Study On The Dynamic Changes Of Main Biochemical Components And Key Enzyme PPO Characteristics During Black Tea Processing

Black tea belongs to fully fermented tea and one of the six traditional tea categories in China.Different tea categories with different colors,flavors and tastes can be produced through varying degrees of control or use of polyphenol oxidase（PPO）activity during tea processing.PPO can catalyze the oxidation of flavanols（catechins）to quinones,which are then oxidized and condensed into more complex fermentation products such as theaflavin,thearubigin and theabrownin,playing a key role in the sense and quality of black tea.To comprehensively analyze the formation of black tea flavor compounds,multi-omics combined with enzyme activity was used to analyze the dynamic changes of flavor compounds in the main metabolic pathways.In order to further explore the formation of flavor substances,the key enzyme（polyphenol oxidase,PPO）was selected during black tea processing in this paper,and further studied its separation and purification,enzyme morphology and enzymatic properties,and enzymatic reactions in vitro.The main results are as follows:（1）The flavor substances undergo significant dynamic changes,and the fermentation process plays a key role in the formation of the color,aroma and taste substances of black tea during the processing.A total of 107 non-volatile compounds and 222 volatile compounds were identified by UHPLC-Q-Exactive/MS and SBSE-GC-MS techniques,among which compounds in these metabolic pathways showed different trends.The levels of flavanols,flavanol glycosides and flavonoid glycosides gradually decreased,while most catechin dimer compounds showed a gradually increasing trend.Tyrosine,phenylalanine,leucine,isoleucine and glutamic acid were significantly increased during the withering process,and aspartic acid,tryptophan and theanine were significantly decreased during the rolling and fermentation process.Aldehydes were higher accumulation than alcohols in fatty acid-derived volatiles（FADVs）during fermentation stage,and amnio acid-derived volatiles（AADVs）with floral,fruity or sweet aroma characteristics were also accumulated in large quantities.The correlation analysis in the dynamic changes of non-volatile substances and volatile substances found that phenylalanine and isoleucine were transformed into derivative volatiles（phenylacetaldehyde,benzaldehyde,phenyl ethanol,benzyl alcohol,and 2-methylbutyraldehyde）,glycosidically bound volatiles（GBVs）are converted into terpenes such as linalool during the rolling and fermentation stages,enriching the tea aroma components.（2）The different changes of enzyme activities in different metabolic pathways,which affected the accumulation of flavor substances.The decreased enzyme activity in the flavonoid synthesis pathway and the increased oxidase（PPO）activity in the theaflavin synthesis pathway,resulting in a decrease in the level of flavonoids and an increase in the level of polymers such as theaflavin;the enzyme activities of chlorophyll-related metabolic enzymes decreased significantly after withering,and the green value gradually decreased and the red value gradually increased,which constituted the characteristics of black tea taste and color.GBVs were catalyzed to volatile terpenoids with sweet and floral characteristics by highly activeβ-glucosidase（β-GC）during the withering process.Lipoxygenase（LOX）shows stronger activity than alcohol dehydrogenase（ADH）in the fermentation stage,so aldehydes are more accumulate than alcohols in FADVs during fermentation stage.（3）A fast and effective method for the separation and purification of tea PPO,the three-phase partitioning（TPP）,was established.tea PPO from tea leaf was purified with 15.76-fold enrichment in the specific activity of 210.46 U/mg.Tea PPO has the strongest affinity for pyrogallol.When catechol is used as the substrate,the optimum temperature and p H of PPO were 45℃and 5.5,respectively.The enzyme had great stability under condition of25℃and p H 4.5～7.0.The most significant inhibitory effect of different compounds on purified tea PPO was ascorbic acid.This work proves that the TPP method can better retain the tea PPO activity,and easy to operate,time-consuming and low-cost for the purification of tea PPO,which is beneficial to industrial applications.（4）Membrane-bound polyphenol oxidase（m PPO）was isolated from tea leaves by TPP.Comparing the enzymatic properties of s PPO and m PPO,both showed increased activity with diphenols as substrates and reduced activity with monophenols as substrates.The optimum p H for catalytic activity of both enzymes was 5.5.The optimum temperatures for the activity of s PPO and m PPO were 30℃and 25℃,respectively.The differential response of both forms to EDTA（s PPO inhibition and m PPO activation）and greater stability of m PPO in the acidic p H range,and its higher affinity for the tested substrates are the major differences between s PPO and m PPO.（5）A tea PPOI was purified and used for the enzymatic synthesis of theaflavin monomers.Tea leaf PPOI was purified 12.90-fold,with recovery yield of 1.30%,via the sequential use of precipitation with 30%～80%saturated（NH₄）₂SO₄,Q-Sepharose Fast Flow anion exchange chromatography,and Sephadex G-75 gel filtration chromatography.Subsequently,PPOI was used in the enzymatic synthesis of theaflavins:under various p H,temperatures,reaction times,and catechin substrate composition and concentration,only the amount of theaflavin components produced changed,and no other theaflavin component apart from the theaflavin monomer was produced.These results highlight the importance of monomeric theaflavin synthesis using purified PPOI isozyme and provide insight into PPO in the context of tea production.