我国淡水鱼低温保鲜中耐冷腐败细菌的种群与谱系以及腐败形成机制研究

The proliferation of psychrotolerant bacteria is the primary cause of putrefaction of fish during storing and transporting. To improve the cryopreservation techniques，extend the shelf life of freshwater fish and its derived products, it is of both important theoretical and practical significance to investigate the ecological distribution of the populations and genetic lineages of psychrotolerant bacteria as well as the spoilage mechanism. Based on the previous study, we intend to collect the major freshwater aquaculture fishes in different regions in China in different seasons, separate psychrotolerant bacteria, discuss the ecological distribution of population structure and genetic genealogies of the bacteria with geography, species and seasons, using conventional physiological and biochemical analysis, combined with the 16S rDNA sequencing、the molecular fingerprint pattern technology of RAPD and rep-PCR in this project. Meanwhile, we plan to analyze and determine growth kinetic parameters, quorum sensing signal molecules, characteristically volatile components causing spoilage and differntially expressed proteins, respectively using the method of spectrophotometry, HS-SPME-GC-MC, thin layer chromatography combined with biosensor technology and 2DE-PAGE-MS. The comparative and statistical analysis based on the above experiment results would be made to explore the spoilage feature and its formation mechanism of psychrotolerant bacteria from different geography, fishes and seasons, so as to provide theoretical basis for establishing a new efficient cold storage strategy and extending the shelf life of freshwater fish and its derived products.

耐冷细菌增殖是鱼类贮藏流通过程中导致腐败变质的主要原因，探索耐冷细菌种群谱系的生态分布及腐败发生机制对促进冷藏保鲜技术进步、延长淡水鱼及其制品的货架期具有重要的理论和实际意义。本项目拟在前期研究基础上于不同季节采集我国不同区域主要淡水养殖鱼种，分离耐冷细菌，并应用常规生理生化分析结合16S rDNA测序鉴定，应用RAPD和rep-PCR分子指纹图谱分型技术探索耐冷细菌种群结构、遗传谱系随地理、鱼种及季节的生态分布规律。同时分别应用分光光度法、薄层层析-生物传感法、HS-SPME-GC-MS联用法与2DE-PAGE-MS联用法分析测定不同种群谱系典型耐冷菌株在低温生长过程中的生长动力学参数、群体感应信号分子、特征挥发性腐败成分与差异表达蛋白，对比、统计分析，探讨不同地理、季节及鱼种耐冷菌在淡水鱼冷藏中的腐败特征及其形成机制。为建立新型高效的冷藏保鲜策略、延长淡水鱼及其制品货架期提供理论基础。

鱼类低温贮藏期间的腐败是由耐冷特定腐败菌增殖引起的，因此围绕耐冷特定腐败菌的分离鉴定、腐败能力、种群谱系的生态分布及其群体感应信号分子的检测进行研究，可为我国淡水鱼低温保鲜技术进步、延长淡水鱼及其制品的货架期提供有力基础。项目以我国江苏扬州、湖南岳阳、内蒙古呼和浩特、辽宁沈阳、广东广州等地的鳙鱼、鲫鱼、草鱼和鲢鱼为研究对象，进行了耐冷特定腐败菌分离鉴定、低温贮藏期间细菌种群结构、遗传谱系随地理、鱼种、时间的生态分布规律、典型耐冷菌株的腐败能力和特征腐败气味以及优势腐败菌的群体感应检测技术及其应用等研究。结果显示，我国不同地域淡水鱼低温贮藏期间均以假单胞菌、气单胞菌和腐败希瓦氏菌为主要特定腐败菌，且腐败希瓦氏菌的腐败能力高于假单胞菌和气单胞菌。同时，利用高通量测序技术揭示了淡水鱼低温贮藏期间其细菌群落结构和多样性变化规律；此外，证实了不同种腐败细菌在腐败过程中产生的特征性腐败物质具有差异性，2-甲酸组胺和三甲胺是淡水鱼腐败臭气的主要来源；最终，成功构建了定量检测3种N-酰基-L-高丝氨酸内酯（AHLs）的新型肥大细胞电化学传感器，证明了导致淡水鱼腐败的耐冷特定腐败菌可产生AHLs，且其在鱼肉腐败检测实际应用中取得了很好的效果。