巯基氧化修饰对肌原纤维蛋白热诱导交联的影响机制

Oxidation is one of the major cause for quality deteriaration of meat products. The reactive thiol side chain of cysteine is highly sensitive to reactive oxygen species.The role of thiol oxidative modifications on the cross-linking pattern of myofibrillar proteins during heating process (denaturation, aggregation and gelation) is still need further study. In this project, myofibrillar proteins of pork will be exposed to different oxidative environments. The redox proteomics techniques with high throughput mass spectrometry analysis will be used for the identification of individual modified residues to compare the differences between unoxidized samples and oxidized samples during heating. The individually modifications, such as sulfhydryls disulfides, sulfenic acid, sulfinic acid, and sulfonic acid residues will be characterized. Circular dichrois, fluorescence spectroscopy and 2D diagonal redox SDS-PAGE will be used to investigate the influence of thiol oxidative modifications on heat-induced denaturation and aggregation. The conformation and protein molecular assembly pattern will be analyzed. In order to evaluate the influence of thiol oxidative modifications on the heat induced protein aggregations, thiol blocking and dissulfide bond breaking stratages will be used. The gel properties will be investigated to evaluate the role of thiol oxidative modifications on the driving force for heat-induced cross-linking. Static light scattering technique will be used to evaluate the the role of non-covalent crosslinking during heating. Moreover, the hydrogen bond, ionic bond and hydrophobic bond content will be analyzed. The aim of this project is to elaborate the role of thiol oxidative modifications on the mechanisms of heat-induced cross-linking pattern of myofibrillar proteins and to provide some fundamental theoretical basis on the control of oxidation for meat products.

原料肉的蛋白质氧化影响肉制品品质，蛋白巯基基团对活性氧敏感，但巯基氧化对蛋白分子的热诱导去折叠、聚集、交联机制有待进一步揭示。本项目以模拟氧化控制体系下猪肉肌原纤维蛋白为研究对象，利用氧化还原蛋白组学（Redox proteomics），追踪氧化和未氧化蛋白在热处理过程中半胱氨酸巯基氧化修饰产物二硫键、次磺酸、亚磺酸、磺酸等位点的动态演变，探究巯基可能的氧化还原途径；采用巯基反应试剂对蛋白巯基进行氧化修饰，利用圆二色光谱、对角线电泳等技术，表征蛋白分子构象和去折叠、聚集行为，阐述巯基修饰介导的蛋白热聚集组装机制；借助巯基封闭、二硫键还原试剂，靶向控制二硫键形成，分析蛋白凝胶特性，确证巯基氧化产物对肌原纤维蛋白热诱导分子交联的驱动效应，采用静态光散射技术，结合氢键、离子键、疏水键测定，明确非共价键交联的作用规律，揭示巯基氧化修饰对肌原纤维蛋白热诱导交联的影响机制，为肉制品氧化控制提供依据。

原料肉的蛋白质氧化影响肉制品品质，蛋白巯基基团对活性氧敏感，蛋白贮藏和加工过程中氧化诱导的氨基酸残基氧化修饰以及蛋白质组分变化规律机制有待进一步揭示。本项目设计了高氧气调包装贮藏实验，采用SDS-PAGE、非依赖性采集定量技术蛋白质组学等方法，阐明了高氧气调包装猪肉与新鲜猪肉蛋白组的变化规律；利用生物信息学方法，确定了氧化过程影响猪肉的蛋白质类型和功能，建立中高低水平脂肪气调包装猪肉组，探究脂肪和蛋白相互作用规律；借助氧化还原蛋白质组学手段和蛋白质非标记定量技术，明确了发生氧化修饰的蛋白质类型和功能信息，解析了猪肉全蛋白高氧包装条件下巯基基团（半胱氨酸和蛋氨酸残基）发生的二硫键、次磺酸、亚磺酸、磺酸修饰类型，氧化修饰蛋白的肽段修饰位点，明确了肉制品实际生产加工过程中可逆性和非可逆性巯基氧化修饰位点的演变规律；进一步深入进行蛋白质网络互作分析，明确了参与氧化过程的功能性蛋白。本项目丰富了传统的蛋白质氧化理论，将宰后肉制品氧化理论深入到了蛋白质修饰的水平，为生产高品质肉奠定了理论基础。