Two and a Bud 59(2):112-118, 2012

RESEARCH PAPER

Characterization of volatile flavour constituents of orthodox black tea of

twenty nine Tocklai released cultivars for Darjeeling

L. P. Bhuyanl, Kula Kamal Senapati, Pranjal Saikia and Mridul HazarikaDepartment of Biochemistry, Tocklai Experimental Station, Tea Research Association, Jorhat - 785008, Assam, India

ABSTRACT

Tea Research Association, Tocklai has released several cultivars to the tea Industry in Darjeeling; however, understandingofthe flavour characteristics of these cultivars is much to be desired. Quantification ofVFC and determination of flavourindex will help the Industry in the choice of right kind of planting materials. The objective of this study was to compare thevolatile flavour constituents of orthodox black teas of29 TRA released cultivars for Darjeeling.

Eighteen aroma constituents were identified and estimated by comparing with available authentic standards in Gas LiquidChromatography. The identified components were hexanal, t-2-hexenal, 1-hexanol, cis-3-hexene-l-01, linalool oxide 1,

linalool oxide II, benzaldehyde, linalool, phenylacetaldehyde, a-terpineol, methyl salicylate, nerol, geraniol, benzylalcohol,2-phenyl ethanol, a-ionone, nonanoic acid and geranic acid. Relative levels oflinalool oxide 11, linalool, methyl salicylate,geraniol and 2-phenyl ethanol were observed to be higher in comparison with other volatile components. The cultivarBennockburn 668 showed the highest flavour index followed by Kopati 1/1, CP 1,AV 2, Bennockburn 777 etc.

Trans-2-hexenal, Iinalool oxide U, Iinalool, methyl salicylate, geraniol and 2-phenyl ethanol are the determining componentsfor quality ofDarjeeling orthodox black tea. High concentration ofthese components except t-2-hexenal produced highflavour index in cultivars Bennockburn 668, Kopati 111, CPl,AV 2 etc. which was in conformity with the flavour potentialoftasters' evaluation.

INTRODUCTION

It is a well-recognized fact that the composition andconcentration of aroma compounds playa vital role indetermining quality and valuation of Darjeeling orthodoxblack tea. Flavour of Darjeeling tea is unique in the worldalthough other teas also have characteristic flavour oftheirown. The peculiar characteristic 'Darjeeling flavour' helpthe Darjeeling tea estates to fetch high prices and is a factorresponsible for consequent well-being of the Darjeelingtea industry. Several factors influence VFC and the endproduct of black tea quality. However, the knowledge ofsuch attractive aroma and flavour is very little but it is wellestablished that high altitude clubbed with peculiar agro­climatic conditions contributes substantially towardsattaining high flavour in Darjeeling teas (Ganguli, 1980).The genetic make-up of the cultivars and conditions ofblack tea processing are also important factors for

I Corresponding author: bhuvanlp@yahoo.co

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development of aroma and flavour and desirable cupcharacters (Takeo, 1981 a; Roberts, 1962; Roberts andFernando, 1981, Bhuyan and Mahanta, 1989). Though thegenetic make-up of the tea cultivars determines the type ofchemical constituents, the biosynthetic reactions in theirformation is controlled mainly by environmental conditions(Wickremasinghe, 1974). The characteristic volatile flavourconstituents are mostly formed by the hydrolytic action ofenzymes on cell constituents during leaf maceration. Inorthodox processing, disruption of cell is less duringmaceration, which is favourable for the production of aromacomponents. (Owuor et al., 1989). Thus the processingtechnique largely determines the quantity of flavourconstituents in made tea (Hazarika and Mahanta, 1983;Takeo, 1981b;Renoldetal., 1974).

Nearly 800 volatile compounds, collectively known as aromacomplex, have been detecTed in various types of tea. Although,

many aroma compounds have been identified in various teas,the biogenetic pathways for the formation of many of thesecompounds have not been fully understood. These aromacompounds in tea can broadly be classified into primary andsecondary products. The primary products are thosesynthesized by the tea plant directly in the growing shoots.On the other hand the secondary products are derived fromcarotenoids, amino acids, terpene glycosides and lipidsespecially from unsaturated fatty acids via hydrolytic andoxidative reactions with certain enzymes during black teaprocessing (Sanderson and Graham, 1973; Hatanaka, 1983;Hatanaka et al., 1982; Motoda, 1979; Wickremasinghe et al.,

1979; Saijo and Takeo, 1973; Mahanta et al., 1993; Sakata etal., 2004, Wang et al., 2000, 2001 and 2004). A good number ofsecondary metabolic products such as hexanal, Z-3-hexanal,hexenal, hexenol, Z-3-hexenal, Z-3-hexenol, E-2-hexenal, E-2­

hexenol, hexanoic acid, linalool, geraniol, a-ionone, a-ionone,3-hydroxy-a-ionone and other terpine aldehydes and ketones,2-methyl-propanol, 2-methyl butanal, pentanal, phenylacetaldehyde, benzyl alcohol etc. have already been identifiedand estimated by different investigators (Hazarika andMahanta, 1983, 1984; Howard, 1978, Wickremasinghe, 1974;Co and Sanderson, 1970; Dev Choudhoury and Bajaj, 1980;Saijo, 1973; Saijo and Takeo, 1972; Hatanaka et aI., 1987;

Selvendran et al., 1978;). The aroma generation of Darjeel ingblack tea is a complex phenomenon consisting offour linalooloxides, linalool, geraniol, hexanoic acid, benzyl alcohol, 2­phenyl ethanol, methyl salicylate, geranic acid, 3,7-dimethyl­1,5,7-octatrien-3-ol and 2,6-dimethyl-3,7-octadiene-2,6-diol(Kawakami et aI., 1995,2004,2010).

Table 1. Characterization of cultivars according tomorphological study, yield potential and tasters'evaluation for Darjeeling (Singh, 1989)

Cultlvars ()Jigin Category Flavour potentialTukdah 145 Assam hybrid Average

Bannockburn 668 ASSrull hybrId •• Very good

Bannockburn 777 Olina hybrid •• Average

K opati III Assam hybrid •• Very goodPhoobsering 312 Olina hybrid Good

Phoobsering 1404 ASSrul1 hybrId Average

Phoobsering 1258 China hybrid Average

Tukdah 78 at ina hybrid GoodTukdah 383 at ina hybrid Very good

Tukdah 246 Clonal cultivar AverageTukdah 135 Assam hybrid Good

Bannockbul11 157 Cambod hybrid Good

Rungli Rungiliot 17i144 Ch ina hybrid GoodTh urbo 3 Ch in a hybrid Goo d

Thurbo 9 China Cambod hybrid • Good

Balasun 7/1N76 China hybrid • Good

Balasun 9/3176 Ch ina hybrid GoodAV2(Balai) China hybrid Very good

TV I China hybrid Average

Sikkim I China hybrid GoodBadamtrull 15/263 Ch ina hybrid Good

TV 14 Assam hybrid GoodNrulda Devi Sl 378 Biclonal seed Good

Tukdah 253 Clonal cultivar ••• Above average

Rungli Rungiliot 4/5 China hybrid ••• Average

CP I Assam hybrid ••• AverageSundaram (13/5/63) ASSrull hybrid ••• Average

Happy valley 36 Assam China hybrid ••• Average

TV 19 Cambod hybrid ••• Good

* Standard: Above average yield and quality with yieldpotential of3000-3500kg made tea/ha/year

** Quality: High quality but average yield with yield potentialof2500-2800kg made tea/ha/year

Chemicals

MATERIALS AND METHODS

Collection of samples

*** Yield: Average quality but high yield with yield potential of4000kg made tea/ha/year

7. Phoobsering 12588. Tukdah 789. Tukdah 38310. Tukdah 24611. Tukdah 13512. Bannockburn 157

Authentic volatile flavour compounds were purchased fromSigma-Aldrich Chemicals Pvt. Ltd. (New Delhi) and otherchemicals from Merck. HPLC grade dichloromethane wasused for VFC extraction.

1. Tukdah 1452. Bannockburn 6683. Bannockburn 777

4. Kopati 1/15. Phoobsering 3 126. Phoobsering 1404

Tea shoots, usually two leaves and a bud, of 29 Tocklaireleased cultivars for Darjeeling were collected from theclonal probing station, Ging T.E., Darjeeling. The clonalprobing station is situated at an altitude of 1200 m abovemean sea level in a south-east slope. The cultivars were:

The mixture of volatile compounds identified in tea may alsobe categorized into two groups, group I and group II. Thecompounds having retention time lower than linalool oxideare ranked as group I, which impart inferior flavour to tea andcompounds having retention time greater than linalool oxideare ranked as group II, which give tea a superior flavour. It hasalso been reported that group II/group I, termed as flavourindex, is the determ ining factor of flavoury black tea (Owuor et

aI., 1986, 1988, 1989; Wickremasinghe et aI., 1973; Yamanishiet al., 1978).

Tocklai Experimental Station has released several cultivars forDarjeeling gardens. All of them have been evaluated at theClonal Probing Station, Ging T.E., Darjeeling, and describedbriefly under following heads: origin morphological descriptionofframe, leaf type, shoot size and pubescence, yield, resistanceto drought, pests and diseases, planting suitability etc., tasters'evaluation for quality and flavour and distinguishing featuresif any (Singh, 1989, Prakash, 1989). A brief description aboutthe origin of cultivars, yield potential and tasters' evaluationof29 cultivars are shown in Table 1. However:there is a gap inthe knowledge of the flavour characteristics of these cultivars.Hence, an investigation was undertaken to study the volatileflavour constituents of orthodox black tea of twenty nineTocklai released cultivars for Darjeeling.

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Extraction and estimation ofVFC

100 g of tea sample was taken in a 3-litre flask with 500mlde-ionized boiling water. The steam distillate was extracted

The black tea processing of all the cultivars was carried outin the miniature-manufacturing factory of Ging T.E.,Darjeeling.

As wither plays the most pivotal role in Darjeelingmanufactures and to get the liquor bright with a yellowishtinge, flavoury and mellow and also to meet the marketrequirement, the leaves were withered to a moisture level ofaround 50%. To maintain it the leaves were spread at athickness 00-4 inches, the hygrometric difference kept ataround 3°-4°C for a period of 16-18 h. The withered leaveswere then rolled for 70 minutes in a single-action roller intwo stages. The condition of the rolling pressure was asfollows:

RESULTS AND DISCUSSIONS

with 50 ml ofdichloro-methane for 45 min by using a modifiedLikens-Nickerson apparatus (Schultz et ai., 1977). Theextract was dried over anhydrous sodium sulphate for 12hours, filtered and the solvent was removed using EyalaCCA-I110 rotary evaporative concentrator. The solvent wasfurther reduced under purified nitrogen atmosphere to 100Jll.

The variations of 18 major volatile flavour components,viz. hexanal, t-2-hexenal, l-hexanol, cis-3-hexene-l-ol,linalool oxide I, Iinalool oxide II, benzaldehyde, linalool,phenylacetaldehyde, a-terpineol, methyl salicylate, nerol,geraniol, benzylalcohol, 2-phenyl ethanol, a-ionone,nonanoic acid and geranic acid of 29 Tocklai releasedcultivars for Darjeeling are presented inTable 2. Well-markedvariations of volatile flavoury components were observedamong the cultivars. Trans-2-hexenal, Linalool oxide-I,Linalool oxide-II, Iinalool, methyl salicylate, geraniol,phenylacetaldehyde, and 2-phenylethanol wereconsiderably higher than the rest of the flavourcomponents. Hexanal and t-2-hexenal were the highest inclone Happy Valley-39 followed by Phoobsering-1404,Tukdah-145, Thurboo-3, Tukdah-246, Bannockburn­668,etc. Both the VFCs contributed to grassy flavour of tealiquor. Higher concentration ofLinalool and Linalool oxideswere observed in cultivars like Bannockburn-668,

Phoobsering-312, Phoobsering-1404, Tukdah-383, Tukdah­145, Nanda Devi st. 378 etc.which produce sweetish flavorof tea liquor. An appreciable amount of Phenyl acetaldehydewhich has rose like sweetie aroma was obtained in TV 19,

Happy valley39, Nanda Devi stock 378, CPl, Tukdah-135,Tukdah-145, Bannockburn-668 and Bannockburn-777.Geraniol, the key component of Darjeeling tea that producesfloral aroma, was found in considerably higher amounts inBannockburn-668, Tukdah-145, Teesta Valley-I and Happy

Valley-39. Another major VFC methylsalicylate which alsocontributes floral aroma to the liquor, was observed in higherconcentration in Bannockburn-668, Tukdah-145,

Tukdah383, Phoobsering-1404 etc.

The concentrate thus obtained was analyzed in Varian GCmodel CP 3800 equipped with flame ionization detector (FID)and 60 m x 0.25 rom CP Wax 52 CB column. Nitrogen wasused as a carrier gas at a flow rate of 12 psi. Both the injectorand detector temperatures were 250°C at the split ratio ofI: 100. The column oven temperature was held at 60°C for 10min and programmed to 200°C at 2°/min and held for 20 minat final temperature. A good number of peaks were obtainedin the GC profile. Out of these peaks, depending on theavailability of standards, a few compounds of significancewere identified for the study. The amount of each componentwas determined and expressed as the ratio of each peakarea to that of internal standard in the chromatogram.

No pressureTouch pressureNo pressureLight pressureNo pressureNo pressureTouch pressureNo pressureMedium pressureNo pressure

22. TV1423. Nanda Devi st. 37824. Tukdah 253

25. Rungli Rungiliot 4/526. CPl27. Sundaram (13/5163)

28. Happy valley 3629. TV19

lOmin.lOmin.5min.10min.5min.5min.5min.5min.10min.5min.

1st roll 40 min.

2nd roll 30 min.

13.Rungli Rungiliot 17/14414.Thurbo 315.Thurbo 916.Balasun 711A/7 617.Balasun 9/3/76

18.AV2 (Balai)19.TVI20.Sikkim 121. Badamtam 1/263

Black tea processing

The rolled leaves were then put on fermentation trays for a

period of 2 hours. The temperature of the entire processwas maintained at 22 to 24°C. The fermented leaves were

then fired in a conventional batch type of dryer with inlettemperature of 100 to 110°C. Samples from different stagesof black tea manufacture were deactivated under steam on

min and dried. The following samples were thus obtainedfrom the different manufacturing stages: i) deactivated driedwithered leaf (DDWL); ii) deactivated dried rolled leaf(DDRL); iii) deactivated dried fermented leaf(DDFL) andiv) black tea (BT).

Conditions of roIling pressure

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Table 2. Volatile flavour components of different cultivars

Cll ItivaI's

--..T-145B-668B-77 7K-l/1P -312P-1404P-12 5 8T-78 T-383 T-246 T-135B -157R.R.-T-3

Components~

171144

Hexanal0.3160.2050.0340.0110.0540.4800.0480.1360.1280.2000.1830.0530.126 0.320

t-2-hexen al0.7770.4 750.2160.0670.3451.6230.1640.6210.6390.6731.0110.3000.339 0.838

I-hexenol0.0670.0550.0300.0460.0330.0560.Q3 5 0.0360.0360.0420.0620.0270.040 0.079

Cis-3-hexene-l-o!0.4260.0780.0390.0320.2250.3390.0410.1200.1360.0810.2040.0790.058 0.359

Linalool oxide [0.699L1180.6600.2660.3 820.6940.194 0.4470.4150.0970.5370.2620.137 0.360

Linalool oxide II2.86943862.00 I1.0211.2013.5080.5151.2501.2290.3371.7540.7770646 0.724

Benzeldehyde0.1730.0090.0750.0040.0660.1340.008 0.0980.0690.0050.2080.0770.060 0.086

L inalool43849.2262.8721.8727.0156.0350.7571.4298.8403.5063.1161.0840.748 3.855

Ph eny lacetaldehyde0.4240.3 860.472. 0.3340.4620.1010.096 0.4440.1120.0900.5620.2620.261 0.\25

Terpeniol

0.1170.4850.4290.2920.3140.1750.034 0.0720.3040.0810.15100590085 0.071Methyl Salicylate

2.5292.8770.4150.8861.0342.0040.273 0.5182.2080.6950.8360.4880.4351.160Nerol

0.1440.3370.1650.0590.0290.0810.040 0.0480.0710.1660.1280.1870.135 0.093Geraniol

16.86520.7383.8432.8510.9161.1710.433 2.1943.7970.4865.4182.93 I3702 2.573Benzylalcohol

1.0712.4000.2050.4590.51213480.1990.4310.2430.3381.7180.3940280 0.924

2-phenyl ethanol

30614.6210.4090.3390.4260.4910.1240.7810.2430.4700.7430.7630.299 0.67\p-ionone

0.1070.1990.0440.0510.0950.0990.128 0.0260.03300140.1080.0420.250 0.210Nonanoic acid

03950.1320.0200.1870.0760.2520.203 0.1370.0930.2560.0350.0880.072 0.(148G eranic acid

0.7890.1560.0560.0480.0610.0520.117 0.3120.0550.0590.0760.0570.4410.243

Cultivars

--+-T-9B- B-

AV-2lV-I5-1BT-TV-ND-T-253 R-415 CP-I

HV-TV-

C omponcnts ~

7/I/N769/3/76 15/26314378 13/5/638 3619Hexanal

0.008 0.0860.1660.0570.1540.024 0.0080.0030.0730.010 0.024 0.005 0.0660.6470.184t-2-hexenal

0.108 0.2420.4720.1220.7710.1110.0210.11 00.4300.028 0.119 0.061 0.3052.3800.771I-hexcnol

0.0210.0260.0400.0240.0350.069 0.0440.0560.0280.030 0.028 0.038 0.0550.2100.040C is-3-hexene-l-ol

0.0510.0820.1700.0490.1710.068 0.Q750.0%0.0670.036 0.052 0.053 0.0700.6340.095Linalool oxide I

O.OOS0.1640.3640.1060.1440.036 0.0170.4070.3430.023 0.133 0.214 0.3350.9330.152Linalooloxide II

0.075 0.5501.2660.4450.9560.191 0.0341.5401.2200.077 0.445 0.7931.%73.9330.522Benzeldehyde

0.004 0.0120.0780.0380.0680.007 0.0040.0810.1250.012 0.038 0.040 0.0410.2130.075Linalool

0.130 4.7565.5973.3311.1790.315 0.1402.4664.5200.210 0.4111.693 1.9913.4561.480Phenylacetaldehyde 0.061

0.3080.2600.0760.1390.053 0.0700.2850.4240.114 0.209 0.458 0.2340.5990.696

Terpeniol

0.0110.2170.2410.0440.0780.040 0.0£40.1410.2390.023 0.015 0.035 0.1440.0690.106M ethyl Salicylate

0.1031.9531.7041.2431.3000.258 0.0361.4441.2810.092 0.136 0.260 (1).7641.6381.166Nerol

0.Q35 0.0910.0940.0510.1640.050 0.0080.0490.1420.014 0.022 0.052 0.cl600.0910.046Geraniol

1.925 1.4721.9963.71811.862 3.208 03690.9265.6720.4310.937 3.353 2.3917.8-580.521Benzylalcohol

0.339 0.41l0.5480.144 0.956-0.173 0.0220.2520.5280.093 0.205 0.417 0.1320.934O.2OS

2-phenyl ethanol

0.113 0.3370.8421.316 0.7400.181 0.0180.2930.9080.146 0.191 0.6150.0711.5870.564~-ionOl1e

0.015 0.0960.1710.027 0.0570.030 0.0080.0060.3510.0110.012 O.l)t5 0.0100.0110.033Nonanoic acid

0.029 0.0440.1740.029 0.02&0.Q35 0.0260.0520.1l80.Q75 0.168 0.117 0.7270.2290.080Geranic acid

0.046 0.0380.0750.0910.2300.028 0.0280.0630.1520.040 0.251 0.0500.1170.1090.160

Trans-2-hexenal, Iinalool oxide II, Iinalool, methylsalicylate, geraniol and 2-phenyl ethanol are thedetermining components for quality ofDarjeeling orthodoxblack tea. High concentrations ofthese components exceptt-2-hexenal produced high flavour indices in cultivarsBennockburn 668, Kopati 1/1, CP I, AV2, Bennockburn777, TVI4, Nanda Devi stock 378 etc. (Fig. I) which isconcomitant with the flavour potential of tasters'evaluation.

The results of Cluster Analysis and Principal ComponentAnalysis (PCA) using 18 aroma components of29 cultivarsare presented in Figs. 2a and 2b.

The cluster table was divided into two clusters and both

the clusters contained essential quality components ofDarjeeling tea.

115

60

so

40

'0

'0

11. J "5 (, 7 ~ l) IOII121'1-41"1(.111~1')2(I!122212~2"~.rI~"~t<2'J

Fig. 1. Flavour Index of 29 Darjeeling cultivars

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Geraniol

Bionone

TerpenioN'onanoic

HexanolBenzalde

Nerol

BenzylalJIIethylSaPhenylet

L1nalo _2L1nalool

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~ r-----]

3

7

12161017

GeranicA 18Hexanal 1C1~3hl!xl! 4

L1.nalo_l 5

Phenylac 9Ttan:sHe.x 2

14

1115

6

B

13

Fig. 2a. Cluster Analysis of 29 Darjeeling cultivars

os..J

N'C

:!!

~ 00oU

~s~I

I

.'0-t,_·'0 00

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I05

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