Study On The Enzymatic Reaction Mechanism Of Flavonol Glycosides In Tea And Its Application

Flavonols and their glycosides are important flavonoid components in tea,the amount of which is second to catechin compounds,being associated with the flavor and infusion color of green tea.To date,more than 20 flavonol glycosides have been determined in tea,which mainly exist in the form of glycosides.Flavonol glycosides with mild and astringent taste have extremely low thresholds and are also considered to exert enhancing effect on the bitterness of caffeine.Previous studies showed that the contents of flavonol glycosides declined during the fermentation of black tea to different extent,suggesting the existence of endogenous enzyme in fresh tea leaves responsible for the conversions of flavonol glycosides.In this study,in vitro studies showed that polyphenol oxidase(PPO)significantly induced the oxidation of flavonol glycosides,and the reaction conditions including reaction time,temperature,pH value and dose effects of PPO and peroxidase(POD)were investigated.The crude enzyme extract was extracted from fresh tea leaves and the contribution of PPO component in the crude enzyme extract to the conversions of flavonol glycosides was verified by an addition of PPO inhibitor.Computer molecular docking simulation technology was used to understand the differential interaction between PPO and flavonol glycosides.Furthermore,PPO was used as an exogenous enzyme in order to improve the quality of summer and autumn green tea through reducing the bitterness or astrigency.The main results were as follows:PPO more effectively induced the conversions of flavonol glycosides,compared with POD and ?-glucosidase.Flavonol glycosides decreased sharply within 30 min under enzymes treatment,and after 3 hours,the concentrations of flavonol glycosides were stable.The reactivity of flavonol glycosides to PPO was highly influenced by pH value but moderately impacted by temperature(25~55?).The oxidation of flavonol glycosides catalyzed by PPO showed PPO-dose dependent in the range of 39~72 U/mL,and the coexistence of POD significantly intensified the PPO-induced oxidation.Myricetin glycosides and quercetin glycosides were most sensitive,while kaempferol glycosides were relatively stable during the enzymatic reaction of PPO.Aglycone was the crucial moiety that impacted the sensitivity of flavonol glycosides to PPO.The PPO inhibitor treatment verified the dominant contribution of PPO component in tea crude enzyme extract to the conversions of flavonol glycosides.The mixture of PPO and inhibitor L-cysteine exerted no obvious enzymatic catalysis on flavonol glycosides,suggesting that PPO component was the crucial enzyme in crude enzyme extract responsible for the conversions of flavonol glycosides.Molecular docking simulation showed that flavonol glycosides was embedded into the hydrophobic pocket of PPO,interacting with the major residues on the active site of PPO through hydrogen bonding.The presence of sugar moiety promoted the interaction between flavonol glycosides and PPO active site.The exogenous PPO treatment was applied to the processing of summer and autumn green tea in order to reduce the astringency and bitterness of tea.The results showed that addition of PPO enzyme moderately reduced the flavonol glycoside contents of tea leaves,and the decrement of flavonol glycosides was related with tea cultivar.The sensory evaluation result showed that the bitter and astringent taste of tea was moderately reduced after PPO treatment.