NO诱导肌原纤维蛋白的亚硝基化修饰在猪肉成熟中的作用机制研究

The high percentage of inferiority and inconsistency of chilled pork meat quality is now becoming the important issue to be considered for meat industry in China. The underlying mechanism for meat quality formation during the postmortem aging is recognized to be the key point and/or the prerequisites for seeking the solution. The biochemical and metabolic pathways in postmortem cell is complex while muscle contraction in rigor and myofiber integrity during meat tenderization is recognized to be critical to affect meat quality. Nitric oxide synthase can generate nitric oxide (NO) in postmortem muscle and NO-induce protein S-nitrosylation modification can potentially affect protein structure and function. The current project is emphasized on the NO-induced myofibrillar protein S-nitrosylation during the postmortem aging in pork. By employing the techniques of biological free radical detection and proteomics, the critical S-nitrosylated myofibrillar proteins and specific modified-cysteines are identified to explore the potential effects on their structure and functions. The S-nitrosylated proteins features and significant metabolic pathways is further investigated using bioinformatics. It is expected to develop the correlation between S-nitrosylated myofibrillar proteins and muscle contraction and proteolysis of myofibrillar proteins to elucidate the underlying mechanism of meat quality formation. This project can facilitate to develop the meat aging theory to a large extent and give a theoretical basis for promotion of pork meat quality in China.

冷却肉的劣质肉发生率高和品质不稳定是我国猪肉产业亟需解决的难题，探究宰后猪肉品质形成的内在机制是解决猪肉品质难题的关键和前提。宰后肌细胞生化代谢复杂，对猪的鲜肉品质起关键作用是僵直期的肌肉收缩程度及嫩化期的肌纤维结构完整性。宰后肌细胞中一氧化氮合成酶催化产生一氧化氮（NO），可诱导蛋白质发生亚硝基化修饰，影响其结构与功能。本项目重点研究宰后猪肉成熟过程中NO及其诱导肌原纤维蛋白的亚硝基化修饰；基于生物自由基检测技术和蛋白质组学等技术，表征猪肉成熟过程中关键肌原纤维蛋白的亚硝基化修饰，并鉴定特异性半胱氨酸修饰位点，解析对蛋白结构和功能的潜在影响；利用生物信息学手段剖析亚硝基化修饰蛋白的特征及显著性代谢途径；建立亚硝基化肌原纤维蛋白与肌肉收缩和肌原纤维蛋白降解的内在联系，从而探究亚硝基化修饰对鲜肉品质形成的影响机制。本项目的开展可丰富和发展宰后猪肉的成熟理论，为阐述猪的鲜肉品质形成奠定基础。

冷却肉的成熟机理一直是肉品领域研究的热点和难点问题，肌原纤维蛋白的降解及修饰影响了猪肉成熟过程中品质的形成。本项目以背部最长肌为研究对象，通过宰后1h的pH值、L*值和宰后24 h贮藏损失等指标作为分组依据对正常肉和PSE肉进行分组，检测猪肉成熟过程中氧化还原状态、蛋白质降解和品质的变化，使用蛋白质组学鉴定肌原纤维蛋白及其亚硝基化修饰，并进行生物信息学分析。研究结果表明，猪肉成熟过程中氧化及亚硝基化程度逐渐增加，还原能力逐渐降低；与正常肉相比，PSE肉肌原纤维更易于断裂，宰后早期肌原纤维蛋白的蛋白降解程度更高；在宰后1h的PSE肉和正常肉中共检测出172个差异蛋白，上调（PSE/正常）的有151个，下调（PSE/正常）有21个。差异蛋白主要分布于细胞骨架中，发挥蛋白结合活性的分子功能，通过糖酵解/糖异生、HIF-1信号通路、钙信号通路、AMPK信号通路和PI3K-Akt信号通路等显著性代谢途径影响肉品质的形成。PSE肉中上调的半胱氨酸亚硝基化修饰位点有109个，下调的位点为30个，对应的上调蛋白为75个，下调蛋白为40个。主要分布于细胞骨架的差异蛋白参与了肌肉收缩、肌原纤维的组装、肌细胞发育等过程，发挥了与细胞骨架蛋白结合的分子功能。亚硝基化修饰蛋白通过氨基酸的生物合成糖酵解/糖质新生、AMPK信号通路、HIF-1信号通路和紧密结合等显著性代谢通路参与了肉品质的形成。综上所述，本项目结果表明NO通过对肌原纤维蛋白的亚硝基化修饰，影响了肌肉收缩和蛋白质降解过程，参与了宰后肌肉的生化代谢，影响肉品质的形成，从而进一步阐释了PSE肉形成的机理。