鲢鱼加工副产物的酶解物中抗冻组分表征及其抗冻机制研究

The most competitive way to utilization of low value freshwater fish is to be processed into frozen surimi, while how to prevent the freezing denaturation of protein is an important and unsolved issue in surimi processing industry. Our previous studies have demonstrated that the enzymatic hydrolysates, which were prepared by limited hydrolysis of by-products from silver carp surimi processing, showed excellent cryoprotective abilities. But until now, the specific components and their cryoprotective mechanisms were still unclear. The present study will be focused on the following issues: (1) Screening out the most cryoprotective hydrolysates through investigating their physicochemical properties, Ca2+-ATPase activities of myofibrillar protein, the proportions of unfrozen water and gel structure properties in the frozen surimi containing these hydrolysates. (2) Isolating and purifying the specific antifreeze components from hydrolysate by multi-step chromatography, and Identifying and characterizing their types and molecular structure using MALDI-TOF mass spectrometry and nuclear magnetic resonance. (3) Exploring their cryoprotective action mode by observing their inhibition on ice crystal growth and interaction with proteins in surimi, combined with differential proteomic analysis and molecular dynamics simulation. The project is expected to illuminate the cryoprotective mechanism of these enzymatic hydrolysates in experimental and theoretical levels, and provide theoretical and technical support for development of natural cryoprotectants using by-products from fish processing, which has an important significance in theoretical study and practical application.

鲢鱼是我国重要的淡水鱼种，占全国淡水鱼产量的15.52% ，加工成冷冻鱼糜产品是其最具竞争力的利用途径，而如何防止蛋白质冷冻变性一直是该产业亟待解决的难题；前期研究表明鲢鱼鱼糜加工副产物的酶解产物具有良好的抗冻性能，但其抗冻组分及机理尚不明确。本项目通过研究：(1)制备鲢鱼加工副产物的系列酶解产物并作用于鱼糜，以鱼糜肌原纤维蛋白的理化特性、Ca2+-ATPase活性、不凝结水含量及鱼糜凝胶特性等为指标筛选出抗冻效果最佳的酶解产物组；(2)采用多级色谱分离纯化出抗冻组分并以质谱、核磁共振等方法鉴定其种类和结构；(3)利用显微镜观察抗冻组分对冰晶的抑制及其与蛋白质的相互作用，并结合差异蛋白质组分析和分子动力学模拟探讨其发挥抗冻作用的模式；期望从实验和理论水平上明晰酶解产物防止鱼糜蛋白质冷冻变性的机制，为利用加工副产物开发天然抗冻剂提供理论与技术支撑，这在理论研究和实际应用上都具有重要意义。

如何防止鱼糜中蛋白质的冷冻变性一直是该产业亟待解决的难题。自立项以来，本项目采用复合蛋白酶水解鲢鱼鱼糜加工副产物，制备了酶解时间15、30、60、120和240 min的酶解产物，将上述酶解产物添加到鲢鱼冷冻鱼糜中，以鱼糜的凝胶强度、不凝结水含量、Ca2+-ATPase 活性、巯基和二硫键含量等为指标分析其抗冻性能，筛选出30min酶解产物组的抗冻效果最佳。分别测定了上述酶解产物的分子量分布、氨基酸组成、表面疏水性、巯基等理化性质和特征，通过对比分析酶解产物的抗冻性能及理化特性，初步明确谷氨酸、天门冬氨酸和赖氨酸等极性氨基酸的含量，亮氨酸、异亮氨酸及苯丙氨酸等疏水性游离氨基酸含量，分子量2000 Da左右的部分、疏水性与抗冻性具有较大的相关性；将30min酶解产物分别用膜分离纯化及凝胶过滤色谱进行纯化，分别得到不同的纯化组分，进行质谱鉴定和结构解析，明确了通过膜分离得到的GVDNPGHPFIM、GVDNPGHPFIMT和IITNWDDMEK三种肽对鱼糜具有抗冻保护活性；通过凝胶过滤得到FAVIDQDKSGFIEEDELKLFLQ、DFSAGARALTDAETKAFLKAGD、FAVIDQDKSGFIEEDELKLFLQDFSAGARALTDAETKAFLKAGDSDGDGKIGVDEFAVLV、FAIIDQDKSGFIEE对酵母菌具有抗冻活性；采用低场核磁、低温显微镜观察、透射电镜结合分子对接揭示酶解产物与冰晶间通过氢键结合，抑制冰晶的生长，减缓机械损伤对鱼糜的结构造成的破坏；酶解产物中的抗冻组分与蛋白质间也存在相互作用，抑制了冻融造成的蛋白质变性。总体而言，完成了原计划的研究内容。研究成果为应用鱼糜加工副产物的酶解产物开发天然抗冻剂奠定了理论基础。