基于胞外蛋白酶分解靶蛋白解析特定腐败菌对冷藏鱼糜制品的致腐机制

Surimi-based product is using frozen storage and distribution at present, with the problem of product quality being badly affected, high energy consumption, high cost and low quality. Refrigerated storage of surimi-based products, can not only keep the food quality, but also reduce energy consumption. However, the growth of microorganisms will cause the surimi-based products to spoil during refrigerated storage. Controling the growth of the microorganisms is an important prerequisite for refrigerating surimi-based products. This project aims to study the dynamic analysis of bacterial microflora and ensuring specific spoilage organisms (SSO), and to explore the mechanism of surimi-based product spoilage lead by SSO, which is caused by SSO hydrolyzing myofibrillar protein secreted extracellular protease (ECPase) in surimi-based products. Firstly, dominant bacteria of refrigerated surimi-based products will be confirmed by using the polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) technology and methods in microbiology, and then identified by molecular biology technology. Secondly, the SSO will be confirmed by statistical analysis of dominant bacteria and conventional spoilage evaluation indexes of surimi-based product. Thirdly, SSO's ECPase will be separated and purified adopting modern biochemical technology and then specific ECPase will be determined. Fourthly, the myofibrillar protein target protein of SSO's ECPase in surimi-based products will be explored by two-dimensional gel electrophoresis and MALDI-TOF-MS combination of proteomics technology. Finally, combined with the structure and the target protein of the myofibrillar protein， the mechanism of SSO leading to decaying of surimi-based product will be revealed. This project is a new theoretical probe into refrigerated storage surimi-based products.

鱼糜制品目前多采用冷冻贮藏和分销模式，产品质量受到严重影响，存在高能耗、高成本、低品质问题。冷藏保鲜鱼糜制品既能保护其食用品质又能降低能耗，但易产生微生物引起的腐败变质问题，控制微生物生长是实现冷藏保鲜的前提。本项目拟对鱼糜制品中细菌菌相构成进行动态分析，确定其特定腐败菌(SSO)，探究SSO胞外蛋白酶分解鱼糜制品肌原纤维蛋白而导致产品腐败变质的机制。首先，利用PCR-DGGE技术和微生物学方法分析冷藏鱼糜制品中细菌菌相构成，明确优势菌，并进行分子生物学鉴定；其次，应用统计学方法对优势菌与鱼糜制品常规腐败指标进行相关性分析，确定SSO；再次，应用现代生化技术分离纯化SSO胞外蛋白酶，确定特征胞外蛋白酶；最后运用双向电泳与MALDI-TOF-MS技术探究SSO特征胞外蛋白酶分解鱼糜制品肌原纤维蛋白靶蛋白并结合肌原纤维蛋白结构，揭示SSO的致腐机制。本项目是鱼糜制品冷藏保鲜理论的一种新探索。

鱼糜制品目前多采用冷冻贮藏和分销模式，产品质量受到严重影响，存在高能耗、高成本、低品质问题。冷藏保鲜鱼糜制品既能保护其食用品质又能降低能耗，但易产生微生物引起的腐败变质问题，控制微生物生长是实现冷藏保鲜的前提。本项目研究冷藏鱼丸在冷藏条件下微生物和理化品质的变化规律，并通过传统分离培养和PCR-DGGE技术相结合，研究了冷藏过程中菌群多样性和优势菌群，分离鉴定了其中的优势腐败菌，并确定了特定腐败菌为海水芽孢八叠球菌、地衣芽孢杆菌和松鼠葡萄球菌。采用双向凝胶电泳体系分别研究了4℃和25℃下，蛋白酶对白鲢鱼肌原纤维蛋白的降解情况。结果表明，随着酶处理时间的增长，蛋白图谱中蛋白点的数量出现了先增加后减少的趋势。最终，通过PDQuest软件找出75个差异点，其中4℃组有45个，25℃组有30个。对割胶成功的24个蛋白点进行质谱鉴定和分析，显示为肌动蛋白、肌球蛋白重链等。利用GO功能注释，对这24个蛋白质进行了生物信息学分析，从细胞组成、分子功能和生物过程层面上，并结合前文生化指标，推测碱性蛋白酶降解肌原纤维蛋白的途径为：首先破坏肌原纤维骨架结构，肌动球蛋白解体为肌动蛋白和肌球蛋白，随后，肌球蛋白及肌动蛋白进一步被分解为其他产物如肌球蛋白重链等。