Interaction Of Tea Polyphenols And Theaflavin With Proteins And Their Effects On The Formation Of Tea Cream

As a beverage whose consumption is second only to water in the world,tea is rich in a variety of nutrients,among which tea polyphenols and theaflavins have health benefits such as anti-oxidation,regulating blood lipid and lowering blood sugar.However,due to the interaction of biochemical components in tea drinks（including brewed tea,bottled pure tea and bottled mixed tea）,tea drinks tend to appear an obvious turbidities（tea cream）,which seriously affects the sensory quality and nutritional characteristics of tea drinks.The interaction of tea polyphenols and theaflavins with protein can also induce the production of tea cream,but the mechanism of this interaction has not been reported yet.At present,filtration centrifugation and enzymatic method are mainly used to remove or inhibit tea cream.However,filtration will reduce the nutritional content of tea,and the enzymatic method is greatly affected by temperature,which is prone to instability.Therefore,this study started from the phenomenon and mechanism of the formation of tea cream.Firstly,the interaction between bovine serum albumin（BSA）and different concentrations of TPs and its effect on the formation of tea cream were studied.Secondly,molecular docking was used to analyze the interaction mechanism between four major catechins in TPs and BSA.Thirdly,the interaction between theaflavin and five protein clarifiers（β-lactoglobulin,zein,soy protein isolate,gelatin,collagen）and their effects on tea cream were studied,and the protein clarifiers were screened.Finally,molecular docking was used to study the interaction mechanism between theaflavin-3,3’-digallate（TFDG）,BSA andβ-lactoglobulin（screened protein clarifier）,which is the most active and highest content of theaflavin-3,3’-digallate（TFDG）,to determine whether protein clarifier is suitable for tea beverage clarification.The results of this study will provide a theoretical basis for the regulation of tea cream in tea drinks.The main research contents and results are as follows:1.Taking TPs and BSA as the main research objects,the light transmittance,particle size,precipitation,catechin content and loss rate of the TPs-BSA mixed solution with different concentrations were determined,and the homogenization analysis of indexes was carried out by using technique for order preference by similarity to an ideal solution（TOPSIS）.The results showed that,there were significant differences in light transmittance,particle size,precipitation amount and catechin content of mixed solutions with different concentrations of TPs and BSA（P<0.05）.TPs concentration had a significant effect on light transmittance,while BSA concentration had a more significant effect on particle size and precipitation amount.The light transmittance was in the range of 99.6%～46.7%,the precipitation was in the range of 0.23～15.10 mg,and the particle size was in the range of 0.41～11.49μm.In the mixed solution,the content of ester catechin changed greatly.When the concentration of TPs was fixed,the loss rate of catechin decreased first and then increased with the increase of BSA concentration.TOPSIS successfully simplified solution selection and selected a concentration combination of 800 mg/L TPS and 40mg/L BSA（C=0.7572）.2.The interaction mechanism of four catechins:epigallocatechin gallate（EGCG）,epicatechin gallate（ECG）,epigallocatechin（EGC）,epicatechin（EC）with BSA was studied.The molecular docking method was used to analyze 22 active sites of BSA with the four catechins,respectively,and the lowest binding free energy of 88 docking sites was calculated.The results showed that:BSA could spontaneously bind EGCG,ECG,EGC and EC,the hydroxyl groups in catechins affect the binding strength with BSA,and the binding free energy showed that the binding strength between ester catechins and BSA was higher than that of non-ester catechins.Hydrogen bonding is the main driving force of the amino acid residues at the active sites of the four catechins and BSA.TYR-30,TYR-84,TYR-137,TYR-139,TYR-156,TYR-160 and TYR-451 in BSA play an important role in binding to catechins.3.Taking theaflavin,β-lactoglobulin,zein,soybean protein isolate,gelatin,and collagen as the research objects,the interaction mechanism of theaflavin and different proteins and their effects on the formation of tea cream were analyzed,and the solution was screened by TOPSIS method based on light transmittance,particle size and antioxidant activity in vitro.Fluorescence spectra,thermodynamic parameters,and synchronous fluorescence spectra were used to analyze the interaction mechanism.The main results were as follows:different protein-theaflavin solution have difference influence on the formation of tea cream,the light transmittance and particle size ofβ-lactoglobulin,zein,and soy protein isolates with theaflavin showed higher clarity.TOPSIS had simplified the choice of sample,theβ-lactoglobulin is expected to be used in the tea beverage clarification.Five kinds of protein can spontaneously combine with theaflavin through a static quenching.The combination of theaflavin affectedβ-lactoglobulin,zein,soybean protein isolate tyrosine micro-environment,changing the gelatin and collagen of the micro-environment of tryptophan.Hydrogen bonding and van der Waals interaction force dominates the combination ofβ-lactoglobulin,soybean protein isolate,collagen with theaflavin,and the combination of zein and gelatin with theaflavin mainly dominated by hydrophobic effect.4.Taking TFDG,BSA,andβ-lactoglobulin as research objects,molecular docking was used to analyze the binding free energy and binding conformation of TFDG-BSA and TFDG-β-lactoglobulin,and Ligplot was used to analyze the interaction force and binding amino acids,which proved the feasibility of using protein to improve the stability and availability of theaflatins.The results showed that:BSA andβ-lactoglobulin could spontaneously bind to TFDG,and hydrophobicity and hydrogen bonding were the main driving forces of the interaction.LES-31,TYR-84,HIS-105,GLN-33,THR-83,GLU-82,GLY-85,and ASP-111 in BSA formed hydrophobic pockets,which wrapped TFDG molecules.MET-87,GLN-32,ASP-86,TYR-30,ASP-107 formed seven hydrogen bonds with TFDG.LEU-140,GLU-137,GLU-60,HIS-132,SER-131,LYS-58,GLY-61 inβ-lactoglobulin formed hydrophobic pockets,which were coated with TFDG molecules.TFDG could form nine hydrogen bonds with GLU-136,GLU-56,GLU-53,LYS-139,PHE-133,TYR-23,GLU-57,and ARG-64 amino acid residues.In the structure of TFDG,the hydroxyl groups on C₂₅,C₂₆,C₁₁,C₄₁,and C₄₂ play an important role in binding with BSA,and the hydroxyl groups on C₂₄,C₂₅,C₂₆,C₁₇,C₃₅,C₃₃,and C₃₇ play an important role in binding withβ-lactoglobulin,which proves that it is feasible to use protein to improve the stability of theaflavins.