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CHAPTER 16

Tea Processing and itsImpact on Catechins,Theaflavin and ThearubiginFormationAnakalo. A. Shitandi, Francis Muigai Ngure, Symon M. MahunguDepartment of Food Science, Egerton University, Egerton, Kenya

Abbreviations

EC ()-epicatechinECG ()-epicatechin-3-gallateEGC ()-epigallocatechinEGCG ()-epigallocatechin-3-gallateGC (þ)-gallocatechin

INTRODUCTION

Tea processing is an operation in which the leaves from the plant Camellia sinensis are altered to

dried leaves. There are a few varieties and hundreds of cultivars within the Camellia sinensis

species, Camellia sinensis var. sinensis and Camellia sinensis var. assamica are the varieties being the most commonly known. The number of cultivars and varieties employed differs between

the locations where tea is propagated. Some are unique and restricted to certain regions, but

others are similar and used on a large scale across different areas. Differences in processing

entail a different path of biochemical change within the tea leaves. Tea types are distinguishedby the processing means, and based on the degree of fermentation can be categorized as green

teas, white teas, yellow teas, oolong teas, black teas and post-fermented teas. All of these

different teas can be further processed by blending, scenting, or flavoring. Others can be mixed with other ingredients as additives or converted to other versions such as instant dissolvable

granules.

Black tea is the type which is most widely produced and consumed worldwide, accounting for

76e78% of the tea produced ( Yang and Landau, 2000; Cabrera et al., 2003). Black tea is

produced by extended fermentation of tea leaves. During processing, a tea shoot consisting of two leaves and a bud is plucked. The quality of the processed tea depends mainly on the raw

tea shoots. The factors which influence the tea quality include plucking standard, age of bush,

stage from previous pruning and plucking. The chemical composition within the shoot of first

leaf, second leaf, other leaves and stem also vary widely. On average the tea shoot contains up

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to 78% moisture and 22% solid matter. The solid matter is insoluble in water and is composed

of crude fiber, lignin, proteins, fats, chlorophyll and pigments, starches, pectins and cellulose.

The basic constituents that have an impact on the taste and color characteristics of tea include

polyphenolic bodies, caffeine, non-caffeine nitrogenous compounds, pectic substances,

minerals and other compounds jointly or separately. The leaf extract of the plant Camellia

sinensis is an important source of dietary polyphenols. During the fermentation of black tea the

monomeric flavan-3-ols undergo polyphenol oxidase-dependent polymerization, leading tothe formation of theaflavins, thearubigins and other complex molecules (Robertson, 1992;Mario et al., 2008). Flavan-3-ols play a major role in green tea and black tea quality (Obanda

et al., 2001) and have been reported to have important pharmacological properties, which

include: antioxidative activity (Satoshi and Hara, 1990; Pekal et al., 2011), antimutagenic

effects ( Yen and Chen, 1994), anticarcinogenic effects (Isao, 1990) and anticariogenic activity

(Hamilton-Miller, 2001). A maximum EGCG content (14.28 mg/g) has been achieved by

fermenting clone 6/8 green leaf at 18 C for 60 minutes and EGCG significantly contributes tothe black tea antioxidant activity among clones ( Tables 16.1 to 16.6). The pharmacological

roles of the catechins can be attributed either to their ability to inhibit enzymes, or to act as

reducing agents ( Adrian and Bolwell, 2000).

THE CHEMISTRY AND BENEFITS OF GREEN TEA Tea contains a number of bioactive chemicals, but it is particularly rich in catechins, of which

epigallocatechin gallate (EGCG) is the most abundant derivative (Higdon and Frei, 2003;

Seeram et al., 2006). Catechins and their derivatives are thought to contribute to the beneficial

effects ascribed to tea (Basu and Lucas, 2007; Pekal et al., 2011). Tea catechins and polyphenols

are effective scavengers of reactive oxygen species in vitro and may also function indirectly asantioxidants through their effects on transcription factors and enzyme activities (Higdon and

Frei, 2003; Basu and Lucas, 2007).

The chemistry of low-molecular-weight polyphenols which have been isolated and charac-terized from black tea has been identified, and the likely mechanisms of their formation

studied (Drynan et al., 2010). Besides the catechins, green tea also contains Kaempferol,

quercetin and myricetins glycosides in small amounts (Balentine et al., 1997). The principalalkaloids are caffeine, theobromine and theophylline, while phenolic acids and amino acids

are also present in small quantities ( Yang and Landau, 2000).

The components of green tea beverage and their average compositions include: catechins

(30e42%), flavonols (5e10%), caffeine (3e5%), theogallin (2e3%), quinic acids (2%),

theanine (4e6%), theophylline (0 .03%), theobromine (0 .1%), carotenoids (0.02%),mineral content (6e8%). The polyphenols occurring in the tea plant are derivative of

gallic acid (C6H2(OH)3COOH) and catechin (C15H14O64H2O). Catechins represent the

extractable solids, and are the most abundant green tea solids. The three most important

chemical substances in fresh tea leaf are caffeine, aromatic or essential oils, and the

TABLE 16.1 Variation in Gallic Acid, Catechins, Total Polyphenols and Antioxidant Activity in Clonal Black Teas (mean ± SD)

CloneGA ( mg/g)

EGC(mg/g)

( þ )C(mg/g)

EC(mg/g)

EGCG(mg/g)

ECG(mg/g)

TTC(mg/g)

TPP(mg/g)

AA (%)

6/8 7.57 0.9a 3.08 1.5a 3.96 1.6a 5.41 3.4a 5.73 3.2a 3.75 2.5a 29.53 12.9a 108.18 11.1a 84.95 5.5a

303/577 5.28 2.2c 2.31 1.2b

n ¼ 24

2.52 0.5b

(n ¼ 27)

1.97 2.0c 4.17 2.3c 2.75 2.0b 16.63 11.2c 96.32 7.1b 80.64 5.8c

311/287 6.86 0.7b 1.86 0.7c 2.52 1.0c 3.23 1.8b 5.49 3.0b 2.38 1.7c 22.36 8.8 b 95.09 9.8 c 83.67 4.4b

CV 6.57 7.11 4.47 8.66 6.08 5.81 3.58 2.82 2.83

Means followed by the same letter are not significantly different at p < 0.05. n ¼ 36 except where otherwise indicated.

(Ngure, 2008)

94

SECTION 3

Manufacturing and Processing



TABLE 16.2 Variation in Gallic Acid, Catechins, Polyphenols and Antioxidant Activity for Clonal Black Teas with Fermentation Temperature and Time

Duration 60 90 120 150

Clone Temp Mean SE Mean SE Mean SE Mean SE

GA ( mmol/g DM)6/8 18 9.01 0.08 8.58 0.05 7.92 0.18 7.61 0.11

24 8.57 0.42 7.49 0.23 7.09 0.28 6.32 0.2130 7.79 0.66 7.36 0.24 6.87 0.46 6.28 0.71

303/57718 7.83 0.01 7.14 0.14 6.57 0.04 5.45 0.0524 7.33 0.05 6.65 0.05 2.74 0.08 2.46 0.0430 6.85 0.04 6.44 0.05 2.15 0.07 1.86 0.07

311/28718 7.79 0.03 6.84 0.25 7.35 0.13 6.85 0.2624 8.07 0.23 7.32 0.29 6.49 0.38 6.47 0.1030 7.4 0.22 6.23 0.23 5.97 0.03 5.62 0.15

EGC (mg/g DM)

6/8 18 7.01 0.11 5.16 0.37 3.56 0.00 2.78 0.0624 3.74 0.25 2.40 0.07 2.07 0.09 1.60 0.0330 2.95 0.09 2.52 0.11 2.08 0.09 1.15 0.01

303/57718 4.77 0.07 2.09 0.06 1.83 0.06 nd nd24 3.38 0.07 2.09 0.04 0.77 0.03 nd nd30 2.76 0.08 0.79 0.07 nd nd nd nd

311/28718 3.74 0.05 2.34 0.04 2.15 0.04 1.49 0.0224 2.56 0.03 1.92 0.06 1.54 0.05 1.15 0.0430 2.15 0.05 1.57 0.05 1.14 0.06 0.64 0.01

( D )-C (mg/g DM)

6/8 18 6.11 0.08 5.92 0.08 4.76 0.01 3.39 0.0624 5.95 0.06 4.19 0.03 2.34 0.03 1.41 0.0230 4.87 0.03 4.25 0.03 3.05 0.02 1.33 0.05

303/57718 3.86 0.04 2.37 0.04 2.14 0.05 nd nd24 3.16 0.04 2.35 0.03 2.22 0.01 nd nd30 2.49 0.01 2.28 0.01 1.89 0.03 nd nd

311/28718 4.74 0.21 3.34 0.21 2.25 0.14 1.6 0.0724 3.76 0.09 2.8 0.11 1.92 0.06 1.49 0.0230 2.91 0.15 2.23 0.05 1.78 0.07 1.43 0.08

EC (mg/g DM)

6/8 18 13.24 0.43 9.02 0.34 6.31 0.22 2.99 0.17

24 8.35 0.07 3.73 0.29 2.84 0.10 1.90 0.1830 7.19 0.43 5.15 0.08 3.18 0.14 1.09 0.17

303/57718 7.47 0.09 3.64 0.05 1.93 0.07 0.52 0.0324 1.38 0.07 0.47 0.06 0.4 0.08 0.21 0.0730 3.58 0.16 2.28 0.08 1.33 0.08 0.54 0.08

311/28718 5.99 0.18 5.30 0.13 4.47 0.04 1.53 0.1824 6.44 0.05 3.28 0.10 2.68 0.21 2.28 0.2530 2.84 0.22 1.96 0.10 1.3 0.11 0.71 0.09

Continued

CHAPTER 1

Tea Processing and its Impact on Catechins, Theaflavin and Thearubigin Formatio



TABLE 16.2 Variation in Gallic Acid, Catechins, Polyphenols and Antioxidant Activity for Clonal Black Teas with Fermentation Temperature andTimedcontinued

Duration 60 90 120 150


EGCG (mg/g DM)

6/8 18 14.28 0.06 9.33 0.33 6.42 0.03 3.99 0.0424 7.40 0.06 3.92 0.06 2.88 0.06 2.12 0.0130 6.26 0.04 4.95 0.06 3.84 0.04 3.47 0.03

303/57718 10.36 0.29 6.06 0.19 4.64 0.18 3.62 0.2324 5.80 0.17 3.75 0.19 3.31 0.15 2.37 0.0630 3.54 0.18 2.86 0.16 2.52 0.24 1.22 0.02

311/28718 12.13 0.47 8.07 0.14 5.67 0.11 4.10 0.2824 9.45 0.29 5.65 0.14 4.23 0.29 3.57 0.0330 6.15 0.16 3.29 0.15 2.45 0.14 1.24 0.07

ECG (mg/g DM)6/8 18 9.83 0.05 6.36 0.04 3.61 0.05 2.88 0.03

24 5.93 0.06 2.93 0.04 1.34 0.06 0.68 0.0730 4.24 0.03 3.79 0.09 2.26 0.03 1.25 0.03

303/57718 7.87 0.03 4.16 0.04 2.28 0.10 1.99 0.0324 4.57 0.02 1.75 0.04 1.15 0.01 0.75 0.3230 4.26 0.09 2.27 0.02 1.23 0.01 0.77 0.07

311/28718 6.44 0.17 3.72 0.10 2.87 0.06 1.29 0.1024 4.58 0.18 2.50 0.10 1.23 0.09 0.71 0.0430 2.52 0.09 1.76 0.27 0.58 0.08 0.40 0.04

Total Catechins (mg/g DM)

6/8 18 59.48 0.42 44.36 1.03 32.58 0.38 23.62 0.2824 39.95 0.79 24.67 0.21 18.56 0.17 14.03 0.1930 33.30 1.07 28.02 0.37 21.28 0.54 14.57 0.69

303/57718 42.15 0.24 25.45 0.32 19.37 0.11 6.12 0.2524 25.61 0.37 17.50 0.37 10.59 0.29 3.32 0.4130 23.47 0.2 16.93 0.12 6.97 0.28 2.52 0.16

311/28718 40.82 0.37 29.62 0.37 24.77 0.29 16.88 0.6324 34.86 0.55 23.46 0.42 18.09 0.74 15.66 0.2430 23.97 0.44 17.05 0.74 13.22 0.23 10.03 0.18

Total Polyphenols (mg/g DM)

6/8 18 127.29 0.55 117.59 2.69 115.08 2.99 105.84 0.5224 118.97 2.63 108.29 0.29 102.67 1.16 97.57 0.4430 114.04 2.40 108.57 0.98 93.42 0.44 88.85 1.11

303/57718 104.15 1.57 102.93 0.81 99.52 0.49 94.41 2.6124 103.23 0.64 100.73 0.27 94.89 2.32 85.57 0.7330 102.23 0.64 93.83 3.50 88.91 1.07 85.43 2.29

311/28718 107.33 1.06 104.33 0.74 98.74 0.49 92.80 1.7624 110.86 1.19 92.37 1.22 89.57 0.49 84.40 0.6830 103.52 2.68 93.12 2.02 82.55 2.48 81.56 0.43

SECTION 3


96



catechins/polyphenols ( Wang and Ho, 2009). The enzymes and polyphones are crucial for

the biochemical changes which occur during processing. The enzymes polyphenol oxidaseand peroxidase are important as they are the catalysts for oxidation. Teas from different

regions have different capacities to oxidize due to the variations on levels of polyphenol

oxidase.

Tea polyphenols demonstrate biochemical and pharmacological properties, which includeantioxidant activities, inhibition of cell proliferation, induction of apoptosis, cell cycle arrest

TABLE 16.2 Variation in Gallic Acid, Catechins, Polyphenols and Antioxidant Activity for Clonal Black Teas with Fermentation Temperature andTimedcontinued

Duration 60 90 120 150


Total Polyphends (mg/g DM)

6/8 18 9.38 0.37 88.68 0.44 86.47 0.93 84.13 0.9424 91.09 0.19 84.63 1.60 81.11 1.18 79.58 1.7830 89.76 2.04 83.21 2.68 82.48 2.92 75.91 3.90

303/57718 87.4 0.81 87.25 0.29 84.55 0.69 81.66 0.6224 85.78 0.51 84.33 0.41 79.91 1.16 72.76 2.5030 82.79 0.51 76.92 0.92 73.42 0.72 70.98 1.46

311/28718 88.38 0.68 87.49 1.68 84.31 0.26 81.48 1.7124 89.4 0.75 86.91 0.28 80.42 0.91 76.29 1.2530 88.2 0.78 82.48 0.39 80.66 0.38 78.04 0.87

( Ngure, 2008 )

TABLE 16.3 Antioxidant Activity Regressed Against Gallic Acid and IndividualCatechins

Linear Model

Antioxidant Activity Parameter Standard Variable Estimate Error t Value Pr > jtj

Intercept 69.426 2.285 30.38 <0.0001GA 1.117 0.380 2.94 0.0042EGC 0.539 0.646 0.83 0.4067C 1.752 0.468 3.74 0.0003EC 0.530 0.277 1.91 0.0590EGCG 0.712 0.243 2.93 0.0043ECG 0.296 0.456 0.65 0.5187

Root MSE 2.849Dependent mean 84.138CV 3.386R-square 0.658 Adjusted R-square 0.635 ANOVA

Source df Sum of squares Mean square F Value Pr > FModel 6 1392.355 232.059 28.59 <0.0001Error 89 722.413 8.117Corrected total 95 2114.769

(Ngure, 2008)

CHAPTER 1




potential biological activity and could also be used as nutraceuticals and for preservationpurposes in food formulations (Borse et al., 2007)

The character of tea is influenced by many factors, including the sub-varieties and cultivar used, elements of the growing environment, and plucking conditions (Owuor and Obanda

2007). The two most well-known varieties used in different production locations are Camellia

sinensis var.

sinensis and

Camellia sinensis var.

assamica.

TEA PROCESSING

Tea production begins with tea plucking, and then the freshly harvested tea leaves require

processing to convert them into green, oolong and black teas. Variables in harvesting andprocessing determine the type and quality of the tea. Tea is traditionally classified based on the

degree or period of oxidation/fermentation the leaves have undergone (Cabrera et al. ,

2003).The degree of processing and oxidation/fermentation determines whether the teabecomes a black, oolong, green or white variety. Inhibiting the interaction of the enzymes and

the catechins produces green tea. The main difference when making green tea is thus the

omission of the oxidation stage, allowing the tea to remain green in color. In order to ensure

that the freshly picked leaf does not oxidize, the leaf is steamed, after which it is rolled.

The enzymes are consequently inhibited and oxidation prevented. Oolong tea on the other hand is produced through partial oxidation of catechins.

Black tea processing typically consists of six distinct stages, namely plucking/picking and

transport to factory; withering, which is physical and biochemical; cell maceration; fermen-

tation/oxidation; drying; sorting and packing. Black tea undergoes various processing andoxidation steps that create the characteristic flavor and color of tea. As a result of the

processing, leaves from the Camellia sinensis plant are transformed into the dried leaves for

brewing tea. Black tea is the type most widely produced and consumed worldwide, and

accounts for 76e78% of the tea produced (Cabrera et al., 2003). The flavor of the tea leaves is

determined by the type of cultivar of the tea, the quality of the plucked tea leaves and the

manner and quality of the production processing they undergo (Obanda et al., 2004).

Tea processing is considered the art of tea as much of the uniqueness in taste, body and overallcharacter is formed at this point. Different ways and degrees of oxidation of the leaves occur before drying ( Adrian and Bolwell, 2000). Oxidation as a process is initiated when the leaf

is broken and the enzymes are exposed to oxygen. The degree of oxidation is dependent upon

how much of the enzymes are exposed and for how long. Essential plant oils, caffeine and

polyphenols are some of the substances specific to tea leaves which contribute to its character.

The essential oils provide the aroma of tea, caffeine is a stimulant, and the polyphenols

contribute to tea’s antioxidant properties. The polyphenols further contribute to the solublematter in the fresh tea leaf. During the fermentation process about one third of the total

constituents in green leaf are oxidized into oxidized and unoxidized polyphenols.

IMPACT OF TEA PROCESSING ON CATECHINS, THEAFLAVIN

AND THEARUBIGIN FORMATIONCatechins are a group of polyphenolic compounds which are found in several plant foods

including tea(Goodin et al., 2002). Theyare known tomakeup 30% ofthe dry weightof greentealeaves, as indicated earlier. The levels of the catechins differ with the nature and extent of

processing, being lowest in black tea, and somewhat higher in oolong, green tea and white tea.

White teas are not fermented and are balanced/steamed to inactivate the enzyme polyphenoloxidase and then dried, hence they contain the highest concentration of catechins. Green tea

processing entailsthe steaming of fresh tea leaves, followedby dryingto inactivate thepolyphenol

oxidase enzyme. This maintains the polyphenols in their monomeric state (Zaveri, 2006). In

a study by Basu and Lucas (2007), green tea,rich in antioxidant and anti-inflammatory catechins,

CHAPTER 1




especially epigallocatechin gallate (EGCG), was shown to reduce surrogate markers of athero-sclerosis and lipid peroxidation, particularly LDL oxidation and malondialdehyde concentra-

tions, in several in vitro, animal and limited clinical studies. Another study compared the

protective effects of polyphenol ()-epigallocatechin gallate (EGCG) and other antioxidants onlipid peroxidation in gerbil brain homogenates. EGCG was observed to be the most potent

antioxidant in inhibiting ferrous ion-induced lipid peroxidation (Lee et al., 2003).

Oolong teas are only partially fermented as they are oxidized for shorter periods than black

teas. They thus have intermediate catechin concentrations.

Black tea is produced by extended fermentation of tea leaves which produces the polymeric

compounds thearubigins and some theaflavins. The levels of these compounds are thus

lowest in white teas, followed by green tea, oolong teas and highest in black tea e theopposite order to the catechins. Temperature has an influence on oxidation, as the uptake of

oxygen increases at temperatures up to 29 C (84 F), thus bringing the enzymes to their peak

activity. Black tea is known to be an important source of dietary phenolics among regular

consumers of tea (Rechner et al., 2002). Epigallocatechin gallate, four theaflavins, as well as

epicatechin gallate, theogallin, quercetin-3-rutinoside and 4-caffeoyl quinic acid are the

main phenolic compounds that have been identified in brewed black tea (Rechner et al.,

2002).

Oolong tea is a partially fermented product and contains a mixture of the monomeric poly-

phenols and higher-molecular-weight theaflavins (Graham, 1992; Zaveri, 2006). All the varieties of tea contain significant amounts of caffeine (3e6%) which is unaffected by the

different processing methods (Zaveri, 2006).

Polyphenols in black tea undergo alteration into other forms of flavonoids and taste/aroma

components during processing. Theaflavin and thearubigin are two compounds which give an

indication of the quality of tea. The type of fermentation process which tea leaves undergodetermines the level and impact of the chemicals found in various tea types (Ngure et al. ,

2009). In research, black teas from the clones studied differed significantly (p < 0.05) in their

levels of gallic acid, individual catechins and total polyphenols ( Tables 16.1 and 16.2). A

significant difference was observed for the antioxidant activity of the resultant clonal black teas(p < 0.05). Of the three clones studied, clone 6/8 black teas had the highest antioxidant

activity, while clone 303/577 black tea had the lowest ( Tables 16.3e16.6). Tea polyphenols act

as antioxidants in vitro by scavenging reactive oxygen and nitrogen species and chelating redox-

active transition metal ions (Frei and Higdon, 2003). They may also function indirectly asantioxidants through the inhibition of the redox-sensitive transcription factors and inhibition

of ‘pro-oxidant’ enzymes (Frei and Higdon, 2003).

There are several polyphenolic catechins in green tea of which the main six are ()-epica-techin (EC), ()-epicatechin-3-gallate (ECG), ()-epigallocatechin (EGC), ()-epigalloca-

techin-3-gallate (EGCG), (þ)-catechin, and (þ)-gallocatechin (GC) (Goodin et al., 2002;

Chiu and Lin, 2005; Zaveri, 2006). The ‘epi’ signifies the structural formula illustrated in

Figure 16.1, with different orientations which determines the ease of oxidization. The three

predominant catechins are EGCG, ECG, and EGC (Goodin et al. , 2002; Zaveri, 2006), of which EGCG is the most abundant, accounting for 65% of the total (Chiu and Lin, 2005;

Chien-Wen et al., 2007).

Flavan-3-ols are a large subclass of plant phytochemicals, known as flavonoids. During the

fermentation of black tea, it is well established that the monomeric flavan-3-ols undergopolyphenol oxidase-dependent polymerization. This results in the formation of theaflavins,

thearubigins, bisflavanols and other complex oligomers (Robertson, 1992; Malik et al., 2003;

Mario et al., 2008). Flavan-3-ols play a major role in green tea and black tea quality (Obanda

et al., 2001) and have been reported to have important pharmacological properties, whichinclude antioxidative activity (Satoshi and Hara, 1990), antimutagenic effects ( Yen and Chen,

SECTION 3


00



1994) and anticarcinogenic effects (Isao, 1990). The pharmacological roles of the catechinscould generally be attributed either to their ability to inhibit enzymes or act as reducing

agents ( Adrian and Bolwell, 2000).

The antioxidant activity of catechins depends on their structure and the number of hydroxyl

groups (Richelle et al., 2001). EGCG is thought to have the highest antioxidant capacity

because it is extensively hydroxylated ( Adrian and Bolwell, 2000). In vitro, the antioxidativecapacity of green tea is much higher than that of black tea (Serafini et al. , 1996).

However, Zeyuan et al., (1998) reported that the antioxidative ability of black tea was better

than that of green tea in aged rats, and it had been reported earlier that black tea produceda response at the same antioxidative intensity to that of green tea in vivo (Serafini et al.,

1996).

Variation in the individual green leaf catechins present in the germplasm of 102 Kenyan tea

cultivars has been reported (Magoma et al., 2001). The study reported the existence of

catechin-rich tea clones with a potential for use in pharmacological preparations. A further study established differences between individual green leaf flavan-3-ols of 11 popular clones

(Owuor and Obanda, 2007). Several studies have been devoted to investigating the quality

potential of diverse clonal teas (Obanda et al., 1992; Owuor and Obanda, 2007) and theeffect of fermentation conditions on black tea quality (Obanda et al., 2004; Ngure et al.,

2009). The black teas produced from the clones studied were found to differ in their levels of

gallic acid, individual catechins, total polyphenols ( Table 16.1) and antioxidant activity

(Obanda et al., 2004; Owuor and Obanda, 2007; Ngure et al., 2009).

FIGURE 16.1

The Flavan-3-Ols (catechins) in Fresh Tea Leaves.

CHAPTER 1




Establishing the optimum fermentation time of black tea has been of interest to researchers. This derives from the premise that developing clone-specific processing technologies would

take advantage of the inherent genetic potential of catechin-rich clones to add value to black

tea by enhancing the health potential of the most consumed type of tea. A catechin-rich black tea of good quality would be an important source of potent antioxidants. Ngure et al. (2009)

investigated the effect of clonal differences and fermentation conditions on black tea residual

catechins and antioxidant activity.

Interactions of fermentation temperature and duration have no marked effect on gallic acid

and antioxidant activity within different clones. The levels of gallic acid and individualcatechins in the black tea do, however, decrease with an increase in fermentation temper-

ature and time for different clones (Obanda et al., 2001; Ngure et al., 2009). During

fermentation, considerable quantities of EGC, EC, EGCG and ECG are oxidized to formtheaflavins and their gallates. The catechins are able to be detected even after 150 minutes of

fermentation at 30 C (Obanda et al., 2001). The residual catechins in black tea can be

attributed to the inactivation of the polyphenol oxidase through the formation of complexes

with oxidized flavanols.

Increasing fermentation temperature and time results in an insignificant decline in the levels of

gallic acid for black tea clones (Muthumani and Kumar, 2006; Ngure et al., 2009). This may bedue to the formation of the formation of free gallic acid from EGCG and ECG by oxidative

degallation during fermentation. The liberation of free gallic acid has also been reported by Coggon et al. (1973).

Antioxidant activity varies between clones, ranging from 75.91 to 92.38% inhibition of a-piphenyl-b-picryl-hydrazyl (DPPH) radical. Gallic acid, (þ)-C and EGCG positively

contribute to the black tea antioxidant activity among clones, as is evident from Table

16.6 (Ngure et al., 2009). However, EGC and EC have negative insignificant contribution

to antioxidant activity. During processing, gallic acid and EGCG show strong linear relationships with antioxidant activity, while the total polyphenols have a strong corre-

lation with antioxidant activity (Ngure et al., 2009).

While gallic acid and individual catechins contribute positively and significantly to antioxidant activity within clones, the concentration of gallic acid declines insignificantly with fermenta-

tion temperature and time. Gallic acid is not a substrate for polyphenol oxidase, but its

quinone can be generated by reaction with quinones derived from some catechins (Luczaj and

Skrydlewska, 2004). During fermentation, free gallic acid and catechin are formed from theother catechin fractions such as EGCG, ECG and EGC, by oxidative degallation.

EGCG makes a weaker contribution to antioxidant activity for certain black tea clones.

However, total polyphenol content accounts for a high percentage of variation in antioxidant activity. Therefore, EGCG cannot entirely be taken as the major determinant black tea anti-

oxidant activity, as other black tea polyphenols contribute to this. The theaflavins present in

black tea possess antioxidative properties, for example, TF3 has been proved to show higher

antioxidative activity than EGCG, which is the strongest antioxidant among all catechins and

a precursor of TF3 (Hamilton-Miller, 2001).

Theaflavin gallates have been shown to have stronger antioxidant properties than free thea-flavins, since they contain more gallic acid residues ( Wang and Helliwell, 2001). (þ)-Catechin,

gallic acid and total polyphenols were better predictors of antioxidant activity than EGCG for

clone 6/8 black tea. Therefore, the radical scavenging activity of the black extracts could not beentirely attributed to unoxidized catechins, since total polyphenols includes other bioactive

molecules in black tea.

The decline in total polyphenol content with fermentation temperature and time for clones

observed by Ngure et al. (2009) suggests that black tea antioxidant activity cannot entirely be

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02



attributed to unoxidized catechins. Further studies on the contribution of individual thea-flavins to black tea antioxidant activity may thus be warranted.

There is an inherent variation in strength and contribution of the black tea constituents toantioxidant activity within the clones, which further indicates the differences between the

clonal black teas. However, these observations do not negate the overall positive and signifi-

cant contribution of gallic acid, (þ)-C and EGCG to black tea antioxidant activity among allthe clones.

The highest mean antioxidant activity is due to the high content of individual and totalunoxidized catechins. A high EGCG content for tea clones tends to result in a high percent

inhibition of the a,a-DPPH radical, especially for tea fermented at low temperature and

shorter duration. Mean gallic acid content and total polyphenols also tend to be high for thesefermentation conditions, emphasizing the importance of EGCG in contributing to antioxidant

activity of black tea (Magoma et al., 2001). Due to the inherent genetic differences, and

therefore catechin profiles, between clones, the yield of unoxidized catechins and other

bioactive polyphenols is seen to vary. Varying fermentation ability among tea clones (Wachira

et al., 2006) causes the individual catechins concentration of the black tea to vary. In addition,

the textural properties of the black tea from the different clones affect the infusion kinetics and

thus the different catechin concentrations of the infusion.

CONCLUSION

A great deal of research has been done on manipulating black tea fermentations to yield tea

that is rich in antioxidants and of good quality. The field of investigation involving thedevelopment of clone-specific processing technologies remains open. Only a few varieties/

subvarieties of the Camellia sinensis species are employed for tea production. There are

hundreds of other cultivars, each possessing unique biological characteristics. The botanical

classification of many tea bushes used in different regions has yet to be undertaken. This wouldadd value to processed tea by enhancing the health potential of the most consumed type of tea

which could be useful in the field of nutracueticals.

SUMMARY POINTS

l During tea processing, gallic acid, (þ)-catechin, EGCG and total polyphenols are the major

components that contribute to antioxidant activity in clonal black tea.l Black tea antioxidant activity is dependent on inherent clone differences in polyphenol

composition and processing conditions.l Manipulating black tea fermentation conditions can yield tea rich in antioxidants and of

perceived good quality.l The exploitation of the genetic diversity of tea could take advantage of the inherent

potential of catechin-rich clones to add value to processed tea by enhancing its health

potential.

Acknowledgment The author gratefully acknowledges the editorial assistance of Joshua Ogucha, Division of Research Kisii University

College (a constituent college of Egerton University) in Kenya.

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