蛋白基载酶颗粒的构建及其皮克林乳液界面稳定和催化机制研究

The interfacial catalysis efficiency of bioenzyme in two-phase interface is relatively low in food processing. The particle-stabilized Pickering emulsion can provide an extremely large two-phase interface to accelerate in situ enzyme-catalyzed modification of fat-soluble components. How to achieve the emulsion stability and the enrichment of the enzyme in the interface without losing their activity at the same time is the key point for constructing Pickering emulsion interface catalysis system. In this project, dual-functional particles with both interfacial and catalytic activity will be constructed by using food grade materials. The influence mechanisms of particle structure and interface behavior on emulsion stability and interface catalytic property will be systematically investigated. The detailed researches are described as follow: (1) Preparation and characterization of enzyme-loaded Pickering particles. The interfacial active particles are formed by strengthen the interaction and co-assembly of the soybean 7S protein active fragments and the polysaccharides or calcium phosphates, and the enzyme loading with the particles is achieved by means of embedding or adsorption. The size, structure, and functional properties of the particles are regulated. The particle formation mechanism will be studied; (2) the interface adsorption kinetics of enzyme-loaded particles and rheological properties and microstructure of interfacial membranes will be characterized; (3) the Pickering emulsion will be constructed using enzyme-loaded particles, and the emulsion stability and interfacial microstructure as well as interfacial catalytic efficiency will be evaluated. This project will reveal the interfacial stability and catalytic mechanism of the enzyme-loaded particles. The intrinsic relationship between the interface behavior and macroscopic emulsion properties will be evaluated, which will provide new ideas for the development of enzyme interface microreactors.

生物酶两相界面催化效率偏低是食品加工中常见问题。颗粒稳定的皮克林（Pickering）乳液可提供极大的两相界面有利于脂溶性成分高效酶促转化。如何同时实现乳液界面稳定和酶在界面富集且不失活是构建载酶乳液的关键点。本项目着眼于利用食品原料构造兼具界面和催化活性的双功能载酶颗粒，系统研究颗粒结构及界面行为对乳液稳定性和界面催化功能两个方面的影响机制。内容包括：(1)载酶颗粒制备及结构表征。通过大豆7S蛋白活性片段与多糖或磷酸钙之间相互作用及共组装形成界面活性颗粒，并以包埋或吸附的方式实现颗粒对酶的荷载，构造尺度、结构及性质可控的载酶颗粒，研究颗粒形成机制；(2)表征载酶颗粒界面吸附动力学、界面膜流变学性质及微结构；(3)构建载酶乳液，表征乳液稳定性及界面微结构，评价其界面催化效率。本项目将揭示载酶颗粒的界面稳定及催化机制，明确界面行为与宏观乳液性质的内在关系，为酶界面微反应器开发提供新思路。

在食品加工中存在生物酶两相界面催化效率偏低的问题。颗粒稳定的皮克林（Pickering）乳液可提供极大的两相界面加速酶催化脂溶性组分原位转化。如何同时实现乳液界面稳定和酶界面富集且不失活是构建载酶乳液的关键点。本项目利用食品级材料之间的相互作用，制备了具有界面活性并可增强酶活性的胶体颗粒，并构建Pickering乳液促进两相催化，明确了颗粒形成机制及对界面稳定及催化性质的影响，在乳液原位相反转控释以及风味物质合成等方面展现出应用前景。本项目主要研究结果如下：.1、制备了一种基于壳聚糖纳米胶的Pickering乳液催化体系。正电荷的壳聚糖纳米胶具有良好界面活性，优先形成水包油型Pickering乳液，并通过静电等非共价力吸附负电荷的脂肪酶。相比游离酶，Pickering乳液体系有效提高脂肪酶水解反应活性，并在pH1.5-7.5范围内保持催化活性，13次批次回收反应后，体系催化活性仍可保持初始活性的55%以上。通过水解甘油三酯油相获得的游离脂肪酸对壳聚糖纳米胶的原位修饰作用，改变壳聚糖纳米胶的润湿性，实现脂肪酶刺激响应的Pickering乳液相反转。.2、以乳液模板法制备一种疏水改性壳聚糖颗粒修饰的明胶微球，脂肪酶包埋于微球内，以微球为颗粒乳化剂构建水包油型Pickering乳液，用于催化己酸己酯合成反应。长碳链脂肪醛（庚醛，壬醛，十一醛）疏水改性的壳聚糖颗粒吸附于明胶微球表面，促使明胶微球润湿性得到改善，能够较好的稳定Pickering乳液。乳液体系提供的较大界面面积使其催化己酸己酯合成反应效能达87.8%，并可通过离心实现产物和酶的分离。载酶明胶微球具有较好的稳定性，8次回收后反应转化率保持为初次反应的52%左右，明胶微球能够有效发挥和保护脂肪酶活性。.3、制备了一种以玉米醇溶蛋白-明胶复合胶体载酶颗粒稳定的Pickering乳液体系。在pH8.0时玉米醇溶蛋白颗粒与明胶颗粒1:1复合，以静电相互作用和氢键等非共价力结合，胶体颗粒表面润湿性好，界面吸附能力较强，颗粒稳定的Pickering乳液粒径较小，稳定性好。相比游离酶，Pickering乳液油水界面吸附脂肪酶能力强，酶水解活力高，耐高温性强。