UHT乳中荧光假单胞菌胞外耐热蛋白酶结构解析及其耐热机理研究

The typical psychrophilic-pseudomonas fluorescens and their heat-stable extracellular proteases is one of the most important factors that cause spoilage of UHT milk. Many of their proteases can survive heat treatments even in UHT-conditions and remain active in derived dairy products. The remaining activities of the proteases may cause some quality defects such as age gelation, bitterness and proteolysis during storage. The proteases have strong ability to resist heat, but the mechanism for heat resistance has not been elucidated and continues to be difficult to control. The proteomics methods such as ESI-MS, N-terminal amino acid sequencing, X-ray diffraction and NMR were used to analyse the structure of a purified protease of Pseudomonas fluorescens. Circular dichroism and fluorescence spectroscopy analyses of the purified protease were carried out at different temperatures, in an attempt to explain how different temperatures influence their conformations; establish thermal denaturation kinetic equation and raw milk enzymatic activity control threshold of UHT milk production. The determination of the amino acid sequence, protease highest tolerable temperature, optimum reaction temperature, simulation of advanced structure, the relativity between advanced structure and heat stability allowed the elucidation of the heat resistance mechanism. Through the above research, we can provide a theoretical basis for effective control of psychrotrophic protease contamination and improving quality and safety of UHT milk.

原料乳中主要嗜冷菌-荧光假单胞菌及其分泌的蛋白酶是造成UHT乳品质劣化最主要的因素之一。荧光假单胞菌胞外蛋白酶即使经UHT热处理也不能使其完全失活，残留的酶会在产品货架期内缓慢作用而发生凝胶、变苦、蛋白水解等质量缺陷。荧光假单胞菌蛋白酶具有极强的耐热性，但其耐热机制尚不明确，给控制造成了一定的难度。本研究拟采用ESI-MS、N-末端氨基酸序列分析、X射线晶体衍射、核磁共振等蛋白质组学技术对荧光假单胞菌蛋白酶的三维结构进行解析，采用圆二色谱法、荧光光谱法分析经不同温度处理前后蛋白酶构象所发生的变化、并建立蛋白酶的热变性动力学方程和生产UHT乳所用原料乳的酶活控制阈值。通过对氨基酸序列及蛋白酶的最高耐受温度和最适反应温度测定、酶分子高级结构的模拟,高级结构和耐热性之间相关分析, 阐明蛋白酶耐热机理，以期为有效控制嗜冷菌蛋白酶污染，提高UHT品质和安全性提供理论基础。

原料乳中主要嗜冷菌-荧光假单胞菌及其分泌的蛋白酶是造成超高温灭菌（UHT）乳品质劣化最主要的因素之一。荧光假单胞菌胞外蛋白酶即使经UHT热处理也不能使其完全失活，残留的酶会在产品货架期内缓慢作用而发生凝胶、变苦、蛋白水解等质量缺陷。荧光假单胞菌蛋白酶具有极强的耐热性，但其耐热机制尚不明确，给控制造成了一定的难度。本研究以分离自原料乳中荧光假单胞菌BJ-10为目标菌株，完成了BJ-10活菌数与其胞外蛋白酶活性相关性分析，建立了BJ-10胞外耐热蛋白酶的分离纯化方法及耐热蛋白酶快速检测方法，构建了耐热蛋白酶三维结构图，明确了蛋白酶的热变性动力学方程和生产UHT乳所用原料乳的酶活控制阈值，进一步验证了耐热蛋白酶是造成UHT 乳货架期品质下降的主要因素，初步探讨了UHT 乳在货架期内陈化凝胶的形成机理， 建立通过原料奶耐热酶活性、体细胞数、嗜冷菌数等指标预测预报UHT 乳安全货架期的数学模型；完成了微滤除菌技术改善超高温灭菌乳品质的研究。研究结果对有效控制嗜冷菌蛋白酶污染，提高UHT品质和安全性具有理论和现实意义。发表文章6篇，其中SCI文章2篇，EI文章1篇；申报发明专利1项。