豌豆蛋白-多糖-皂苷相互作用对乳液界面性质和聚集稳定性的影响机制

The protein emulsions often undergoes destabilization during processing and shelf life, which seriously affects its quality. This destabilization presents a technical problem that needs to be solved urgently in the food industry. In the previous work, we found that the combination of protein, polysaccharide and low-molecular weight (LMW) surfactant plays an important role in the prevention of random protein aggregation and inhibition of competitive adsorption behavior at interface between protein and LMW surfactant, thus long-term stability of emulsion can be effectively achieved. However, the mechanism of interaction among protein, polysaccharide and LMW surfactant was unclear. Therefore, pea protein, polysaccharides and saponins were selected as research subjects in this project, and the effect of environmental factors (including combination order, pH, ionic strength, etc.) on the phase separation behavior of complexes in bulk were firstly investigated; the correlation between synergistic/competitive adsorption behavior and aggregation stability of emulsions was then studied in oil-water interface; in order to reveal the mechanism how the interaction of complexes affecting the interface properties and aggregation stability of emulsions, isothermal titration calorimetry, molecular docking and molecular dynamic simulation were further used to speculate the thermodynamic binding rules of the complexes, as well as the spatial conformation change of hydrophobic and charged groups around binding sites to establish the structure-effect relationship between the interface properties and the spatial conformation change. These studies would provide some important scientific basis for developing techniques to prepare stabilized pea protein emulsion and other vegetable protein emulsions.

蛋白乳液体系在加工和货架期内常发生失稳，严重影响其品质，是食品工业中亟待解决的技术问题。前期研究发现，蛋白质、多糖和小分子活性剂三者结合对防止蛋白质无规聚集以及抑制蛋白质-小分子活性剂界面竞争性吸附行为具有重要作用，能有效地实现乳液的长期稳定性，但其相互作用机制却不清楚。基于此，本项目拟以豌豆蛋白、多糖、皂苷为研究主体，首先考察环境因素（结合顺序、pH、离子强度等）对复合物在体相中的相分离行为的影响规律；然后结合静电吸附和共价交联模式，研究复合物油-水界面协同/竞争吸附行为与乳液聚集稳定性的相关性；最后基于等温滴定量热、分子对接及动态分子模拟等技术研究三者结合过程的热力学规律，推测结合位点处疏水及带电基团构象变化等，建立其与复合物界面性质的构-效关系，揭示相互作用影响乳液界面性质和聚集稳定性的机制。项目的顺利实施，以期为豌豆蛋白及其他植物蛋白稳定乳液的研发提供重要的理论支撑。

对于植物蛋白饮料来说，为了实现乳液的短期和长期稳定性，往往复合使用蛋白质、多糖以及小分子表面活性剂，使其发挥各自优点，降低界面张力，并形成保护层阻止乳状液滴凝聚、聚合和脱稳。本项目通过研究豌豆蛋白结合多糖及皂苷复合物的体相行为，以及界面动力学吸附及界面膜扩张流变学，获得物理稳定的豌豆蛋白乳液体系；通过等温滴定量热、分子模拟等揭示豌豆蛋白与多糖及皂苷的分子机制，解释影响乳液稳定性的机理。项目研究发现，豌豆分离蛋白（PPI）/多糖体系的临界pH值与蛋白质/多糖比、多糖官能团和电荷密度密切相关，硫酸多糖体系的临界pHφ和pHmax高于羧基多糖。豌豆乳清蛋白与阴离子多糖存在选择性复凝聚行为，其中PA2优先参与复凝聚，其选择性分离度取决于蛋白多糖比例和多糖电荷密度，羧甲基纤维素体系对PA1a回收率最高（纯度>90%）。PPI与大豆皂苷不能形成有效的静电相互作用，随着皂苷含量的增加，浊度曲线的浊度值不断降低，临界pH值没有发生变化。就乳液性质而言，当DS含量超过≥0.4%时，PPI-DS形成稳定的可溶性复合物，通过静电斥力和空间位阻效应，实现了PPI在酸性pH条件下的乳化稳定性。构建了具有不同微观结构的PPI-卡拉胶（CG）微凝胶颗粒和混合物溶液，结果表明，微凝胶颗粒具有更大的接触角（超过70°），其乳化稳定性更高；混合乳液形成了凝胶网络结构，微凝胶乳液没有形成凝胶形态，乳液流动性较高。研究了PPI-大豆皂苷乳液的性质，结果表明，随着大豆皂苷浓度增加，PPI/大豆皂苷的界面张力曲线衰减速率变小，乳液在pH 7和6下保持稳定，PPI与大豆皂苷发生了协同效应，说明皂苷的复配增加了乳液的酸性稳定性。ITC结果表明，清蛋白PA2与多糖的亲和常数高于PA1a，表明PA2可优先结合多糖发生选择性复凝聚行为。AlphaFold2结合PDB2PQR和APBS模型分析表明，7S、PA2、PA1a的分子表面存在不同数量的“正电荷补丁”，PA2的正电荷区域要多于PA1a，因此PA2与多糖的亲和能力更强，导致界面层蛋白组成、界面层厚度和电荷性质存在不同，从而影响乳液的稳定性。