蛋白质非共价结合蓝莓花色苷稳定性的影响机理

Anthocynanins are recognized as one of the most functional bioactive compounds. Improving the stability of anthocyanins is the most effective method to solve its low utilization. However, traditional methods, such as co-pigmentation, molecular modification or chemical modification, will alter the natural colors of anthocyanins, and thus are difficult to be applied. Our previous study has found that the antioxidant activity of blueberry anthocyanins changed after binding to proteins. In this study, we propose a hypothesis that non-covalent interaction between proteins and phenolic hydroxyl group of anthocyanin B-ring could improve the stability of blueberry anthocyanins and will discuss it in three parts: (1) to understand the mechanism of anthocyanin degradation by using malvidin-3-galactoside and delphinidin 3-galactoside chloride to build a Thermal Degradation Kinetics Model; (2) to utilize lactoprotein and bovine serum albumin as protective carriers in order to analyze the mechanisms of interaction, analyze protein structure change and explain the properties and stability of non-covalent interaction between proteins and blueberry anthocyanins through methods including fluorescence spectrum, FTIR and circular dichroism; (3) to explore the mechanism of how the protein-anthocyanin compound functions in vitro by testing antioxidant activity and then setting a Caco-2 cell absorption model. We believe that this study will not only be a critical fundamental basis for future study of anthocyanin, but also has an impact in improving anthocyanin stability in blueberry processing and storage.

花色苷是自然界最重要的功能性色素。提高花色苷的稳定性是解决其利用率低最有效的方法，传统依靠分子修饰、化学改性等方法存在影响其自身颜色的问题，难以实际应用。本团队在发现蓝莓花色苷与蛋白质结合后，抗氧化性发生变化的基础上，围绕花色苷B环上的酚羟基非共价结合蛋白质会提高花色苷稳定性这一关键问题开展研究：（1）以蓝莓富含的锦葵色素-3-半乳糖苷为研究对象，建立降解动力学模型，揭示降解途径和机制；（2）以酪蛋白和牛血清蛋白为保护载体，通过荧光光谱、红外光谱及圆二色谱等光谱法结合分子模拟技术，解析结合反应机制、探索蛋白质各级结构变化机理，阐明蛋白质非共价结合蓝莓花色苷稳定性的影响机理；（3）以蛋白质-花色苷复合物为模拟对象，通过抗氧化活性的测定，建立Caco-2细胞吸收模型，探讨体外模拟消化过程中复合物作用机理。以上成果将奠定花色苷领域的重要基础，对蓝莓贮藏和加工过程中提高花色苷稳定性具有重要意义。

花色苷是蓝莓中主要的天然活性物质，具有强抗氧化能力，但稳定性较差的问题极大地限制了蓝莓花色苷的应用。本研究探究了蓝莓花色苷的降解动力学，揭示了酪蛋白和牛血清蛋白对蓝莓花色苷的保护作用及机制，具体结果如下：（1）蓝莓花色苷的降解符合一级动力学，pH值越大，温度越高，加热时间越长，蓝莓花色苷降解速率越大。光照和H2O2处理均会加快蓝莓花色苷的降解，0.2% Na2SO3对花色苷的降解起到抑制作用，而0.05%、0.1%、0.15% Na2SO3会促进花色苷的降解反应；（2）采用荧光光谱、圆二色谱等方法，揭示了蓝莓花色苷中含量最高的单体-锦葵色素-3-半乳糖苷分别与牛血清蛋白通过静电作用力，与酪蛋白通过氢键、范德华力发生非共价结合，并且结合后蛋白质构象发生改变。分子模拟显示蛋白质特有的结构腔能够作为保护腔，将蓝莓花色苷包裹其中；（3）在加工条件和体外模拟消化的条件下，牛血清蛋白和酪蛋白有效地提高了蓝莓花色苷的稳定性，减少抗氧化活性的损失；（4）选取保护效果突出的α-酪蛋白进行大鼠药代动力学实验，验证了α-酪蛋白能够促进蓝莓花色苷以其原型及代谢产物的形式吸收到大鼠血液中，并减少花色苷因排泄出体外造成的损失。该研究对解决蓝莓花色苷易降解、生物利用率低的难题具有重要意义。