共价交联修复损伤蛋白成膜性机制研究

In practical system of foods, proteins often were damaged by the causes of severe heating or oxidation, which lead to impair various functional properties of protein and reduce their values. The work involves in restoring functional losses of damaged proteins via covalent cross-linking and consequently provides important scientific interests and potentially practical applications in food industry. Whey proteins and film formation (one of the most important functional properties to a protein) are used to serve as a model protein and a typical feature, respectively. Given that transglutaminase efficiently catalyzes cross-linking reactions, intra- and intermolecular SH/SS exchange reactions regulated by cysteine, this proposal is to elucidate the effect of protein restoring on the capacity of film formation of the damaged proteins through the aforementioned reactions. A simulated system (thermal and oxidative) to practical protein processing is employed to investigate the heating and oxidative effects on the constituent, structural properties and physiochemical performance related to film forming of whey proteins. The mode of action and regulatory strategies involved in restoring film formation of the damaged protein via covalent cross-linking are also to explore. The significance of this study is to pay attention to the utilization of damaged proteins, and focus on developing the theory of restoring functionality like a film formation of the damaged proteins. It births a novel insight into the further efficiently utilization of protein resources, promotes the development of protein-based materials endowed with diverse functional properties, and provides fundamentally scientific evidence for innovation of proteins-based materials.

在食品体系中，热和氧化压力造成蛋白质的损伤，进而影响蛋白质的许多功能特性和价值。通过蛋白质共价交联对损伤蛋白功能性修复的研究，具有重要的科学意义和应用价值。本研究以乳清蛋白为模式蛋白以及蛋白众多功能性中之一的成膜性能为例，基于谷氨酰胺转移酶高效催化蛋白交联以及半胱氨酸调节巯基和二硫键相互转化的特性，对损伤蛋白成膜性能进行修复研究。通过构建模拟热、氧化损伤体系，研究热、氧化等损伤效应对蛋白组分、结构和成膜性行为的影响，深入探讨谷氨酰胺转移酶与半胱氨酸组合共价交联损伤蛋白的分子行为以及揭示这种组合对损伤蛋白成膜性的修复机制和调控策略。该研究关注损伤蛋白的利用，将发展共价交联对损伤蛋白成膜性的修复理论，为蛋白资源的进一步有效利用提供新思路，同时也为蛋白基质材料的功能多样性开发提供重要的科学依据。

食品蛋白质在提取、加工与贮藏过程中因为氧化造成蛋白质功能的变动，影响其营养价值和加工功能性。本研究基于乳清蛋白及其成膜性为模式，首先通过将羟基自由基氧化体系引入乳清蛋白以模拟蛋白实际氧化，再经历不同温度（70-90 °C）加热后利用TGase进行修复，通过表征蛋白质结构和分子量、成膜液行为及所成膜的机械性能，发现氧化度在10 mM H2O2以下的蛋白可被温度的提升与TGase交联修复其机械强度，归结于蛋白分子具有可利用性的氨基和巯基可供酶共价交联和分子间二硫键的形成。考虑到加热/氧化序列对蛋白成膜性的影响，将上述氧化、加热体系引入蛋白制备蛋白膜，发现氧化促进蛋白低聚物的共价物形成，但破坏蛋白所成膜的微观结构，超过5 mM H2O2以上氧化度则显著提高膜蛋白浸出率。对于热损伤蛋白代表性的案例就是脂类氧化产物MDA进入蛋白体系并反应从而影响蛋白成膜功能性。通过MDA产生模拟体系引入蛋白中，测定二者的反应性，发现MDA与氨基和巯基有很强的反应性，共价交联蛋白后形成耐水性多孔性膜，提高膜的机械强度和耐热性，并显著抑制膜蛋白的溶出性。上述三种试验中均发现巯基/二硫键在成膜性中的作用，为此在蛋白中加入NEM以屏蔽巯基和还原剂Cys/DTT，结果显示Cys能够促进SH→SS和分子重排而形成聚合物，TGase的加入促进蛋白进一步交联形成分子量更大的聚合物，并具有“叠加效应”，所成蛋白膜的机械性能、热力学及吸水得到显著提高，而蛋白浸出率则降低，这归结于上述处理时分子聚合形成质地细腻紧密结构有关。本研究结果阐明了共价交联损伤蛋白成膜机制，并初步发现了氧化、热损伤对蛋白的结构及成膜性影响机制，为蛋白资源的综合利用及功能多样性开发提供理论依据。