| As an ancient and popular health drink,tea infusion have a variety of health benefits such as helps produce saliva and slake thirst,and keeps you a cool head and refreshment.The previous study mainly focused on the effect of single chemical substances,such as tea polyphenols,but little research on this typical multiphase disperse system.In recent years,studies have shown that the micro/nanoparticles formed during the boiling process of tea infusion are closely related to their physiological functions.This discovery found a new way for exploring the medicinal mechanism of tea infusion.In this study,the protein of Lapsang souchong was studied,the changes of protein components were investigated during the processing of black tea,and the heat-stable protein were identified,separated,purified and characterized.The optimal condition for the protein formation of micro/nanoparticles and the morphology of micro/nanoparticles were explored.The main results are shown as below:(1)The molecular weight of heat-stable proteins in tea leaves was determined to be 66 kDa and 47 kDa,respectively,by comparing the components of tea infusion,tea infusion ultrafiltration,tea infusion filtration and fresh tea leaves by SDS-PAGE.By determining the protein concentration of the crude extract under different single factor conditions,the optimal condition for extracting the protein from the fresh leaves of Lapsang souchong was:a suitable amount of fresh leaves,solid-liquid ration(W/V)1:3,added Tris-HCl buffer(0.05 M,pH 7.2),which was precooled at 4?,10%PVPP(g/fresh tea leaves weight g),5 M urea,1 mM PMSF,1 mM EDTA,grinding in ice bath,slurry extraction(4?,3 h),gauze filtration,and then was centrifuged(4?,10000 g,30 min),the supernatant was the crude protein extracted from fresh leaves.(2)By using the urea assisted UNOsphere Q ion exchange chromatography,the protein profile of crude protein extract from the fresh leaves,tea infusions,retention components of tea infusion were obtained.The results showed that there were relative heat-stable proteins in the three samples(named P66,P47,P18),and the molecular weight is about 66.0 kDa,47.0 kDa,18.0 kDa.Among them,P66 and P47 had polyphenol oxidase activity,P66 reached electrophoresis purity,respectively,and P66 reached electrophoresis purity.Then P66 was isolated,purified and concentrated according to its specificity and feasibility.(3)After SDS-PAGE determination,the molecular weight of P66 was 66.0 kDa,and the result of high performance liquid phase-multi angle light scattering showed that P66 protein was polymer,According to the periodic acid-Schiff(PAS)staining identified P66 as glycoprotein,isoelectric focusing results showed that the isoelectric point of P66 was 4.82,With catechol as substrate for P66 polyphenol oxidase enzymology characteristics are studied,the results show that the optimum temperatures is 35?,the optimum pH is 6.2,the optimal concentration of Cu2+ was 0.25 × 10-7 M,the inhibitory effect is cysteine>EDTA>sodium sulfite>ascorbic acid.The enzyme kinetic parameter Km was 6.9729 mM,and Vmax was 15.7878.The observation of P66 protein aqueous by transmission electron microscopy shows that it has a self-aggregation trend.(4)The aggregation behavior of P66 protein in aqueous solution under 30? was investigated by the use of a Malvern particle size analyzer,and the results showed that the optimum condition of P66 to form micro/nanoparticles was:30?,pH 5.05,holding 30 min,protein concentration was 0.11 mg/mL,the particle size distribution was about 75 nm,Zeta potential was-24.7 mV,Transmission electron microscopy observation showed that the surface structure of the micro/nanoparticles was spherical. |
PDF Full Text Request