Food Flavor - American Chemical Society

Chapter 8

Identification of Aroma Components during Processing of the Famous Formosa Oolong Tea Oriental Beauty" Downloaded by UNIV OF OKLAHOMA on April 29, 2013 | http://pubs.acs.org Publication Date: September 30, 2008 | doi: 10.1021/bk-2008-0988.ch008

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Miharu Ogura , Tomomi Kinoshita , Bun-ichi Shimizu , Fumiharu Shirai , Kazuhiko Tokoro , Mu-Lien Lin , and Kanzo Sakata 1

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3

1

Central Research Laboratory, TAKASAGO International Corporation, 1-4-11 Nishi-yawata, Hiratsuka city, Kanagawa 254-0073, Japan Tea Research and Extension Station, 324 Chunghsin Road, Yangmei, Taoyuan, 326, Taiwan Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan 2

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Oriental Beauty (Pom-Fong tea) is a high-grade tea that has a pronounced honey aroma and a rich flavor of ripe fruit. It is produced via a higher degree of fermentation from tea leaves infested by tea green leafhoppers (Jacobiasca formosana) in Taiwan. We have carried out aroma analyses of tea samples obtained during processing to know the molecular basis of production of the characteristic aroma. Two series of tea samples were prepared by the same method from healthy leaves and those infested by the insect. As a result of aroma analysis, the amount of the aroma components produced from the infested tea leaves was higher than the non-infested tea leaves as the fermentation progressed. 2,6-Dimethylocta-3,7diene-2,6-diol (DOD) in the infested tea leaves was found to have been generated even in the leaves and at early stages of the manufacturing processes. Chiral analysis of DOD showed high enantiopurity and its absolute configuration was the same as linalool and 3,7-dimethylocta-l,5,7-trien-3-ol (hotrienol), suggesting that DOD and hotrienol are biosynthesized from linalool.

© 2008 American Chemical Society In Food Flavor; Tamura, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

87

Downloaded by UNIV OF OKLAHOMA on April 29, 2013 | http://pubs.acs.org Publication Date: September 30, 2008 | doi: 10.1021/bk-2008-0988.ch008

88 There are various aroma types of oolong tea. Oriental Beauty, in particular, has a very unique flavor compared with other teas, and is also called Formosa oolong tea, Pom-Fong tea, Champagne oolong tea, White Tip oolong tea, etc. This is one of the tea products representing Taiwan. Aroma analyses of oolong tea has been reported by several researchers (7, 2). The major components of Chan Pin oolong tea were reported by Kawakami et al (5). In our study, the key odorous compounds of Oriental Beauty and the change of aroma profile at each step of the manufacturing process were investigated by GC/MS and aroma extract dilution analysis using a sniffmg-GC (4, 5). As hotrienol and DOD are characteristic aroma components in this oolong tea, aroma formation should be focused on the tea manufacturing procedures or the leaves. In comparison with other oolong tea, a unique manufacturing process enables a preparation of this oolong tea. The characteristic points are in the tea leaves and the process via the higher degree of fermentation. The leaves infested by the tea green leafhopper, J. formosana, are used as raw materials for tea manufacturing. The leafhoppers suck the juvenile leaves using their needle-like mouthparts that cause the leaves to become curved with tiny yellow wounding spots. By the continuous infestation, especially under the dry conditions in the season, the leaves become stunted. The infested leaves with severe symptoms are used for manufacturing Oriental Beauty. This is a very unique and important point. Another unique point is the manufacturing process. The steps of solar withering are longer, more times of turning over are applied, and the additional steps, wetting and softening, are carried out before rolling. Thus, the degree of fermentation becomes higher. The infusion of Oriental Beauty has a unique and rich aroma like a ripenedfruitand honey after these manufacturing processes. We think that these unique features induce synthesis of many volatile compounds in the leaves in response to different stresses such as insect infestation, light stress, drought stress, and wounding. To understand the biochemical process of flavor formation during manufacturing, we have investigated the changes of tea aroma formation throughout the manufacturing process as well as the differences of volatile components between the tea samples prepared in the same manner from healthy tea leaves and infested ones.

Materials and Methods Tea Sample Preparation Two types of leaves (with/without insect infestation) were used for tea sample production: leaves of cv. Chinshin Dahpan cultivated under the best quality control and those of the same variety plucked from tea plants to be intentionally infested by leafhoppers were harvested in June 2004. The non-

In Food Flavor; Tamura, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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infested tea samples were prepared from leaves of the same cultivar plucked in September 2004 because the healthy leaf samples obtained in June 2004 were found to be slightly infested. Each tea sample was prepared via general manufacturing process of Oriental Beauty using these tea leaves after panning process. Run 7 shows the ordinary manufacturing process for the tea. To examine the change of aroma profile at each step of earlier processing where enzyme reactions possibly occur, two series of seven kinds of tea samples were prepared as shown in Table 1: e.g., AF and BF samples were prepared by subjectingfreshlyplucked leaves to panning and the following processes without solar and indoor witherings as well as turning over from infested and healthy leaves, respectively.

Preparation of Aroma Extract Each tea sample (5.0 g) was brewed with 75 g of deionized boiling water for 10 min. After filtration, the filtrate volume was adjusted to 50 mL, and 50 \xL of an ethanol solution containing 0.01% 3-octanone as an internal standard was added. The solution was saturated with sodium chloride and was extracted with 20 mL of dichloromethane. The extract was dried over anhydrous sodium sulfate for 12 h. The solvent was carefully removed with a Kuderna-Danish evaporative concentrator. The aroma extracts were analyzed by GC and GC/MS.

Instrumental Methods Gas Chromatography (GC). GC analysis was performed on a HP 6890 gas chromatograph (Agilent Technologies, Inc., Palo Alto, CA), equipped with an FID and a HP-20M fused silica capillary column (25 m x 0.2 mm i.d., film thickness 0.1 nm, Agilent Technologies). The oven temperature was programmed from 55 °C to 215 °C at 4 °C/min. Helium was used as the carrier gas and the linear velocity was 18 cm/s. The split ratio was 1:50. The injector and detector temperatures were 250 °C. Gas Chromatography/Mass Spectrometry (GC/MS). GC/MS analysis was performed on a GCMS-QP-2010 (Shimadzu), equipped with a BC-WAX fused silica capillary column (50 m x 0.2 mm i.d., film thickness 0.15 jum, GL Sciences, Inc., Tokyo, Japan). The oven temperature was programmed from 70 °C to 220 °C at 4 °C /min. Helium was used as the carrier gas and the linear velocity was 28 cm/s. The split ratio was 1:50. Mass spectra were obtained at 70 eV (EI) with an ion source temperature of 200 °C. The identification of the components was made by comparison of their GC retention times and mass spectra to those of authentic compounds. Enantio-MDGC/MS System. Enantio-MDGC/MS analysis was performed with a multidimensional gas chromatograph, MDGC-2010 (Shimadzu Corporation, Kyoto, Japan). The two capillary columns of the MDGC were



a

Softening

and

Rolling

:F, fresh leaves; SW, solar withering; T1-T5, 1-5 times of indoor-withering and turning-over.

Withering

Solar

Wetting

Table I. Preparation of Tea Samples


Infested

(not infested)

Healthy

Tea leaves"

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coupled with a switching device. The precolumn was identical with the column of the GC/MS system described above. The oven temperature was programmed from 70 °C to 220 °C at 9 °C /min. Helium was used as the carrier gas and the linear velocity was 45 cm/s. Split (1:20) and splitless injections were used. The injector temperature was 250 °C. The detector was an FID and the temperature was 250 °C. The main column used was a Beta-DEX™ 225 (25 m x 0.25 mm i.d., film thickness 0.25tim, Supelco, Bellefonte, PA). The oven temperature was programmed from 70 °C to 100 °C at 1 °C /min. Mass spectra were obtained at 70 eV (EI) with an ion source temperature of 200 °C. Constituents were identified by comparison of their mass spectra and retention times with those of authentic compounds.

Results and Discussion Volatile Components To identify aroma components induced by the stresses in tea leaves and during the manufacturing process, the infusion of each tea leaf sample was extracted and analyzed by GC and GC/MS. Gas chromatograms of these aroma extracts are shown in Figure 1. Most of the aroma components gradually increased as the fermentation progressed. The rate of increase of the volatile components in the tea produced from infested leaves was much higher than that of the tea sample from healthy leaves. Twenty-four main aroma compounds were selected from aroma components shown in Fig. 1 and the relative ratio of each GC area to internal standard was examined. To compare these components between tea samples with and without insect infestation at different steps of the tea manufacturing process, four samples (AF, BF, AT5, BT5) were summarized in Table II. Alcoholic aroma compounds, especially benzyl alcohol, 2-phenylethanol and hexanol, were increased as the fermentation progressed as well as the linalool oxides and hexenoic acids. The increasing ratio of these compounds in the infested tea leaves was almost the same as those in the non-infested tea leaves. When the aroma components between the infested (AF) and healthy leaves (BF) were compared to elucidate the effect of insect infestation on volatiles, the prominent difference was observed in compound number nineteen, namely, 2,6dimethylocta-3,7-diene-2,6-diol (D6D). The amount of DOD in AF is about 15 times higher than that in BF. The level of hotrienol in AF is also about 70 times higher than that in BF. These compounds were confirmed to be diagnostic for the insect infestation as previously reported (6). Moreover, the complicated manufacturing procedures, which are considered to cause the stress-responsive biochemical reactions in tea leaves for their self-defense against many kinds of stresses such as draught stress during withering, injuring stress during turning over and so forth, was demonstrated to be necessary for the rich aroma of Oriental Beauty.


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Enantioselective Analysis We found large differences in the amounts of hotrienol and DOD between the healthy and infested tea leaves. DOD was a characteristic compound in infested tea leaves. Simultaneous enantioselective analysis of DOD and related compounds, linalool and hotrienol, was achieved by using an enantioMDGC/MS system equipped with an achiral precolumn and a chiral main column. The optically active hotrienol and DOD as authentic references were synthesized to identify the absolute configuration of these compounds. The Rform of hotrienol was synthesized from commercial available (/?)-linalool (7). The /?-form of DOD was synthesized by epoxidation and reduction of the optically active hotrienol. DOD and hotrienol had high enantiopurity in both the infested and the noninfested tea samples, while linalool showed lower optical purity as shown in Table III. The predominant absolute configurations of these three compounds were the S-forms. No significant racemization was observed during the tea processing. The biosynthesis of DOD has not yet been elucidated though DOD is considered to be derivedfromthe oxidation of linalool.

Conclusion The profiles of aroma components of Oriental Beauty were investigated during the manufacturing process. As the fermentation progressed, the amounts of the aroma components produced in infested tea leaves became much higher than those in non-infested tea leaves. We confirmed that insect infestation and higher fermentation were necessary to produce Oriental Beauty rich in the characteristic aroma. DOD was even contained in fresh tea leaves, indicating that DOD was a key volatile compound produced by insect infestation. DOD was present in high optical purity in the tea leaves and its absolute configuration was the same as those of hotrienol and linalool. This clearly indicates that DOD is produced via enzyme reactions. The identification of the enzymes and cDNAs involved in the biosynthesis of DOD is now in progress.

Acknowledgments We thank Kuo-Renn Chen and Chun-Liang Chen of Tea Research and Extension Station for the tea sample preparation, and Yukihiro Kawakami, Makoto Emura, Yoshifiimi Yuasa, Ikuo Terada of Takasago for their kind help and valuable discussions, and Masaharu Mizutani and Jeong-yong Cho of Kyoto University for their useful advice.



Figure 1-1. Gas chromatograms of tea aroma extracts. Upper chromatograms are Infested samples, and lower are healthy (not infested) samples. IS. is internal standard (3-octanone).

Time (min)



0

11 /

20

10

18

igTime (min)

20

17

40

50

BT3

50

AT3

60

o

10

20

ill

14

13

19 Time (min)

171

AT4

Figure 1-2. Gas chromatograms of tea aroma extracts. Upper chromatograms are Infested samples, and lower are healthy (not infested) samples. IS. is internal standard (3-octanone).

20

30

Time (min)

Till

10

19

-1

1

13

JLiL J LliJI

6 I

if)

17|

18

1819



Compound

Hexanal l-Penten-3-ol Isoamyl alcohol Amyl alcohol Hexanol (Z)-3-Hexenol (£)-2-Hexenol trans -Linalool 3,6-oxide (Linalool oxide I) cis -Linalool 3,6-oxide (Linalool oxide II) Benzaldehyde Linalool 3,7-Dimethylocta-l,5,7-trien-3-ol (hotrienol) trans -Linalool 3,7-oxide (Linalool oxide III) cis -Linalool 3,7-oxide (Linalool oxide VI) Caproic acid Geraniol Benzyl alcohol 2-Phenylethanol 2,6-Dimethylocta-3,7-diene-2,6-diol (DOD) (Z)-3-Hexenoic acid (£)-2-Hexenoic acid 3,7-Dimethylocta-l,7-diene-3,6-diol 2,6-Dimethylocta-2,7-diene-1,6-diol Methyl jasmonate

nd: not detected

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

No. 0.0046 0.0057 nd 0.0047 nd nd nd trace 0.016 nd 0.014 0.0029 trace 0.052 0.063 nd 0.038 0.065 0.12 nd nd nd nd 0.12

BF 0.052 0.057 0.034 0.074 0.17 0.46 0.11 0.36 0.72 0.075 0.20 0.060 0.43 0.61 1.3 0.87 3.5 3.6 0.43 1.6 1.1 0.049 0.46 0.080

Area/I. S. Area BT5

Table II. Comparison of Tea Aroma Components


nd 0.0027 0.0024 0.010 nd nd 0.0016 0.040 0.067 0.0014 0.022 0.20 0.11 0.14 0.043 0.036 0.11 0.11 1.8 nd nd nd 0.073 0.038

AF

AT5 0.040 0.047 0.20 0.064 0.18 0.71 0.13 1.8 1.7 0.24 0.24 0.16 1.6 1.2 0.60 0.87 4.3 4.4 3.1 0.47 0.68 0.22 0.44 trace


nd: not detected

Healthy leaves

Infested leaves

Tea samples >99.9 95.5 >99.9 94.1

AF AT5 BF BT5

S

R

5.9

nd

4.5

nd

Hotrienol

98.0

>99.9

98.7

99.1

S

DOD

2.0

nd

1.3

0.9

R

S

94.3

80.7

82.5

77.6

Table III. Enantiomer Ratio of Hotrienol, DOD and Linalool


R

5.7

19.3

17.5

22.4

Linalool

97

References 1. 2. 3. 4.

Sakata, K.; Mizutani, M. Food & FoodIngred. J. Jpn. 2003, 208, 991-1003. Yamanishi, T. Koryo 2001, 211, 129-136. Kawakami, M.; Ganguly, S. N.; Banerjee, J.; Kobayashi, A. J. Agric. Food Chem. 1995, 43, 200-207. Ogura, M.; Otsuka, M.; Yamazaki, Y.; Shimizu, T.; Kawakami, Y.; Shirai, F. The 47 Symposium on the Chemistry of Terpenes, Essential Oils, and Aromatics, 2003, pp 13-15. Kinoshita, T.; Sakata, K. Koryo 2006, 229, 113-120. Chen, Z.; Xu, N.; Baoyu, H.; Zhao, D. 2004 International Conference on OCHA (tea) Culture and Science, Shizuoka, 2004, pp 90-93. Yuasa, Y.; Kato, Y. J. Agric. Food Chem. 2003, 51, 4036-4039.


th

5. 6. 7.


Food Flavor - American Chemical Society

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