【作者】 刘睿；

【导师】 谢笔钧；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2004， 博士

【摘要】 高粱在我国种植广泛，作为酿酒用原料已有千余年的历史，人们对于高粱中淀粉、蛋白质和脂肪等成分在酿酒中的作用研究甚多，但对具有抑菌和调节酶活特性的原花青素对小曲酒挥发性化合物产生影响及其机制的研究，却未见相关的文献报道。尽管原花青素的研究已经有40多年的历史，但是对高粱中原花青素的研究仅国外有少数报道。为此，本文在分析和鉴定我国产高粱外种皮中原花青素的基础上，采用GC-MS、DSC等现代分析技术，首次研究了高粱外种皮中原花青素对小曲酒挥发性化合物的贡献，通过分析和讨论高粱原花青素对小曲酒酿造中典型微生物和酶的影响，探讨其作用机制，为进一步深入研究高粱中原花青素对我国白酒酿造的影响和提高我国白酒的品质与风味提供理论基础和科学依据。同时也为高粱外种皮的深加工和利用提供实验依据和思路。 主要研究结果如下： 1．高粱中原花青素含量测定和提取纯化 首次对我国湖北、安徽、黑龙江等11个主产地高粱中原花青素的含量进行了测定，结果显示，原花青素主要分布在高粱的外种皮中，约占高粱中原花青素含量的96％；不同产地高粱外种皮中原花青素含量差异较大(1.24％～8.42％)，其中以安徽产高粱中原花青素含量最高，达8.42％。这表明地域品种对高粱种子外种皮中原花青素含量影响较大。 比较了水、不同体积比的丙酮／水溶液、甲醇／水溶液和乙醇／水溶液对黑龙江产高粱中原花青素(以下简称SPC)提取率的影响，在单因素实验的基础上，通过正交实验优化，确定以50％乙醇水溶液作为浸提溶剂，最佳提取工艺为：浸提温度80℃，pH4，料液比1：20(W／V)，提取时间90min。 采用大孔树脂柱层析对SPC进行纯化，先用水洗去杂质后，再用30％乙醇洗脱可得到SPC含量为96.01％的产物，得率为3.76％。 2．黑龙江产高粱原花青素的分离鉴定和结构表征 2．1 SPC的初步结构分析 采用UV、IR、MS等现代分析技术对SPC进行了初步结构分析。SPC显示原花青素典型的UV和IR光谱特性，ESI-MS分析表明SPC是由原花青素单体(儿茶素或表儿茶素)、二聚体、三聚体、四聚体、五聚体和表儿茶素没食子酸酯及表没食子儿茶素组成，其分子量分别是290、578、866、1154、1442、466和306。华中农业大学2004届博士学位论文2.2 SPC的分离和SPC一45的结构鉴定 通过SePhadex LH一20凝胶色谱以阶梯梯度洗脱方式分离sPC，采用Porter’s法和Esl一MS对每个组分中原花青素含量和组成进行测定。发现以不同体积比乙醇/水溶液作流动相阶梯梯度洗脱后，再用70%丙酮/水溶液等度洗脱，分别得到SPC一15一5s和SPC一65共6个组分，各组分中原花青素含量和聚合度均随着乙醇浓度的增加而增大，30%乙醇洗脱组分SPC一25中为表没食子儿茶素和表儿茶素格酸单酷;50%乙醇洗脱得到的SPC一35为表没食子儿茶素和原花青素二聚体，而SPC一45是由表没食子儿茶素、原花青素二聚体和三聚体组成;70%乙醇洗脱组分SPC一5s为原花青素二聚体和三聚体的混合物，70%丙酮洗脱组分SPC一6s主要是由原花青素单体、二聚体、三聚体和四聚体组成。 采用ESI一MS、NMR、IR等分析技术对SPC一4S的结构进行了初步表征，结果表明SPC一45为儿茶素与表儿茶素(或儿茶素)通过C4~C8连接的原花青素二聚体、三聚体和四聚体及儿茶素单体。进一步通过PR一HPLC一MS分析，结果表明采用ZothaxSB一C18的色谱柱(50XZ.lrnm，sum)，流速0.3mL/min，进样量4.ouL，检测波长280nm，流动相(A)乙酸:水=10:90，(B)去离子水，进行梯度洗脱，原花青素三聚体被分离鉴定了。 此外还采用NP一HPLC方法对SPC一45和以0~95%乙醇作流动相线性梯度洗脱的组分SPC一SL进行分离鉴定，结果表明可使SPC按聚合度大小分离。3.高粱原花青素对小曲酒挥发性化合物影响的研究 采用GC一MS方法对添加不同量SPC(0%、0.05%、0.1%、0.3%)酿造的酒样中挥发性化合物进行了分析，鉴定了乙酸乙酷、乳酸乙酷、丁二酸二乙酷、乙酸、3-甲基丁酸、异丁醇、异戊醇、正丙醇、乙醛、糠醛、2一丁酮等16种挥发性成分，结果表明四个酒样皆是以乙酸乙酷为典型香型的小曲酒，不同酒样中挥发性成分基本相同，但组成比例有差异，可见SPC对小曲酒中挥发性化合物的产生有影响。尤以对主要酷类影响的结果最为明显，添加SPC可使小曲酒中乙酸乙酷的含量明显增加，其中以添加0.1%SPC的酒样中乙酸乙醋含量最高，为对照组的8倍;乳酸乙酷的含量同样以添加0.1%SPC的酒样最高，约为对照组的1 .8倍，而且添加0.05%、0.1%和0.3%SPC酉良造的酒样中乙酸乙酷和乳酸乙酷的比值分别为2.85:1，1.“:1和2.01:1，也以添加0.1%SPC酒样中两者的比例最为协调;对小曲酒中酸的影响表现为，添加SPC后有利于降低酒样中乙酸的含量，各酒样中乙酸的量随着SPC添加量高粱外种皮中原花青素及其对小曲白酒挥发性成分影响机制研究的增加而降低;对醛类的影响为，添加SPC可降低酒样中糠醛的含量，其中以添加0.1%SPC酒样中含量最低，为对照组的39.3%，而4个酒样中乙醛含量则无明显差别;低SPC添加量有降低酒样中杂醇油含量的趋势。上述结果表明，添加0.1%高粱?更多还原

【Abstract】 Sorghum is widely planted in China, and it has a long history of being used for liquor-making material. So far, there has been a great amount of research about the effects of its ingredients, including starch, protein and fat on liquor quality. However, relatively little research explores the effect and its mechanism of SPC featuring the activity of suppressing bacteria and mediating enzyme on the volatile ingredients of distilled liquor. The research on procyanidins has lasted over 40 years, but there are only a few literatures about SPC in sorghum. Based on analyzing and identifying the SPC content of sorghum from different region of China, the research was conducted (1) to study the effect of SPC in sorghum on the volatile ingredients of distilled liquor, (2)to probe its mechanism by analyzing the effect of SPC in sorghum on the classical microbes and enzymes in distilled liquor, (3) to provide scientific evidence for delving into the effect of SPC in sorghum on white spirit-making and improving its flavor, and (4) to offer supporting resources for deep processing and application of outer husk of sorghum. The main results are as follows: 1. Methods for extraction, purification and determination of content of SPCThe analytical result of SPC in sorghum collected from 11 main planting region, such as Hubei, Anhui, Heilongjiang showed that (i) SPC was mainly distributed in the episperm of sorghum, reaching 96% of total procyanidins, (ii) SPC content differed (1.24%~8.42%) in the episperm of sorghum from different locations, in which the maximum SPC content was 8.42% from Anhui. This indicated that SPC content in sorghum varied from region to region.The extraction rations of procyanidins from soghum (SPC) was compared using different solvents, such as water, different volume rates of acetone to water, methanol to water and ethanol to water. Through single factor and orthorhombic analysis, ethanol/water (50:50v/v) was selected as extracting solvent and the best extracting process. At the same time, the control conditions were obtained, including temperature (80), pH (4), the ratio of material to extracting solvent (1:20W/V), extracting time (90min).Then SPC was purified by chromatography with macro-porous resin as stationary phase. Water was used to elute foreign matter, and then 30% ethanol was used to elute SPC,whose content was 96.01% and the yield was 3.76%.2.Separation, identification and chemical structure characteristics of procyanidins insorghum from Heilongjiang2.1 Structural Analysis of SPCStructure of SPC was analyzed with UV> IR and MS. SPC showed UV and IR spectrogram of procyanidins. ESI-MS spectrogram indicated that SPC consisted of monomer, dimmer, trimer, tetramer, pentamer of procyanidins, epicatechin gallate ester and epicatechin. Their molecular weights are 290, 578, 866, 1154, 1442, 466 and 306 respectively.2.2 Separation of SPC and chemical structure characteristics of SPC-4SSPC were separated with gradient of step elution model in Sephadex LH-20 gel chromatogram. Porter’s method and ESI-MS was used to determine the content and constitute of procyanidins in each fraction. Different ethanol to water ratio mobile phase was used with gradient of step elution followed by 70% acetone/ water isocratic elution, and gained 6 fractions (SPC-1S-5S and 6S respective). The content and degree of polymerization of procyanidins in each fractions increased with the rising of ethanol concentration. The SPC-2S fraction with 30% EtOH were constituted epicatechin and epicatechin gallate ester ; The SPC-2S fraction with 50% EtOH contained epicatechin and dimeric procyanidins, another SPC-3S contained epicatechin ,dimeric procyanidins and trimeric procyanidns ; Dimeric procyanidins and trimeric procyanidns constitute SPC-5S fraction; monomer dimer trimer tetramer pentamer of procyanidins were the major fractions in 70% acetone elute solution.ESI-MS NMR and IR was used in determining SPC-4S fraction of gradient of step elution model, the results showed the dimeric , trimeric, tetrameric procyan更多还原