是一种重要的食源性致病菌，易在食品、设备表面形成耐受性极强的生物被膜，给食品安全造成重大隐患。乳酸链球菌素作为天然抗菌肽对单增李斯特菌有较好的抑制作用，但是难以渗透生物被膜发挥抑菌作用。基于脱氧核糖核酸酶(DNaseI)对被膜中胞外DNA的降解，利用DNaseI修饰的壳聚糖纳米粒作为载体,旨在提高乳酸链球菌素对单增李斯特菌生物被膜的消除作用。项目首先构建DNaseI修饰型壳聚糖-乳酸链球菌素纳米粒，明确纳米粒理化性质与抗被膜活性之间的关系。其次，分析纳米粒对胞外聚合物成分和被膜结构的影响，动态解析纳米粒的吸附特性与被膜中的空间分布规律，阐明纳米粒渗透生物被膜的作用机制。最后，基于纳米粒与被膜细胞的吸附作用和被膜菌的灭活规律，结合纳米粒对菌体形态和细胞膜功能的影响，阐释纳米粒灭活被膜菌的作用机制。项目成果将为食品科学领域构建新型、高效的生物被膜清除策略提供科学依据与技术支撑。

Listeria monocytogenes is an important food-borne pathogen, which easily forms biofilms with high tolerance to antimicrobials on surfaces of food and equipment, causing great safety concern for food industry. Nisin can inhibit growth of L. monocytogenes as a natural antibacterial agent, but it cannot penetrate biofilms. Based on the degradation of extracellular DNA in extracellular polymeric substances （EPS）by deoxyribonuclease (DNaseI), surface modification of chitosan nanoparticles by DNaseI is to be used as carrier to stimulate the elimination of biofilms. In this project, DNase-modified nisin-loaded chitosan nanoparticles is firstly to be constructed, and the relationship between the physicochemical properties and anti-biofilm activity of nanoparticles is determined. Secondly, the composition of EPS and spatial structure of biofilms treated with nanoparticles are to be analyzed, and the EPS-adsorption characteristics and spatial distribution of nanoparticles in biofilms are to be determined, elucidating the mechanisms of how nanoparticles penetrate into biofilms. Thirdly, the attachment of nanoparticles with biofilm cells is to be studied as well as the inactivation principles of nanoparticles in terms of cell viable and metabolic activity. By explaining the effects on microbial morphology and membrane function of biofilm cells by nanoparticles, the bactericidal mechanisms of nanoparticles are to be elucidated. In sum, the results of this project will provide scientific basis and technical support for the establishment of a new and efficient biofilm removal strategy in the field of food science.