| Tieguanyin is highest grade of oolong tea, and its unique aroma is known at home andabroad. About studies on the chemical mechanism of tieguanyin aroma formed are many, but itsenzymology, molecular biology mechanism research reported rarely.In this study were used astieguanyin materials to study biochemistry and molecular mechanism of tieguanyin aromaformation.It determined and analysised tieguanyin's aroma substances, components, the totalamount in different processing phase,and explored the dynamic change of tieguanyin's aromasubstances, components, the total amount.Tieguanyin's β-glucosidase activity in differentprocessing phase was determed and analysised,and it explored the dynamic change oftieguanyin's β-glucosidase activity and the correlations among β-glucosidase and aromasubstances, components,the total amount.Tea aroma gene of cβ-glucosidase was cloned fromTieguanyin and its biological information was forecasted and analysised. The quantitativeexpression of Tieguanyin's cβ-glucosidase in different processing phase was analyzed.On thebasis of the studies above, the correlations of Tieguanyin tea aroma change, β-glucosidaseactivity, cβ-glucosidase expression were analysised comprehensively,and aroma formation'smechanism was revealed.The main results shows as follows:1. The dynamic change of tieguanyin aroma in different processesThe dynamic change of tieguanyin aroma in different process was explored in this study.The results shows that:(1) The sun-withering stage: In the process of tea leaves-makingtechnology of the degree of light fermentation in low temperature,tieguanyin main aromacompositions increased in sun-withering lightly processing, and the sun-withering lightlyprocessing promoted effectively main aroma substances to form in quantity,such as alphaFarnesene,beta Ionone,geranylacetone, Jasmone,cedrene. In the process of traditional tealeaves-making technology in the natural conditions,the sun-withering heavily processing alsopromoted tieguanyin main aroma to form, and the increase rate was inferior to the sun-witheringlightly.(2) The shaking tea leaves stage: In the process of tea leaves-making technology of thedegree of light fermentation in low temperature,tieguanyin main aroma compositions increasedin shaking tea leaves lightly processing, and shaking tea leaves lightly processing promotedeffectively main aroma substances to form in quantity,such as Nerolidol,alpha Farnesene,betaLonone,Geranylacetone,Jasmone,trans-2-Hexenyl butyate,cis-3-Hexenyl benzoate.In the processof traditional tea leaves-making technology in the natural conditions,the shaking tea leavesheavily processing also promoted effectively main aroma substances to form in quantity,such asNerolidol,alpha Farnesen,bate Farnesene,Cedrene,L-Menthol,beta Lonone,Geranylacetone,trans-2-Hexenyl butyate,cis-3-Hexenyl benzoate,Phenethyl acetate,Hexyl hexanoate. The Total aromaforming in shaking tea leaves heavily processing was that forming in shaking tea leaves lightlyprocessing1.39times,then the former more effectively than the latter promotes the formation oftieguanyin aroma.(3) The spreading tea leaves stage: In the process of tea leaves-makingtechnology of the degree of light fermentation in low temperature,spreading tea leaves thinly fora long time promoted effectively main aroma substances to form in quantity,such as bate Elemene, Jasmone, trans-2-Hexenyl butyate, Phenethyl acetate,2-Propyl-1-heptanol. Afterspreading tea leaves for24h, although total aroma reached a high level, but the maincharacteristics aroma such as Nerolidol aroma,alpha Farnesene content reduced down.2. Differences of tieguanyin tea aroma in different seasons and different stylesDifferences of tieguanyin tea aroma in different seasons and different styles were explored. Theresults shows that:(1) Differences of different seasons tieguanyin aroma: the aroma contents ofautumn processing tieguanyin was higher than spring. The main characteristics aroma of autumnprocessing tieguanyin was significantly higher than spring,such as Nerolidol, alpha Farnesene,trans-2-Hexenyl butyate, Phenethyl acetate, Methyl dihydrojasmonate, Hexyl hexanoate, Indole,Geranylacetone, beta Lonone, Jasmone,N-Eicosane.(2) Differences of different style tieguanyinaroma: the fragrant and lingering charm type, the tea leaves fagrant and lingering charm type, thesour fragrant type and the traditional and highly scented type tieguanyin had two majorconstituents of alcohol and Alkenes.They had common aroma substances and highlycontents,such as Nerolidol, alpha Farnesene,trans-2-Hexenyl butyate,Phenethyl acetate,betaLonone. The main aroma substances of the traditional and highly scented type tieguanyin werehigher than the fragrant type such as Nerolido, alpha Farnesene, trans-2-Hexenyl butyate, Hexylhexanoate, Indole, Geranylacetone, cis-3-Hexenyl benzoate, Eicosane.The aroma substances ofthe fragrant and lingering charm type tieguanyin were higher than other types such as alphaFarnesene,bate Farnesene, N-Eicosane.The Ocimene content of the tea leaves fagrant andlingering charm type was more prominent.The Phenethyl acetate, Jasmone,3,7-methyl-1,5,7-three octene-3-alcohol and Cedrene content of the sour fragrant type were more prominent.3.The relations of different Tieguanyin styles' aroma substances and sensory evaluationThe relations of different Tieguanyin styles' aroma substances and sensory evaluation wereexplored. The results showed that: The significant characteristics aroma Nerolidol and alphaFarnesene contents of the traditional and highly scented type were the highest.Other aromasubstances contents were higher than the fragrant type.Thus its aroma's concentration, intensityand persistance performed excellently,but elegant degrees and purity performed poorly. Thesignificant characteristics aroma Nerolidol and alpha Farnesene contents of the traditional andhighly scented type were the highest. The relations of different Tieguanyin styles' aromasubstances and sensory evaluation were explored. The results showed that: The significantcharacteristics aroma Nerolidol and alpha Farnesene contents of the traditional and highlyscented type were the highest.Other aroma substances contents were higher than the fragranttype.Thus its aroma's concentration, intensity and persistance performed excellently,but freshdegrees and purity performed poorly.The significant characteristics aroma Nerolidol and alphaFarnesene contents of the fragrant and lingering charm type were higher than other the fragranttypes.But it had many aroma substances' contents were lower than other types such astrans-2-Hexenyl butyate,Phenethyl acetate,Indole,Methyl dihydrojasmonate,Hexyl hexanoate,beta Lonone,Ocimene.So its fresh degrees and purity performed excellently. The aromasubstances' contents of the tea leaves fagrant and lingering charm type got moderate level.Thetotal aroma contects of the sour fragrant type was highest,but the significant characteristicsaroma Nerolidol and alpha Farnesene contents were lower and many others aroma substancescontects were higher than other types.So its concentration was better but purity and fresh degreesand intensity were bader,then it has miscellaneous characteristics.The Research obtained the main aroma substances of tieguanyin were Nerolidol,alphaFarnesene,trans-2-Hexenyl butyate,Phenethyl acetate,Indole,Methyl dihydrojasmonate,Hexylhexanoate,Geranylacetone,cis-3-Hexenyl benzoate,betaLonone,Ocimene,N-Eicosane,Jasmone,Bate Farnesene,3,7-methyl-1,5,7-three octene-3-alcohol,Cedrene and so on. The chemical basisthat Tieguanyin aroma giving ardent tea people feeling of " fragrance of flower and fruit aroma " lied in: Orchid aroma contains Methyl dihydrojasmonate. Osmanthus aroma contains Ocimene,Monoterpenoids and Sesquiterpenoids. Gardenia aroma contains Linalool, Geraniol and Methylbenzoate..Plum aroma contains Benzyl acetate.Honey peach aroma contains lactone.Thetieguanyin aroma substances determined were the identical with them, or proceed heterogeneousand transformation to form them.4. Differences of tieguanyin β-glucosidase enzyme activity changed in differentseasons,different processing and different places' leavesDifferences of tieguanyin β-glucosidase enzyme activity changed in differentseasons,different processing and different places' leaves were explored. The results showed thatdifferences of tieguanyin β-glucosidase enzyme activity changed in different seasons were asfollows:In the process of the tea leaves-making technology of the degree of light fermentation inlow temperature, β-glucosidase enzyme activity of spring tieguanyin mature leaves was autumn's1.4-2.6times and rangeability was larger than autumn's.Then in two seasons tieguanyin matureleaves beta glucosidas enzyme activity change trend were a little different,and themperformed"as one falls another rises" change characteristics. In the process of the traditional tealeaves-making technology in the natural conditions, β-glucosidase enzyme activity of springtieguanyin mature leaves was autumn's1.7-2.2times and rangeability was larger than autumn's.Then in two seasons tieguanyin mature leaves beta glucosidas enzyme activity change trend wereidentical. Therefore, β-glucosidase enzyme activity of spring tieguanyin mature leaves washigher than autumn's and rangeability was larger than autumn's.Differences of tieguanyin β-glucosidase enzyme activity changed in different processingwere as follows:In the process of the tea leaves-making technology of the degree of lightfermentation in low temperature, β-glucosidase enzyme activity was higher than the traditionaltea leaves-making technology in the natural conditions.In spring, β-glucosidase enzyme activitychange of different processings were identical,which ascended in the sun-withering stage,appeared the change trend"drop-rise-drop-(rise)"in the shaking tea leaves stage. In autumn,β-glucosidase enzyme activity change of different processings performed"as one falls anotherrises" change characteristics,and the rangeability of the traditional tea leaves-making technologywas larger than the tea leaves-making technology of the degree of light fermentation.Differences of tieguanyin β-glucosidase enzyme activity change in leaves at the differentplaces were as follows:In spring, differences of β-glucosidase enzyme activity of leaves at thedifferent places were small. In autumn, differences of β-glucosidase enzyme activity of leaves atthe different places performed that mature leaves>old leaf> young leaf. β-Glucosidase enzymeactivity of leaves at the different places got the highest in former midium-term and β-glucosidaseenzyme activity of young leaves has large variation range in two seasons and technologys.5.cDNA clonging of tieguanyin β-glucosidaseThe sequence's length of tieguanyin cβ-glucosidase cloned was1477bp,including openreading frame (ORF) which contained1284base composition and encoded428amino acids. Inthe3'RACE and conservative base area overlaped for267bp, the coding areas' RACE was186bp, with other plant homologous rate was60%-80%.This genetic sequence was registered inGenBank, and the registration number was HQ679938.2.6. Analysis of bioinformatics of tieguanyin cβ-glucosidaseBioinformatics of tieguanyin cβ-glucosidase was analyzed. The results shows that, theprotein of tieguanyin cβ-glucosidase was alkaline, stable and belong to hydrophilic protein.Tieguanyin cβ-glucosidase located in cytoplasm and existed transmembrane domain so belong totransmembrane protein, nevertheless existed no signal peptide so wasn't secretory protein.Inaddition, tieguanyin cβ-glucosidase had20phosphorylation sites, and Ser of phosphorylationsite lied in C-terminal. Ser of phosphorylation site was forecasted relating with tieguanyin cβ-glucosidase 's protein conformation change, nuclear localization regulation and cell cycle.7. Differences of tieguanyin cβ-glucosidase expression level in different processingDifferences of tieguanyin cβ-glucosidase expression level in different processing wereexplored. The results shows that: In the process of the tea leaves-making technology of thedegree of light fermentation in low temperature and the traditional tea leaves-making technologyin the natural conditions, there had been a gradual rise in tieguanyin cβ-glucosidase expressionlevel in sun-withering and shaking tea leaves stage. No significant differences existed betweentieguanyin cβ-glucosidase expression level in sun-withering and shaking tea leaves stage andcβ-glucosidase expression level rised significantly in shaking tea leaves stage end.In earlierspreading tea leaves stage, cβ-glucosidase expression level changed significantly,and whenspreading tea leaves stund up to6-10h, cβ-glucosidase expression level reached the highest. Allthat tieguanyin tea processing technology promoted cβ-glucosidase to express,and spreading tealeaves stage to a certain extent.cβ-Glucosidase expressed level highestly,and as the continuationof spreading tea leaves, cβ-Glucosidase expression weakened.8.The relations of different tieguanyin aroma change, β-glucosidase enzyme activity andcβ-glucosidase expression level in different processingThe relations of different tieguanyin aroma change, β-glucosidase enzyme activity andcβ-glucosidase expression level in different processing were explored. The results shows that:Tieguanyin β-glucosidase enzyme activity and aroma contents presented index negativecorrelation. In the process of the tea leaves-making technology of the degree of lightfermentation in low temperature and the traditional tea leaves-making technology in the naturalconditions,the relations of β-glucosidase enzyme activity and aroma amount, alcoholic aromacontent, the significant characteristics aroma Nerolidol content perform that:In sun-witheringstage, β-glucosidase enzyme activity improved with aroma amount, alcoholic aroma content,andthe significant characteristics aroma Nerolidol content;In former midium-term of shaking tealeaves stage, β-glucosidase enzyme activity improved with Pretreated aromatic substanceslargely;In spreading tea leaves stage, the change trend of enzyme activity and the changetendency of the aroma were identical.The relations of β-glucosidase enzyme activity and cβ-glucosidase expression level indifferent processing perform that:In the process of the tea leaves-making technology of thedegree of light fermentation in low temperature and the traditional tea leaves-making technologyin the natural conditions, cβ-glucosidase expression level increased and β-glucosidase enzymeactivity improved significantly. In the end shaking tea leaves stage,cβ-glucosidase expressionlevel increased persistently,and β-glucosidase enzyme activity descended significantly.Inspreading tea leaves stage, the change trends of β-glucosidase enzyme activity andcβ-glucosidase expression level were identical.The relations of cβ-glucosidase expression level and aroma contents in different processingperform that: In the sun-withering and shaking tea leaves processes of the tea leaves-makingtechnology of the degree of light fermentation in low temperature and the traditional tealeaves-making technology in the natural conditions, cβ-glucosidase expression level and aromaamount, alcoholic aroma content, the significant characteristics aroma Nerolidol content keptrising trend.In spreading tea leaves stage, the change trend of aroma amount consistented withcβ-glucosidase expression level. However, cβ-glucosidase expression level increased persistentlywhich didn't cause alcoholic aroma content change correspondly.They appeared the changetrend"drop-rise-drop-(rise)" in the spreading tea leaves stage. |
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