花色苷C环4位限制对果蔬汁维生素C与花色苷协同降解的调控与机制研究

Anthocyanin and Vitamin C (VC) are critical nutrients in fruit and vegetable juices, with great physiological health benefits. However, the synergetic degradation of the two lead to the reduced quality of juice appearance and nutrition. Previous studies showed efforts to control synergetic degradation via classic anthocyanin stabilization technologies, and the results turned out to be not very satisfactory. In addition, the synergetic degradation mechanism is still unclear. The 4 position at C ring (C4) is a typical active reaction site for anthocyanin. To better understand the effects of C4 steric hinderance on synergetic degradation control and its mechanism, the most abundant anthocyanins in nature, cyanidin-3-glucoside (cy-3-glu), is selected as model pigment. Different levels of anthocyanin C4 steric hinderance is achieved via glycosylation, acylation and pyranization. The anthocyanins were evaluated for their quality improvements in clear apple juice. Then representative anthocyanins for each C4 modification were sent to juice model for detailed quantitative analysis. Anthocyanins and VC are quantified by HPLC-PDA-MS/MS, while their degradation products are identified through untargeted metabolomics analysis. The possible mechanism for anthocyanin C4 regulation is to be investigated by comparing degradation kinetics of different anthocyanin structures, as well as correlating color and antioxidant capability changes with degradation kinetics. This study will provide insights on anthocyanin-VC synergetic degradation control, which would guide to production of higher quality fruit and vegetable juices.

花色苷和VC皆有重要生理活性，是果蔬汁饮料的重要营养成分。然而二者易发生协同降解，导致果蔬汁外观与营养品质的双重降低。研究显示，通过经典的花色苷稳色方法调控协同降解的效果比较有限，且二者协同降解机制尚不明确。C环4位是花色苷的活性位点之一，为探明花色苷C环4位限制对果蔬汁花色苷与VC协同降解的调控作用与机制，本实验选用自然界中最广泛存在的矢车菊素-3-葡萄糖苷（cy-3-glu）为模型花色苷，通过构建糖基化、酰基化、吡喃化修饰的cy-3-glu，按“定性探索—定量分析—机制解析”的思路，通过HPLC靶向定量和非靶向代谢组学方法，深度解析花色苷与VC协同解降的动态变化，研究过程中果蔬汁颜色和生理活性变化，通过动力学模型建立和关联性分析评价花色苷C环4位限制对其与VC协同降解的调控机制效果，并推断其调控机制，为提高果蔬汁外观与营养品质、实现有效调控果蔬汁花色苷与VC协同降解提供理论与技术指导。

花色苷（ACN）和维生素C（VC）是果蔬中具有重要生理活性的营养成分，但果蔬汁饮料体系中二者易发生互作降解，导致饮料外观与营养品质的双重降低。本研究聚焦ACN潜在反应位点C环4位，以矢车菊素-3-葡萄糖苷（C3G）为模型花色苷，分离制备了C环4位活性由大到小的系列C3G衍生物：矢车菊素-3,5-二葡萄糖苷（C35G）、矢车菊素-3(6”-丙二酰葡萄糖苷）（MAL）、芳香酸酰化的C3G衍生物纯化物（ARO）和10-儿茶酚-吡喃花色苷-3-葡萄糖苷单体（PACN）。评估了系列C3G衍生物在苹果清汁中对ACN和VC同步降解的干预效果，结果显示ACN的5位糖基化、酰基化、吡喃化修饰通过对VC潜在攻击位点C4构建逐级增强的空间阻碍，能够减缓苹果清汁中的ACN降解，ACN稳定性效果PACN＞ARO＞C35G，MAL＞C3G，推测ACN的C环4位是ACN和VC相互作用的关键位点；多酚代谢组学分析显示不同C3G衍生物间特有差异代谢物主要是酚酸类、黄酮类、木质素类和香豆素类。解析了模拟果汁中不同C环4位活性的C3G衍生物对ACN和VC的干预机制，ACN聚合色指数研究支持ACN和VC的互作很可能是基于ACN C环4位的缩合反应，VC的引入加速了以ACN为底物的聚合物的生成；一级反应动力学拟合结果显示VC加速ACN降解的作用大小取决于ACN的C环4位活性，C4的空间位阻越大，VC加速ACN降解作用和ACN的半衰期缩短越不显著；ACN的吡喃化修饰通过吞并C4位点成环，从源头上限制了VC对C4位进攻的可行性，故PACN添加的体系稳定性显著优于其他结构，VC的引入也未大幅缩短PACN的半衰期；但相同条件下5位糖基化和酰基化的修饰对C3G的半衰期延长效应非常有限，提示花色苷的C环4位虽然是ACN和VC相互作用的首选关键位点，但非唯一位点。本研究成果将为限制果蔬产品加工与贮藏过程中ACN和VC同步降解的限制技术研发提供新思路，研究结果对提高食品的外观与营养品质，实现营养素的精准加工有重要意义。