发酵桑果浆黄酮醇改善花色苷稳定性的作用机制研究

Mulberry (Morus atropurpurea Roxb.) is a characteristic berry and has homology of medicine and food. However, anthocyanins in mulberry are extremely unstable and are susceptible to degradation, causing the fading of mulberry products during the processing and storage. Consequently, the quality of mulberry products is declined. Comparing with mulberry puree without any treatment, there are more nutrients and bioactive compounds in fermented mulberry puree which could be used for producing mulberry products with high quality. Our previous results showed that a* value of the mulberry puree fermented by Saccharomyces cerevisiae was higher than those in the control, and this variation was related to the flavonols in fermented mulberry puree. Therefore, this project focuses on the inter-relationship among flavonols components or content, total anthocyanins content and the value of color difference of mulberry puree. The stability characteristics of anthocyanins in the mulberry puree before and after fermentation, such as half-time, activation energy, UV absorption spectra and hydration equilibrium constant will be compared. The parameters representing the reaction process between the main flavonols and the main anthocyanins (cyanidin-3-O-glucoside or cyanidin-3-O-rutinoside), such as entropy change, enthalpy change and Gibbs free energy will be calculated to reveal the effect of flavonols on the copigmentation of anthocyanins. The interaction force and acting sites between flavonols and anthocyanins will be analyzed using infrared spectrum, nuclear magnetic resonance spectrum and theoretical calculations of molecular structure parameters and molecular dynamics. The mechanism of flavonols on improving anthocyanins stability in fermented mulberry puree will be illustrated. These results will provide a theoretical basis and reference for producing high quality mulberry products and the processing of other fruits and vegetables containing anthocyanins.

桑葚是药食同源的特色浆果，但其花色苷稳定性差、极易降解，导致桑葚产品褪色、品质下降。桑葚打浆（桑果浆）、发酵后活性成分增加，可用于生产营养价值更高的桑葚产品。申请人前期研究发现，桑果浆经酿酒酵母发酵后，色差值a*显著升高，色泽稳定性增强，该变化与黄酮醇相关。本项目通过探究发酵桑果浆中黄酮醇组分与花色苷含量、果浆色泽的内在联系，考察发酵前后花色苷稳定特性（半衰期、活化能、水合平衡常数等）的变化规律，分析主要黄酮醇与主要花色苷（矢车菊素-3-O-葡萄糖苷和矢车菊素-3-O-芸香糖苷）形成稳定呈色物的熵变、焓变和吉布斯自由能，研究黄酮醇对花色苷辅色的作用规律；利用红外光谱和核磁共振光谱技术，结合分子结构参数计算和分子动力学分析，解析黄酮醇与花色苷之间的作用力类型和作用位点，阐明发酵桑果浆黄酮醇改善花色苷稳定性的作用机制。研究结果可为生产高品质桑葚产品和其它富含花色苷的果蔬加工提供理论基础和参考。

桑葚是药食同源的特色浆果，不耐贮藏，除鲜食外，大部分用于加工果汁、果酱等产品。桑葚富含花色苷，但花色苷结构和性质不稳定，花色苷降解引起的褪色或褐变是影响桑葚产品品质的关键原因。本研究首先测定了发酵前后桑葚浆中花色苷和黄酮的种类和含量，通过相关性分析，明确了桑葚浆中与桑葚浆红色值相关的黄酮醇为芦丁、金丝桃苷、异槲皮苷和山奈酚（r > 0.7，P < 0.01）。通过研究不同黄酮对桑葚汁中花色苷含量、A520nm和色差值a*的影响，结果表明能够维持桑葚汁色泽稳定的黄酮为山奈酚、芦丁和异槲皮苷，金丝桃苷和槲皮素次之，儿茶素和槲皮苷的辅色作用较弱。以桑葚中的主要花色苷矢车菊素-3-O-葡萄糖苷（Cyanidin-3-O-glucoside，C3G）为研究对象，将C3G和不同黄酮醇按照摩尔比1：20混合，测定其降解速率常数k、半衰期t1/2、水合平衡常数pKh、活化能Ea、焓变（ΔH）、熵变（ΔS）和吉布斯自由能（ΔG）。结果表明山奈酚、芦丁、金丝桃苷、槲皮素、异槲皮苷显著提高了C3G稳定性，如降解速率常数k降低了8.6%~50.0%，半衰期t1/2延长7.4%~99.0%，pKh升高8.3%~37.8%，Ea提高60.0%~95.7%。焓变ΔH和熵变ΔS也有不同程度升高，表明花色苷要变成活跃状态所需克服的能量升高，体系中的无序状态升高，体系稳定性增加。槲皮苷和儿茶素对C3G稳定性的提升不显著。紫外全波长扫描图谱显示，添加山奈酚、金丝桃苷、异槲皮苷、芦丁、槲皮素后的紫外图谱发生显著红移，且在最大紫外吸收波长处的吸光值增加，而添加儿茶素、槲皮苷的体系中红移现象不明显。通过分子对接模拟计算，黄酮醇与C3G可以通过氢键和范德华力结合，并形成结构较稳定的复合物，但由于各个黄酮醇结构特点不同，他们与C3G之间的相互作用区域和强度也不同，按照作用强度排序依次为C3G-KAE > C3G-HYP > C3G-RUT > C3G-QTI > C3G-IQTR> C3G-QTR > C3G-CAT，计算结果与实验结果基本一致。本研究结果将为改善花色苷结构稳定性、提高富含花色苷食品的色泽稳定性提供理论基础和技术支撑。