货架期黑莓果酒二次沉淀的蛋白质和酚类物质基础及聚合机制解析

In recent years, more and more consumers pay attention to blackberry wine because of its considerable health benefits, such as anti-oxidative, anti-cancer, anti-inflammation, anti-diabetes or anti-cardiovascular. However, the applicant found that the secondary precipitation phenomenon frequently appeared during shelf life of blackberry wine, which caused the quality deterioration of the blackberry wine and the commercial value lost, and restricted severely the development of blackberry wine industry. The factor caused secondary precipitation were preliminary investigated by the applicant, and the results showed that the proteins and phenolic compounds were the main abiotic factors. Based on the previous research, this project plans to attain the main components of proteins leading to secondary precipitation by analyzing the dynamic changes of the protein components of blackberry wine and precipitation during shelf life and identify key proteins using iTRAQ (Isobaric tag for relative and absolute quantitation) technology, analyzing the changes of phenolic substances in wine and precipitation by HPLC-DAD - MS/MS method. The blackberry wine simulation system will be constructed using water, malic acid, lactic acid, succinic acid, ethanol and sodium hydroxide according to the characteristics of blackberry wine. Then the reaction mechanisms of proteins and phenolic in the blackberry wine simulation system will be analyzed via Isothermal Titration Calorimetry (ITC introduce) analysis, Fourier Transform Infrared Spectrometer (FTIR spectroscopies) and Atomic Force Microscope (AFM) technology. The variation of turbidity of blackberry wine simulation system will be analyzed during the polymerization process and the threshold range of precipitate will be determined. The successful implementation of this study will give the clear target for solving the secondary precipitation of blackberry wine during storage, and also provide theoretical foundations and new technical thinking for solving precipitation problem of other wine.

黑莓果酒因具有抗氧化、抗炎、抗肿瘤等功能活性而备受关注。申请者在黑莓果酒加工研究中发现，在货架期易出现二次沉淀，导致品质下降，商业价值丧失，因此二次沉淀成为亟待解决的关键问题。申请者对果酒沉淀形成因素进行初步研究，发现果酒中蛋白质和酚类是导致沉淀的两个主要非生物因素。在此基础上，本项目拟利用iTRAQ技术分析货架期黑莓果酒及沉淀中蛋白质组分动态变化并鉴定关键蛋白；利用HPLC-DAD-MS方法对果酒及沉淀中的酚类物质的动态变化进行分析，明确导致沉淀的主要蛋白和酚类物质基础；构建黑莓果酒模拟体系，模拟货架期条件，采用MALDI-TOF-MS、原子力显微镜分析等技术解析关键蛋白和酚类物质在模拟体系的聚合状态，明确结合物导致沉淀的阈值范围；利用等温滴定量热技术和傅里叶红外光谱分析等技术解析关键蛋白和酚类物质分子间的聚合机制。研究结果为针对性解决黑莓果酒货架期沉淀问题提供明确靶标，具有科学意义。

黑莓果酒因具有抗氧化、抑菌、抗炎、抗肿瘤等功能活性而备受关注。在货架期易出现二次沉淀，导致品质下降，商业价值丧失，严重制约该产业健康发展。申请者对果酒沉淀形成的非生物因素及其聚合机制进行了详细研究。首先，对黑莓果酒沉淀过程中酚类物质、蛋白质含量和花色苷含量的动态变化，研究结果显示整体上随着储藏期的延长和沉淀的出现呈现下降趋势。其次，对黑莓果酒沉淀中酚类物质进行了详细研究，确定了雾霾中多酚为地榆酸，利用液质TOF分析确定地榆酸(M/Z 469)的结构是由一个鞣花酸(M/Z 301)通过醚键与没食子酸基团连接形成。第三个方面的研究是分析果酒内源性物质对果酒体系稳定性的影响，结果表明与酒体氧化相关的因素导致了酒体的不稳定性。第四个研究内容围绕黑莓果酒沉淀蛋白的提取与组成分析展开研究，研究结果提供了一个果酒沉淀蛋白提取的新方法；蛋白组分结果显示黑莓新酒和陈酿14个月的黑莓酒中共鉴定出503种肽段属于178种蛋白。其中存在差异的蛋白有125种和无差异蛋白有53种。从种类上分析，在全部126种差异蛋白中，最大的一类是来源于酵母蛋白，共有49种。来源于黑曲霉和其它霉菌的蛋白有37种，第三类蛋白来源于植物的蛋白有23种。第五个方面围绕构建黑莓果酒模拟体系，解析鞣花酸和果酒沉淀蛋白在该模拟体系中的结合机制，研究结果显示20和 37℃下黑莓蛋白的猝灭速率常数Kq分别为1.2224×1012和0.8376×1012，均远高于2.0×1010mol/(L•s),表明鞣花酸与黑莓蛋白的相互作用机制为静态结合。鞣花酸与黑莓酒蛋白的结合位点n 均在１附近，且远小于2，并且受温度的影响不大。因此，鞣花酸与黑莓酒蛋白的结合比例约为 1:1，并且鞣花酸与黑莓酒蛋白作用的结合常数 Ka值以 1012为数量级，均远大于104数量级，说明鞣花酸与黑莓酒蛋白之间的结合作用较强。因此，导致黑莓果酒二次沉淀的蛋白和酚类物质的聚合机制为两者之间是以较强的分子间作用力的静态结合机制。研究结果为针对性解决黑莓果酒货架期沉淀问题提供明确靶标，具有重要科学意义。