肌原纤维蛋白氧化羰基化诱发的蛋白交联对牦牛肉质构特性的影响机制

The myofibrillar protein oxidation will lead to the formation of intramolecular or intermolecular cross-links which include disulfide cross-links, dityrosine cross-links and carbonyls cross-links. The potential influence of cross-links induced by myofibrillar protein oxidation on texture traits of yak meat has been found in previous study, but the intrinsic mechanisms remain unknown. The myofibrillar protein oxidation model of yak meat will be established in this project to investigate the effect of oxidation systems (MetMb+H2O2 system and Fe3++H2O2 system) under different oxidation strength on characteristics of oxidation (special carbonyls formation, thiol groups loss and Ca2+-ATPase activity loss), three kinds of cross-links (disulfide cross-links, dityrosine cross-links and carbonyls cross-links) and advanced structure changes which could be measured through DSC, Fourier spectrum and Raman spectrum. While three potential influence paths of cross-links induced by myofibrillar protein oxidation on texture traits will be confirmed through induced oxidation in both vivo model and vitro model: ① signal myofiber breaking strength and strain will be analyzed as well as MFL and MFI to confirm the influence path by strengthening muscle fiber structure; ② morphology of muscle fiber shrinkage will be observed through SEM and TEM with evaluation of effect of cross-bridge (incubation of ATP and Mg2+) to prove the influence path by affecting shrinkage; ③ myofibrillar protein degradation patterns and its relationship with cross-links and calpain and cathepsin activity will be analyzed to confirm the influence path by alternating myofibrillar protein hydrolyzing sensitivity. The key myofibrillar protein origin and cross-linking structure of critical protein aggregation in each potential influence path will be determined through proteomics method. Finally, the influence mechanism of cross-links induced by protein oxidation carbonylation on texture traits of yak meat will be illuminated in order to provide fundamental basis for control of yak meat texture and research and development of new product with special texture.

肌原纤维蛋白氧化羰基化会导致3类交联结构（二硫键、二酪酸、羰基交联）的形成，前期研究发现这对牦牛肉质构特性具有潜在影响，但其内在机制尚不清楚。本项目拟组建牦牛肌原纤维蛋白氧化模型，分析氧化系统（MetMb和Fe3+），在不同氧化强度下，对氧化状态（特定羰基、巯基、ATPase活力）、3类交联、高级结构（DSC、傅里叶光谱等）的影响，并通过诱导氧化，验证氧化交联对质构的3种可能影响途径：①分析单根肌纤维强度、断裂应变及组织学特点，结合MFL和MFI指数，验证强化途径；②利用电镜观察肌纤维收缩，配合ATP、Mg2+离体孵化，验证收缩途径；③分析蛋白降解模式，及其与交联结构和蛋白酶活力的关系，验证水解敏感度影响途径，采用蛋白组学方法确定各影响途径中的关键肌原纤维蛋白和交联方式，从而阐明肌原纤维蛋白氧化羰基化对牦牛肉质构特性的影响机制，为牦牛肉质构特性控制及具备独特质构的新产品研发提供理论基础。

蛋白质氧化会导致肌原纤维蛋白生化特性改变及内源酶活性降低，进而使肌原纤维蛋白的降解受到限制，最终阻碍了肉品的宰后嫩化。本项目研究发现，同一氧化强度下，随着成熟时间的延长，各处理组均表现出ROS和羰基含量增加，总巯基含量减少，caspase-3、caspase-6和calpain 1活性在0-1 d增强，随后1-14 d减弱，肌钙蛋白-T和肌间线蛋白均发生不同程度的降解，蛋白质氧化阻碍了牦牛肉宰后成熟过程中嫩度的改善，尤其是成熟5 d后肉的嫩化。羟自由基氧化体系下，随着氧化强度的增大，羰基含量、二硫键含量和二聚酪氨酸相对含量均显著升高，总巯基含量和表面疏水性均显著降低，肌球蛋白重链的交联加剧，同时产生20-25 kDa肌球蛋白轻链降解产物，此外，副肌球蛋白、原肌球蛋白、肌动蛋白和肌钙蛋白-T均发生不同程度的降解。此外，羟自由基氧化显著降低了caspase-3、caspase-6和calpain 1的活性。结合原位与体外试验发现，不同氧化强度下牦牛肉宰后成熟过程中蛋白质氧化会导致其生化特性改变和交联物形成，加强了肌原纤维蛋白的结构；同时，宰后成熟过程中内源酶（caspase-3、caspase-6和calpain 1）的氧化降低了其活性，进而降低了其对肌原纤维蛋白的降解作用，阻碍了牦牛肉宰后成熟过程中嫩度的改善。