Does Enzymatic Hydrolysis of Glycosidically Bound Volatile Compounds Really Contribute to the Formation of Volatile Compounds During the Oolong Tea Manufacturing Process?

Abstract

It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles (GBVs). In this study, most GBVs showed no reduction during oolong tea manufacturing process. beta-Glycosidases either at protein or gene level were not activated during the manufacturing process. Subcellular localization of beta-primeverosidase provided evidence that beta-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active manufacturing process. After the leaf cell disruption, GBVs content reduced. These reveal that during the enzyme-active process of oolong tea, non-disruption of the leaf cell walls resulted in impossibility of interaction of GBVs and -glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing process. Interestingly, the three volatiles contents reduced after the leaf cell disruption, suggesting that mechanical damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In addition, 11 volatiles with flavor dilution factor ≥44 were identified as relative potent odorants in the oolong tea. These results propose that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic volatiles with potent odorants were produced from the manufacturing process.

Full text

Does Enzymatic Hydrolysis of Glycosidically Bound Volatile
Compounds Really Contribute to the Formation of Volatile
Compounds During the Oolong Tea Manufacturing Process?
Jiadong Gui,
†,‡,§
Xiumin Fu,
†,∥,§
Ying Zhou,
†,∥
Tsuyoshi Katsuno,
⊥
Xin Mei,
†,∥
Rufang Deng,
†
Xinlan Xu,
†
Linyun Zhang,
#
Fang Dong,
¶
Naoharu Watanabe,
○
and Ziyin Yang*
,†,‡,∥
†
Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden,
Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China
‡
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
∥
Provincial Key Laboratory of Applied Botany South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723,
Tianhe District, Guangzhou 510650, China
⊥
Tea Research Center, Shizuoka Prefectural Research Institute of Agriculture and Forestry 1706-11 Kurasawa, Kikugawa 439-0002,
Japan
#
College of Horticultural Science, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China
¶
Guangdong Food and Drug Vocational College, Longdongbei Road 321, Tianhe District, Guangzhou 510520, China
○
Graduate School of Science and Technology, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu 432-8561, Japan
*
SSupporting Information
ABSTRACT: It was generally thought that aroma of oolong tea resulted from hydrolysis of glycosidically bound volatiles
(GBVs). In this study, most GBVs showed no reduction during the oolong tea manufacturing process. β-Glycosidases either at
protein or gene level were not activated during the manufacturing process. Subcellular localization of β-primeverosidase provided
evidence that β-primeverosidase was located in the leaf cell wall. The cell wall remained intact during the enzyme-active
manufacturing process. After the leaf cell disruption, GBV content was reduced. These findings reveal that, during the enzyme-
active process of oolong tea, nondisruption of the leaf cell walls resulted in impossibility of interaction of GBVs and
β-glycosidases. Indole, jasmine lactone, and trans-nerolidol were characteristic volatiles produced from the manufacturing
process. Interestingly, the contents of the three volatiles was reduced after the leaf cell disruption, suggesting that mechanical
damage with the cell disruption, which is similar to black tea manufacturing, did not induce accumulation of the three volatiles. In
addition, 11 volatiles with flavor dilution factor ≥44were identified as relatively potent odorants in the oolong tea. These results
suggest that enzymatic hydrolysis of GBVs was not involved in the formation of volatiles of oolong tea, and some characteristic
volatiles with potent odorants were produced from the manufacturing process.
KEYWORDS: aroma, glycosidically bound volatiles, indole, β-primeverosidase, tea, volatile
■INTRODUCTION
Tea (Camellia sinensis) aroma (volatility and odor-activity) is
one of the main sensory properties which are decisive for the
quality of the final product. The tea volatile compounds can be
divided into four major classes according to their metabolic
origin: terpenoids, phenylpropanoids/benzenoids, fatty acid de-
rivatives, and carotenoids.
1
Monoterpenoids are enzymatically
synthesized de novo from acetyl CoA and pyruvate provided by
the carbohydrate pools mainly in plastids and the cytoplasm,
2
such as linalool (fresh floral odor), linalool oxides (floral), and
geraniol (floral, rose-like).
3
Most volatile benzenoids and
phenylpropanoids are primarily derived from phenylalanine,
4
such as 2-phenylethanol (floral, rose-like), benzyl alcohol (weak
floral), and phenylacetaldehyde (floral, rose-like).
3
Fatty acid-
derived straight-chain alcohols, aldehydes, ketones, acids, esters,
and lactones are basically formed by three processes, α-oxidation,
β-oxidation, and the lipoxygenase pathway,
4
such as hexanol
(green note), (Z)-3-hexen-1-ol (green leaf-like), and methyl
jasmonate (floral, jasmine-like).
3
The oxidative cleavage of
carotenoids leads to the production of apocarotenoids through
catalysis of a family of carotenoid cleavage dioxygenases,
5
such as
β-ionone (floral).
3
Moreover, volatiles occur in tea leaves not
only as free forms but also as glycosidically bound forms, which
are more water-soluble and less reactive than their free aglycon
counterparts.
6
Many glycosidically bound volatiles (GBVs)
have been isolated and identified in tea leaves, such as the
β-primeverosides and β-glucopyranosides of alcoholic volatiles
including benzyl alcohol, 2-phenylethanol, 1- phenylethanol, methyl
salicylate, (Z)-3-hexenol, linalool, linalool oxides, and geraniol, and
some GBVs that are hydrolyzed to form nonalcoholic volatiles
including benzaldehyde, coumarin, and damascenone.
7−15
Received: March 17, 2015
Revised: July 24, 2015
Accepted: July 26, 2015
Published: July 26, 2015
Article
pubs.acs.org/JAFC
© 2015 American Chemical Society 6905 DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914

Many attempts have been done to improve or modify the
volatiles of tea leaves either by treating leaves during growth
of the plants (raw materials) or by postharvest treatment of
leaves during the tea manufacturing process.
1
Biotic stress
(insect attack) and abiotic stress (light) have been utilized for
the improvement of volatiles in raw tea leaves. As a typical
example of biotic factors, the famous Taiwan oolong tea
(Oriental Beauty) has a unique aroma reminiscent of ripe fruit
and honey that is induced by the attack of tea green leaf-
hoppers.
16
As an example of abiotic factors, Gyokuro and
Tencha, known as the finest teas in Japan, are produced from
tea leaves under shading treatment. It was found that tea leaves
kept in darkness significantly increased levels of volatiles,
especially volatile phenylpropanoids/benzenoids.
17
Besides the
modification of raw materials, the manufacturing processes have
significant influences on the compositions of volatiles in final
tea products. In general, fermented teas including oolong tea
and black tea have more volatiles and aroma properties than
nonfermented green tea. This was generally thought to be due
to the involvement of hydrolysis of GBVs in the manufac-
turing process.
18
The GBVs are present within vacuoles, while
β-primeverosidase, one major glycosidase involved in the hy-
drolysis of GBVs, was presumed to be localized in cell walls.
18
This compartmentation of substrates and enzymes in plant cells
implies that interactions between the enzyme and the substrate
do not happen in intact tea leaves.
19
However, during the
manufacturing process, tea leaf tissues and cells are disrupted,
which results in the possibility of interactions between the
glycosidase and the GBVs and liberation of the free volatiles.
During the manufacturing process of black tea, the levels of
primeverosides of the volatiles decreased greatly, and the
glycosidase activities showed a peak during withering but were
drastically reduced after rolling, suggesting that enzymatic hy-
drolysis of GBVs mainly occurred during the stage of rolling.
20
Therefore, enzymatic hydrolysis of GBVs is proposed as an
important process during the manufacturing of black tea. In
contrast, most GBVs showed no reductions in their contents
during the manufacturing process of oolong tea. The contents
of GBVs increased after the solar-withering stage, and reached
their highest levels in the final stages of oolong tea produc-
tion.
21
This result led us ask whether enzymatic hydrolysis of
GBVs really contributes to the formation of volatiles during the
oolong tea manufacturing process. To answer this question, we
monitored changes in contents of free volatiles and GBVs,
glycosidase enzyme activities, and β-glycosidase gene expression
level during the manufacturing process of oolong tea. We
attempted to give direct evidence of subcellular localization of
β-primeverosidase, and also investigated the effect of the manu-
facturing process on the leaf cell structure. Finally relatively
potent odorants occurring in oolong tea product were evaluated
by gas chromatography−mass spectrometer/olfactometry
(GC−MS/O). The results help us find out the truth of forma-
tion of volatiles in oolong tea, and advance our understanding
of differences of formation of volatiles between oolong tea and
black tea.
■MATERIALS AND METHODS
Chemicals. Benzaldehyde, benzyl alcohol, β-damascenone,
δ-decalactone, ethyl decanoate, Furaneol, 2-hexen-1-ol, 3-hexenyl
acetate, indole, β-ionone, jasmine lactone, 3-methylnonane-2,4-dione,
methyl salicylate, 2-phenylethanol, and vanillin were purchased from
Wako Pure Chemical Industries Ltd., Japan. α-Farnesene, (Z)-3-
hexen-1-ol, linalool, linalool oxides, methyl jasmonate, trans-nerolidol,
geraniol, polyvinylpolypyrrolidone (PVPP), XAD-2, and β-glucosidase
were purchased from Sigma-Aldrich Company Ltd., USA. β-Prime-
verosidase and p-nitrophenyl-β-primeveroside were purchased from
Amano Enzyme Inc., Japan. p-Nitrophenyl-β-D-glucopyranoside
was purchased from Aladdin Industrial Co. Shanghai, China.
[15N]Anthranilic acid (15N% = 98%) was purchased from Cambridge
Isotope Laboratories Inc., Cambridge, MA. Porapak Q cartridge was
purchased from Waters Corporation, USA. Coomassie Blue R250,
30% acrylamide/Bis solution, N,N,N′,N′-tetramethylethylenediamine
(TMEMD), sodium dodecyl sulfate (SDS), and 2×SYBR Green
Universal PCR Mastermix were purchased from Bio-Rad Laboratories,
USA. Quick RNA isolation Kit was purchased from Huayueyang
Biotechnology Co., Ltd., China.
Plant Materials and Manufacturing Process of Oolong Tea.
One bud and two or three leaves of Camellia sinensis var. Jinxuan were
plucked at the Tea Experiment Station in the South China Agricultural
University (Guangzhou, China) in October, 2014. The manufacture of
oolong tea was carried out according to the general method,
21
as
shown in Figure S1 (Supporting Information). The freshly plucked tea
leaves (P) were exposed to sunlight (43 °C and 93,500 Lux) for
70 min as solar withering (SW). Afterward, the tea leaves were indoor-
withered (IW) for 2 h at temperature of 27 °C and a relative humidity
of 70%, and subsequently turned over by 5 times (T1−T5) every
1.5 h. Then the tea leaves were parched in a tea-firing roller machine at
250 °C for 2−3 min to inactivate the enzymatic activity and fix the
sample (F). Finally, the tea leaves were rolled at room temperature for
15 min and dried at 105 °C for 1.5 h. The samples from P, SW, IW,
and T1−T5 were immediately frozen by liquid nitrogen for enzyme
activity and gene expression profiling. Three replicates were processed
according to the oolong tea manufacturing.
Extraction and Analysis of Volatiles in Tea Samples. One
gram of samples (finely powdered) was extracted by 2.5 mL of dichlo-
romethane containing 5 nmol of ethyl decanoate as an internal
standard in a shaker at room temperature overnight. The extraction
solution was dried over anhydrous sodium sulfate and concentrated to
400 μL using nitrogen stream (MIULAB NDK200-1, MIU Instru-
ments CO., Ltd., Hangzhou, China). One microliter of the concentrate
was subjected to GC−MS QP2010 SE (Shimadzu Corporation, Japan)
equipped with a SUPELCOWAXTM 10 column (30 m ×0.25 mm ×
0.25 μm, Supelco Inc., Bellefonte, PA, USA). The injector temperature
was 240 °C. The splitless mode was used with a splitless time of 1 min,
and helium was the carrier gas with a velocity 1.0 mL/min. The GC
oven temperature was 60 °C for 3 min, ramp of 4 °C/min to 240 °C,
and then 240 °C for 20 min. The mass spectrometry was operated
with full scan mode (mass range m/z40−200).
Extraction and Analysis of GBVs in Tea Samples. Analysis of
GBVs used the enzymatic hydrolysis combined with the GC−MS
analysis method, which was the same as described previously.
15,22
Five
hundred milligrams (fresh weight) of sample (finely powdered) was
extracted with 2 mL of cold methanol by vortexing for 2 min followed
by ultrasonic extraction in ice-cold water for 10 min. The extracts were
mixed with 2 mL of cold chloroform and 0.8 mL of cold water for
phase separation. The resulting upper layer was dried and dissolved in
1 mL of water. The resulting solution was mixed with 30 mg of PVPP,
stood for 90 min, and was centrifuged (16400g,4°C, 10 min), and
repeated with the supernatant. The final supernatant was loaded to an
Amberlite XAD-2 column (1 mL) and eluted with 5 mL of water,
5 mL of pentane:dichloromethane (2:1), and 5 mL of methanol.
The methanol eluent was dried using nitrogen stream, and redissolved
in 400 μL of 50 mM citric acid buffer (pH 6.0) containing
β-primeverosidase and β-glucosidase, and reacted at 37 °C for 14 h.
Afterward, 144 mg of sodium chloride was added to the reaction
solution, and stood for 15 min followed by addition of 5 nmol of ethyl
decanoate as an internal standard. The solution was extracted with
400 μL of dichloromethane and centrifuged, and the dichloro-
methane fraction was dried over anhydrous sodium sulfate, followed
by GC−MS analysis. The GC−MS conditions were the same as
described above.
Determination of Enzyme Activities of β-Glucosidase and
β-Primeverosidase. The enzyme assay method referred to the previous
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6906

reports.
20,23
One gram of finely powdered tea leaves was ground in ice-
cold acetone and filtered using a suction pump (T-50.2L, Tianjin
Jinteng Experiment Equipment Co., Ltd., Tianjin, China). The
acetone-insoluble powder on the filter paper was washed with ice-
cold acetone several times until clean and colorless acetone filtrate was
obtained. The acetone-insoluble powder was dried by nitrogen stream
to remove residual acetone and stored at −80 °C. For each 0.25 g of
acetone-insoluble powder, 6.25 mL of ice-cold 50 mM sodium citrate
buffer (pH 6.0) was added to dissolve the powder, accompanied by
0.125 g of PVPP. After vortexing twice for 30 s at 0 °C and
centrifuging at 10000g(4 °C, 20 min), the supernatant was used as
the crude enzyme solution. The substrate, either p-nitrophenyl-β-D-
glucopyranoside or p-nitrophenyl-β- primeveroside, was dissolved in
90 μL of 50 mM sodium citrate buffer (pH 6.0) and incubated at
37 °C for 5 min. Ten microliters of crude enzyme solution was
added, and then the whole reaction mixture was incubated at 37 °C
for 60 min. The reaction was stopped by adding 140 μL of 0.2 M
sodium carbonate, resulting in yellow color of the mixture, which was
Figure 1. Changes in free volatiles during the manufacturing process of oolong tea. P, freshly plucked tea leaves; SW, solar withering; IW, indoor
withering; T1−T5, turn over by 5 times; F, fixing. Data represent the mean value ±standard deviation of three in independent experiments
performed in triplicate. The y-axis unit is peak area ratio of analyte to internal standard per g dry weight tea leaves. The volatiles in the pane showed
significant increase during the manufacturing process of oolong tea.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6907

measured at 400 nm by a spectrophotometer (Infinite M200 PRO,
TECAN, Switzerland). The amount of p-nitrophenol was determined
according to a calibration curve.
Transcript Expression Analysis of β-Primeverosidase,9/13-
Lipoxygenase (LOX), and Terpene Synthases (TPS). Total RNA
was isolated immediately after dissection. The reactions were perfor-
med using Power SYBR Green PCR Master Mix in a 20 μL volume
containing 10 μL of Power SYBR Green PCR Master Mix (2×),
0.2 μM each specific forward and reverse primer. Two microliters of
10-fold diluted template was used for a 20 μL PCR reaction. The
encoding elongation factors (EFs) were used as internal reference
gene.
24
The EFs,β-primeverosidase,
15
LOX,
25
and TPS
26
specific primers
of qRT-PCR are shown in Table S1 (Supporting Information). The
qRT-PCR was carried out on Roche LightCycle 480 (Roche Applied
Science, Mannheim, Germany) under the condition of one cycle of
95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min.
A melt curve was performed at the end of each reaction to verify PCR
product specificity. The 2−ΔΔct method was used to calculate the
relative expression level.
27
Changes in mRNA levels of the target genes
were normalized to that of EFs.
Observation of Tea Leaf Cell Structure. The tea leaf samples of
P, T1, T2, and T5 stages were cut into approximately 1 mm ×2mm
pieces and fixed in 0.1 M phosphate buffer (pH 7.2) containing 2%
glutaraldehyde and 2.5% paraformaldehyde. After 6 times washing with
0.1 M phosphate buffer, the leaf samples were postfixed in 1% osmium
tetroxide for 4 h and washed with 0.1 M phosphate buffer. Then the
fixed leaf samples were dehydrated and embedded in flat molds using
EPON812 resin. Ultrathin sections (80 nm) were cut by ultramicro-
tome (Leica UC7, Leica Microsysteme GmbH, Wetzlar, Germany),
which then were stained by 4% uranyl acetate and 2% lead citrate.
Ultrathin sections were observed by a transmission electron micro-
scope (JEOL JEM-1010, Tokyo, Japan) operating at 100 kV.
Subcellular Localization of β-Primeverosidase in Tobacco.
To construct vectors for expression of GFP-fusion protein, the stop
codon of β-primeverosidase coding sequence was replaced by XmaI
restriction site, with the following primers: 5′-GTCGACATGATGG-
CAGCGAAAGGG-3′(forward), 5′-CCCGGGGCTTGAGGAGGA-
ATTTCTT-3′(reverse). The PCR product of β-primeverosidase
cDNA fragment, in which the stop codon was eliminated, was cut with
SalI/XmaI and then cloned into the SalI/XmaIsitesofthe
pCAMBIA3300-GFP vector. The reorganization vector was trans-
formed into Agrobacterium GV3101 by freeze−thaw. The overnight
Agrobacterium cultures were sedimented at 5000gfor 1 min. The pellet
was resuspended in a solution containing 10 mM MgCl2,10mM
morpholineethanesulfonic acid (pH 5.6), and 100 μM acetosyringone
to OD600 = 0.4.
28,29
Leaves of Nicotiana benthamiana were infiltrated
by using a syringe without a needle, and then the tobacco was grown
on a 16 h light/8 h dark under 25 °C regime for 4−5 days. Protoplasts
were prepared as follows: tobacco leaves were cut into 2−3cm
2pieces
Figure 2. Effect of disruption of tea leaf cell on amounts of free
volatiles and total GBVs. CK-1, The 4th turn over, at which stage tea
leaf cell is not disrupted; CK-2, the 5th turn over, at which stage tea
leaf cell is not disrupted; cell disruption-1, after turn over 1 (T1), parts
of tea leaves were completely crushed to cause disruption of leaf cell,
and indoor-withered at room temperature for 4.5 h; cell disruption-2,
after turn over 1 (T1), parts of tea leaves were completely crushed to
cause disruption of leaf cell, and indoor-withered at room temperature
for 6.0 h. Data represent the mean value ±standard deviation of three
independent experiments performed in triplicate. The y-axis unit is
peak area ratio of analyte to internal standard per g dry weight tea
leaves.
Figure 3. Effect of disruption of tea leaf cell on the transformation
from [15N]anthranilic acid to [15N]indole. (A) GC−MS chromatog-
raphy (m/z118) of [15N] indole from CK and leaf cell disruption. The
[15N]anthranilic acid feeding experiment started from the turn over 1
(T1). CK, the 5th turn over, at which stage tea leaf cell is not
disrupted; leaf cell disruption, after T1, parts of tea leaves were
completely crushed to cause disruption of leaf cell, and indoor-
withered at room temperature for 6.0 h. (B) Data represent the mean
value ±standard deviation of three independent experiments
performed in triplicate. The relative content was calculated based on
peak area of m/z118 of [15N] indole.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6908

with a sterile scalpel blade. Two grams of leaf pieces was transferred to
50 mL tubes containing 10 mL of buffer solution (50 mM 2-(N-
morpholino)ethanesulfonic acid, 0.6 M mannitol, and 10 mM calcium
chloride), containing 1% cellulose and 0.05% macerozyme. The tubes
were incubated at 25 °C on a reciprocating shaker (50 rpm) for 4 h.
Protoplasts were washed and held in buffer solution. Confocal
Figure 4. Changes in GBVs during the manufacturing process of oolong tea. P, freshly plucked tea leaves; SW, solar withering; IW, indoor withering;
T1−T5, turn over by 5 times; F, fixing. Data represent the mean value ±standard deviation of three independent experiments performed in
triplicate. The y-axis unit is peak area ratio of analyte (free volatiles from enzymatic hydrolysis of GBVs) to internal standard per g dry weight tea
leaves.
Figure 5. Changes in β-glycosidase enzyme activity, β-Primeverosidase,LOX, and TPS expression levels during the manufacturing process of oolong
tea. P, freshly plucked tea leaves; SW, solar withering; IW, indoor withering; T1−T5, turn over by 5 times. Data represent the mean value ±standard
deviation of three independent experiments performed in triplicate. (A, B) One unit was defined as the formed amount of p-nitrophenyl by the
action of per mg protein per minute. (C, D, E, F, G) Transcript abundance was calculated based on the difference in cycle threshold (Ct) values
between target gene and internal reference gene transcripts by the normalized relative quantification 2−ΔΔCt method. The expression level of gene
from P sample was defined as 1.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6909

microscope images were taken using a Zeiss LSM 510 META confocal
laser microscope (Cal Zeiss, Jena, Germany) with a 100×oil objective
under 488 nm excitation wavelength for GFP detection.
Effect of Disruption of Tea Leaf Cell on Amounts of Free
Volatiles and GBVs. After turn over 1 (T1), parts of tea leaves were
completely crushed to cause disruption of leaf cell, and indoor-
withered at room temperature for 4.5 and 6.0 h, respectively. After-
ward, 1 g of treated sample was used for analyses of free volatiles and
GBVs, which methods are described as above. The amounts of free
volatiles and GBVs of the treated samples were compared with those
of T4 and T5, respectively.
Administration of [15N]Anthranilic Acid into Tea Leaves and
Analysis of [15N]Indole. Fifty microliters of solution of [15N]-
anthranilic acid (20 mM) was injected into each tea leaf at the T1
stage. One part of the tea leaves with [15N]anthranilic acid was com-
pletely crushed to cause disruption of leaf cell and indoor-withered at
room temperature for 6.0 h, while the other part of the tea leaves with
[15N]anthranilic acid followed the oolong tea manufacturing and was
processed to the T5 stage. The amount of [15N]indole was analyzed by
GC−MS as described above. The characteristic ion of [15N]indole is
m/z118.
Identification of Relatively Potent Odorants of Oolong Tea.
The relatively potent odorants were evaluated using GC−MS/O and
aroma extraction and dilution analysis (AEDA), as described
previously.
30−33
The oolong tea product powders (2 g) were incubated
in hot distilled water (40 mL, 80−90 °C) for 5 min and centrifuged for
10 min at 3000g. The resultant supernatant (30 mL) was loaded on a
Porapak Q cartridge (2 mL volume, 50−80 mesh), eluted with 3 mL
of water, and 3 mL of pentane:diethyl ether (1:1, v/v) as aroma
extract. Afterward, the aroma extract was added with ethyl decanoate
(20 μg/20 μL) as an internal standard, dried over anhydrous sodium
sulfate, and concentrated to 100 μL in a stream of nitrogen to give an
original odor concentrate sample.
The original odor concentrate of the sample infusion was stepwise
diluted with pentane:diethyl ether (1:1, v/v) 1:4 = 41, 1:16 = 42,
1:64 = 43, 1:256 = 44, 1:1024 = 45, 1:4096 = 46, and 1:16384 = 47. The
aliquots (2 μL) of each sample were analyzed by GC−MS/O. The
flavor dilution (FD) factors of the odorants were determined by
AEDA.
The GC/MSD (5975C, Agilent Technologies Inc., USA) equipped
with a DB-WAX capillary column (60 m ×0.25 mm i.d., 0.25 μmfilm
thickness, Agilent Technologies Inc., USA) was used for mass spectro-
metric identification. The splitless mode was used with a splitless time
of 1 min. The split ratio at the time of the split vent liberation was
20:1. Helium was used as a carrier gas with a flow rate of 2 mL/min.
The injector temperature was 240 °C. The GC oven was maintained at
40 °C for 2 min, increased by 5 °C/min to 240 °C, and was kept at
this temperature for 25 min. The mass spectrometer was operated in
full scan mode with a mass range of m/z20−280. For GC−MS/O, the
effluent was split into the olfactory detection port (Gerstel K.K.,
Japan) and MS using two deactivated, uncoated fused silica capillaries
(106 cm ×0.15 mm i.d., 139 cm ×0.1 mm i.d.) at the end of the
capillary. Volatile compounds were identified by direct comparison
with the Kovats GC retention indices (RI), mass spectra, and those of
the authentic specimens.
■RESULTS AND DISCUSSION
Indole, Jasmine Lactone, and trans-Nerolidol Were
Significantly Increased at the Turn Over Stage. To find
out which volatiles, especially those also occurring in glyco-
sidically bound form, have big changes during the manufactur-
ing process, we monitored changes of free volatiles from the
plucking process to fixing process (enzymatic reaction termi-
nation) of oolong tea. Among the manufacturing processes, the
turn over process (T1−T5) had a significant influence on the
contents of volatiles (Figure 1). In the present study, not all the
free volatiles had big changes during the manufacturing process
of oolong tea. The three volatiles including indole, jasmine
lactone, and trans-nerolidol had significantly big increase at the
turn over process (Figure 1). In addition, most fatty acid-
derived volatiles such as 1-hexanol, (E)-2-hexen-1-ol, (Z)-
hexen-1-ol, etc., linalool oxides, and α-farnesene increased at
T1−T5, whereas some free volatiles such as linalool, geraniol,
diendiol I, benzyl alcohol, 2-phenylethanol, and methyl
salicylate, which also occur in glycosidically bound form, did
not show significant changes during the process (Figure 1).
Wang et al. first reported that the contents of free alcoholic
aroma compounds remained almost unchanged or slightly
decreased, but jasmine lactone and indole significantly
increased in the final oolong tea products compared to dried
fresh tea leaves.
21
In addition, increase of indole during the
oolong manufacturing process was also demonstrated by a
direct analysis in real time mass spectrometry.
34
Our previous
study on comparison of volatiles of green teas, oolong teas, and
black teas from different cultivars and regions also proposed
that jasmine lactone and indole were characteristic volatiles in
oolong tea products.
35
Furthermore, the present study indi-
cated that these volatiles mostly accumulated at the turn over
Figure 6. Subcellular localization analysis of GFP-β-primeverosidase. Upper photos: GFP-β-primeverosidase is visible in cell wall structures at the
tobacco leaf cell. Lower photos: Green fluorescence was not shown in the protoplast cell prepared from the tobacco leaves (the upper photos), which
proved that the GFP-β-primeverosidase protein is localized in the cell wall.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6910

process.
36,37
During the oolong tea manufacturing process, tea
leaves are exposed to various stresses, such as plucking (wounding),
solar withering (drought, heat, and UV/light radiation), indoor
withering (drought), and turn over (wounding).
16
Therefore, the
aroma formation in tea leaves during the manufacturing process of
oolong tea is proposed to be the stress-responsive biochemical
reactions of juvenile leaves of tea plants. Indole is a very repre-
sentative stress-responsive volatile compound in the manufacturing
process of oolong tea. Indole occurs with very trace amount in
intact tea leaves. However, indole level increases rapidly after
plucking.
38
Also indole increased during the withering process
of green tea leaves even at a relatively low temperature (15 °C,
for 16 h).
33
In the present study, the turn over led to more
accumulations of indole (Figure 1). This evidence suggests that
wounding stress from the turn over process is a key factor
regulating formation of indole. Interestingly, black teas contain
much less indole,
35
although wound stress is also involved in
the manufacturing process of black tea. Tea leaves are still
relatively intact and alive from the plucking to the turn over
process of oolong tea, whereas the structure of the leaf cells is
disrupted during the rolling process and the contents of the
cells are completely mixed during the rolling process of black
tea.
20,21
Amounts of indole, jasmine lactone, and trans-nerolidol
reduced after tea leaf cell disruption (Figure 2), suggesting that
mechanical damage with tea leaf cell disruption, which is similar
to the manufacturing process of black tea, does not induce the
accumulation of indole, jasmine lactone, and trans-nerolidol. As
anthranilic acid is one of precursors of indole,
33
we investigated
the formation of indole by feeding of stable isotope-labeled
compound [15N]anthranilic acid to the tea leaves. It was found
that the amount of [15N] indole in tea leaf with cell dis-
ruption was much lower than that in tea leaf without disruption
(Figure 3). This suggests that indole may be oxidized by P450
enzymes after the leaf cell disruption.
39
Therefore, the pathway
leading from anthranilic acid to indole may be a key step of
formation of indole during the oolong tea process. As the genes
involved in the pathway leading from anthranilic acid to indole
are still unknown in plants, we are attempting to identify the
involved genes and will investigate their responses to the
stresses from the manufacturing process of oolong tea. In addi-
tion, it would be very interesting to investigate whether dis-
ruption of leaf cell and complete mixture of contents of cell
may lead to the pathway competition between indole, jasmine
lactone, and trans-nerolidol and other metabolites.
Most GBVs Did Not Show Reduction during the
Manufacturing Process. To confirm whether enzymatic
hydrolysis of GBVs occurs during the manufacturing process of
oolong tea, we monitored changes of GBVs from the plucking
process to fixing process (enzymatic reaction termination) of
oolong tea. No GBVs were reduced in their contents during the
enzyme-active process (Figure 4), which is consistent with the
previous report.
21
This suggests that enzymatic hydrolysis of
GBVs may not happen during the manufacturing process of
oolong tea. The GBVs occur in vacuoles, whereas hydrolases
such as β-primeverosidase were presumed to be localized in cell
walls.
18
This compartmentation of substrates and enzymes in
plant cells leads to little possibility of hydrolysis of GBVs in
nondisrupted tea leaf cells. After additional treatment was done
to disrupt tea leaf cell, GBVs were reduced (Figure 2), further
demonstrating that lack of disruption of tea leaf cell during the
oolong tea process resulted in unavailability of interaction of
Figure 7. Transmission electron micrograph of the tea leaf cells during
the oolong tea manufacturing process. P, plucking; T1, turn over 1;
T2, turn over 2; T5, turn over 5; V, vacuole; CW, cell wall. (A, D, G, J)
Multicells on the edge of sample tea leaves in P, T1, T2, and T5
respectively. Bar, 5 μm. (B, E, H, K) Single cell on the edge of sample
tea leaves in P, T1, T2, and T5 respectively. Bar, 2 μm. (C, F, I, L) Cell
wall of the cell on the edge of sample tea leaves in P, T1, T2, and T5
respectively. Bar, 0.1 μm.
Table 1. Identification of Potent Odorants (FD Factor ≥44)
in Oolong Tea
RI identified compound odorant quality FD factor (4n)
1538 linalool fresh floral 5
1716 3-methylnonane-2,4-dione fresh floral 5
1821 β-damascenone honey 5
1840 unidentified
a
minty 5
1883 unidentified
a
fresh floral 4
1945 β-ionone floral 4
2017 Furaneol
a
sweet 7
2200 δ-decalactone floral, sweet 6
2266 jasmine lactone floral, sweet 6
2431 indole animal-like, floral 4
2546 vanillin sweet 5
a
The MS spectrum is provided as Figure S2.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6911

substrates and enzymes and no hydrolysis of GBVs. Inter-
estingly, the previous report
21
and the present study found that
the amounts of some GBVs increased (Figure 4). Currently,
most studies on GBVs have concentrated on glycosidases,
whereas little information on glycosyl transferases is available.
1
Recently glycosyl transferases in tea leaves have been func-
tionally characterized;
40
it will be helpful for our understanding
of the accumulation of GBVs during the oolong tea manu-
facturing process.
β-Glycosidases Were Not Activated during The
Manufacturing Process, and Interaction of β-Glycosi-
dases and GBVs Were Unavailable. To investigate whether
β-glycosidases were activated during the manufacturing process,
we monitored enzyme activities and gene expression levels of
β-glycosidases from the plucking process to the fifth turn over
process of oolong tea. The enzyme activities of β-glucosidase
and β-primeverosidase did not show increase during the manu-
facturing process (Figures 5A and 5B). Moreover, expression
level of β-primeverosidase showed reduction trend at the turn
over process (Figure 5C). Although many stresses including
drought, heat, wounding, and UV/light radiation are involved
in the oolong tea manufacturing process, our study indicates
that β-glycosidases were not activated under these stresses.
This also reveals that contact of substrate (GBVs) and enzyme
(β-glycosidases) is a key point determining whether enzymatic
hydrolysis of GBVs happens in the tea manufacturing process.
Previously, Mizutani et al. found that β-primeverosidase
is N-glycosylated, and has an N-terminal signal sequence, thus
presumed that β-primeverosidase may locate in the cell
wall.
18
However, a direct evidence of subcellular localization of
β-primeverosidase in tea leaves is still unavailable. In the present
study, subcellular localization of β-primeverosidase provided
more evidence that β-primeverosidase is located in the leaf cell
wall (Figure 6). Moreover, we investigated the cell structures
of tea leaves from the manufacturing process of oolong tea.
From the P to T1 process, the leaf cells showed complete and
unwounded. The content was divided into several parts and
showed the clear boundary (Figure 7A−F). From the T2 to T5
process, the cells shrank and distorted intensely, while the cell
walls remained well and unwounded (Figures 7G−L). These
results strongly support that lack of disruption of tea leaf cell
during the oolong tea process resulted in unavailability of inter-
action of GBVs (substrates located in vacuole) and β-glycosidases
(enzymes).
Besides β-primeverosidase, we also investigated the expression
levels of LOX that is a key gene that involved in formation of
fatty acid-derived volatiles and jasmine lactone,
1,25
and TPS
that are possibly involved in formation of trans-nerolidol in
tea leaves.
26
LOX is proposed as a wounding stress-response
gene.
25
LOX gene expression level significantly increased at
the turn over process (Figure 5D), which may be one of the
reasons that most free fatty acid-derived volatiles and jasmine
lactone increased at that process (Figure 1). Similarly, TPS1, 2,
and 3 gene expression levels also significantly increased at the
turn over process (Figures 5E,F,G). The present study provides
important hints that the wounding stress at the turn over
process may activate some key genes involved in formation of
volatiles.
Furaneol, Jasmine Lactone, δ-Decalactone, Linalool,
Vanillin, β-Ionone, β-Damascenone, 3-Methylnonane-
2,4-dione, and Indole Were Identified as Relatively
Potent Odorants in Oolong Tea. The above evidence shows
that enzymatic hydrolysis of GBVs did not significantly con-
tribute to the formation of volatiles of oolong tea. To further
find out which volatiles contribute to flavor of oolong tea,
GC−MS/O and AEDA were employed to determine the
contribution of each volatile to the quality of the tea’s aroma
and flavor. The results revealed 49 odor-active peaks with FD
factors between 41and 47in the final oolong tea product.
Among them, the 11 compounds showed as relatively potent
odorants with the higher FD factor (≥44)(Table 1). AEDA is
most frequently used for identifying ordor-active compounds in
Figure 8. Hypothetical model of different formations of volatiles between the oolong tea process and the black tea process.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6912

tea, and evaluating the flavor changes that occur during the
manufacturing of tea beverages.
33,41−45
Based on this technique,
many volatile compounds were identified as the most important
components in Japanese and Chinese green teas and black tea
beverages. However, little information on potent odorants in
oolong tea is available. In the present study, Furaneol, jasmine
lactone, δ-decalactone, linalool, vanillin, β-ionone, 3-methylno-
nane-2,4-dione, β-damascenone, indole, and two other com-
pounds were identified as relatively potent odorants in oolong
tea (Table 1). Although linalool was one of the potent odorants,
its formation was not from enzymatic hydrolysis of glycosides of
linalool (Figures 1 and 4). Jasmine lactone and indole were
identified as relatively potent odorants, further suggesting that
they were characteristic compounds derived from the
manufacturing process of oolong tea (Figures 1 and 2)and
partly contributed to the aroma of oolong tea. Other compounds
showed high FD factors, suggesting that some compounds with
low concentrations may have a comparatively low olfactory
threshold detected by human and contribute to the characteristic
aroma of oolong tea.
Formation of Volatiles Is Different between Oolong
Tea and Black Tea. Oolong tea and black tea are fermented
teas and have more aroma characters than green tea. Therefore,
analysis and identification of volatile compounds during the
manufacturing process of oolong tea and black tea have been
well done. Based on the previous reports and the present study,
we propose a hypothetical model of different formations of
volatiles between the oolong tea process and the black tea
process (Figure 8). (1) For oolong tea, turn over is a key pro-
cess to produce tea aroma and increase of wounding intensity
could lead to accumulations of indole, jasmine lactone, and
trans-nerolidol, which may be characteristic aromas of oolong
tea. However, such wounding is not sufficient for disruption of
tea leaf cell, which is essential for interaction of substrates and
enzymes. (2) For black tea, rolling is a key process to produce
tea aroma. At this process, complete disruption of tea leaf
cell leads to interaction of substrates and enzymes and many
GBVs are hydrolyzed to release free alcoholic aroma com-
pounds. The results help us find out the direct evidence of
formation of volatiles in oolong tea, and advance our un-
derstanding of differences of formation of volatiles between
oolong tea and black tea.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b02741.
Table of studied primers of qRT-PCR, flow chart of
oolong tea manufacturing process, and mass spectra
(PDF)
■AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-38072989. E-mail: zyyang@scbg.ac.cn.
Author Contributions
§
J.G. and X.F. equally contributed to this work.
Funding
This study was supported by “100 Talents Programme of the
Chinese Academy of Sciences”(Y321011001 and 201209), and
the Foundation of Science and Technology Program of
Guangzhou (2014J4100219).
Notes
The authors declare no competing financial interest.
■ABBREVIATIONS USED
AEDA, aroma extraction and dilution analysis; EFs, elongation
factors; FD, flavor dilution; GBVs, glycosidically bound
volatiles; GC−MS/O, gas chromatography−mass spectrome-
ter/olfactometry; IW, indoor-withered; LOX, 9/13-lipoxyge-
nase; P, freshly plucking tea leaves; PVPP, polyvinylpolypyrro-
lidone; RI, retention indices; SW, solar withering; T1−T5,
turned over by 5 times; TMEMD, N,N,N′,N′-tetramethyle-
thylenediamine; TPS, terpene synthases
■REFERENCES
(1) Yang, Z. Y.; Baldermann, S.; Watanabe, N. Recent studies of the
volatile compounds in tea. Food Res. Int. 2013,53, 585−599.
(2) Nagegowda, D. A. Plant volatile terpenoid metabolism:
biosynthetic genes, transcriptional regulation and subcellular compart-
mentation. FEBS Lett. 2010,584, 2965−2973.
(3) Wang, X.; Wang, D.; Li, J.; Ye, C.; Kubota, K. Aroma
characteristics of cocoa tea (Camellia ptilophylla Chang). Biosci.,
Biotechnol., Biochem. 2010,74, 946−953.
(4) Schwab, W.; Davidovich-Rikanati, R.; Lewinsohn, E. Biosynthesis
of plant-derived flavor compounds. Plant J. 2008,54, 712−732.
(5) Auldridge, M. E.; McCarty, D. R.; Klee, H. J. Plant carotenoid
cleavage oxygenases and their apocarotenoid products. Curr. Opin.
Plant Biol. 2006,9, 315−321.
(6) Winterhalter, P.; Skouroumounis, G. K. Glycoconjugated aroma
compounds: Occurrence, role and biotechnological transformation. In
Biotechnology of Aroma Compounds; Berger, R. G., Ed.; Advances in
Biochemical Engineering/Biotechnology, Vol. 55; Springer: Berlin,
Heidelberg, 1997; pp 73−105.
(7) Nishikitani, M.; Kubota, K.; Kobayashi, A.; Sugawara, F. Geranyl
6-O-α-L-arabinopyranosyl-β-D-glucopyranoside isolated as an aroma
precursor from leaves of a green tea cultivar. Biosci., Biotechnol.,
Biochem. 1996,60, 929−931.
(8) Nishikitani, M.; Wang, D.; Kubota, K.; Kobayashi, A.; Sugawara,
F. Z)-3-Hexenyl and trans-linalool 3, 7-oxide β-primeverosides isolated
as aroma precursors from leaves of a green tea cultivar. Biosci.,
Biotechnol., Biochem. 1999,63, 1631−1633.
(9) Guo, W.; Hosoi, R.; Sakata, K.; Watanabe, N.; Yagi, A.; Ina, K.;
Luo, S. S)-Linalyl, 2-phenylethyl, and benzyl disaccharide glycosides
isolated as aroma precursors from oolong tea leaves. Biosci., Biotechnol.,
Biochem. 1994,58, 1532−1534.
(10) Guo, W.; Sakata, K.; Watanabe, N.; Nakajima, R.; Yagi, A.; Ina,
K.;Luo,S.Geranyl6-O-β-D-xylopyranosyl-β-D-glucopyranoside
isolated as an aroma precursor from tea leaves for oolong tea.
Phytochemistry 1993,33, 1373−1375.
(11) Wang, D. M.; Yoshimura, T.; Kubota, K.; Kobayashi, A. Analysis
of glycosidically bound aroma precursors in tea leaves. 1. Qualitative
and quantitative analyses of glycosides with aglycons as aroma
compounds. J. Agric. Food Chem. 2000,48, 5411−5418.
(12) Guo, W.; Sasaki, N.; Fukuda, M.; Yagi, A.; Watanabe, N.; Sakata,
K. Isolation of an aroma precursor of benzaldehyde from tea leaves
(Camellia sinensis var. sinensis cv. Yabukita). Biosci., Biotechnol.,
Biochem. 1998,62, 2052−2054.
(13) Yang, Z. Y.; Kinoshita, T.; Tanida, A.; Sayama, H.; Morita, A.;
Watanabe, N. Analysis of coumarin and its glycosidically bound
precursor in Japanese green tea having sweet-herbaceous odour. Food
Chem. 2009,114, 289−294.
(14) Kinoshita, T.; Hirata, S.; Yang, Z. Y.; Baldermann, S.; Kitayama,
E.; Matsumoto, S.; Suzuki, M.; Fleischmann, P.; Winterhalter, P.;
Watanabe, N. Formation of damascenone derived from glycosidically
bound precursors in green tea infusions. Food Chem. 2010,123, 601−
606.
(15) Zhou, Y.; Dong, F.; Kunimasa, A.; Zhang, Y.; Cheng, S.; Lu, J.;
Zhang, L.; Murata, A.; Mayer, F.; Fleischmann, P.; Watanabe, N.;
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6913

Yang, Z. Y. Occurrence of glycosidically conjugated 1-phenylethanol
and its hydrolase β-primeverosidase in tea (Camellia sinensis) flowers. J.
Agric. Food Chem. 2014,62, 8042−8050.
(16) Cho, J. Y.; Mizutani, M.; Shimizu, B.; Kinoshita, T.; Ogura, M.;
Tokoro, K.; Lin, M. L.; Sakata, K. Chemical profiling and gene
expression profiling during the manufacturing process of Taiwan
oolong tea “Oriental Beauty. Biosci., Biotechnol., Biochem. 2007,71,
1476−1486.
(17) Yang, Z. Y.; Kobayashi, E.; Katsuno, T.; Asanuma, T.; Fujimori,
T.; Ishikawa, T.; Tomomura, M.; Mochizuki, K.; Watase, T.;
Nakamura, Y.; Watanabe, N. Characterization of volatile and non-
volatile metabolites in etiolated leaves of tea (Camellia sinensis) plants
in the dark. Food Chem. 2012,135, 2268−2276.
(18) Mizutani, M.; Nakanishi, H.; Ema, J. I.; Ma, S. J.; Noguchi, E.;
Inohara-Ochiai, M.; Fukuchi-Mizutani, M.; Nakao, M.; Sakata, K.
Cloning of β-primeverosidase from tea leaves, a key enzyme in tea
aroma formation. Plant Physiol. 2002,130, 2164−2176.
(19) Sarry, J. E.; Günata, Z. Plant and microbial glycoside hydrolases:
Volatile release from glycosidic aroma precursors. Food Chem. 2004,
87, 509−521.
(20) Wang, D. M.; Kurasawa, E.; Yamaguchi, Y.; Kubota, K.;
Kobayashi, A. Analysis of glycosidically bound aroma precursors in tea
leaves. 2. Changes in glycoside contents and glycosidase activities in
tea leaves during the black tea manufacturing process. J. Agric. Food
Chem. 2001,49, 1900−1903.
(21) Wang, D. M.; Kubota, K.; Kobayashi, A.; Juan, I. M. Analysis of
glycosidically bound aroma precursors in tea leaves. 3. Changes in
glycoside contents during the oolong tea manufacturing process. J.
Agric. Food Chem. 2001,49, 5391−5396.
(22) Dong, F.; Yang, Z. Y.; Baldermann, S.; Kajitani, Y.; Ota, S.;
Kasuga, H.; Imazeki, Y.; Ohnishi, T.; Watanabe, N. Characterization of
L-phenylalanine metabolism to acetophenone and 1-phenylethanol in
the flowers of Camellia sinensis using stable isotope labeling. J. Plant
Physiol. 2012,169, 217−225.
(23) Yano, M.; Okada, K.; Kubota, K.; Kobayashi, A. Studies on the
precursors of monoterpene alcohols in tea leaves. Agric. Biol. Chem.
1990,54, 1023−1028.
(24) Hao, X.; Horvath, D. P.; Chao, W. S.; Yang, Y.; Wang, X.; Xiao,
B. Identification and evaluation of reliable reference genes for
quantitative real-time PCR analysis in tea plant (Camellia sinensis
(L.) O. Kuntze). Int. J. Mol. Sci. 2014,15, 22155−22172.
(25) Liu, S.; Han, B. Differential expression pattern of an acidic 9/13-
lipoxygenase in flower opening and senescence and in leaf response to
phloem feeders in the tea plant. BMC Plant Biol. 2010,10, 228.
(26) Liu, J.; Wang, F.; Liu, G.; He, Z.; Yang, H.; Wei, C.; Wan, X.;
Wei, S. Correlation between spatiotemporal profiles of volatile
terpenoids and relevant terpenoid synthase gene expression in
Camellia sinensis (in Chinese). Acta Hortic. Sin. 2014,41, 2094−2106.
(27) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene
expression data using real time quantitative PCR and 2−ΔΔct method.
Methods 2001,25, 402−408.
(28) Liu, Z. Z.; Wang, J. L.; Huang, X.; Xu, W. H.; Liu, Z. M.; Fang,
R. X. The promoter of a rice glycine-rich protein gene, Osgrp-2,
confers vascular-specific expression in transgenic plants. Planta 2003,
216, 824−833.
(29) Zhou, Y.; Zhang, L.; Gui, J. D.; Dong, F.; Cheng, S.; Mei, X.;
Zhang, L. Y.; Li, Y. Q.; Su, X. G.; Baldermann, S.; Watanabe, N.; Yang,
Z. Y. Molecular cloning and characterization of a short chain
dehydrogenase showing activity with volatile compounds isolated
from Camellia sinensis.Plant Mol. Biol. Rep. 2015,33, 253−263.
(30) Schmid, W.; Grosch, W. Identifizierung flüchtiger Aromastoffe
mit hohen Aromawerten in Sauerkirschen (Prunus cerasus L.). Z.
Lebensm.-Unters. Forsch. 1986,182, 407−412.
(31) Ullrich, F.; Grosch, W. Identification of the most intense volatile
flavour compounds formed during autoxidation of linoleic acid. Z.
Lebensm.-Unters. Forsch. 1987,184, 277−282.
(32) Schieberle, P.; Grosch, W. Evaluation of the flavor of wheat and
rye bread crusts by aroma extract dilution analysis. Z. Lebensm.-Unters.
Forsch. 1987,185, 111−113.
(33) Katsuno, T.; Kasuga, H.; Kusano, Y.; Yaguchi, Y.; Tomomura,
M.; Cui, J. L.; Yang, Z. Y.; Baldermann, S.; Nakamura, Y.; Ohnishi, T.;
Mase, N.; Watanabe, N. Characterisation of odorant compounds and
their biochemical formation in green tea with a low temperature
storage process. Food Chem. 2014,148, 388−395.
(34) Fraser, K.; Lane, G. A.; Otter, D. E.; Harrison, S. J.; Quek, S. Y.;
Hemar, Y.; Rasmussen, S. Monitoring tea fermentation/manufacturing
by direct analysis in real time (DART) mass spectrometry. Food Chem.
2013,141, 2060−2065.
(35) Baldermann, S.; Yang, Z. Y.; Katsuno, T.; Tu, V. A.; Mase, N.;
Nakamura, Y.; Watanabe, N. Discrimiantion of green, oolong, and
black teas by GC-MS analysis of characeristic volatile flavor
compounds. Am. J. Anal. Chem. 2014,5, 620−632.
(36) Yamanishi, T. Tea (in Japanese). Koryo 1989,161,57−72.
(37) Huang, F. P.; Chen, R. B.; Liang, Y. R.; Chen, W.; Lu, J. L.;
Chen, C. S.; You, X. M. Changes of aroma constituents during zouqing
procedure and its relation to oolong tea quality (in Chinese). J. Tea Sci.
2003,23,31−37.
(38) Yang, Z. Y.; Mochizuki, K.; Watase, T.; Kobayashi, E.; Katsuno,
T.; Asanuma, T.; Tomita, K.; Morita, A.; Suzuki, T.; Nakamura, Y.;
Watanabe, N. Influences of light emitting diodes irradiations and shade
treatments on volatile profiles and related metabolites of leaves of tea
(Camellia sinensis) plants and postharvest tea leaves. In Advances and
Challenges in Flavor Chemistry &Biology; Hofmann, T., Meyerhof, W.,
Schieberle, P., Eds.; Deutsche Forschungsanstalt für Lebensmittelche-
mie: Germany, 2010; pp 207−213.
(39) Gillam, E. M. J.; Notley, L. M.; Cai, H.; De Voss, J. J.;
Guengerich, F. P. Oxidation of indole by cytochrome P450 enzymes.
Biochemistry 2000,39, 13817−13824.
(40) Ohgami, S.; Ono, E.; Horikawa, M.; Murata, J.; Totsuka, K.;
Toyonaga, H.; Ohba, Y.; Dohra, H.; Asai, T.; Matsui, K.; Mizutani, M.;
Watanabe, N.; Ohnishi, T. Volatile glycosylation in tea plants:
Sequential glycosylations for the biosynthesis of aroma β-primevero-
sides are catalyzed by two Camellia sinensis glycosyltransferases. Plant
Physiol. 2015,168, 464.
(41) Kumazawa, K.; Masuda, H. Identification of potent odorants in
Japanese green tea (Sen-cha). J. Agric. Food Chem. 1999,47, 5169−
5172.
(42) Kumazawa, K.; Masuda, H. Change in the flavor of black tea
drink during heat processing. J. Agric. Food Chem. 2001,49, 3304−
3309.
(43) Kumazawa, K.; Masuda, H. Identification of potent odorants in
different green tea varieties using flavor dilution technique. J. Agric.
Food Chem. 2002,50, 5660−5663.
(44) Baba, R.; Kumazawa, K. Characterization of the potent odorants
contributing to the characteristic aroma of Chinese green tea infusions
by aroma extract dilution analysis. J. Agric. Food Chem. 2014,62,
8308−8313.
(45) Schuh, C.; Schieberle, P. Characterization of the key aroma
compounds in beverage prepared from Darjeeling black tea:
Quantitative differences between tea leaves and infusion. J. Agric.
Food Chem. 2006,54, 916−924.
Journal of Agricultural and Food Chemistry Article
DOI: 10.1021/acs.jafc.5b02741
J. Agric. Food Chem. 2015, 63, 6905−6914
6914