LC-MS Based Non-targeted And Targeted Metabolomics Analysis Revealed The Marker Compounds Of Pu-erh Tea During Pile-fermentation Processing

Pu-erh tea has attracted more attentions because of its special flavor and quality.The chemical basis and health benefits were also discussed widely.The special quality and feature of pu-erh tea is mainly formed during the pile-fermentation,therefore,it is very important to study the chemical changes of pu-erh tea samples during pile-fermentation.So far,there have been many researches about the pu-erh tea,but systematic studying on the compounds'changes during pile-fermentation is insufficient.The traditional pilefermentation has some uncontrolled factors,such as the temperature and relative humidity.Therefore,the quality of Pu-erh tea is often difficult to maintain consistency.The modern numerical controlling pile-fermentation can accurately control the above-mentioned factors,and which should be new developing direction of standardization and clean production.However,the chemical changes and sensory evaluation of pu-erh tea produced by numerical controlling pile-fermentation are not studied in depth.Therefore,it is of great significance to study the variation of the chemical composition of Pu-erh tea in the process of numerical controlling pile-fermentation,and to optimize the fermentation process and improve the quality of the product.In the present study,the appearance,infusion color,flavor,taste and leaves after brewing of pu-erh tea samples(CPET?1st-PF?2nd-PF?3rd-PF?RPET)were evaluated.The contents of catechins,gallic acid,caffeine,theobromine,and compounds with high molecular weights were determined by high performance liquid chromatography.The tea samples during pilefermentation were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS)based non-targeted metabolomics and liquid chromatography triple quadrupole mass spectrometry(LC-QQQ-MS)based targeted metabolomics.The multivariate analysis gave 53 marker compounds.The contents' change of flavonol glycosides,procyanidins,puerins(N-ethyl-pyrrolidone substituted flavan-3-ols)were determined.The inhibition effects of pu-erh tea samples on ?-glucosidase and ?amylase were detected.The correlation analysis of marker compounds and inhibitory effects on ?-glucosidase and ?-amylase were analyzed to find the critical bioactive marker compounds.The main results of this research were listed as below:1)The sensory evaluation directly presented the changes of appearance,infusion color.With the prolonging of pile-fermentation,the infusion color,flavor and taste were changed.At the end of pile-fermentation,the final pu-erh tea can show the red,brown and bright color of infusion,lower astringent taste,normal aged flavor.2)Through non-targeted metabolomics analysis,53 marker compounds are catechins,flavonol glycosides,phenolic acids and hydrolysable tannins were confirmed.The pyrogallol,quinic acid and theogallin,galloyl-HHDP-glucose,caffeoyl-quinic acid were also firstly recognized as the main marker compounds.At the beginning of pile-fermentation,the content of catechins,flavonoid glycosides and hydrolyzable tannins was significantly reduced,while the content of gallic acid and catechin derivatives teadenol A and teadenol B increased.3)Through the targeted metabolomics,the flavonol glycosides,procyanidins,puerins were quantitatively determined.The results showed that the myricetin-3-O-galactoside,quercetin-glucoside,kaempferol-3-O-rutinoside,rutin,kaempferol-glucoside were marker compounds.The results suggested that the contents'change of flavonol glycosides is the main difference between crude and ripened pu-erh tea after pile-fermentation.4)With the prolonging of pile-fermentation,the inhibitory effects of tea samples on ?glucosidase were decreased.The correlation analysis of chemical and biological data showed that EGCG,EGC,theogallin were positively correlated with the biological effects.Meanwhile,serine,gallic acid,pyrogallol,methoxy-hydroxyphenyl propionic acid were negatively correlated with the bioactivities.Through this study,the sensory evaluation and chemical constituents of pu-erh tea during numerical controlling pile-fermentation were studied,and 53 compounds were marker compounds for pile-fermentation.The contents of flavonol glycosides,procyanidins,and puerins were quantitatively detected,and the hypoglycemic effects of high-molecular weight compounds were also analyzed.These results will provide theoretical basis for chemical changes,and scientific supports for the improvement of numerical controlling pilefermentation of ripened pu-erh tea...