本文選題：茶紅素 + 分離純化�。� 參考：《湖北工業(yè)大學(xué)》2017年碩士論文

【摘要】：茶紅素作為紅茶加工過(guò)程中形成的紅褐色酚類物質(zhì),是紅茶湯色和滋味的主要關(guān)鍵因子。茶紅素是由兒茶素類物質(zhì)在多酚氧化酶和過(guò)氧化物酶作用下氧化聚合而成,分子量差異較大,其組分結(jié)構(gòu)尚不明晰,至今未制備出茶紅素單體。關(guān)于茶紅素分離的研究亦鮮有報(bào)道,茶紅素檢測(cè)僅停留在Roberts系統(tǒng)分析法。茶紅素研究是目前茶葉界的焦點(diǎn)問(wèn)題,而且茶紅素作為天然色素和其生物學(xué)活性,都使得茶紅素有著非常好的應(yīng)用前景,所以茶紅素研究和利用亟需解決。本文對(duì)茶紅素浸提進(jìn)行優(yōu)化,通過(guò)分級(jí)萃取、柱層析和高速分離色譜進(jìn)行純化制備出茶紅素,并采用紅外色譜、質(zhì)譜聯(lián)用、核磁共振對(duì)茶紅素進(jìn)行初步鑒定,最后從不同參數(shù)變化對(duì)茶紅素穩(wěn)定性進(jìn)行研究。主要內(nèi)容如下:(1)以紅碎茶為原料,采用熱水浸提法從紅碎茶提取茶紅素,考察料液比、提取溫度和提取時(shí)間最優(yōu)工藝。結(jié)果表明,料液比為1:30、提取溫度80℃、提取時(shí)間30 min時(shí),茶紅素提取率最高,可達(dá)10.57%。然后在此工藝參數(shù)下提取,經(jīng)有機(jī)試劑分級(jí)萃取,制備出三類茶紅素粗品。(2)采用反相C18柱層析進(jìn)行初純化,結(jié)合Sephadex LH-20再純化,洗脫級(jí)分經(jīng)HPLC分析,研究結(jié)果表明:反相C18柱純化,依次為30%、50%、70%甲醇梯度洗脫,能除去粗品中所含葉綠素等非酚類雜質(zhì),而且較大程度除去兒茶素組分和茶黃素,能完全去除咖啡堿;而Sephadex LH-20葡聚糖凝膠再純化,依次以甲醇和丙酮洗脫,所得茶紅素部分,經(jīng)HPLC分析下,純度較高且無(wú)兒茶素和茶黃素等雜質(zhì),且三部分茶紅素出峰位置均在3-5min。(3)采用反相C18柱層析結(jié)合高速逆流色譜分離茶紅素,研究表明,溶劑體系選擇確定為乙酸乙酯層采用乙酸乙酯-正己烷-甲醇-水(1:3:1:5,v/v/v/v)四元溶劑體系,其分配系數(shù)為1.34,固定相保留率為42%;而正丁醇層和酸性正丁醇層采用乙酸乙酯-正丁醇-水(3:2:5,v/v/v)三元溶劑體系,其分配系數(shù)為1.65和1.71,固定相保留率為51%和52%。高速逆流色譜參數(shù)選擇上樣量為20mg/mL、主機(jī)轉(zhuǎn)速700r/min、流動(dòng)相流速2.0mol/L時(shí),能進(jìn)一步除去部分兒茶素雜質(zhì),達(dá)到純化目的。(4)茶紅素進(jìn)行結(jié)構(gòu)鑒定,采用紅外光譜、質(zhì)譜和核磁共振。結(jié)果表明:三部分茶紅素均發(fā)現(xiàn)存在酚類分子間-O-H、-C=O、苯環(huán)、芳香醚結(jié)構(gòu),而苯環(huán)上取代基位置和數(shù)量各不相同。質(zhì)譜分析以ESI電噴霧在負(fù)離子模式下檢測(cè),研究表明乙酸乙酯層茶紅素分子量為855,正丁醇層和酸性正丁醇層分子量為742。而三部分茶紅素核磁共振氫譜分析,因物質(zhì)無(wú)法達(dá)到所需純度,僅能看到7ppm處為苯環(huán)上H和化學(xué)位移為9-10時(shí),為C=O中C位上的H,無(wú)法準(zhǔn)確定位每個(gè)H的位置推出基本結(jié)構(gòu)。茶紅素保留兒茶素所具有的基本結(jié)構(gòu),綜上得出茶紅素可能為兒茶素三聚體化合物。通過(guò)以保留率和Lab色差系a值作為指標(biāo),探究茶紅素在不同光照、pH值和溫度條件下茶紅素穩(wěn)定性。研究發(fā)現(xiàn):遮光處理對(duì)茶紅素貯藏更好,pH=3酸性條件下穩(wěn)定性高,溫度在60℃以下穩(wěn)定性高。溫度和強(qiáng)堿性不利于其穩(wěn)定性,導(dǎo)致茶紅素發(fā)生氧化褐變。
[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.

【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS264.4

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