Does Enzymatic Hydrolysis of Glycosidically Bound Volatile

Jul 26, 2015 - College of Horticultural Science, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China. ¶ Guangd...
5 downloads 14 Views 8MB Size
Article pubs.acs.org/JAFC

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 S Supporting 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 enzymeactive 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 ≥44 were 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



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

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 derivatives, 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 acidderived 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 © XXXX American Chemical Society

Received: March 17, 2015 Revised: July 24, 2015 Accepted: July 26, 2015

A

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

geraniol, polyvinylpolypyrrolidone (PVPP), XAD-2, and β-glucosidase were purchased from Sigma-Aldrich Company Ltd., USA. β-Primeverosidase 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 indoorwithered (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 dichloromethane 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 Instruments 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/z 40−200). 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 dichloromethane 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

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 leafhoppers.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 manufacturing process.18 The GBVs are present within vacuoles, while β-primeverosidase, one major glycosidase involved in the hydrolysis 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 hydrolysis 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 production.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 manufacturing 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 formation 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)-3hexen-1-ol, linalool, linalool oxides, methyl jasmonate, trans-nerolidol, B

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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. reports.20,23 One gram of finely powdered tea leaves was ground in icecold 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 icecold 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-Dglucopyranoside 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 C

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 3. Effect of disruption of tea leaf cell on the transformation from [15N]anthranilic acid to [15N]indole. (A) GC−MS chromatography (m/z 118) 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 indoorwithered 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/z 118 of [15N] indole. 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.

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 × 2 mm 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 ultramicrotome (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 microscope (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′-GTCGACATGATGGCAGCGAAAGGG-3′ (forward), 5′-CCCGGGGCTTGAGGAGGAATTTCTT-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/XmaI sites of the pCAMBIA3300-GFP vector. The reorganization vector was transformed into Agrobacterium GV3101 by freeze−thaw. The overnight Agrobacterium cultures were sedimented at 5000g for 1 min. The pellet was resuspended in a solution containing 10 mM MgCl2, 10 mM 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 2−3 cm2 pieces

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, 9/13Lipoxygenase (LOX), and Terpene Synthases (TPS). Total RNA was isolated immediately after dissection. The reactions were performed 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,15LOX,25 and TPS26 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 D

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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. 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-(Nmorpholino)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 E

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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. 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/z 20−280. 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.

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 indoorwithered at room temperature for 4.5 and 6.0 h, respectively. Afterward, 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 completely 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/z 118. 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 μm film thickness, Agilent Technologies Inc., USA) was used for mass spectrometric 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



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 glycosidically bound form, have big changes during the manufacturing process, we monitored changes of free volatiles from the plucking process to fixing process (enzymatic reaction termination) 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 acidderived 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 indicated that these volatiles mostly accumulated at the turn over F

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. Identification of Potent Odorants (FD Factor ≥44) in Oolong Tea

a

RI

identified compound

odorant quality

FD factor (4n)

1538 1716 1821 1840 1883 1945 2017 2200 2266 2431 2546

linalool 3-methylnonane-2,4-dione β-damascenone unidentifieda unidentifieda β-ionone Furaneola δ-decalactone jasmine lactone indole vanillin

fresh floral fresh floral honey minty fresh floral floral sweet floral, sweet floral, sweet animal-like, floral sweet

5 5 5 5 4 4 7 6 6 4 5

The MS spectrum is provided as Figure S2.

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 disruption 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 addition, it would be very interesting to investigate whether disruption 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.

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 representative 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 G

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 8. Hypothetical model of different formations of volatiles between the oolong tea process and the black tea process.

substrates and enzymes and no hydrolysis of GBVs. Interestingly, the previous report21 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 functionally characterized;40 it will be helpful for our understanding of the accumulation of GBVs during the oolong tea manufacturing process. β-Glycosidases Were Not Activated during The Manufacturing Process, and Interaction of β-Glycosidases 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 manufacturing 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 interaction 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-Methylnonane2,4-dione, and Indole Were Identified as Relatively Potent Odorants in Oolong Tea. The above evidence shows that enzymatic hydrolysis of GBVs did not significantly contribute 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 41 and 47 in 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 H

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry 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-methylnonane-2,4-dione, β-damascenone, indole, and two other compounds 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 process 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 compounds. The results help us find out the direct evidence of formation of volatiles in oolong tea, and advance our understanding of differences of formation of volatiles between oolong tea and black tea.



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 spectrometer/olfactometry; IW, indoor-withered; LOX, 9/13-lipoxygenase; P, freshly plucking tea leaves; PVPP, polyvinylpolypyrrolidone; RI, retention indices; SW, solar withering; T1−T5, turned over by 5 times; TMEMD, N, N,N′,N′-tetramethylethylenediamine; TPS, terpene synthases



(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 compartmentation. 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. Geranyl 6-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.;

ASSOCIATED CONTENT

S Supporting 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)



REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-20-38072989. E-mail: [email protected]. 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). I

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry 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 nonvolatile 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 9/13lipoxygenase 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 Lebensmittelchemie: 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-primeverosides 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.

J

DOI: 10.1021/acs.jafc.5b02741 J. Agric. Food Chem. XXXX, XXX, XXX−XXX