片球菌素PA-1与乳酸协同损伤酸耐受嗜水气单胞菌内外膜的机理

G- pathogenic bacteria are responsible for most of the aquaculture diseases. Aquaculture probiotic Lactic Acid Bacteria produce antibacterial substances such as lactic acid and bacteriocins, those may work synergistically against gram negative (G-) pathogens, by an unknown process and mechanism. The present research aims to elucidate the synergistic functioning of bacteriocins and lactic acid on G- bacteria inhibition, specific for class IIa bacteriocin pediocin PA-1 and G- aquaculture pathogen Aeromonas hydrophila, which will be induced to be weak acid tolerant (named as WATAh for shot). First prove the synergistic inhibitory/bactericidal effect of PA-1 and lactic acid and their dose-effect relationships; Then illuminate outer membrane damages caused by weak concentrations of lactic acid, specific as changes of structure, composition, surface charge, permeability and bacteriocin penetration; Further focus on inner membrane damaging mechanism by pediocin PA-1 in the assistance of lactic acid, and lock the location and target molecules of PA-1 in WATAh cells. The results will complete the synergistic inhibition mechanism of bacteriocin and lactic acid, and provide a foundation for the application of bacteriocin producing Lactic Acid Bacteria in aquaculture, especially as a substitute for antibiotics.

革兰氏阴性（G-）致病菌是水产养殖的主要病原。水产益生乳酸菌的抗菌产物乳酸和细菌素有协同抑制水产G-致病菌的潜能，但其机理不明。本研究以乳酸菌IIa类细菌素的典型代表片球菌素PA-1，结合亚抑制/致死浓度乳酸，处理重要的G-水产病原菌嗜水气单胞菌（菌株经驯化为低酸耐受菌株，简称WATAh菌），旨在揭示细菌素和乳酸对G-菌的协同抑制机理。首先明确片球菌素PA-1和乳酸对WATAh菌的协同抑杀作用及量效关系；进一步深入解析乳酸对WATAh菌外膜结构、成分、表面电荷、通透性及细菌素穿透力的影响；重点探讨乳酸协助下片球菌素PA-1对WATAh菌内膜的损伤效应，确定片球菌素PA-1在WATAh菌上的作用位点及靶分子。研究结果将完善细菌素与乳酸协同抑杀G-菌的机理，并为产细菌素乳酸菌作为抗生素替代品应用于水产养殖提供理论依据。

片球菌素PA-1是一种安全性较高的食品级抗菌肽，但其本身对革兰氏阴性菌作用有限。通过本项目研究，明确了片球菌素PA-1和乳酸可协同抑制水产革兰氏阴性病原菌-嗜水气单胞菌，并阐明了其作用机理。量效关系研究发现30-50 μM PA-1与3.5 mM L-乳酸可完全抑制嗜水气单孢菌的生长，处理12 h可造成60%以上的死亡率。片球菌素PA-1和乳酸可协同损伤嗜水气单胞菌的细胞壁（外膜）和细胞膜（内膜）。乳酸可使嗜水气单胞菌外膜脂多糖释放，造成细胞壁损伤，进而允许PA-1接触并作用于菌体细胞质膜，造成细胞膜渗漏，膜电动势和pH动势消散，细胞内膜以及其他内部结构损坏。SEM和TEM研究表明，乳酸和PA-1诱导菌体产生囊泡，内外膜破裂，出现细胞质渗漏甚至细胞裂解。PA-1可与嗜水气单胞菌细胞膜膜脂、膜蛋白、胞质蛋白结合，暂未发现特异性受体。片球菌素PA-1和乳酸协同处理，可抑制嗜水气单胞菌内稳态维持系统以及药物外排系统的表达，同时引起抵抗性响应。项目成果的延伸和应用研究证明I类细菌素nisin，抗菌肽epinecidin-1与乳酸协同对水产革兰氏阴性病原菌有强烈抑制作用，在水产养殖和水产品保鲜应用中有良好效果。本项目的研究结果完善了细菌素与乳酸协同抑杀G-菌的理论基础，并为抗菌肽及其产生菌作为抗生素或防腐剂替代品应用于水产养殖和水产品保鲜提供了依据。