Article pubs.acs.org/JAFC
Characterization of the Potent Odorants Contributing to the Characteristic Aroma of Chinese Green Tea Infusions by Aroma Extract Dilution Analysis Ryoko Baba* and Kenji Kumazawa Ogawa & Company, Ltd., 15-7 Chidori Urayasushi, Chiba 279-0032, Japan S Supporting Information *
ABSTRACT: The volatile fractions of three famous Chinese green tea cultivar infusions (Longjing, Maofeng, and Biluochun) were prepared by a combination of the adsorptive column method and the SAFE techniques. The aroma extract dilution analysis (AEDA) applied to the volatile fractions revealed 58 odor-active peaks with flavor dilution (FD) factors between 41 and 47. Fortysix of the odorants, which included six odorants that have not been reported in the literature in Chinese green tea (2-isopropyl-3methoxypyrazine, 2-ethenyl-3,5-dimethylpyrazine, cis-4,5-epoxy-(E)-2-decenal, 4-ethylguaiacol, (E)-isoeugenol, and 3-phenylpropionic acid), were identified or tentatively identified by GC-MS and GC-O. Among the perceived odorants, 4-hydroxy-2,5dimethyl-3(2H)-furanone, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, coumarin, vanillin, geraniol, (E)-isoeugenol, and 2methoxyphenol showed high FD factors in all of the cultivars, irrespective of the cultivar or harvesting season, suggesting that these seven odorants are essential for the aroma of Chinese green tea. On the other hand, the contents of the odorants, FD factors of which were uneven between the cultivars, were suggested to influence the characteristic aroma of each cultivar. In addition, the formation mechanism of (E)-isoeugenol, one of the odorants which have not been reported in the literature with a high FD factor common to all the cultivars, was investigated, and it was suggested that the (E)-isoeugenol content of the tea products has a close correlation with the manufacturing process of the tea leaves. KEYWORDS: Chinese green tea, flavor, aroma, AEDA, isoeugenol, coniferyl alcohol
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INTRODUCTION
(withering, heating, etc.), and each of them has its own characteristic aroma. A number of volatile compounds in Chinese green tea have been identified by gas chromatography (GC) and GC/mass spectrometry (MS).1−6 A number of potent odorants in green tea have also been identified (Japanese green tea,4,7 Darjeeling green tea, 8 and Chinese green tea 4−6). However, the investigations of Chinese green tea were only intended for some of the cultivars, despite there being many.4−6 Therefore, they cannot fully explain the characteristic aroma of Chinese green tea in general. In this investigation, in order to elucidate the potent odorants common to Chinese green tea in general, the three famous Chinese green tea cultivar infusions (Longjing, Maofeng, and Biluochun) were investigated by aroma extract dilution analysis (AEDA). Simultaneously, the characteristic odorants of each cultivar were also investigated. In addition, the formation mechanism of (E)-isoeugenol, which has not been reported in the literature in Chinese green tea, was studied.
Tea is one of the most widely consumed beverages in the world. Being differentiated by the degrees of fermentation by the endogenous enzymes in the leaves, three main types of teas are produced; i.e., fermented tea (black tea), semifermented tea (oolong tea), and unfermented tea (green tea). Each of them has its own characteristic aroma, despite being made from the same plant (Camellia sinensis). Among them, green tea, the leaves of which are immediately heated after being plucked to inactivate the endogenous enzymes, is the most popular tea in China, Japan, and other East Asian countries. The manufacturing process of green tea is mainly divided into two types according to the heating process to inactivate the endogenous enzymes in the leaves, i.e., pan-firing and steaming, which are common in China and Japan, respectively. The difference in the heating process is said to be one of the factors which influences the aromas of the tea products; the steamed tea (Sen-cha), which is not affected by the enzymes because they are immediately inactivated, has a green, marine aroma. On the other hand, pan-fired tea, affected by the enzymatic reactions because the temperature of the leaves slowly rises and the enzymes are not immediately inactivated, has a sweet, floral aroma like fermented teas, besides the characteristic pan-fired aroma. Furthermore, in China, the leaves are generally withered before being pan-fired, during which the leaves are affected by the enzymes. In addition, there are numerous Chinese green tea cultivars classified by the subtle differences in the producing area, varieties of tea, and/or the manufacturing process © XXXX American Chemical Society
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MATERIALS AND METHODS
Materials. Tea samples: Chinese green tea products, Longjing, Maofeng, and Biluochun (spring and summer crops), were produced in the Zhejiang, Anhui, and Jiangsu provinces (China) in 2012. Sencha was produced in the Shizuoka prefecture (Japan) in 2008 and Received: May 15, 2014 Revised: July 28, 2014 Accepted: August 3, 2014
A
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2012. The oolong tea products, Pouchong tea, Dongding oolong tea, Oriental Beauty tea, Tie Guan Yin tea, Golden Cassia, and Robe tea, were produced in Taiwan or China in 2001. The black tea products, Dimbura, Java tea, Kenyan tea, and Assam tea, produced in 2005, were purchased from Mitsui Norin Co., Ltd. (Tokyo, Japan). All of these products were of high grade based on the market price and stored at −80 °C until needed. Chemicals. The following compounds were synthesized according to the literature procedures: 2-acetyl-1-pyrroline;9 (Z)-1,5-octadien-3one;10 4-mercapto-4-methyl-2-pentanone;11 2-ethenyl-3,5-dimethylpyrazine;12 3-methyl-2,4-nonanedione;13 2-acetyl-2-thiazoline;14 and cis- and trans-4,5-epoxy-(E)-2-decenal.15 Compounds (Table 2) 3, 6, 12, 14, 16−19, 21, 23−26, 29, 32, 33, 35, 38, 40, 41, 43, 47, 48, 52− 55, 57, and 58 were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); compounds 4, 15, 30, 45, 46, and 56 were purchased from Sigma-Aldrich Japan (Tokyo, Japan); 39, 44, and coniferyl alcohol were purchased from Wako Pure Chemical Industries (Osaka, Japan); 50 was purchased from the Zeon Corporation (Tokyo, Japan); cellulose and cinnamyl alcohol were purchased from Nacalai Tesque (Kyoto, Japan); and pectriase was purchased from the Shinnihon Chemicals Corporation (Chiba, Japan). Isolation of the Volatiles from Chinese Green Tea Infusion. Distilled water (90 °C, 1500 g) was added to 75 g of Chinese green tea, and the leaves were filtered after standing for 5 min. The filtrate (1000 g) was immediately cooled in tap water and passed through a column packed with 20 mL of SP700 (Mitsubishi Chemical Corporation). 16 The adsorbed compounds were eluted with methylene chloride (100 mL). As the internal standard solution, 0.1% 2-octanol in methylene chloride (100 μL) was added to the eluate. The eluate was dried over anhydrous sodium sulfate and distilled under reduced pressure (4 and RSD 1) in Chinese Green Tea log4 (FD factor) Longjing a
no.
R.I.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
946 1009 1076 1132 1189 1294 1334 1366 1374 1380 1407 1422 1447 1449 1459 1492 1521 1530 1538 1551 1581 1674 1625 1633 1660 1698 1700 1756 1807 1817 1842 1842 1851 1940 1961 1981 1996 2020 2023 2025 2075 2146 2157 2174 2190 2196 2213 2236 2253 2259 2299 2338 2389 2432 2446 2540 2551 2610
compound
b
unknown unknown hexanal (Z)-3-hexenalc unknown 1-octen-3-one 2-acetyl-1-pyrroline (Z)- 1,5-octadien-3-onec 4-mercapto-4-methyl-2-pentanonec unknown unknown 2-isopropyl-3-methoxypyrazinec,d unknown methional 2-ethyl-3,5-dimethylpyrazine 2,3-diethyl-5-methylpyrazine 2-isobutyl-3-methoxypyrazinec (E)-2-nonenal linalool 2-ethenyl-3,5-dimethylpyrazinec,d (E,Z)-2,6-nonadienal unknown 2-acetylpyrazine phenylacetaldehyde isovaleric acid (E,E)-2,4-nonadienal 3-methyl-2,4-nonanedione 2-acetyl-2-thiazolinec (E,E)-2,4-decadienal β-damascenonec unknown geraniol 2-methoxyphenol unknown maltol cis -4,5-epoxy-(E)-2-decenald trans -4,5-epoxy-(E)-2-decenal 4-ethylguaiacold 4-nonanolide 4-hydroxy-2,5-dineuhyl-3(2H)-furanone p -cresol unknown eugenol 2-methoxy-4-vinylphenol 3-hydroxy-4,5-dimethyl-2(5H)-furanonec 5-decanolidec o -aminoacetophenone methyl anthranilate unknown jasmin lactone unknown (E)-isoeugenold (Z)-methyl jasmonate indole coumarin phenylacetic acid vanillin 3-phenylpropionic acidc,d
odor quality
e
stimulus fruity, green green, grassy green nutty mushroom-like roasty metallic green, meaty green nutty, green pea-like nutty potato-like nutty nutty earthy, musty green, sweet floral nutty green, cucumber-like roasty, sour, smoky nutty sweet, honey-like sweaty, rancid fatty green roasty fatty sweet, honey-like sweaty, green floral, green burnt, rubber-like green, sweaty sweet sweet, juicy sweet, juicy, metallic spicy sweet sweet, caramel-like phenolic sweet, green, floral spicy spicy seasoning-like, caramel-like lactone-like grape-like grape-like phenolic sweet, burnt green floral, spicy floral animal-like sweet sweet vanilla-like animal-like D
Maofeng
Biluochun
spring
summer
spring
summer
spring
summer
2
1
2
2
1
1
1
1 2
1 1 1 2
1 1
1
2 1 1
2 1
1 3 3
1 3
2
3 3 3 2 2 2 3
2 2 1 2 2 1
2
1 1 1 1
1
1 1 3
3 2 2 1 1 4 1 2
2 2 2 2 2 1 1 2
1 2 1 1
2 3 3 3 3 3 1 3 2
2 1 3 2 1 2 2 1
1
2 2
2
1
1
1 4 1 2
2 4 2 1 1
2 4 4 2 1 2
2 3 1 1 1
4 6
5 5
4 7
4 5
5 5
1
2
2 1 3
2
2
2
1 1 1 2 3 1 2 1 1 5 4 1 2
2 4
7
1 6
3 7
4
3
5
5 2
5 2 5
1 1 2 5 2 1 6 4 5 3
3 1 1 1 2 5 2 5 4 4 2
2 4 2 3 5 3 5 3
5
2 7
2 4
3
5 2 2 4
4 1 2 1
3
2 1 5 2 3 5 3 5 2
2
1 1 3
4 3 2 4 4 5 3
5 3 4 4 3 5 2
2 1
4 2
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Table 2. continued a Retention index on DB-Wax column (30 m × 0.25 mm i. d.; coated with a 0.25 μm film) observed for GC-O. bThe compound was identified by comparison with the reference substance on the basis of the following criteria: retention index (R.I.) on stationary phase DB-Wax, mass spectra, and odor quality. cThe MS signals were too weak for unequivocal interpretation. The compound was tentatively identified by comparison with the reference substance on the basis of the following criteria: retention index (R.I.) on stationary phase DB-Wax and odor quality. dThe compound has not been reported in the literature in Chinese green tea. eOdor quality assigned during AEDA.
Figure 1. Semiquantified (E)-isoeugenol content of various tea (three Japanese green teas, six Chinese green teas, six oolong teas, and four black teas) infusions. Error bars show standard deviations. (JP, Japanese green tea (Sen-cha); CH, Chinese green tea; OT, oolong tea; BT, black tea.)
contents were correlated with those of 52, suggesting that coniferyl alcohol might be an important intermediate in the formation of 52, also in the tea leaves. The formation of 52 from coniferyl alcohol was assumed to be caused by heating during the manufacturing process, not by an enzymatic reaction, since coniferyl acetate was not detected in the tea infusions, and 52 was reported to be generated from coniferyl alcohol by heating. To confirm this assumption, coniferyl alcohol mixed with cellulose was heated under the same conditions as the pan-firing and drying process of the tea leaves (150 °C, 30 min), and then 52 and eugenol (43) were generated. It was assumed that 52 is formed from coniferyl alcohol during the reducing dehydration reaction. The mechanism presumed for this reaction includes the corresponding quinone methide intermediate since coniferyl alcohol has a hydroxyl group at the C-4 position on the benzene ring. To confirm this, the requirement of the hydroxyl group at the C-4 position on the benzene ring for this reaction was indirectly tested, i.e., cinnamyl alcohol, which has no hydroxyl group on the benzene ring like coniferyl alcohol, was heated (150 °C, 30 min). As a result, the corresponding product, 3-phenylpropene, was not generated, indicating that the hydroxyl group on the benzene ring is required for this dehydration reaction. Therefore, it was suggested that this reaction includes a quinone methide intermediate which is relatively stable, and thus this reaction would proceed under such moderate heating conditions (Figure 2). On the other hand, coniferyl alcohol is known as a precursor of lignin, an essential substance of plants, and known to be accumulated as a glycoside called coniferin in plants because of its instability. In addition, coniferin is found in Camellia taliensis, a close relative of Camellia sinensis.23 Considering that most of the alcohols in tea aroma are known to be generated from glycosides by enzymatic reactions during the manufacturing process,19 a glycoside like coniferin is assumed to be involved in the formation of coniferyl alcohol. To confirm this assumption, a hydrolysis enzyme, pectriase, was added to the nonvolatile fraction of the Sen-cha infusion, in which glycosides were assumed to be included. As a result, coniferyl alcohol was generated.
Figure 2. Proposed pathway for the formation of (E)-isoeugenol in tea leaves during the manufacturing process of Chinese green tea.
On the basis of these results, the formation mechanism of 52 in tea leaves during the manufacturing process was indirectly suggested as follows: first, coniferyl alcohol is generated from its glycoside like coniferin by an enzymatic reaction, and then 52 is formed from coniferyl alcohol by heating. In other words, it was suggested that coniferyl alcohol is generated from the precursor like coniferin by the endogenous enzymatic reaction during the withering and pan-firing processes and that then 52 is formed from coniferyl alcohol via the quinone methide intermediate by heating during the pan-firing and drying processes in tea leaves during the manufacturing process (Figure 2). These results indicated that the formation of 52, which is assumed to be the important compound for the characteristic floral aroma of Chinese green tea, is closely correlated to the specific manufacturing process of Chinese green tea: withering the plucked leaves and slowly inactivating the endogenous enzymes in the leaves by pan-firing. However, the coniferyl alcohol content showed a tendency to be higher in the semifermented tea (oolong tea) than in the fermented tea (black tea), suggesting that the coniferyl alcohol content is not necessarily in proportion to the degree of fermentation. It seems possible that coniferyl alcohol once generated in the tea leaves by fermentation changes into other compounds as the E
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(9) Buttery, R. G.; Ling, L. C.; Juliano, B. O.; Turnbaugh, J. G. Cooked rice aroma and 2-acetylpyrroline. J. Agric. Food. Chem. 1983, 31, 823−826. (10) Swoboda, P. A. T.; Peers, K. E. Metallic odour caused by vinyl ketones formed in the oxidation of butterfat. The identification of octa-1,cis-5-dien-3-one. J. Sci. Food Agric. 1977, 28, 1019−1024. (11) Guth, H. Identification of character impact odorants of different white wine varieties. J. Agric. Food. Chem. 1997, 45, 3022−3026. (12) Czerny, M.; Wagner, R.; Grosch, W. Detection of odor-active ethenylalkylpyrazines in roasted coffee. J. Agric. Food. Chem. 1996, 44, 3268−3272. (13) Guth, H.; Grosch, W. 3-Methylnonane-2,4-dione − An intense odour compound formed during flavour reversion of soya-bean oil. Fat Sci. Technol. 1989, 93, 225−230. (14) Cerny, C.; Grosch, W. Evaluation of potent odorants in roasted beef by aroma extract dilution analysis. Z. Lebensm.-Unters. Forsch. 1992, 194, 322−325. (15) Kumazawa, K.; Wada, Y.; Masuda, H. Characterization of epoxydecenal isomers as potent odorants in black tea (Dimbula) infusion. J. Agric. Food. Chem. 2006, 54, 4795−4801. (16) Kaneko, S.; Kumazawa, K.; Nishimura, O. Studies on the key aroma compounds in raw (unheated) and heated Japanese soy sauce. J. Agric. Food. Chem. 2013, 61, 3396−3402. (17) Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation − a new and versatile technique for the careful and direct isolation of aroma compounds from complex food matrices. Eur. Food Res. Technol. 1999, 209, 237−241. (18) Schieberle, P. New Developments in Methods for Analysis of Volatile Flavor Compounds and Their Precursors. In Characterization of Food: Emerging Methods; Goankar, A., Ed.; Elesevier: Amsterdam, The Netherlands, 1995; pp 403−431. (19) Yang, Z.; Baldermann, S.; Watanabe, N. Recent studies of the volatile compounds in tea. Food Res. Int. 2013, 53, 585−599. (20) Kobayashi, A.; Tachiyama, K.; Kawakami, M.; Yamanishi, T.; Juan, I.; Chiu, W. T. Effects of solar-withering and turn over treatment during indoor-withering on the formation of pouchong tea aroma. Agric. Biol. Chem. 1985, 49, 1655−1660. (21) Koeduka, T.; Fridman, E.; Gang, D. R.; Vassao, D. G.; Jackson, B. L.; Kish, C. M.; Orlova, I.; Spassova, S. M.; Lewis, N. G.; Noel, J. P.; Baiga, T. J.; Dudareva, N.; Pichersky, E. Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10128−10133. (22) Sakai, K. Organosolv delignification. Kami Parupu Gijutsu Kyokai shi 1994, 48, 1003−1012. (23) Gao, D.; Zhang, Y.; Yang, C.; Chen, K.; Jiang, H. Phenolic antioxidants from green tea produced from Camellia taliensis. J. Agric. Food. Chem. 2008, 56, 7517−7521.
fermentation proceeds because coniferyl alcohol is an unstable compound. Therefore, further studies and elucidation of the precursor of coniferyl alcohol in tea leaves are necessary to clarify the relationship between the 52 contents of the tea leaves and the tea manufacturing process. In this study, the essential volatile compounds and characteristics of the aroma of three famous cultivars of Chinese green tea were identified. It is assumed that the contents of these compounds are affected by the precursor contents of the tea leaves or their manufacturing process. However, the details about the formation mechanism of these compounds have not been clarified. Now that green tea has become increasingly popular around the world, it would be important to elucidate the potent odorants, their formation mechanism, and the effect of the manufacturing process on their contents to produce better-smelling and highly palatable green tea.
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ASSOCIATED CONTENT
S Supporting Information *
Correlation between the content of coniferyl alcohol and that of (E)-isoeugenol in various tea infusions; extracted-ion chromatogram of (E)-isoeugenol formed from coniferyl alcohol by heating; heating reaction of coniferyl alcohol and cinnamyl alcohol; and total ion chromatogram of coniferyl alcohol released from the crude glycosidic fraction of the Japanese green tea (Sen-cha) infusion. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +81-47-305-1423. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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REFERENCES
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