【作者】 李春阳；

【导师】 许时婴；

【作者基本信息】 江南大学 ， 食品科学， 2006， 博士

【摘要】 葡萄是世界上经济价值较高的一种水果,为葡萄科(Vitaceae)葡萄属(Vitis)植物。葡萄果实中含有大量的酚类物质,其中30-40%存在于葡萄皮中,60-70%存在于葡萄籽中。原花青素(Proanthocyanidin,简称PC)是葡萄籽中含量最大的酚类物质,具有高度羟基化的结构,能够抗氧化、清除自由基、治疗心血管疾病,有很大的开发利用价值。经改良的香草醛-盐酸法测定原花青素含量的精密度、准确度比原方法有明显提高,而且大大提高了显色稳定性,缩短了分析时间。改良后的测定方法快速、简便、有效,可用于葡萄皮、籽、梗及其提取物中的游离态原花青素含量测定。在正丁醇-盐酸液中加入SDS和2-巯基乙醇,能够将结合态中的原花青素分离出来,通过显色反应确定结合态原花青素的含量,其测定结果的精密度和稳定性较好,可用于葡萄籽、梗中蛋白结合态和纤维素结合态原花青素含量的测定。葡萄籽中游离态、蛋白结合态、纤维素结合态原花青素含量分别为3.86%、2.93%、2.80%。在低温条件下贮藏,铝箔真空包装可减少多酚和游离态原花青素的损失以及向结合态原花青素的转化。以真空干燥葡萄籽为原料,用70%乙醇提取葡萄籽原花青素,其最佳工艺参数为葡萄籽粉粒度80-150目、搅拌时间55min、提取温度15℃、加酸量0.75%。20℃时,HP-2MGL树脂对葡萄籽原花青素的纯化效果最好,吸附率为91.1%,解吸率为95.8%。经HP-2MGL纯化的GSPP3组分的原花青素含量为96.5%,明显高于GSPP1和GSPP2。LC-MS分析结果表明,GSPP3质谱总离子流共有27个峰,说明GSPP3组分中原花青素的组成较为复杂,其中2号峰和4号峰的质谱及碎片信息表明,它们是原花青素二聚体。用SephadexLH-20对GSPP3组分进一步纯化,用70%丙酮洗脱得到GSPP3-SP1和GSPP3-SP2两个组分。LC-MS分析表明,GSPP3-SP1有四个峰,GSPP3-SP2有七个峰,通过质谱及碎片信息分析,它们主要含有单体和二聚体原花青素。根据葡萄籽原花青素提取物GSP和纯化级分GSPP1、GSPP2、GSPP3红外谱图分析,发现葡萄籽原花青素基本结构单元主要为原花青定。利用LC-MS对葡萄籽原花青素醇解产生的花色素进行分析测定,确定为矢车菊素,从而证明葡萄籽原花青素主要结构单元为花青定,即其B-环上含有两个羟基。激光光散射与GPC联用测定了葡萄籽原花青素纯化分级级分的重均相对分子质量,乙酰化GSP的平均相对分子质量为9.39×104Da,相对分子质量分布范围较宽,多分散系数为1.65;乙酰化GSPP3的重均相对分子质量为9.64×103Da, GSPP3的重均相对分子质量为4.26×103Da,平均聚合度为13,多分散系数为1.56,相对分子质量分布曲线呈对称分布,以高聚合度原花青素为主;乙酰化GSPP3-SP的重均相对分子质量为4.25×103Da,GSPP3-SP的重均相对分子质量更多还原

【Abstract】 Grape is a fruit with a high economic value in the world and belongs to Vitaceae Visit. There are a lot of phenolic compounds found in the grape; 30-40% is found in the grape skin and 60-70% in the grape seed. The major constituent of phenolic compound is proanthocyanidin (PC) which has a highly hydroxyl structure. It can act as an antioxidant, removes free radicals, and reduces risk of chronic diseases such as cardiovascular disease and cancer.The precision and accuracy of the measurement of PC content had been improved by the modifidied Vanillin-HCL assay, the coloring stability has been increased greatly, and the analysis time had been shortened. It is an instant and convenient assay, and can be applied to the measurement of free PC content in the grape skin, seed, stem and its extracts. The PC can be separated from the bound form with protein or fibre in the butanol-HCL solution of SDS and 2-mercaptoethanol, and the bound PC content of grape seed and stem could be measured after coloring with better precision and accuracy. The PC content of free form, bound form with protein or fibre in the grape seed was 3.86%, 2.93% and 2.80% respectively. The bound PC exists in the form of hydrophobic bond, hydrogen bond and covalent bond. It can reduce the loss of polyphenol and free PC, and also inhibits its transformation to bounded PC with vacuum foil package of grape seed at lower temperature.The grape seed was vacuum dried at 40℃, and the PC was extracted from it with 70% ethanol. The best technical parameters were 80-150 mesh particle size of grape seeds, 55 min of stirring, 15℃as extracting temperature, and 0.75%(v/v) acid content. The adsorption yield of PC in HP-2MGL was 91.1%. However the desorption yield was 95.8%. The PC content of GSPP3 was 96.5% which was higher than that of GSPP1 and GSPP2. The result of LC-MS analysis of GSPP3 showed that there were 27 peaks of GSPP3 in the total ion current of mass chromatogram. This meant the compose of GSPP3 was very complex. The peaks 2 and 4 were all dimmers of PC according to their mass spectra. GSPP3 was further purified by SephadexLH-20, and two components GSPP3-SP1 and GSPP3-SP2 were got by elution with 70% acetone. LC-MS analysis showed that there were four peaks for GSPP3-SP1, and seven peaks for GSPP3-SP2. According to their mass chromatogram and fragment information, they contained the monomer and dimmer of PC.On analysis of infrared spectra of GSP、GSPP1、GSPP2 and GSPP3, it was found that the basic structural unit of grape seed PC was procyanidin. Alcoholyzing the grape seed PC and analysing it by LC-MS, it was proved to be cyanidin. Hence the main structural unit of grape seed anthocyanidin is procyanidin which has two hydroxyl groups with a B-ring.On determining the relative molecular weight of grape seed PC by laser light scattering with GPC, it was found that the average relative molecular weight for更多还原