茶红素分离纯化及稳定性研究

发布时间：2018-05-11 19:09

本文选题：茶红素 + 分离纯化　；参考：《湖北工业大学》2017年硕士论文

【摘要】：茶红素作为红茶加工过程中形成的红褐色酚类物质,是红茶汤色和滋味的主要关键因子。茶红素是由儿茶素类物质在多酚氧化酶和过氧化物酶作用下氧化聚合而成,分子量差异较大,其组分结构尚不明晰,至今未制备出茶红素单体。关于茶红素分离的研究亦鲜有报道,茶红素检测仅停留在Roberts系统分析法。茶红素研究是目前茶叶界的焦点问题,而且茶红素作为天然色素和其生物学活性,都使得茶红素有着非常好的应用前景,所以茶红素研究和利用亟需解决。本文对茶红素浸提进行优化,通过分级萃取、柱层析和高速分离色谱进行纯化制备出茶红素,并采用红外色谱、质谱联用、核磁共振对茶红素进行初步鉴定,最后从不同参数变化对茶红素稳定性进行研究。主要内容如下:(1)以红碎茶为原料,采用热水浸提法从红碎茶提取茶红素,考察料液比、提取温度和提取时间最优工艺。结果表明,料液比为1:30、提取温度80℃、提取时间30 min时,茶红素提取率最高,可达10.57%。然后在此工艺参数下提取,经有机试剂分级萃取,制备出三类茶红素粗品。(2)采用反相C18柱层析进行初纯化,结合Sephadex LH-20再纯化,洗脱级分经HPLC分析,研究结果表明:反相C18柱纯化,依次为30%、50%、70%甲醇梯度洗脱,能除去粗品中所含叶绿素等非酚类杂质,而且较大程度除去儿茶素组分和茶黄素,能完全去除咖啡碱;而Sephadex LH-20葡聚糖凝胶再纯化,依次以甲醇和丙酮洗脱,所得茶红素部分,经HPLC分析下,纯度较高且无儿茶素和茶黄素等杂质,且三部分茶红素出峰位置均在3-5min。(3)采用反相C18柱层析结合高速逆流色谱分离茶红素,研究表明,溶剂体系选择确定为乙酸乙酯层采用乙酸乙酯-正己烷-甲醇-水(1:3:1:5,v/v/v/v)四元溶剂体系,其分配系数为1.34,固定相保留率为42%;而正丁醇层和酸性正丁醇层采用乙酸乙酯-正丁醇-水(3:2:5,v/v/v)三元溶剂体系,其分配系数为1.65和1.71,固定相保留率为51%和52%。高速逆流色谱参数选择上样量为20mg/mL、主机转速700r/min、流动相流速2.0mol/L时,能进一步除去部分儿茶素杂质,达到纯化目的。(4)茶红素进行结构鉴定,采用红外光谱、质谱和核磁共振。结果表明:三部分茶红素均发现存在酚类分子间-O-H、-C=O、苯环、芳香醚结构,而苯环上取代基位置和数量各不相同。质谱分析以ESI电喷雾在负离子模式下检测,研究表明乙酸乙酯层茶红素分子量为855,正丁醇层和酸性正丁醇层分子量为742。而三部分茶红素核磁共振氢谱分析,因物质无法达到所需纯度,仅能看到7ppm处为苯环上H和化学位移为9-10时,为C=O中C位上的H,无法准确定位每个H的位置推出基本结构。茶红素保留儿茶素所具有的基本结构,综上得出茶红素可能为儿茶素三聚体化合物。通过以保留率和Lab色差系a值作为指标,探究茶红素在不同光照、pH值和温度条件下茶红素稳定性。研究发现:遮光处理对茶红素贮藏更好,pH=3酸性条件下稳定性高,温度在60℃以下稳定性高。温度和强碱性不利于其稳定性,导致茶红素发生氧化褐变。
[Abstract]:As the red and brown chromite substances formed during the processing of black tea, the tea erythropoietin is the main key factor of the color and taste of the black tea. The tea erythropoietin is formed by the oxidation and polymerization of catechin under the action of polyphenol oxidase and peroxidase. The difference of the molecular weight is great, and the composition of the composition is not clear. There are few reports on the separation of teanin. The detection of teanin only stays in the Roberts system analysis. The research of teanin is the focus of the tea industry, and as a natural pigment and its biological activity, teanin has a very good application prospect. Therefore, the research and utilization of teanin needs to be solved. Tea erythropoietin extraction was optimized by fractionated extraction, column chromatography and high speed separation chromatography. The tea erythropoietin was prepared by infrared chromatography, mass spectrometry and nuclear magnetic resonance. Finally, the stability of the tea erythropoietin was studied from different parameters. The main contents are as follows: (1) red broken tea is used as the raw material. A hot water extraction method was used to extract the teanin from red broken tea. The optimum process of extraction temperature and extraction time was investigated. The results showed that the extraction rate of tea erythropoietin was the highest when the ratio of material and liquid was 1:30, the extraction temperature was 80, and the extraction time was 30 min, and the extraction rate of the tea erythropoietin was 10.57%., and then the crude extracts of three kinds of tea erythropoietin were extracted by organic reagent. (2) primary purification by reverse phase C18 column chromatography, Sephadex LH-20 re purification and elution grade by HPLC analysis. The results show that the reverse phase C18 column is purified by 30%, 50%, and 70% methanol gradient elution, which can remove the chlorophyll and other non phenol impurities in the crude products, and the catechin components and theaflavins can be removed to a large extent, and the caffeine can be completely removed. The Sephadex LH-20 glucan gel was repurified and eluted with methanol and acetone in turn. After HPLC analysis, the purity of the tea erythropoietin was higher and no catechin and theaflavin and other impurities, and the three part of theanin was found in 3-5min. (3) by reverse phase C18 column layer analysis combined with high speed counter flow chromatography. The selection of the agent system is that the ethyl acetate layer uses ethyl acetate n-hexane methanol water (1:3:1:5, v/v/v/v) four element solvent system, its distribution coefficient is 1.34, the retention rate of fixed phase is 42%, while the n-butyl alcohol layer and acid n-butanol layer use ethyl acetate n-butanol water (3:2:5, v/ v/v) three element solvent system, its distribution coefficient is 1.65 and 1.71, fixed The phase retention rate is 51% and the 52%. high speed counter current chromatography parameter selection is 20mg/mL, the main engine speed 700r/min, the flow phase flow velocity 2.0mol/L, can further remove some catechin impurities, achieve the purification purpose. (4) the tea erythrocyte structure identification, infrared spectrum, mass spectrometry and nuclear magnetic resonance. The results show that the three parts of the tea erythropoietin all exist The structure of -O-H, -C=O, benzene ring and aromatic ether are different between the molecules of the phenols, and the substituents and the number of substituents on the benzene ring are different. The mass spectrometry analysis shows that the molecular weight of the ethyl acetate is 855, the n-butanol layer and the acid n-butanol layer are 742. and the three part of the 1H NMR spectra of the tea erythropoietin by the mass spectrometry analysis. Because the substance can't achieve the desired purity, it can only be seen that when 7ppm is H on the benzene ring and the chemical shift is 9-10, it is H in the C position in C=O. It can not accurately locate the basic structure of each H position. The basic structure of the tea erythropoietin retention catechin is that the tea erythropoietin can be a catechin tripolymer. By the retention rate and L The a value of AB color difference system is used as an indicator to explore the stability of teanin under different light, pH and temperature conditions. It is found that shade treatment is better for the storage of tea erythropoietin, high stability under pH=3 acid condition, high stability below 60 C. Temperature and strong alkalinity are not good for its stability, causing oxidation browning of the tea erythrocyte.

【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS264.4

【参考文献】