The Formation Mechanism Of Methoxybenzenes In Fermentation Of Pu-erh Tea

The contents of methoxybenzenes including 1,2,3-trimethoxybenzene and 1,2-dimethoxybenzene were increased during fermentation,which are responsible for the stale aroma of Pu-erh tea.However,the formation mechanism of methoxybenzenes during fermentation needs to be studied.Aspergillus luchuensis is a dominant fungus in the fermentation of Pu-erh tea,which produced catechol-O-methyltransferase during fermentation.Therefore we suggested this enzyme catalyzes catechol and catechins to form methoxybenzenes.In this thesis,the enhanced fermentation(EF)of Pu-erh tea with inoculation of A.luchuensis were developed,and the normal fermentation(NF)without inoculation was developed as the control test.And three studies were developed:(1)the metabolism of methoxybenzenes in Pu-erh tea fermentation was studied using head-space solid-phase microextraction gas chromatography-mass spectrometry;(2)the formation mechanism of methoxybenzenes was surveyed through metatranscriptomics analysis;(3)catecholO-methyltransferase of A.luchuensis was prepared by genetic engineering method,and the catalysis of this enzyme was verified preliminary.The main results are as follows.(1)Metabolism of methoxybenzenes in fermentation with inoculation of A.luchuensis.Contents of theaflavin,catechin,epicatechin-3-gallate,and catechin-3-gallate in the enhanced fermentation were significantly higher than those in the normal fermentation(p < 0.05);Contents of tea polyphenols,free amino acids,soluble sugars,gallic acid,and caffeine were significantly lower than that in the control fermentation(p < 0.05).126 volatile components belonging to nine categories were identified using HS-SPME GC/MS.The relative contents of hydrocarbons,alcohols,aldehydes,heterocycles,and aromatic compounds were decreased,while the contents of ketones,esters,phenols,and methoxy compounds were increased during fermentation.Contents of methoxybenzenes and ketones in the enhanced fermented samples were significantly higher than that in the control fermentation(p < 0.05),indicating that inoculation of A.luchuensis can increase the content of methoxybenzenes in tea leaves.(2)Formation mechanism of methoxybenzenes in Pu-erh tea fermentationThe V3-V4 of bacterial 16 S r DNA and fungal ITS region in fermenting samples were analyzed through amplicon sequencing technology.The microbial abundance and diversity index was increased in the early stage,while decreased and then increased in the medium and final stages of fermentation.Dominant bacteria in the early stage of the two fermentations were Serratia;and dominant bacteria in the medium and final stages were Staphylococcus,Kocuria,and Bacillus.Dominant fungi at the early stage of both fermentation were Aspergillus,and at the medium stage of fermentation was Aspergillus,Thermomyces,Blastobotrys,Debaryomyces,and Rasamsonia.Metatranscriptomes in the early stage and medium stage of fermentation were sequenced by Illumina Hi Seq TM 2500 technology.It was found that the abundance of A.luchuensis in enhanced fermentation was significantly higher than that in normal fermentation,indicating that A.luchuensis became the dominant microbes in the early stage of fermentation and changed the microbial community of fermentation.360 genes encoding enzymes involved in the metabolism of phenolic compounds were identified through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations,which were produced by A.luchuensis,A.kawachii,A.fumigatus,A.tubingensis,R.emersonii,Syncephalastrum racemosum,A.eucalypticola,A.piperis,etc.The expression levels of five catechol-Omethyltransferase encoding genes were 10.98-45.38 fpkm,which were produced by A.luchuensis,A.fumigatus,and R.emersonii,respectively.The expression levels of these genes in EF were higher than that in NF,indicating that microorganisms such as A.luchuensis produce catechol-O-methyltransferase during fermentation to catalyze the formation of methoxybenzene.(3)Preparation of catechol-O-methyltransferase of A.luchuensisThe catechol-O-methyltransferase of A.luchuensis was prepared using the genetic engineering method,which has a molecular weight of 28.3 k Da and an activity of 0.686 U/mg.It can catalyze phenolic compounds such as catechol,gallic acid,and epicatechin,but the reaction products need further identification.In summary,we found that A.luchuensis was grown to dominate microbes in the early stage of fermentation after inoculation.It produced catechol-Omethyltransferase with other microorganisms,which catalyzes the formation of methoxybenzenes in fermentation.New knowledge about the formation of the stale aroma of Pu-erh tea was discovered.