鱼糜中特征腥味物质与蛋白质分子结合作用机制的研究

The presence of special fishy odor substances is the bottleneck that constraints freshwater fish surimi industrial development. There is no effective deodorization method in the present. Key troubled this question was that most of the fishy odor substances in the surimi binds with surimi protein and they are hard to be removed by rinsing. The concept of protein-ligand interaction was introduced in the present project, to eluciadate the binding mechanism of typical surimi fishy odor substances with fish muscle proteins, and propose the theoretical basis for effective rinsing de-odorzing by controlling the binding and releasing. For this aim, silver carp will be chosed as the study object. The fishy odor substances, both in free and protein-bound states, in fush surimi will be extracted and indentified, and the characteristic fishy odor substances will be determined. The binding properties of fishy odor materials with fish sarcoplasmic protein, actin and myosin, including equilibration time, binding sites, binding constants and thermodynamic parameters will be studied by surface plasmon resonance and diffusion-ordered (DOSY) NMR studies. The effects of rinsing medium characteristics on protein conformation, fishy odor substances spectral characteristics, and the binding properties will also be studied and analyzed by bioinformatics-based molecular docking technology. According the above studies, the binding mechanism of typical surimi fishy odor substances with fish muscle proteins, and the effect of rinsing medium on binding and releasing will be eluciadated. The results of the present projest will provide necessary scientific theoretical basis for the effective removal of surimi fishy odor substances.

特殊鱼腥味物质的存在是制约淡水鱼糜产业发展的瓶颈，但目前国内外尚无有效脱腥的方法。困扰这一问题的关键是鱼糜中腥味物质大部分与蛋白质以吸附作用存在而难以有效漂洗脱除。本项目引入蛋白质-配体作用理念，通过腥味物质与蛋白质分子结合机制及影响因素的阐明，提出通过控制其结合和解离而漂洗脱腥的理论依据。研究拟以鲢鱼为研究对象，提取和鉴定鲢鱼鱼糜中本底游离态和蛋白质结合态特征腥味物质，确定典型特征腥味物质，通过表面等离子共振和核磁扩散序谱等技术研究腥味物质与鱼肌原纤维蛋白中肌动蛋白和肌球蛋白结合作用的平衡时间、结合位点、亲合指数及热动力学参数，同时研究漂洗介质特性对蛋白构象、腥味物质波谱特性及腥味物质与蛋白结合作用的影响，并通过基于生物信息学的分子对接技术进行模拟分析，建立鱼糜中典型特征腥味物质与蛋白质分子结合作用机制，阐明漂洗介质对其结合和解离的影响，为鱼糜中腥味物质的有效脱除提供必要的科学理论依据。

本研究着重阐明了鱼糜加工过程中特殊鱼腥味物质与鱼肉蛋白质结合的作用机制。主要取得以下三方面的研究结果：（1）采用同时蒸馏萃取、固相微萃取和整体材料吸附萃取三种风味分析技术分析了白鲢鱼肉的挥发性风味成分，鉴别了鱼糜中的本底特征腥味物质，确定了四种特征腥味物质（己醛、庚醛、辛醛和1-辛烯-3-醇）；（2）基于液上气体分析法研究了特征腥味物与肌球蛋白的结合作用，运用多光谱分析（紫外-可见、荧光）和圆二色谱技术研究了肌球蛋白与特征腥味物的结合作用机制，表明特征腥味物质可以引起肌球蛋白的荧光淬灭，淬灭机制为静态淬灭，通过构建Stern-Volmer 方程确定了肌球蛋白与特征腥味物的结合常数和结合位点数，经热力学计算推测两者间的作用力以氢键为主，与肌球蛋白相比，两者形成的复合物中α-螺旋含量显著升高，证实了氢键结合作用的存在；（3）比较了不同漂洗介质脱腥效果以及对多种鱼糜蛋白质的影响，考察了不同漂洗参数（pH、盐离子种类、离子强度）对特征腥味物与肌球蛋白结合作用的影响，确定了较优的鱼糜漂洗工艺。该研究可为实现淡水鱼糜的有效脱腥提供理论支持，对鱼糜及其制品的高值化加工具有重要现实意义。