糟鲭鱼中微生物多样性分析及其对组胺消长机制的研究

Histamine is one of the important unsafe factors in food consumption. The accumulation of histamine can easily occur in fatty red-meat fish and fermented aquatic products. And the problem may cause food safety issues and economic losses. This project will focus on microbial diversity and histamine dynamics in the fermentation process of pickled mackerel（Pneumatophorus japonicus）. The microbial community structure and diversity will be analyzed by the MiSeq sequencing method. will be isolated by a trap of a diffusion chamber covered by top and bottom membrane. The growth and enzyme producing characteristic will be determined. and the characteristics of histamine degradation will be investigated as following: enzyme active sites, enzyme type, enzymology properties and degradation kinetics. The effects of histidine decarboxylase producing bacteria and hitamine-degrading bacteria on histamine content will be compared. Also the interaction of two kinds of microorganism will be studied. Meanwhile the influence of various factors and utilization of starter on dynamics of two kinds of microorganism and histamine content will be investigated in fermentation system. Thus the mechanism of increase and decline of histamine in pickled mackerel will be explained. The research achievements are of important guiding significance for histamine biodegradation technique, and also will provide important basis for safety control in food processing and preservation. Therefore it has high theoretical and social economic benefits.

组胺是食品中重要的不安全因素，在青皮红肉鱼和发酵水产品中极易发生组胺的积累现象，可能导致食品安全问题和经济损失。本项目以糟鲭鱼为研究对象，研究发酵过程中微生物群落和组胺的动态变化；以MiSeq测序方法对微生物群落结构及多样性进行分析，以双层膜固定化方法分离产组氨酸脱羧酶微生物和组胺降解菌，确定生长特性和产酶特性，探索组胺降解酶的酶活性部位、产酶类型、酶学性质及降解动力学等降解特性；比较产组氨酸脱羧酶微生物和组胺降解菌以及这两类微生物之间对组胺含量的交互影响；并探究发酵体系中各种环境因素和发酵剂对两类微生物变化及组胺含量的影响，明确糟鲭鱼发酵过程中组胺产生、累积和消长的机制。本项目不仅对发酵水产品中组胺的微生物定向控制研究具有指导作用，也将为食品加工与保藏中组胺相关质量安全控制提供重要依据，因此具有较高基础理论价值和潜在经济社会效益。

研究鲭鱼贮藏与发酵过程中微生物多样性和组胺的动态变化规律。主要成果包括：结合平板筛选、酶活力和组胺测定，从糟鲭鱼中筛选得到多株组胺降解菌和产生菌。针对降解能力较好的Pseudomonas azotoformans V-TB-2-5，明确其生长、发酵条件、产酶特性及组胺降解特性。在糟鲭鱼中人工接种后，组胺降解率为18.2～40.7%；同时，对糟鲭鱼中亚精胺和精胺含量有显著影响。针对水产品基质复杂，传统检测方法操作复杂等特点，分别建立了超高效合相色谱-质谱法和固相微萃取法结合气相色谱-质谱联用法，快速简便的对水产品中生物胺进行准确的定性和定量。利用Illumina高通量测序技术研究了鲭鱼贮藏与发酵过程中鱼肉和酒酿的微生物群落结构的变化和多样性，并与组胺的动态变化进行相关性分析。其中，组胺、尸胺和腐胺是鲭鱼贮藏中的主要胺类，储存第2天含量均超过FDA标准。腐胺和尸胺是鲭鱼发酵过程中主要的生物胺，在发酵过程中含量增加。贮藏过程中的菌群结构和发酵过程中存在较大差异，贮藏的优势菌为厚壁菌门和变形菌门，变形菌门为优势菌。发酵过程中以厚壁菌门、变形菌门、放线菌门和拟杆菌门为主。其中厚壁菌门占主导地位，相对丰度不断增加，发酵结束时达到98.32%。储藏期间细菌群落的变化与生物胺的产生显著相关。细菌群落的变化与组胺、色胺、精胺高度相关，酪胺的产生与精胺显著相关。发酵过程中，具有组胺生产能力的微生物受到抑制，与功能基因和代谢模式的预测结果一致。研究结果有助于探索发酵过程中微生物群落与生物胺之间的关系和相互作用，为进一步研究发酵鱼的形成机理和质量改进提供参考。