基于速酿鱼露风味的嗜盐古菌筛选及其胞外蛋白酶特性研究

At present, because the traditional craft of fermentation time is too long (6-18 months), which greatly limits the production of Chinese fish sauce. Shorten the fermentation time has become a urgently problem that the fish sauce production industry have to solve. How to quickly fermented fish sauce production and still keep the traditional craft unique flavor to become the bottleneck problem in the research field. Our preliminary research showed that the halophilic archaea with independent property rights can speed up the fish sauce production, but the specific mechanism is still unclear. The project is proposed on this basis, further study on the metabolism characteristics of the halophilic archaea which may produce extracellular protease. Key research will focus on the separation and purification of extracellular protease. And the enzymology characteristics will be clarified. The role of protease to the flavor substances and precursors will be explained. This work will provide theoretical basis and technical guidance to improve the quick brew fish sauce flavor substances, which keeps the unique flavor of the traditional fish sauce.

目前中国鱼露的传统工艺由于发酵时间过长（6-18个月以上），大大限制了鱼露的生产，因此缩短发酵时间成为我国鱼露生产行业迫切解决的问题。如何在鱼露快速发酵生产中依然能保持传统工艺所特有的风味成为该研究领域的瓶颈问题。课题组前期研究筛选出了能加快鱼露生产的具有自主产权的嗜盐古菌，但其具体的作用机制仍不清楚。本项目拟在此基础上，进一步研究筛嗜盐古菌产胞外蛋白的代谢特性，重点研究所产的胞外蛋白酶的分离纯化和酶学特性，探讨其对风味物质及前体物质产生的作用机制，以期为速酿鱼露风味物质形成和增强并保持鱼露的传统独特风味提供理论基础和技术指导。

本课题筛选出一株产蛋白酶的嗜盐古生菌Halogranum rubrum RO2-11，先对其粗酶特性进行初步研究，后采用超滤、离子层析、凝胶层析等方法对其所产的蛋白酶进行分离纯化，对纯化后的蛋白酶特性进行了研究。该蛋白酶为一种对氯化钠具有广泛适应性的碱性蛋白酶，并且对金属离子Ca2+有依赖性，可以耐受一定浓度的有机溶剂，肽指纹图谱鉴定及MS/MS 离子搜索结果显示此蛋白酶与Halogranum salarium所产的丝氨酸蛋白酶存在共同的性质特点。另外比较了4株筛选的嗜盐古菌发酵的龙头鱼鱼露特性。项目的实施为嗜盐古生菌的在鱼露生产中的应用开发提供了理论基础和实验数据，对鱼露的快速发酵生产具有重要意义。