Scholarly article on topic 'Changes in flavor volatile composition of oolong tea after panning during tea processing'

Changes in flavor volatile composition of oolong tea after panning during tea processing Academic research paper on "Chemical sciences"

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Academic research paper on topic "Changes in flavor volatile composition of oolong tea after panning during tea processing"

Food Science & Nutrition



Changes in flavor volatile composition of oolong tea after panning during tea processing

Ershad Sheibani1, Susan E. Duncan1, David D. Kuhn1, Andrea M. Dietrich2, Jordan J. Newkirk1 & Sean F. O'Keefe1

department of Food Science and Technology, Virginia Tech, Blacksburg, Virginia 24061 2Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia 24061


Flavor analysis, GC-MS, GC-O, oolong tea, panning


Sean F. O'Keefe, Food Science and Technology, Virginia Tech, 402-A HABB1 (0924), Blacksburg, VA 24061. Tel: +(540) 231-4437; Fax: 540-818-9293; E-mail:

Funding Information

Virginia Agricultural Experiment Station, and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture. Publication funding was provided by Virginia Tech's Open Access Subvention Fund.

Received: 26 July 2015; Revised: 28 September 2015; Accepted: 30 September 2015


Panning is a processing step used in manufacturing of some varieties of oolong tea. There is limited information available on effects of panning on oolong tea flavors. The goal of this study was to determine effects of panning on flavor volatile compositions of oolong using Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Olfactometry (GC-O). SDE and SPME techniques were applied for extraction of volatiles in panned and unpanned teas. A total of 190 volatiles were identified from SDE and SPME extractions using GC-MS and GC-O. There were no significant differences (P > 0.05) in aldehyde or terpene contents of unpanned and panned tea. However, alcohols, ketones, acids and esters contents were significantly reduced by panning. Among 12 major volatiles previously used for identification and quality assessment of oolong tea, trans nerolidol, 2- hexenal, benzaldehyde, indole, gernaiol, and ben-zenacetaldehyde contents were significantly decreased (P < 0.05) by panning. Panning increased (P < 0.05) contents of linalool oxide, cis jasmone, and methyl salicylate. The GC-O study also showed an increase of aroma active compounds with sweet descriptions and decrease of aroma active compounds with fruity and smoky descriptions after panning. Panning significantly changes the volatile compositions of the tea and created new aroma active compounds. Results from this study can be used in quality assessment of panned oolong tea.

doi: 10.1002/fsn3.307


Oolong tea is manufactured predominantly in southeast China and Taiwan (Lee et al. 2008). Less than 2% of tea manufactured in the world is semi-fermented oolong tea (Hara et al. 1995); however, due to the complex processing steps and the limited supply, oolong teas usually have a higher unit price than green or black teas in the international tea market. Current increases in oolong tea consumption might result from the recent studies on health benefits of tea polyphenols, and also the unique taste and aroma of this tea variety.

Oolong tea categorized as a semi-fermented tea. For tea, fermentation refers to the natural browning reactions induced by oxidative enzymes in the cells of tea leaves. This process is mainly the oxidative polymerization of catechins catalyzed by polyphenol oxidase and peroxidase (Chaturvedula and Prakash 2011). Oolong tea is generally fermented from about 10% to 60% to create a taste and color somewhere between green and black teas. Fermentation is responsible for creation of many flavor compounds. During the fermentation process, the tea leaves are injured and, consequently, an increase in enzyme activity is seen with the creation of aromatic alcohols

© 2015 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. This is an open access article under the terms of 1

the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

(Ma et al. 2014). Wang et al. (2008) found that fermentation can cause the loss of grassy or green flavors and the formation of fruity/floral flavors, providing much of black tea's sweet and bold flavor. There are no standard recipes or procedures on how to manufacture oolong tea. The processing and the level of oxidation are decided by each tea garden or tea master. In Taiwan, Baozhong tea would typically be fermented around 10%, ball rolled Donding oolong from 15-25%, and Baihao oolong (Oriental Beauty) around 60-80%.

Panning is a processing step in the manufacturing of some varieties of oolong tea that is performed after fermentation. There are two steps often called panning: first, exposure of tea leaves to heat in order to inhibit fermentation by inactivating the enzymes, and second, heating after drying to develop unique flavors in oolong tea (Hui et al. 2003; Zhen 2003; Info Taiwan 2014). During this first process, tea leaves lose moisture and thus are softer making the rolling into string shapes and dehydration easier (Hui et al. 2003). Panning can be done using rotary pan or panning machine, convection oven, or via pan-frying. The exact temperature and time depends on various teas and determined by tea masters. The panning period also depends on the variety of tea leaf and loading quantity. Panning is known to eliminate grassy odor while leaving a nutty smell and taste. Panning also prevents tea leaves from breaking before they are rolled and the result is soft and flexible leaf texture with a strong pleasant aroma (Info Taiwan 2014). However, there is limited information available on the effects of panning after drying on volatile compounds of oolong tea. In addition, the compounds responsible for the aroma of panned tea still need clarification.

Flavor analysis of oolong tea is important for variety authentication and quality assessment. Oolong varieties are sold at a premium price compared to lower-grade varieties. Some oolong varieties are very similar in appearance and flavor and accurate identification and differentiation is only possible for tea experts or experienced tea tasters (Zhang et al. 2013). Thus, there is significant interest in developing accurate chemical methods for quality assessment and identification of oolong tea varieties. Composition of the volatile compounds extracted by extraction methods SDE and SPME may differ, and it is advisable that two methods used together for effective volatile analysis (Thompson-Witrick et al. 2015).

Nonvolatile components are generally responsible for taste, while volatile components give the aroma (Rawat et al. 2007). The unusual taste of oolong tea infusion depends on the degree of fermentation, the elevation and growing conditions as well as the tea bush. Nonaka et al. (1983) (as cited in Chaturvedula and Prakash 2011) reported the fruity and sweet taste of oolong tea infusion

are the integrated taste of nonoxidized catechins, thearubigins, some secondary polyphenolic compounds, caffeine, free amino acids, sugars, and volatile compounds. Compared to green tea, the astringency of oolong tea is lower and the sweetness is higher. Volatile flavor compounds of tea are mostly composed of nonterpenoids or terpenoids, which are responsible for sweet flowery aroma of tea (Rawat et al. 2007). Previous studies showed volatile compounds such as trans-nerolidol, trans-2-hexenal, benzaldehyde, methyl-5-hepten-2-one, methyl salicylate, indole (Wang et al. 2008; Pripdeevech and Machan 2011), cis-jasmone (Pripdeevech and Machan 2011), (E)-geraniol, (E)-P-damascenone, linalool oxide B, benzaldehyde (Kawakami et al. 1995; Wang et al. 2008; Wang et al. 2011; Zhang et al. 2013), (E,E)-2,4-heptadienal and (Z)-3-hexenol (Wang et al. 2008, 2011) are the key odorants, and indicators of high quality oolong tea.

The objectives of this study were to investigate effects of panning on flavor volatile compositions of oolong tea and to determine changes in aroma active compounds of panned compared to unpanned oolong tea using Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Olfactometry (GC-O).

Materials and Methods

Panning process

Three batches of unpanned Jin Xuan (Chin-Hsuan, or Zhu Shan) oolong tea samples were purchased from Tea of Life® Health Inc. in Rosedale, NY before the experiment and stored at room temperature. To pan the tea leaves, 680 grams of oolong tea was placed on a metal baking dish. Then, the dish was heated/panned in a convection oven at 120°C for 6 h. The method and selected condition for the panning process was based on our preliminary study and literature. After heating, the tea leaves were cooled to room temperature. The panning and flavor analysis were performed for the three batches separately.

Volatile extraction using SDE

In preliminary experiments, we optimized conditions for SDE extraction including solvent and extraction time for flavor analysis of tea, and we applied the optimal extraction condition to this study. A simultaneous distillation, extraction (SDE), Likens-Nickerson, apparatus was used. Tea leaves (50 g) were placed in a 1 L round bottom flask containing 400 mL of distilled water. One hundred milliliter of HPLC grade diethyl ether (Sigma-Aldrich Co., St. Louis, MO) with 0.5 mL of 100 ppm ethyl decanoate (internal standard) (Sigma-Aldrich Co.) was placed into

a 250 mL extraction flask. Two electric heating plates were used to maintain boiling for the tea and solvents in the SDE apparatus. The volatiles were steam-distilled and extracted into diethyl ether for 40 min. After extraction, the solvent was dried over anhydrous sodium sulfate (Fisher Scientific, Pittsburg, PA) and filtered. Then, the extract was concentrated to 2 mL in a vacuum rotary evaporator and nitrogen gas. The concentrates were analysed using Gas Chromatography - Mass spectrometry (GC-MS) and Gas Chromatography Flame Ionization Detection - Olfactometry (GC- FID/O) for volatile analysis.

Volatile extraction by SPME for GC-MS analysis

Four grams of tea leaves were placed in 200 mL of hot distilled water (98°C) and brewed for 5 min. Then, 5 mL of the filtered tea infusion and 1 g of NaCl were placed into 10 mL headspace vials with Teflon-fined silicon septa (Chromacol, Fisher Scientific). SPME was used to extract volatiles and volatiles were analysed by injection into the GC-MS using an AOC-5000 Plus (Shimadzu Scientific, Columbia, MD) SPME auto-sampler. Samples were equilibrated for 2 min prior to extraction. A DVM/Carboxen/ PDMS SPME fiber (2 cm 50/30 um) (Supelco, Bellefonte, PA) was exposed to the headspace above the tea extract in glass vials for 30 min at 40°C with an agitation speed of 250 rpm.

Volatile extraction by SPME for GC-FID/GC-O analysis

The extraction and injection were performed manually for GC-O analysis. Five milliliter of tea aqueous infusions (which were prepared similar to GC-MS analysis) was placed in a 15 mL amber glass vial with a Teflon-lined cap. A "RTC basic" heater with an ETS D4 Fuzzy Logic Controller (IKA Werke, Wilmington, NC) was used to heat samples at 40°C while being stirred using a 4 mm stir bar. A 50/30 ^m SPME fiber (DVB/CAR/PDMS) on a 2 cm StableFlex fiber (Supelco Bellefonte, PA) was inserted into the vial and was exposed approximately 1 cm above the headspace for 30 min while a magnetic bar continued to stir the sample.

GC-MS analysis

The volatile constituents of each sample were analyzed using a Shimadzu GCMS-QP2010 Ultra gas chromatograph with mass selective detector (Shimadzu) equipped with GCMSsolutions 2.53 and capillary nonpolar column (SHRXI-5MS, Shimadzu, 30 m * 0.25 mm id * 0.25 ^m

film thickness). The oven temperature was initially held at 50°C for 5 min and then increased at 4° C/min to final temperature of 250°C. The injector temperature was 200°C and injections were made in splitless mode. Ultra high purity helium used as a carrier gas at a flow rate of 0.69 mL/min (approximately 25 cm/sec linear flow velocity). The mass spectra were collected at m/z 40-400 and were performed every 0.3 sec. The ion source and quadrupole were set at 230 and 200°C respectively. Identification of the volatile components was performed by combined matching standardized retention time (LRI values) for a DB-5 column (Flavornet and Pherobase) and fragmentation spectra of standards from NIST 11 (Scientific Instrument Services, Ringoes, NJ) and the Wiley 2010 libraries (John Wiley and Sons Inc.). Confirmation of the identification was sought by matching the mass spectra of the compounds with the reference mass spectra present in the NIST 11 and Wiley libraries. The results were compared with our control, unpanned samples.

GC-O analysis

Approval for use of human subjects in research was obtained from the Virginia Tech Institutional Review Board before GC-O experiments began (IRB #13-580). GC-O analysis was carried out using a HP 5890A gas chromatograph (Hewlett-Packard Co., Palo Alto, CA) equipped with a flame ionization detector (FID), a sniffing port (ODOII; SGE Inc. Austin, TX), and a DB-5 ms column (30 m x 0.25-mm i.d. x 0.25 ^m film thickness) (J&W Scientific, Folson, CA). The detector and injector were set to 250°C and 275°C, respectively; all injections were made in the splitless mode. The initial oven temperature was 50°C and increased at 10°C/min until reaching a final temperature of 200°C. Chromatograms were recorded using a HP 3396A integrator (Hewlett-Packard Co., Palo Alto, CA). Hydrogen was used as the carrier gas with a flow rate of 1.0 mL min-1 (linear flow velocity ~ 25 cm/ sec). The GC column effluent was split 1:1 between the FID and the ODOII using deactivated fused silica capillaries (1-m length x 0.32 ^m i.d.). Two trained assessors were selected for GC-O analysis. The assessors sniffed tea extracts from SDE or SPME methods for 20 min from each batch. Aroma descriptions, times and intensity were recorded for every sample. The assessors indicated aroma intensity in scale 1-5 where 1 was the lowest intensity and 5 was the highest.

Mean aroma intensities for each odorant were calculated by averaging the reported intensity by panelists. Aroma-active compounds were defined as the ones that were detected by the panelists fifty percent of the time with similar descriptions and retention times or those scored higher than 3 by panelists. Kovats or Linear Retention

Index (LRI) values were determined using a series of al-kanes (C5-C26) which were run under identical conditions. Identification of volatile compounds was based upon their odor descriptions and RI values from DB-5 column. The databases Flavornet ( html) and Pherobase ( were used to aid in identifying the compounds based upon standardized retention and aroma.

Statistical analysis

We conducted similar experiments on unpanned oolong tea with three replications and the results from GC-MS and GC-O were compared with the panned tea from this study. The data from GC-MS were analyzed by JMP 11.0 (SAS, Cary, NC). Two way analysis of variance (ANOVA) and mean comparisons using Tukey's test with the 5% significance level were conducted on different compound categories: alcohols, aldehydes, ketones, terpenes, acids and ester results from SDE and SPME techniques of panned and unpanned teas.

One way ANOVA was also used to find significant differences in 12 volatiles (previously reported to be major flavor compounds in oolong tea) extracted with SDE and SPME techniques in panned and unpanned tea. Means were compared by using Fisher's least significant difference (LSD) method with significance at P < 0.05.

Results and Discussion GC-MS analysis

A total of 190 volatile compounds were identified using SDE and SPME with GC-MS and GC-O. We identified 200 volatile compounds in unpanned oolong tea, of which only 79 of these compounds were found in panned oolong tea (Table 1); this shows the significant impact of panning on flavor volatiles of oolong tea. We also observed that the compounds identified using SDE and SPME differed and were complementary. Therefore, we used the same approach to analyze and discuss our results from GC-MS and GC-O.

A total of 121 volatiles were extracted from panned oolong tea using SDE. Among these compounds, 18 alcohols, 11 aldehydes, 16 ketones, 23 terpenes and 13 acids were identified. The most abundant compounds were furfural (10.8%), trans-nerolidol (8.5%), a-farnesene (4.8%), ethyl-pyrrole-2-carboxaldehyde (3.9%), benzyl nitrile (3.5%), 5-methyl-2-furancarboxaldehyde (2.8%), indole (2.5%), 4-methoxy-2-methyl-benzenamine (2.3), 3-methyl-butanenitrile (2.1%) and 1-(2-furanyl)-ethanone (2.1%). Only trans-nerolidol, a-farnesene, indole and benzyl nitrile also appeared as most abundant compounds

in unpanned tea. Similar to unpanned tea, trans- nerolidol (43.9% of total alcohols) and indole (13.4%) were the two major alcohols. Major ketones were 1-(2-furanyl)-ethanone (21.5% of total ketones) and dihydro-2-methyl-3(2H)-furanone (11.0%); however, the major ketones in unpanned tea were jasmine lactones and trans-P-ionone. These two compounds were identified in panned tea, but at much lower concentrations. Furfural (49.5% of total aldehydes) was the most abundant aldehyde in panned tea, but hexanal and benzeneacetaldehyde were the most abundant aldehydes in unpanned tea. Among 23 identified terpenes in panned tea, a-farnesene (24.2%) and linalool oxide (9.1%) were the compounds with highest peak areas. For unpanned tea, a-farnesene was the most abundant terpene, followed by geraniol and linalool. Sesquiterpenes in oolong tea may be present as glucosides that can be hydrolyzed to form various aromatic compounds during the manufacturing process (Guo et al. 1996). These glucosides may also be obtained by biosynthesis during the manufacturing process (Wang et al. 2001).

A total of 48 volatile compounds were detected using SPME, including eight alcohols, eight aldehydes, four ketones, seven acids, and 11 terpenes. The compounds with highest peak area were indole (9.3%), furfural (7.1%), 1-ethyl-1-pyrrole-2-carboxaldehyde (5.5%), benzyl nitrile (4.1%), 1,1,5-trimethyl-1,2-dihydronaphthalene (TDN) (3.4%), 4-methoxy-2-methyl-benzenamine (2.9%), 5,9,9-trimethyl-spiro[3.6]deca-5,7-dien-1-one (2.8%), meth-oxy-phenyloxime (2.5%), 3,4-dimethylcyclohexanol (2.4%), and 3-amino-4-methylbenzyl alcohol (2.4%). Indole (44.7% of total alcohols) was the most abundant alcohol for panned and unpanned teas. Similar to what was observed for SDE, furfural (31.6% of total aldehydes) had the highest peak area in panned tea, but for un-panned tea the most abundant aldehyde was 2,4-deca-dienal. Hotrienol (18.5% of total terpenes) had the highest peak areas among terpenes, but this compound was not identified in unpanned tea. The most abundant terpene in the unpanned tea was geraniol.

Results from ANOVA showed there was a significant difference (P < 0.05) between the summed peak percentages of alcohols between panned and unpanned teas (Fig. 1). The percentage of alcohols in unpanned tea were significantly higher (P < 0.05) than panned tea from SDE; however, there was no significant difference (P > 0.05) in alcohols percentages between unpanned and panned tea with SPME. Fermentation results in increased enzyme activity that leads to creation of aromatic alcohols (Ma et al. 2014); however, during panning, many of these enzymes are destroyed by heat, which results in less formation of aromatic alcohols. There were no significant differences (P > 0.05) in aldehyde percentages of panned

Table 1. Identified volatiles in the panned oolong tea with method of identification, LRI and comparisons with the unpanned tea.

No. Compound LRI SDE SPME Unpanned

1 Butanenitrile, 2-methyl- 637 MS

2 Butanenitrile, 3-methyl- 646 MS X

3 2H-Pyran, 3,4-dihydro-6-methyl- 644 MS

4 Pyrrole 646 MS

5 1-Pentanol 657 MS X

6 Pentane, 1-chloro- 662 MS

7 2-Penten-1-ol, (Z)- 661 MS X

8 Cyclopropane, 1,1,2,3-tetramethyl- 695 MS

9 Hexanal 700 MS X

10 3(2H)-Furanone, dihydro-2-methyl- 703 MS X

11 1 H-Pyrrole, 1-ethyl- 711 MS MS

12 Pyrazine, methyl- 717 MS MS

13 Maleic anhydride 724 MS

14 Furfural 727 MS MS X

15 2-Hexenal, (E)- 747 MS X

16 2-Furranmethanol 750 MS MS

17 18 p-Xylene Benzene, 1 ,3-dimethyl- 764 767 MS MS

19 1,5-Heptadiene, 2,6-dimethyl- 781 MS

20 Oxime-, methoxy-phenyl-_ 802 MS X

21 Ethanone, 1-(2-furanyl)- 808 MS

22 1-(3H-Imidazol-4-yl)-ethanone 810 MS

23 Pyrazine, ethyl- 811 MS

24 1 H-Pyrrole-2-carboxaldehyde, 1 -methyl- 824 MS MS

25 Benzaldehyde 857 MS MS X

26 2-Furancarboxaldehyde, 5-methyl- 860 MS MS

27 Methyl 2-furoate 873 MS

28 1-Octen-3-ol 877 MS X

29 Sulcatone 884 MS X

30 P-Myrcene 889 MS MS

31 Pyrazine, 2-ethyl-6-methyl- 895 MS X

32 2,4-Hexadienal 928

33 Bicyclo[2.2.1]heptane, 2-butyl- 909 MS

34 Furan, 2-propyl- 910 MS

35 1 ,3-Cyclohexadiene, 1 -methyl-4-(1 -methylethyl)- 920 MS

36 Mesitylene 930 MS X

37 o-Cymene 931 MS MS X

38 D-Limonene 938 MS MS X

39 Benzyl alcohol 944 MS X

40 trans-P-Ocimene 951 MS MS X

41 Benzeneacetaldehyde 957 MS MS X

42 1 H-Pyrrole-2-carboxaldehyde, 1 -ethyl- 965 MS MS X

43 Cyclohexene, 1-(3-ethoxy-1-propenyl)-, (Z)- 973 MS

44 Ethanone, 1-(1 H-pyrrol-2-yl)- 980 MS MS

45 Acetophenone 989 MS

46 1-Octanol 997 MS

47 Linalool oxide 1000 MS MS X

48 Pyrazine, 3-ethyl-2,5-dimethyl- 1019 MS

49 Linalool oxide(furanoid) 1053 MS X

50 3,5-Octadien-2-one 1066 MS X

51 R-Linalool 1091 MS MS X

52 Hotrienol 1102 MS MS

53 3,4-Dimethylcyclohexanol 1103 MS X

54 Benzenamine, 4-methoxy-2-methyl- 1110 MS MS

55 1,5,9-Undecatriene, 2,6,10-trimethyl-, (Z)- 1115 MS

56 Isophorone 1118 MS X

57 2,4,6-Octatriene, 2,6-dimethyl-, (E,Z)- E,Z-Alloocimene 1128 MS

Table 1. Continued

No. Compound LRI SDE SPME Unpanned

58 Benzyl nitrile 1136 MS MS X

59 1,3-Cyclopentadiene, 1,2,3,4-tetramethyl-5-methylene- 1151 MS

60 1-[2-Aminoethyl]hypoxanthine 1154 MS

61 2-Nonenal, (E)- 1158 MS

62 1 H-Pyrrole-3-carboxylic acid, 2,4-dimethyl-, methyl ester 1161 MS

63 Benzeneacetic acid,.a.-oxo-, ethyl ester 1163 MS

64 2H-Pyran-3-ol, 6-ethenyltetrahydro-2,2,6-trimethyl- 1168 MS X

65 Benzeneacetic acid, methyl ester 1176 MS

66 3-Amino-4-methylbenzyl alcohol 1181 MS MS

67 Butanoic acid, 3-hexenyl ester, (E)- 1184 MS

68 a-Terpineol 1190 MS MS X

69 Methyl salicylate 1193 MS MS X

70 1,3-Cyclohexadiene-1 -carboxaldehyde, 2,6,6-trimethyl- 1199 MS

71 Decanal 1202 MS X

72 (Z)-4-Decenal 1203

73 1 H-Indene, 2,3-dihydro-1,1,5,6-tetramethyl- 1206 MS

74 Benzene, (ethenyloxy)- 1208 MS

75 1,3-Cyclohexadiene-1 -methanol, 4-(1 -methylethyl)- 1211 MS

76 P-Cyclocitral 1211 MS

77 4a(2H)-Naphthalenol, octahydro-, trans- 1213 MS

78 Benzene, 1 -(1,5-dimethylhexyl)-4-methyl- 1216 MS X

79 Prop-2-en-1 -one, 1 -(6,6-dimethylbicyclo[3.1. 1 ]hept-2-en-2-yl)- 1218 MS

80 Citral 1221 MS X

81 Geraniol 1227 MS MS X

82 Acetic acid, 2-phenylethyl ester 1229 MS MS X

83 Isocyclocitral 1230 MS

84 Nonanoic acid 1234 MS X

85 2,6-Octadienal, 3,7-dimethyl-, (E)- 1236 MS

86 2(1 H)-Naphthalenone, 3,4,4a,5,6,7-hexahydro-1,1,4a-trimethyl- 1242 MS

87 Ionone 1242 MS MS X

88 4-Acetamido-2-methallylphenol 1244 MS

89 Indole 1248 MS X

90 Formic acid, (2-methylphenyl)methyl ester 1250 MS X

91 Pyrazine, 2,5-dimethyl-3-propyl- 1253 MS

92 Cyclohexane, 1,2-diethenyl-4-(1-methylethylidene)-, cis- 1256 MS

93 4-Hydroxy-3-methylacetophenone 1258 MS

94 Spiro[3.6]deca-5,7-dien-1-one,5,9,9-trimethyl 1259 MS MS

95 2H-Pyran-3-ol, 2-ethoxy-3,4-dihydro-, acetate 1262 MS

96 2,6-Octadienoic acid, 3,7-dimethyl-, methyl ester 1263 MS

97 6-Hydroxynicotinic acid di-methyl derivative 1264 MS

98 Pentanoic acid, 4-methyl-, ethyl ester 1270 MS

99 Benzene, 2-(2-butenyl)-1,3,5-trimethyl- 1277 MS

100 1,1, 5-Trimethyl-1, 2-dihydronaphthalene 1278 MS MS

101 Naphthalene, 1,2,3,4-tetrahydro-1, 1,6-trimethyl- 1280 MS

102 Bicyclo[3.1.0]hexan-3-ol, 4-methyl-1-(1 methylethyl)- 1282 MS

103 Phenol, 2-(1,1 -dimethyl-2-propenyl)-3,6-dimethyl- 1284 MS

104 cis-anti-cis-Tricyclo[,6)]-7-dodecene 1286 MS

105 Hexanoic acid, hexyl ester 1292 MS

106 Decanoic acid, ethyl ester 1297 MS MS X

107 108 os-Jasmone Naphthalene, 1,2,3,4-tetrahydro-2,5,8-trimethyl- 1299 1402 MS MS MS X

109 Cyclopropanecarboxylic acid, 2,2-dimethyl-3-(2-methyl-1-propenyl)-, 2-methyl-4-oxo-3-(2-pentenyl)-2-cyclopenten-1-yl ester, [1R 1423 MS

110 111 a-Ionone 6,7-Dimethyl-1,2,3,5,8,8a-Hexahydronaphthalene 1428 1432 MS MS

112 Coumarin 1437 MS X

113 P-Phenylethyl butyrate 1440 MS X

114 (E)-Geranyl acetone) 1451 MS X

E. Sheibani et al. Table 1. Continued

No. Compound LRI SDE SPME Unpanned

115 cis- ß-Farnesene 1456 MS X

116 4-(2,4,4-Trimethyl-cyclohexa-1,5-dienyl)-but-3-en-2-one 1484 MS

117 trans-. ß.-Ionone 1488 MS X

118 Jasmin lactone 1493 MS MS X

119 1 H-Benzocyclohepten-7-ol, 2,3,4,4a,5,6,7,8-Octahydro-1,1,4a,7-tetramethyl-, cis- 1499 MS

120 Gamma.-Muurolene 1504 MS

121 a-Farnesene 1508 MS MS X

122 Bicyclo[2.2.1 ]heptan-2-one, 1-(bromomethyl)-7,7-dimethyl-, (1S)- 1512 MS X

123 Butylated Hydroxytoluene 1514 MS X

124 cis-Thujopsene 1519 MS X

125 Levomenol 1545 MS

126 trans-Nerolidol 1564 MS MS X

127 3-Hexen-1-ol, benzoate, (Z)- 1572 MS X

128 Benzoic acid, hexyl ester 1579 MS

129 Farnesene epoxide, E- 1599 MS X

130 Cyclopentaneacetic acid, 3-oxo-2-(2-pentenyl)-, Methyl ester, [1 .a.,2.a.(Z)]- 1649 MS

131 2-Furanmethanol, tetrahydro-.a.,.a.,5-trimethyl-5-(4-methyl-3-cyclohexen-1-yl)-, [2S-[2.a.,5. 1660 MS


132 Phytol 1836 MS

and unpanned tea in both extraction techniques. Analysis of ketones showed significant differences (P < 0.05) in panned and unpanned tea in both extraction techniques. Additionally, the peak percentages of panned tea for ketones were higher (P < 0.05) in SDE compared to SPME. No esters were identified in panned tea. There were no differences (P > 0.05) between the acids contents of panned and unpanned tea in both SDE and SPME. The percentages of terpenes in both extraction techniques were not different (P > 0.05) between panned and unpanned tea; however, terpene percentages of unpanned tea in SDE were higher (P < 0.05) than for SPME.

Most of the published studies on oolong tea volatiles have either investigated major compounds that differentiate oolong with fully fermented teas or nonfermented teas or have studied the compounds that are indicator of quality in oolong tea. During fermentation, several enzymatic reactions are responsible for formation of tea aroma compounds. The main precursors for tea aroma are amino acids and carotenoids, including P-carotene, lutein, neox-anthin, and violaxanthin (Yamanishi 1978). During fermentation, oxidation results in the significant reduction of carotenoids, particularly P carotene, resulting in the formation of ionone and terpenoid carbonyls (Yamanishi 1978). After oxidation and secondary epoxidation reactions, other carotenoids give rise to ionone, linalool and substituted hydroxy- and epoxy-ionones (Sanderson and Grahamm 1973). Generally, grassy or green flavors are diminished during fermentation, but fruity, floral and other fermented characters are increased (Wang et al. 2008).

Pripdeevech and Machan (2011) used a's-jasmone (woody, herbal), trans-nerolidol (floral), indole (pungent)

and hotrienol to differentiate semifermented tea from nonfermented tea. They showed the content of the first three volatiles were increased significantly while hotrienol (green, sweet) was decreased after fermentation. In our study, the content of a's-jasmone was significantly higher (P < 0.05) in panned tea while the content of indole and trans-nerodiol were significantly decreased (P < 0.05) by panning. The content of indole is very low in nonfermented tea, but its level increases quickly at the beginning of fermentation in oolong tea and then slowly decreased by continuing fermentation (Wang et al. 2008). Indole precursors might be destroyed by the heat treatment during panning and lead to changes in indole contents in panned tea. GC-MS analysis was able to identify hotrienol only in the panned tea, suggesting perhaps that heat treatment resulted in formation of hotrienol in oolong tea.

Other studies showed other compounds may be important in distinguishing oolong from other varieties of teas. Other than indole, Wang et al. (2008) found flavor compounds such as trans-2-hexenal (green), benzaldehyde (almond), and methyl salicylate (peppermint) are important to distinguish unfermented teas from fermented ones. Trans-2-hexenal and methyl salicylate also may be used to classify the semi from fully fermented teas. Others have also reported the contents of compounds such as (E)-geraniol (floral, rose), (E)-P-damascenone (not identified in our study), and linalool oxide B (floral) increase with degrees of fermentation (Kawakami et al. 1995; Wang et al. 2008, 2011; Zhang et al. 2013). In our study, trans-2-hexenal, methyl salicylate and geraniol contents were decreased significantly (P < 0.05) by panning, but linalool oxide content was significantly (P < 0.05) increased.

SDE unpanned SDE panned SPME unpanned SPME panned

50 -40 -30 -20 -10 -

Alchohols Aldehydes


Figure 1. Mean comparison of peak percentages of chemical composition the unpanned and panned tea using SDE and SPME. Means within a class of compounds with the same letter are not significantly different (P > 0.05). Bars represent standard deviations.

Trans-2-hexenol (grassy, green) is a product of lipid degradation and result in inferior quality to tea (Pripdeevech and Machan 2011). Usually, a higher amount of trans-2-hexenal is detected in nonfermented tea whereas in semifermented tea concentrations are reportedly significantly lower (0.04-0.08%) (Pripdeevech and Machan 2011). Our results suggest that panning in oolong tea promotes formation of some of the flavor characteristics of non-fermented as well as fermented teas.

Several studies suggest that volatile flavor compounds affect the perceived quality of oolong tea. Trans-nerolidol was reported as one of the key odorants and can be

considered as an indicator for the high quality oolong tea flavor (Kai et al. 2008; Pripdeevech and Machan 2011; Wang et al. 2011; Zou et al. 2011; Ma et al. 2014). We found that trans- nerolidol was the most dominant volatile in unpanned oolong tea; however, during panning the concentration of these compound significantly decreased (P < 0.05) (Fig. 2). Similar to our results, Ma et al. (2014) reported decreases in the nerolidol content during other thermal process steps in oolong tea manufacturing such as fixation, shaping, and drying. Nerolidol has a floral aroma (Lapczynski et al. 2008) and exists at a relatively high concentration in oolong tea. Even though the

Unpanned ■ Panned

& ^ ^ # J*

/ / ^ </ N/ / <

Figure 2. Mean comparison of peak percentage of 12 major volatiles in the panned and unpanned tea identified from SDE and SPME techniques. Means with the same letter within each compounds are not significantly different (P > 0.05). Bars represent standard deviations.

Table 2. The aroma active compounds in the panned oolong tea using time-intensity GC-O with SDE extraction.

No. Compound LRI Confirmed3 Aroma Description Intensity4

1 n.i.5 561-580 Nutty, Chocolate, caramel 4

2 Methylbutenol 620 620 Green, herb 2

3 Methylbutanal1-2 647 641 Popcorn, nutty, chemical 2

4 Isobutyraldehyde 655 662 Green 2

5 Methyl methylbutanoate 675 674 Fruity 2

6 Pentenone1-2 682 780 Fruity 2

7 a.y-Dimethylallyl alcohol 702 712 Green, sweet, fruit 3

8 Methylcyclohexane 715-723 7162 Sweet, nutty, cookies 1

9 Methyl butanoate1-2 727 723 Sweet 3

10 Pentanal1-2 740 732 Smoky, nutty 4

11 Methyl-2-butenal2 747 752 Green pepper 1

12 Methyl-2-butenol1 774 775 Celery, herb 2

13 3-Hexenal 798 800 Green, earthy 2

14 Furfural1-2 825 829 Nutty, chocolate 2

15 n.i.1-5 830-837 Waxy, smoky 1

16 (E)-2-Hexenal 851 844 Fruity 2

17 Furfuryl alcohol 855 851 Smoky, burnt 1

18 2-Hexenal2 859-865 854 Fruity 2

19 Heptanal 882 8852 Burnt plastic, smoky 2

20 Benzaldehyde1 964 960 Nutty 2

21 (E)-2-Penten-1-ol 965 973 Mushroom 2

22 Filbertone 972 972 Nutty 2

23 6,6-Dimethyl-2-methylenebicyclo[3.1.1]heptane 984 9812 Nutty, musty, sweet 2

24 Acetylthiazole 1017-1024 1020 Nutty, Waxy 2

25 1.8-Cineole 1025-1029 1032 Nutty, floral, sweet 2

26 (E)-2-Heptenal 1041 1041 Waxy, fatty 3

28 2-Octenal 1055 10602 Nutty, 2

29 a-Ocimene2 1058 1056 Fruity, floral 1

30 p-Cresol 1076 1075 Smoky 1

31 3-5-Octadienone 1096 1095 Citrus, fruity, sweet 2

32 (E)-Linalool oxide 1176 1172 Sweet, floral, fruity 2

33 Isobutylmethoxypyrazine 1189 1186 Green, smoky 2

34 Decanal 1211 12112 Waxy 3

35 Linalool oxide1-2 1217 12122 Floral, fruity, earthy 2

36 Nerol2 1239 1233 Sweet 2

37 Benzothiazole1 1246 1240 Smoky 3

38 Isobornyl formate 1249 1245 Earthy 2

39 Dihydromethylcyclopentapyrazine1 1255 1248 Nutty, smoky 4

40 Linalyl acetate1 1264-1271 1261 Sweet, floral 1

41 Safrole1 1281 1280 Spicey, smoky 2

42 cis-Linalool pyran oxide 1401 1402 Citrus 2

43 n.i.5 1417-1421 Smoky, cooked meat 4

44 Coumarin 1433 1439 Sweet, waxy 3

45 Linalyl isovalerate 1477 1478 Fruity, waxy 2

46 Citronellyl butyrate 1536 1528 Fruity 3

47 (Z)-Nerolidol 1570 1565 Waxy 3

Bold Compounds were also detected with GC-MS. 11dentified with SDE in GC-O analysis of unpanned tea. identified with SPME in GC-O analysis of unpanned tea.

3LRI values confirmed with databases Flavornet and Pherobase to identify the compounds based upon standardized retention and aroma. 4The average aroma intensity score by panelist on a scale of 5 where 1 = low intensity and 5 = high intensity. 5Not identified compound.

concentration of this compound was significantly decreased by panning, it was the second most abundant compound in the panned tea in our study. The content of nerolidol

is low in fresh leaves, but the content was greatly increased and reaches to its highest level during the fermentation stage of manufacturing (Ma et al. 2014).

Table 3. The aroma active compounds in the panned oolong tea using time-intensity GC-O with SPME extraction.

No. Compound LRI Confirmed3 Aroma description Intensity4

1 Methylbutenol 609 620 Spice, herb 1

2 Methylbutanal1 654 641 Sweet, vanilla, almond 1

3 Methyl 2-methylpropionate 687-688 6852 Fruity, sweet 2

4 a,y-Dimethylallyl alcohol 718 712 Green 1

5 Methyl butanoate1,2 723 723 Fruity, sweet 3

6 Pentanal1-2 728 732 Nutty 1

7 Pentanol1 765 759 Fruity 2

8 Methyl-2-butenol 788 779 Spicy, herb 1

9 1-Hexenol 792 789 Green pepper 3

10 n.i.5 800-807 - Smoky, earthy 3

11 Propyl propanoate1 817 812 Sweet 1

12 Furfural1,2 829 829 Nutty, earthy 2

13 Methyl pyrazine 833-835 828 Nutty, popcorn 1

14 n.i.2,5 839-846 - Chocolate, nutty 1

15 Isopropyl butanoate1 854 847 Fruity, floral 2

16 (Z)-3-Hexenol 861 858 Green 1

17 2-Methylbutyl acetate2 883-885 880 Sweet, fruit 3

18 2,4-Hexadienal 906 910 Grassy 2

19 Dimethylthiazole1-2 928 928 Plastic, smoky 1

20 Heptanol2 970 962 Green 2

21 Ethyl isohexanoate2 971 968 Fruity 2

22 1,5-Octadienone 983 988 Musty 1

23 Ethylmethyl pyrazine1-2 986 993 Fruity 2

24 2,4-Heptadienal2 1019-1023 1011 Nutty, sweet 1

25 (E)- ß-Ocimene 1038 1038 Sweet, vanilla 3

26 2-Acetylpyrrole 1051 1045 Nutty 1

27 a-Ocimene 1062 1056 Apple, banana, fruity 3

28 Ethyldimethylthiazole 1073 1078 Earthy 1

29 Limonene oxide 1129 1132 Fruity 4

30 Y-Heptalactone 1130 1130 Nutty 2

31 3,5-Diethyl-2-methylpyrazine1 1160 11602 Nutty, chocolate 1

32 Dihydrocarveol 1191 1190 Spicey, mint 1

33 Ethyl octanoate 1197 1198 Floral, fruity 3

34 (Z)-4-Decenal 1203 1200 Green, musty 1

35 Linalool oxide2 1214 1212 Green, floral 1

36 3-Phenylpropan-1-ol 1220 1219 2 Fruity 1

37 5-Methyl-2-furancarboxaldehyde 1222 12242 Musty 1

38 Nerol 1226 1233 Sweet 1

39 Methyl salicylate 1234 1234 Nutty, floral 2

40 Isobornyl formate 1246 1245 Green 2

41 n.i.5 1252-1261 - Nutty, chocolate 3

42 Linalyl acetate2 1263 1261 Sweet 2

Bold Compounds were also detected with GC-MS.

11dentified with SPME in GC-O analysis of unpanned tea.

identified with SDEE in GC-O analysis of unpanned tea.

3LRI values confirmed with databases Flavornet and Pherobase to identify the compounds based upon standardized retention and aroma.

4The average aroma intensity score by panelist on a scale of 5 where 1 = low intensity and 5 = high intensity.

5Not identified compound.

Wang et al. (2011) reported that perceived aroma score positively correlated with concentrations of benzyl alcohol (sweet, flower), benzeneacetaldehyde (honey, floral), linalool (flower), phenylethyl alcohol (honey), linalool oxide (flower), indole (pungent), a's-jasmone (herbal, woody), nerolidol (flower), and methyl jasmonate (flower). In addition, they found the total quality score positively correlated with

concentrations of benzyl alcohol, benzeneacetaldehyde, ge-raniol, indole and toluene (not identified in our study), but negatively correlated with the concentrations of (E,E)-2,4-heptadienal (identified by GC-O in our study). Other studies also showed benzaldehyde (almond) (Zhang et al. 2013), jasmine lactone (floral and fruity) (Wang et al. 2001, 2008, 2011; Zhang et al. 2013), and a-farnesene (woody)

(Kawakami et al. 1995; Wang et al. 2008, 2011; Zhang et al. 2013) play important roles in aroma of oolong tea and have high correlation with the aroma of quality oolong tea. Compounds such as (E)-P-damascenone (Zhang et al. 2013) and 5-methyl-hepten-2-one (Wang et al. 2008) are also reported as major flavor compounds, but they were not detected in our study. In addition, phenylethyl alcohol was identified in high concentration in the unpanned tea but was not detected in the panned tea. There were significant reductions (P < 0.05) in amounts of trans-nerolidol, 2-hexenal, benzaldehyde, indole, geraniol, and benzenacet-aldehyde as a result of panning. However, panning caused significant increases (P < 0.05) in contents of linalool oxide, a's-jasmone, and methyl salicylate. There were no significant differences (P > 0.05) in content of linalool, jasmine lactone and benzyl alcohol between panned and unpanned tea.

Furfural was the most dominant volatile in the panned tea analysed using SDE extraction and was also identified as the second most abundant compound using SPME. Furfural was not identified in unpanned tea with either extraction technique using GC-MS analysis, but we did identify it as aroma active compound with GC-O. Furfural has been reported in flavor profile of oolong tea in previous studies (Wang et al. 2008; Pripdeevech and Machan 2011; Zhang et al. 2013). Furfural has been found in an extensive range of teas, coffees, fruits, and wine and has been used as an ingredient for flavor enhancements in food (Rega et al. 2009). Furfural odor is like baked bread, almond and sweet (Rega et al. 2009). Furfural is formed by the heat treatment or acid hydrolysis of polysaccharides, which contain hexose and pentose fragments (IARC 1995). Panning can create compounds that are generated in the thermal degradation of cellulose and hemicellulose such as furfurals, 5-methyl-2-furancarboxaldehyde and other furans (Guillen and Manzanos 1997; Sung 2013).

Pripdeevech and Machan (2011) also indicated that the process of steaming or panning at high temperature in non-fermented tea method may produce lipid degradation products such as heptanoic acid, 2,6,6-trimethyl-1-cyclohexene-1-carb oxaldehyde, or nonanoic acid. In comparison to unpanned tea, many pyrrole compounds were identified in the panned tea such as 1H-pyrrole,1-ethyl-pyrrole, 1-methyl-1H -pyrrole-2-carboxaldehyde, 1 - (1 H-pyrrol-2-yl) -ethanone, 2-acetylpyrrole, and 2,4-dimethyl-1H-pyrrole-3-carboxylic acid methyl ester. The generation of these nitrogen-containing heterocyclic compounds is assumed to be caused by the Strecker degradation of theanine and amino acids during the tea preparation, and is responsible for the aroma of the heat treated teas (Yu et al. 1999).

GC-O analysis

In SDE extraction, 47 aroma active volatiles were tentatively identified based on the combination of LRI and odor descriptors in panned oolong tea and nine of these compounds were also identified by GC-MS analysis (Table 2). Among these compounds, 17 compounds were previously identified by both extraction techniques in the unpanned tea. However, in SPME, we identified 42 compounds that possessed aroma activity, but only 10 of these compounds were identical to extracted aroma compounds from SDE (Table 3). Among these 42 compounds, 17 compounds were shared with unpanned tea and only six of these 42 compounds were detected by GC-MS analysis.

The identified aroma components of panned and un-panned tea for both extraction techniques were grouped in six categories based on their aroma description: fruity, sweet, floral, nutty, green, and smoky/burnt. The total aroma intensities of identified aroma compounds from SDE and SPME for each aroma group are shown in Figs. 3 and 4 respectively. The most important features that were

Figure 3. Radargram of aroma profile of panned oolong tea using SDE obtained from grouping of identified compounds using GC-O with similar aroma characteristics.

Figure 4. Radargram of aroma profile of panned oolong tea using SPME obtained from grouping of identified compounds using GC-O with similar aroma characteristics.

consistent between these two Figures were the increase of sweet aroma and decrease of fruity and smoky in the panned tea. Floral aroma was not considerably affected by panning. Green aroma was the most different feature between two Figures. Previously, our panelists were unable to detect any green aroma in unpanned tea from SPME extraction; however, for the panned tea, a number of compounds responsible for green and grassy flavor were identified. On the other hand, with SDE the green aroma was slightly decreased. The other inconsistent result was related to nutty aroma. In SDE, there were more intense aroma active compounds with nutty smells detected in the unpanned tea. In contrast with SDE results, total aroma intensity for nutty smell was increased by panning which is more consistent with the literature. The differences in two methods' capabilities have been indicated in some other flavor studies. A SPME extraction technique was reportedly unsuitable for the isolation of high molecular compounds or for those with a strong affinity to the matrix; however, some compounds cannot be detected in SDE due to the presence of solvent (Majcher and Jeleii 2009), and also some artificial compounds can be generated during the extraction.

Among the identified aroma active compounds in the panned tea using SDE, pentanal (smoky), dihydromethylcy-clopentapyrazine (nutty), and one unidentified compound (nutty) had the highest aroma intensity in the panned tea. Dihydromethylcyclopentapyrazine and the unidentified compound had nutty flavors and were also detected among the most intense aroma compounds in the unpanned tea. However, limonene oxide (fruity) was the only compound that scored 4 in SPME. We were able to identify (E, E)-2, 4-heptadienal (nutty) and (Z)-3-hexenol (green) in our GC-O analyses, which have been shown to be increased by degree of fermentation (Wang et al. 2008, 2011). Similar to the unpanned tea, trans-2-hexenol was identified, which is considered an off-flavor in oolong tea. We also detected pyrazines such as ethylmethyl pyrazine and dihydromethylcyclopen-tapyrazine, which are known as thermally generated product of amino acids and sugars (Kato and Shibamoto 2001; Wang et al. 2010); however, both of these compounds were found in unpanned tea as well. Jin Xuan oolong is well-known for its milky aroma. Previously, we identified dieactyl with butter aroma and suggested that the milky aroma of this variety of tea is associated with this compound. However, this compound was not detected by our panelists in the unpanned tea and panning might have resulted in elimination of the milky aroma in Jin Xuan oolong.


Despite a few similarities in the most abundant identified compounds from GC-MS analysis and aroma active

compounds from GC-O analysis between the unpanned and panned tea, panning significantly changed the aroma volatile components of oolong tea. The abundance of alcohols, ketones, acids and esters were significantly changed by panning; however, there were no changes in contents of terpenes and aldehydes. Since over-heating/panning the leaves may result in a burnt odor and underpanning may result in a greenish odor and red central vein (Hui et al. 2003), optimization of time and temperature in panning to manufacture best quality tea need to be investigated for the future studies. Moreover, conducting sensory studies to better understand consumer perception of panning effects on quality of oolong tea is necessary for large-scale manufacturing and commercialization of panned tea.

Conflict of Interest

None declared. References

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