沙门氏菌噬菌体聚集介导的抗菌蛋白膜形成及其释放抑菌机制研究

Active packaging materials with antimicrobial properties have gained significant interest over the last decade. Currently, most of the antimicrobial active packaging materials are broad spectrum antimicrobials which do not target bacterial pathogenic species specifically. The need for specificity in antimicrobial activity is important as pathogens may be a small fraction of the total microbial load present in food systems. Thus, developing pathogen specific antimicrobial active packaging materials may improve the antimicrobial efficacy by reducing interactions with non-targeted microbes. Bacteriophages are viruses of bacteria that have high specificity for their host strain and multiply upon infecting their host for up to 1000 viral progeny per bacteria. Based on these advantages and increases in antimicrobial resistance among common pathogens, phage can have significant applications as additives to packaging material formulations. A bacteriophage against Salmonella enteritidis has been isolated and characterized, this study will complete the whole genome sequencing and analyze the molecular structures of main structural proteins of the bacteriophage strain, such as the molecule, charge, amphiphilic, hydrophobic and helix. Then, we will develop a subclass of bacteriophage encapsulated protein films, and characterize its antimicrobial efficacy, stability and release of encapsulated phages. The formation mechanism of antimicrobial protein films encapsulated phages will be clarified at molecular level after thoroughly examining the relationship between the micro-film formed on interface of protein solution and the macro-film coated. Additionally, the phages release from protein films will be studied in an aqueous environment and food surface incubation. Furthermore, the antimicrobial efficacy will be evaluated for fresh chicken coated by the active packaging materials. This study will demonstrate integration of phages with edible packaging materials to develop novel active packaging materials for biocontrol applications.

抗菌活性蛋白膜不仅可持续地抑制微生物生长繁殖，还能有效地阻控交叉污染的发生，是食品活性包装领域重要的发展方向。本项目在前期研究分离获得的一株沙门氏菌烈性噬菌体的基础上，通过噬菌体全基因组测序，解析其主要结构蛋白氨基酸序列、两亲性、疏水性和电荷电位等，探明其作为生物大分子维持活性稳定的分子基础；以成膜蛋白为基材，制备噬菌体抗菌蛋白膜，利用界面技术比较研究噬菌体与成膜蛋白溶液微观膜和宏观膜的界面性能，表征噬菌体聚集及其与成膜蛋白分子相互作用的成膜规律，优化膜的制备体系；进一步探讨抗菌蛋白膜中噬菌体在液-液、液-固界面的释放扩散动力学及其分子机制，评价其对冷鲜鸡肉污染沙门氏菌的抑菌效果。预期研究结果可为开发新型噬菌体类食品抗菌剂提供新的思路，为研制特异性强、抑菌性好的噬菌体抗菌蛋白膜提供理论依据。

本项目以前期建立的食源性致病菌非预增菌快速检测方法，应用于市场上采集的食品快速检测筛查，并对阳性样品进行目标菌分离、纯化、鉴定；以分离到的沙门氏菌、大肠杆菌O157:H7为宿主菌，分离相应的烈性噬菌体并鉴定；进一步以胶原蛋白为成膜基材，探明噬菌体作为生物大分子在基材中维持活性稳定的分子基础；表征噬菌体聚集及其与成膜蛋白分子相互作用的成膜规律，优化膜的制备体系；探讨抗菌蛋白膜中噬菌体的释放扩散动力学及其分子机制，评价其对冷鲜肉污染沙门氏菌的抑菌效果。项目组主要完成的内容：以本项目组建立的蒙脱石封闭活性炭、免疫磁珠等食品样品前处理技术联合沙门氏菌、大肠杆菌特异性环介导等温扩增方法，从市场食品中分离、纯化到近100株沙门氏菌菌株和20余株大肠杆菌菌株；以分离的沙门氏菌、大肠杆菌为宿主菌，从不同污水样品中分离、纯化、鉴定了19株烈性沙门氏菌噬菌体和2株大肠杆菌O157: H7、多重耐药产志贺毒素大肠杆菌烈性噬菌体，对噬菌体进行了一步生长曲线、宿主谱、MOI、pH和温度稳定性、抑菌性能等生物学特性鉴定；测定了其中5株噬菌体全基因组序列，解析它们主要结构蛋白氨基酸序列、电荷电位、疏水性、螺旋性、热学性质，噬菌体头、尾两极性的分子结构基础；制备、优化了噬菌体-明胶、噬菌体- PCA-明胶、噬菌体-姜黄素-明胶和PCA-明胶等生物活性蛋白膜，对膜的理化、机械、阻隔等特性和抗氧化、抑菌活性进行了较系统的研究，表征了新型噬菌体抗菌蛋白膜的界面性质、微观结构、分子互作以及噬菌体扩散动力学、释放机制，探索膜在冷鲜肉中细菌的抑菌性能、机制与保鲜效果。相关成果发表SCI论文11；授权发明专利1项；培养硕士研究生4名，在读博士研究生1名、硕士12名。本课题的研究成果为开发新型噬菌体类食品抗菌剂提供新的思路，为研制特异性强、抑菌性好的噬菌体抗菌蛋白膜提供理论依据。