以AHLs为切入点研究固态发酵鱼糜中特定腐败菌群体感应及其调控机制

Solid-state fermentation is an important way for the development of high quality aquatic products that rich in peptides and amino acids, but there is no mature technology in the present. Key troubled this question is that surimi is easy to spoilage during this process. Studies showed that the sopilage of aquatic product was mainly regulated by quorum sensing phenomenon of specific spoilage organisms (SSO) mediated by N-acyl-homoserine lactones (AHLs). The present study aims to analyze the SSO during surimi fermentation, and to eluciate the AHLs mediated quorum sensing phenomenon and regulation mechanisms. Studies to be conducted including analysis of the dynamic changes of AHLs during the fermentation and its correlation with surimi spoilage;Analysis of the changes of bacterial microflora during the fermentation by traditional isolation method and metagenomic technique, identification of 2-3 typical SSO with strong ability to spoilage, and analysis of the correlation between AHLs-producing activity and phenotypic spoilage characteristics including biofilm formation and hemolysin production; Study of the effects of exogenous AHLs and interfering agents on microflora composition and luxI and luxR gene expression levels. The results of the present study will provide necessary scientific theoretical basis for the control of the spoilage of surimi during ferementation through regulating the AHLs mediated quorum sensing of the specific spoilage bacteria.

鱼糜固态发酵是制备富蛋白肽和氨基酸的水产动物蛋白深加工产品的一个重要发展方向，但目前国内外尚无成熟的相关技术。困扰这一问题的关键是鱼糜在发酵过程极易腐败变质。初步研究表明，水产品的腐败主要受AHLs信号分子介导的特定腐败菌（SSO）群体感应现象调控。鉴于此，本项目拟在分析鱼糜发酵过程中SSO的基础上，进一步阐明该过程中AHLs介导的群体感应现象致腐及其调控机制。研究内容包括：分析该过程中AHLs的动态变化及其与鱼糜腐败的相关性；通过选择性分离培养和宏基因组测序技术分析该过程细菌菌相变化，鉴别致腐性较强的典型SSO 2-3株，并分析菌株产AHLs及其与生物膜形成、产溶血素等表型致腐特性的关联度；研究外源AHLs及其干扰剂对鱼糜菌相变化调控及其群体感应相关基因luxI和luxR表达的影响。相应研究结果将为调控鱼糜固态发酵过程中SSO的群体感应以及选择性抑制鱼糜腐败现象的发生提供必要的理论依据。

本研究着重阐明了以AHLs为切入点研究固态发酵鱼糜中特定腐败菌群体感应及其调控机制。主要取得以下四方面的研究结果：（1）通过传统微生物学方法及高通量测序技术分析了固态鱼糜发酵过程中菌相变化，确定了鱼糜发酵过程中的特定腐败菌；（2）运用生物报告菌、反相薄层层析及液相质谱技术分析了腐败菌产群体感应信号分子情况，筛选了2株高AHLs活性的菌株，并通过比较两株细菌的腐败特性发现，A. veronii bv. veronii具有较强的产TVB-N和腐胺的能力；（3）分析了特定腐败菌A. veronii bv. veronii分泌AHLs的动力学，考察了环境因素（pH、NaCl）对其群体感应系统的影响；（4）分析了外源添加AHLs及大蒜提取物作为群体感应效应抑制剂对特定腐败菌腐败特性相关指标的影响，探讨了群体感应对细菌腐败特性的调控机制。结果显示1.20 mg/mL的大蒜提取物能显著减少细菌的TVB-N和腐胺的产生。该研究可为调控鱼糜固态发酵过程中特定腐败菌的群体感应以及选择性抑制鱼糜腐败现象的发生提供必要的理论依据。