细菌导致的冷藏牡蛎鳃部生物胺的形成和累积机制研究

As a filter-feeding shellfish, oyster accumulates many bacteria in its gills. The applicant has confirmed that oyster gills had high quantity and complex community structure of bacteria, and high contents of biogenic amines at the end of shelf life of chilled storage. The accumulation of biogenic amines in oyster gills will seriously affect the shelf life and safety of oyster. To explore the mechanism of the formation and accumulation of biogenic amines in oyster gills under chilled storage, the high performance liquid chromatography and the amplification of decarboxylase gene will be applied to study the type and capacity of the amine production for isolates, and then reveal the amine producing characteristics for the bacteria from oyster gills. Metagenome of microbiota in chilled storied oyster gills will be sequenced by the high-throughput sequencing technology. The bacterial community structure and metabolic pathway of precursor amino acids for biogenic amines would be analyzed with the results of EGTs database and Contig assembly DNA sequencing, and gene annotation and functional classification. The collaborative relationships of among strains will also be revealed. The dynamic changes of amine-producing bacteria and the relationships among precursor amino acids, amine-producing bacteria and biogenic amines will be studied in gills of oyster during chilled storage. The results would be conducive to explain the role of microbial amine-production in the gills of chilled storied oyster. It can provide a theoretical basis for the development of preservation technology and the study of antiseptic mechanism in oyster.

牡蛎作为海洋滤食性贝类，其鳃部易聚集微生物，申请人已证实牡蛎冷藏货架期终点鳃部微生物不仅数量多、菌群结构复杂，而且生物胺含量高。鳃部生物胺的累积会严重影响牡蛎的货架期和安全性。为探究冷藏牡蛎鳃部生物胺的形成和累积机制，本项目拟采用高效液相色谱和扩增脱羧酶基因研究已分离菌株的产胺种类及能力，揭示鳃部细菌的产胺特性；应用宏基因组高通量测序技术，获得冷藏牡蛎鳃部菌群的宏基因组序列，通过EGTs序列分析及contig序列组装和基因组功能注释分析，全面解析牡蛎鳃部细菌群落结构及生物胺的前体氨基酸代谢途径，揭示细菌间相互协作关系；研究产胺菌群动态变化规律，探讨冷藏过程鳃部前体氨基酸、产胺菌与生物胺的关系。研究结果将有助于深入了解牡蛎鳃部冷藏过程的微生物产胺作用，为牡蛎保鲜技术开发和防腐机理研究提供理论基础。

牡蛎是一种滤食性双壳软体贝类，在鳃部聚集大量微生物。牡蛎开壳后鳃部微生物利用营养物质大量繁殖，影响牡蛎的食用品质和货架期。牡蛎腐败过程的菌群作用及其与腐败产胺的关系累积机制有待阐明。本项目首先采用HPLC 分析冷藏牡蛎鳃部微生物产胺种类及能力，并建立腐败微生物与产胺相关性。结果表明，185株分离菌株主要产尸胺、腐胺和酪胺，主要的产胺菌为肠杆菌科、气单胞菌属、乳杆菌属、乳球菌属、肠球菌属、明串珠菌属和梭菌属。HPLC结合产胺基因 (hdc基因，tdc基因，odc基因)分析气调调控下主导菌菌株产胺特性，全面揭示了主导菌乳酸菌产胺种类和能力。在微生物离体产胺能力分析的基础上，进一步研究冷藏期间鳃部微生物腐败代谢产物及产胺，采用DGGE及Illumina 宏基因组测序技术深度分析了菌群构成和动态变化规律，以及菌间的协作；证实了牡蛎鳃部APC与挥发性盐基氮，乳酸菌与酪胺，假单胞菌与腐胺，肠杆菌和腐胺之间相关性最大，这些菌相互协作可导致冷藏鳃部生物胺的累积。而在气调冷藏过程中，特征产胺基因qPCR定量分析和HPLC检测结果都表明酪氨酸酶基因表达量及酪胺在牡蛎鳃部腐败过程中不断累积，阐明了气调调控下牡蛎鳃部的腐败产胺主要由优势菌乳酸菌引起的，且产生以酪胺为主的生物胺。本研究结果可为牡蛎及其他水产品保鲜技术开发和防腐机理研究提供理论基础。