Efficient Synthesis,Efficient Separation,Taste Of Theasinensins And Their Mechanisms

Based on the research technology and experience of phytochemistry,computational chemistry and taste evaluation,the main purpose of this study is to clarify the mechanism of efficient preparation of theasinesins and the chemical mechanism of bitter and astringent taste quality.The synthesis,coupling and separation of theasinesins,the characteristics of bitter and astringent taste and the mechanism of action were systematically studied.The research results can provide a theoretical basis for the high-efficiency synthesis of catechin dimers,flavor chemistry and terminal products development.Theasinesins were efficiently prepared by chemical synthesis.The effect of the amount of cupric chloride,the volume fraction of methanol and the temperature on the yield of theasinesin A were very significant.The main factor effect was that the volume fraction of methanol>the amount of cupric chloride>the temperature.The optimal condition were that the amount of cupric chloride was 43%,the volume fraction of methanol was 26%,the temperature was 15?,and the yield of theasinesin was 59.12%,which was closed to the model prediction value 59.34%.The chemical synthesis process of theasinesin optimized by PBD and RSM was feasible and predictable.It can provided reference and theoretical basis for the efficient chemical synthesis of other catechin polymers.The molecular mechanism of efficient synthesis of theasinesins was explored.The space distance between EGCG,EGC substrate and copper active center of polyphenol oxidase was far greater than that between substrate and inorganic copper ion in the process of chemical synthesis.The hydrogen bonding and hydrophobic effects of catechin substrate and polyphenol oxidase protein blocked the binding of EGCG,EGC and copper active sites in polyphenol oxidase macromolecular protein.These might be the reasons why chemical synthesis was more efficient than enzymatic synthesis.The adsorption and desorption of theasinesin A,theasinesin B and theasinesin C by fifteen different types of resin fillers were compared and analyzed.The column chromatography packing suitable for separation and purification of theasinesins was screened out.The adsorption capacity of theasinesin A,theasinesin B and theasinesin C increased with the increase of sample concentration.However,too much sample loading might cause leakage.The adsorption capacity of theasinesins increased significantly with the increase of elution flow rate and the proportion of eluting agent.The addition of formic acid or acetic acid helped to improve the separation of theasinesins.The adsorption rate and desorption rate of theasinesins were relatively high under the temperature of 20-30?.Theasinesin A,theasinesin B and theasinesin C monomers were separated by macroporous adsorption resin and gel chromatography coupled method.The purity of the theasinesin A,theasinesin B and theasinesin C were over 98%.The mothod of UV-VIS,HPLC,NMR,IR identified it as theasinesins in tea.The solubility,UV-VIS absorption spectrum,color rendering characteristics,temperature stability and stability of metal ions of theasinesins were analyzed.The mechanism of selective separation based on adsorption thermodynamics was analyzed.The adsorption of theasinesins processed by HX resin was in accordance with Freundlich adsorption isotherm equation(R~2 value was 0.9968-0.9995).The adsorption affinity of HX resin for theasinesin A was stronger than that of theasinesin B and theasinesin C.The adsorption process of theasinesin A,theasinesin B and theasinesin C by fillers belonged to spontaneous physical reaction process.The higher the temperature,the stronger the driving force of spontaneous reaction.In the process of adsorption,the entropy change of theasinesins were negative.The degrees of freedom of theasinesin A,theasinesin B and theasinesin C on the filler decreased.The bitterness and astringency threshold,DOT value and taste intensity of theasinesins were determined.The evaluation model of theasinesins bitterness and astringency were established.The astringency threshold of theasinesin A is 65.6?mol/L,the bitterness threshold of theasinesin A is 109.4?mol/L.The DOT value of theasinesin A is significantly higher than that of other catechins except EGCG,and the main theaflavins such as TF,TF-3-g,TF-3'-G,TFDG.In the astringency material system of tea soup,theasinesin A is significantly higher than that of other catechins except EGCG.The contribution to astringency of theasinesins was stronger than that of non ester catechins,ECG,GCG and main theaflavins.It was of great scientific significance to explore the key factors and mechanism of astringency of tea soup.The model of the intensity of bitterness and the intensity of astringency of theasinesins are established,and the correlation coefficients are0.971 and 0.963.The bitterness time intensity curve and astringency time intensity curve of tea theasinesins were established.The molecular mechanism of bitterness and astringency taste of theasinesins was preliminarily clarified.Theasinesin A had the highest affinity with human salivary protein receptor,which is-10.9,followed by theasinesin B and theasinesin C.Theasinesin A was mainly bound to a half open cavity at the N-terminal of human salivary protein receptor.The cavity had greater hydrophobicity,and there were multiple hydrophobic benzene rings in small molecules,which can formed hydrophobic effect and facilitate the binding of small molecules.The number of hydrogen bond between human salivary protein receptor TAS2R13 and theasinesin A,B and C were different.The hydrogen bond played a key role in the bitterness and astringency taste of theasinesin A,B and C.The above research results have important reference value for enriching the chemical theory of flavor and quality of tea,tea beverage processing technology and diversified utilization of tea deep processing.