氧化修饰调控鱼糜凝胶特性机理研究

The degradation of myofibrillar proteins by cathepsins is the main cause for surimi modori. In addition, both cathepsins and myofibrillar proteins in skeletal muscle have been proved that they can be oxidized by radicals during postmortem. Oxidation will regulate the surimi gel properties by affecting the degradation of myofibrillar proteins via cathepsins, but the mechanism is still unknown. Therefore, the present project, which takes bighead carp as experimental materials, aims to analyze the effect of oxidation on the activity of cathepsin B and L, the degradation of myofibrillar proteins, and the surimi gel properties. The relationship between the three above factors will be studied accordingly. The promotion effect of mild oxidation on the surimi gel properties will be confirmed. Based on the detached incubation model, the effects of oxidized cathepsins, oxidized myofibrillar proteins, and both oxidized cathepsins and myofibrillar proteins on the degradation of myofibrillar proteins via cathepsin B and L will be investigated, as well as the interaction, the conjugation area and sites between cathepsins and myofibrillar proteins, which will reveal the mechanism of cathepsin B and L degrading myofibrillar protein. Finally, the effects of oxidation on the secondary and tertiary structure of cathepsin B and L and myofibrillar proteins will be analyzed, and the effects of structural changes on the surimi gel properties will be revealed. Above all, the effects of oxidation on the surimi gel properties through impacting both the structure of myofibrillar protein and degradation of myofibrillar proteins by cathepsins will be confirmed, thus providing the theoretical basis for the regulation mechanism of oxidation on surimi gel properties according to the conclusions of this project.

组织蛋白酶对肌原纤维蛋白的降解是造成鱼糜凝胶劣化的重要原因，宰后肌肉中组织蛋白酶及肌原纤维蛋白均存在由氧自由基引起的氧化修饰现象，氧化修饰参与调控组织蛋白酶降解肌原纤维蛋白，从而调控鱼糜凝胶特性，但调控机理尚不清楚。本项目以宰后鳙鱼肉为实验材料，明确氧化对组织蛋白酶B、L活性、肌原纤维蛋白降解及鱼糜凝胶特性的影响以及三者之间的相关性；确证温和的氧化有利于凝胶特性。利用离体孵育模型，分别从酶氧化、底物氧化及酶和底物均氧化三个角度，解析氧化修饰对组织蛋白酶降解肌原纤维蛋白的影响程度、影响区域及酶与蛋白结合靶点变化，揭示氧化修饰调控组织蛋白酶B、L降解肌原纤维蛋白作用机理；解析氧化对组织蛋白酶B、L及肌原纤维蛋白二、三级结构的影响，揭示结构变化对凝胶特性的影响，确证氧化通过调控肌原纤维蛋白结构及影响组织蛋白酶对肌原纤维蛋白的降解进而影响凝胶特性的作用，为氧化修饰调控鱼糜凝胶特性提供理论依据。

组织蛋白酶对肌原纤维蛋白的降解是造成鱼糜凝胶劣化的重要原因，宰后肌肉中组织蛋白酶及肌原纤维蛋白均存在由氧自由基引起的氧化修饰现象，氧化修饰参与调控组织蛋白酶降解肌原纤维蛋白，从而调控鱼糜凝胶特性，但调控机理尚不清楚。本项目以宰后鳙鱼肉为实验材料，明确氧化对组织蛋白酶B、L活性、肌原纤维蛋白降解及鱼糜凝胶特性的影响以及三者之间的相关性。结果表明，温和的氧化条件（10mM H2O2）能促进凝胶的凝胶强度及持水力。组织蛋白酶B 及L活性随着H2O2浓度升高而下降，但是仍能在0.1-50mM H2O2下发挥降解蛋白作用。10mM H2O2下，组织蛋白酶使更多肌原纤维蛋白降解，同时使蛋白的疏水相互作用降低，二硫键含量升高，造成凝胶强度及持水力劣化。利用蛋白组学方法解析酶和蛋白未氧化(PC)、仅蛋白氧化(OPC)及酶和蛋白均氧化(OPOC)三组样品的差异蛋白，GO及KEGG富集分析发现与PC-OPC组相比， PC-OPOC组的差异蛋白参与生物合成及代谢的负调控、杂环化合物及有机环状化合物的结合以及氧化磷酸化的较少，参与膜组分相关蛋白的降解、水解酶活性、氧化还原酶活性、肌动蛋白细胞骨架的调节、酪氨酸、缬氨酸、亮氨酸、异亮氨酸、赖氨酸降解等代谢通路的较多。这揭示了氧化前后组织蛋白酶降解作用的差异，这些差异与myosin-9-like isoform X1/X2/X3、tropomodulin-4-like、tropomyosin alpha-1 chain isoform X1/X3、pyruvate kinase PKM-like、histone-lysine N-methyltransferase SMYD1-like等差异蛋白相关。本项目还解析了氧化及组织蛋白酶L对肌原纤维蛋白二、三级结构的影响，揭示结构变化对凝胶质构的影响。结果表明氧化使蛋白的α-螺旋含量上升，β-折叠含量下降，对无规则卷曲及β-转角无显著影响。氧化使的表面疏水性升高，使荧光强度先上升再下降。在氧化情况下，蛋白质的二、三级结构与凝胶的质构呈现显著线性相关。组织蛋白酶的水解使得蛋白及凝胶的α-螺旋及β-折叠含量下降，无规则卷曲含量上升，使表面疏水性下降，使凝胶荧光强度下降。组织蛋白酶对蛋白三级结构的影响与鱼糜质构相关。本项目为氧化修饰调控鱼糜凝胶特性提供理论依据，为生产和贮藏高品质淡水鱼鱼糜提供科学依据。