乳杆菌合成苯乳酸抑制水果腐败真菌作用位点分析

Berry fruits are easily contaminated with spoilage fungi, which need for a safe and effective preservation method to extend the shelf life. Phenyllactic acid (PLA) pruduced by lactic acid bacteria (LAB) to prevent spoilage and to extend the shelf life of foods has gained the interest of producers due to consumers' demands. PLA is a novel biopreservative with broad spectrum activity against bacteria and fungi. The potential application of PLA as food preservatives requires a clear mechanism of how they exert their antifugal effect. Scanning and transmission electron microscopy will be used to obtain the targets of PLA against spoilage fungi. Changes of extracellular N-acetyl glucosamine concentrition will be used to mesure the damage of cell wall peptidoglycan. PLA target cites will be defined by proteomics technology. Two different proteomic approaches were carried out to define differences of cell wall proteins. First, proteins loosely associated or S-S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol (DTT), separated by 2D-PAGE and identified by mass spectrometry (MS). Second, cell surface-exposed proteins were digested with trypsin and DTT from whole intact cells, and analyzed by LC-MS/MS. The differences of intracellular proteins can be defined with similar proteomic approaches. The intracellular proteins extraction can be grinding in liquid nitrogen using a mortar and pestle. Different proteins of fungi between added PLA and no added PLA are isolated and identified using 2DE-PAGE-MS and LC-MS/MS methods. Composition and interaction of these different proteins are analyzed to determine antifungal target cites and to deduce the antifungal mechanism which PLA acts.

浆果类水果易受霉菌污染而腐烂，亟需一种安全有效的保鲜方法延长此类水果的保鲜期，乳酸菌产生的苯乳酸具有很大的应用潜力。苯乳酸是一种新型生物防腐剂，对细菌和真菌都有很强的抑制作用。苯乳酸的抑菌能力已得到确认，但其抑菌机理尚不明确，使苯乳酸在食品中应用受到极大限制。本项目拟采用扫描电镜和透射电镜技术观察苯乳酸抑制真菌的作用靶位；测定真菌胞外N-乙酰葡糖胺的含量变化判断苯乳酸对细胞壁肽聚糖的破坏作用；通过蛋白质组学技术研究苯乳酸抑制真菌的相关作用位点。分析苯乳酸抑菌过程中真菌细胞壁和细胞内蛋白表达的差异，富集和提取苯乳酸作用过程中真菌细胞壁和细胞内的蛋白，比较苯乳酸作用前后经双向电泳质谱法和液相色谱串联质谱法获得的蛋白图谱找出和鉴定差异明显的蛋白点，通过分析真菌细胞壁和细胞内蛋白的变化及其相互作用并结合苯乳酸的结构推断苯乳酸抑制真菌的作用位点及可能的作用机理。

核果类和浆果类水果易受霉菌污染而腐烂，亟需一种安全有效的保鲜方法延长此类水果的保鲜期，乳酸菌产生的苯乳酸具有很大的应用潜力。苯乳酸是一种新型生物防腐剂，对细菌和真菌都有很强的抑制作用。苯乳酸的抑菌能力已得到确认，但其抑菌机理尚不明确，使苯乳酸在食品中应用受到极大限制。.本项目从自制樱桃酒分离的乳酸菌中筛选出一株高产苯乳酸的植物乳杆菌LD1.0001，培养基中添加发酵底物苯丙酮酸钠，30℃培养44h，LD1.0001发酵液中苯乳酸含量达到1852.36mg/L。LD1.0001产生的苯乳酸经乙酸乙酯萃取后用反相色谱法纯化，使用手性柱分离纯化后的苯乳酸，发酵液中D-苯乳酸和L-苯乳酸的比例约为43:57。商品化L-苯乳酸对霉菌的抑制效果稍强于D-苯乳酸的抑菌效果。. 应用扫描电镜和透射电镜观察了苯乳酸作用前后霉菌菌丝表观形态和超微结构的变化。霉菌经苯乳酸作用后，细胞壁没有明显破损，细胞外出现胞外多糖；霉菌菌丝内部出现质壁分离，细胞膜破裂，细胞内分区消失，细胞质固缩成颗粒状。说明苯乳酸的主要作用靶位应在细胞膜上或细胞内，而不在细胞壁上。. 苯乳酸作用前后霉菌上清液中N-乙酰葡萄糖胺含量无明显变化可推断出霉菌细胞壁不是苯乳酸的主要作用靶位；通过荧光双染色法观察发现苯乳酸可以破坏霉菌细胞膜的完整性，并且可以使霉菌细胞失活，可推断出苯乳酸的作用靶位应为细胞膜或者细胞质。. 应用同位素标记相对与绝对定量技术（iTRAQ）技术研究D-和L-苯乳酸处理霉菌前后蛋白的差异。3个样品共鉴定得到2967个蛋白，其中666个蛋白产生了明显的差异，通过分析差异蛋白，确定苯乳酸对霉菌的杀菌机理与其分子立体构型（D-型和L-型）无关；苯乳酸抑制真菌的主要作用位点应位于细胞膜、内膜或细胞内，而不是在细胞壁上。苯乳酸的杀菌机制之一是本身以分子形式进入霉菌细胞，在细胞内水解放出氢离子，进而造成霉菌细胞内物质和能量发生变化，导致细胞死亡；另外苯乳酸可能具有弱酸质子型解偶联剂的功能，破坏霉菌细胞的正常呼吸链，造成能量供应不足导致细胞凋亡。