EGCG共价修饰巯基对肌原纤维蛋白乳化凝胶特性的影响及机制研究

Tea polyphenols are commonly added to meat products in order to reduce oxidation during production and storage. Epigallocatechin gallate (EGCG) is the most abundant and powerful antioxidant in tea catechins, which are main phenolic compounds in tea polyphenols. Polyphenols can convert to quinones, which are able to react with thiol groups in meat proteins to form covalent ‘thiol–quinone’ adducts. However, the effect of ‘thiol–quinone’ adducts on the gel quality of meat products is scarcely documented. In this project, water holding capability, emulsifying property, texture property and microstructure et al. are measured to analyze the effects of EGCG on the properties of myofibrillar protein emulsion gel. Disulfide interactions, total sulfhydryl content, and disulfide content are analyzed in order to understand the mechanisms of the influence of EGCG on the properties of myofibrillar protein emulsion gel. Further, thermo Q-exactive is used to qualitatively and quantitatively analyze the ‘thiol–quinone’ adducts. The accurate reaction site of adduction reaction between EGCG and myofibrillar proteins is to be determined by proteomic analysis as well. In order to regulate the ‘thiol–quinone’ adduct and provide theoretical guidance and technical support for improving the gel properties of myofibrillar protein emulsion gel, the effects of EGCG dose, oxidative stress, ionic strength and pH value on the ‘thiol–quinone’ adducts formation and the properties of myofibrillar protein emulsion gel are further studied. The regulation system is established to regulate the influence of ‘thiol–quinone’ adducts on the properties of myofibrillar protein emulsion gel. The results of this project are important for the application of polyphenols as antioxidants in meat batters during production.

茶多酚是肉制品加工过程中常用的天然抗氧化剂，主要由结构和功能相似的儿茶素类多酚组成，其中EGCG含量最多，生物活性最强。多酚清除自由基后生成的醌类物质可以通过“巯基-醌”加成反应修饰蛋白质的巯基。然而，基于“巯基-醌”加成反应探究多酚对肉制品凝胶特性的影响还尚未报道。本项目通过分析保水性、乳化性、质构特性及微观结构等指标，阐明EGCG对肌原纤维蛋白乳化凝胶特性的影响。通过分析巯基含量、二硫键含量以及二硫键作用力等指标，并采用液质定性定量分析加成反应产物，利用蛋白质组学技术分析蛋白质上发生加成反应的位点，揭示“巯基-醌”加成反应影响肌原纤维蛋白乳化凝胶功能特性的机制。进一步研究EGCG添加量、氧化应激强度、离子强度以及pH值等条件对“巯基-醌”加成反应和蛋白乳化凝胶特性的影响，建立EGCG通过加成反应影响蛋白乳化凝胶特性的调控体系，为多酚在肉糜类制品加工过程中的应用提供理论指导和技术支持。

多酚及富含多酚的天然植物提取物常被做肉制品的抗氧化剂。此外，富含多酚的天然植物提取物也常用作肉制品的风味改良剂和着色剂。然而多酚与蛋白之间的相互作用及其对蛋白理化、结构、功能特性的影响还不清楚。因此，经常造成肉制品的品质不稳定。.项目研究了多酚对肌原纤维蛋白共价和非共价相互作用的规律，研究了相互作用对肌原纤维蛋白理化、结构、流变特性、热力学稳定性的影响，进一步探究了多酚对蛋白凝胶和乳化特性的影响，对凝胶微观结构和脂质分布的影响。研究了多酚类型、添加量、NaCl浓度对蛋白质性质以及乳化凝胶特性的影响规律。.研究发现添加表没食子儿茶素没食子酸酯（EGCG）可以显著阻止羰基含量的提高，说明多酚降低了肌原纤维的氧化水平。添加1000ppm剂量时，EGCG通过“巯基-醌”和“氨基-醌”迈克尔加成反应，对肌原纤维蛋白造成了不可逆转的修饰，显著降低了肌原纤维蛋白的游离巯基和氨基水平，表面疏水性由于蛋白的聚集而显著降低，蛋白的热稳定性变差，流变特性显著改变。不可逆的修饰，致使肌原纤维蛋白乳化凝胶的微观结构和乳化特性受到损伤，最终导致肌原纤维蛋白凝胶的保水性和凝胶强度变差。其次研究发现添加12 µM/g的迷迭香酸（RA）可以显著阻止巯基、氨基的减少和蛋白质的解折叠。但是，300 µM/g RA可以通过共价和非共价的方式与肌原纤维发生相互作用，从而导致巯基、氨基的减少和蛋白质的聚集，引起溶解性降低，导致肌原纤维蛋白乳化特性变差，具有较高的蒸煮损失和较低的凝胶强度。此外，300 µM/g RA损伤了肌原纤维蛋白的乳化特性。此外研究发现，较高浓度的NaCl可以使肌原纤维蛋白充分的吸水溶胀，从而更利于同EGCG和RA发生相互作用，EGCG和RA对肌原纤维蛋白理化、结构特性的影响越显著，从而对肌原纤维蛋白乳化凝胶特性的影响越显著。.项目研究成果为控制多酚与肌原纤维蛋白互作控制体系的建立提供了科学指导。