大黄鱼特定腐败菌西瓦氏菌的群体感应及定植机制研究

Microbial growth and metabolish is a major cause of fresh aquatic products. The specific spoilage organisms (SSOs) is an important factor resulting in spoilage due to rapid growth and high quantitative ability to produce spoilage metabolites. Some studies about SSOs in the farmed fish focused on the isolation and identification on the end of shelf-life and microbial growth kinetics model. Comparatively little is known about the ecology of food spoilage bacteria and the biochemical mechanisms behind spoilage at the molecular level. In the study, the microbial population of yellow croaker (Pseudosciaena crocea) was characterized by PCR-DGGE combined with the culture method at the cold storage. The SSOs of Shewanella was identified. Based on the analysis models for the biofilm formation and quorum sensing built, bacterial growth, biofilm formation and N-acyl-homoserine lactones (AHLs) produced by Shewanella were determined in vitro during the single and mixed bacterial culture. Attachment and colonization of Shewanella was assessed in the different tissue of yellow croaker. The effect of tea polyphenols and chitosan as good preservatives on the attachment and quorum sensing of Shewanella isolated from yellow croaker was analyzed. The role of the signal molecules AHLs in the shewnalla attachment, colonization and spoilage in the cold yellow croaker was investagted. Thus, this work will provide the theorial fundation for research of new preservation techniques, and contribute to improvement of the freshness and quality in the framed fish.

新鲜水产品品质劣变主要是由微生物的生长代谢引起的，其中特定腐败菌（SSOs）具有生长繁殖快、产生腐败产物活性强，是导致产品腐败的重要因素。目前养殖鱼类中特定腐败菌的研究侧重在货架期终点分离鉴定及生长动力学研究工作，而隐藏在食品腐败背后的腐败菌群生态及分子水平的生化机制研究尚少。鉴于此，本项目拟采用PCR-DGGE结合培养方法，分析大黄鱼低温贮藏过程中腐败菌群变化规律，鉴别特定腐败菌西瓦氏菌，建立生物膜形成和群体感应分子测定模型，研究鱼源西瓦氏菌株在体外培养单菌和混合菌中生长、AHLs分子和黏附特性的关联，分析西瓦氏菌在大黄鱼不同组织的黏附和定植动态过程，查明保鲜效果良好的茶多酚和壳聚糖对西瓦氏菌黏附特性和群体感应的影响。研究旨在查明西瓦氏菌群体感应在大黄鱼冷藏过程中品质劣变和腐败中的作用机制，为研发海产养殖鱼类源头保鲜新技术提供理论基础，对提高养殖鱼类鲜度和品质具有重要意义。

微生物的生长代谢是鲜活水产品品质劣变的重要原因，其中特定腐败菌（SSOs）生长繁殖快、腐败活性强，是控制的关键点。大黄鱼是我国重要的出口海水养殖经济鱼类，研究采用PCR-DGGE结合培养阐明4℃冷藏大黄鱼腐败中细菌菌群多样性下降，产H2S菌西瓦氏菌（Shewanella）为优势腐败菌，分离株在鱼汁中致腐能力存在差异，而波罗的海西瓦氏菌（S.baltica）生长快速，产高含量三甲胺、生物胺、挥发性盐基氮，表达高水平torA基因，为SSOs。建立了腐败菌群体感应（QS）分子高丝氨酸内酯（AHLs）、二酮哌嗪化合物（DKPs）和AI-2的报告菌、LC-MS/MS、GC-MS检测技术，构建了细菌生物被膜分析的结晶紫染色、荧光、电子和共聚焦显微镜观察方法。研究显示，西瓦氏菌体外培养有两种DKPs和AI-2活性，无AHLs，产生较高的生物被膜，低温促进被膜形成。相对于弱致腐株，强致腐株S.baltica 的AI-2和DKPs 活性及生物被膜更高，提示QS和生物被膜与致腐能力存在正相关。评价了QS标准品体外添加对西瓦氏菌生长和腐败表型的影响，发现cyclo-(L-Pro-L-Leu)能显著刺激S.baltica 在LB和鱼汁中生物被膜和腐败产物的形成，促进腐败基因表达，而cyclo-(L-Pro-L-Phe)和DPD无明显作用；添加cyclo-(L-Pro-L-Leu)还能加速大黄鱼匀浆液混合菌中产H2S菌的生长及代谢物积累；转录组学揭示cyclo-(L-Pro-L-Leu)能促进鞭毛、趋化、c-di-GMP及腐败相关基因表达上调。亚抑菌浓度茶多酚和壳聚糖能有效减少QS活性及生物被膜形成和粘附，为QS抑制剂。西瓦氏菌在玻璃、不锈钢和塑料片上迅速粘附，在TSB和鱼汁介质中生长和粘附相似；S.baltica还能快速地粘附到大黄鱼背部、腹部、尾部和鳃，不锈钢片上的细菌在30min能定植到鱼块中，其中不带皮鱼块总粘附量高于带皮鱼块，特别是深层定植，表明鱼皮能有效抑制腐败菌定植能力。QS抑制剂茶多酚和白藜芦醇能有效减少西瓦氏菌在鱼肉上的粘附，特别是白藜芦醇处理显著降低深部肌肉的定植。因此，S.baltica是冷藏大黄鱼的特定腐败菌，QS-DKPs参与调控细菌致腐、生物被膜及粘附定植作用，研究为揭示水产品致腐机制奠定基础。研究发表论文16篇，其中SCI收录6篇，授权专利2项，培养研究生7名。