18

Chapter 1

Introduction

19

1.1 Black tea

Tea is one of the most widely consumed beverages worldwide and is made from

the processed leaves of evergreen shrub Camellia sinensis. The beverage got

its significance owing to the presence of high amounts of polyphenolic

compounds and their associated antioxidant properties. In 2009, world tea

production reached over 3.87 million tonnes (Table 1.1). The largest producers

of tea are the People's Republic of China (35.5%) and India (20.7%), followed by

Kenya, Sri Lanka and Turkey (Fig. 1.1). Traditionally, tea produced is classified

into black (fully fermented), oolong (partially fermented) and green (unfermented)

tea based on the period of fermentation, the leaves and buds have undergone

during processing. This process is not a true fermentation but an enzymatic

oxidation, herein simple polyphenols undergo an enzymatic polymerization by tea

polyphenol oxidase leading to formation of complex condensation compounds.

The amount of polyphenols in fresh leaf, green and black teas are in the range

30-35%, 10-25% and 8-21%, respectively (Lunder, 1992). The polyphenolic

composition of green, oolong and black tea leaves is mainly responsible for the

taste, colour, astringency and delightful aroma of their infusion. Black tea

production accounted for 75% of global tea production in 2009, with India as the

major producer as well as the largest consumer (http://www.agritrade.cta.int/en).

The other three major black tea producing countries are Sri Lanka, Kenya and

Indonesia (Table 1.2).

Considerable interests have been developed in the past decade in

unraveling the beneficial health effects of tea, particularly its polyphenolic

20

Table 1.1

World production of tea and major producing countries (2009)

Country Production (tonnes)

China 1,375,780

India 800,000

Kenya 314,100

Sri Lanka 290,000

Turkey 198,601

Vietnam 185,700

Indonesia 160,000

Japan 86,000

Argentina 73,425

Iran 40,000

Bangladesh 60,000

Malawi 52,559

Uganda 48,663

Other countries 185,409

Total 3,870,237

Source: FAOSTAT, 2009

Fig 1.1 Distribution of world tea production (2009)

21

Table 1.2

Black tea production by major producing countries (2009)

Country Production (tonnes)

India 815

Sri Lanka 305

Kenya 238

Indonesia 131

China 65

Bangladesh 54

Source: FAOSTAT, 2009

components and its antioxidant activity. Catechins, TFs and TRs are the three

important groups of polyphenols present in tea. The formation and mechanism

of these compounds during processing as well as their respective biological

activities are of great importance and of scientific and commercial interest.

Consumption of tea flavonoids has been linked to lower incidences of chronic

diseases such as cardiovascular disease and cancer. The antioxidant activity of

phenolic compounds is due to their redox properties, allowing them to scavenge

reactive oxygen species, such as superoxide radical, singlet oxygen, hydroxyl

radical, nitric oxide, nitrogen dioxide and peroxynitrite, which play important roles

in carcinogenesis (Wan et al., 2008).

Green tea extracts are powerful antioxidants, mainly owing to the

presence of high catechins content and reported to have stronger antioxidant

activity and lower toxicity than synthetic antioxidants like butylated

hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and DL- tocopherol

(Chen and Wan, 1994). In the case of black tea, the process used in the

22

manufacture, is known to decrease the levels of monomeric catechins to a much

greater extent of polymerization that leads to the formation of TFs and TRs which

are also known to possess antioxidant activities (Lin and Lin-Shiau, 2008). Satoh

et al. (2005) compared the antioxidant activities of various types of teas and

showed that 2,2-Di-phenyl-1-picrylhydrazyl (DPPH) scavenging activity

decreased from steamed-green tea, roasted-green tea, oolong tea to black tea.

Black tea possesses many biological effects besides being an effective

antioxidant (Vasundhara and Jaganmohan Rao, 2009). The fermented teas,

including oolong, black and pu-erh teas are more effective than unfermented

green tea in suppressing the body weight and lipogenesis in rats (Lin and Lin-

Shiau, 2008).

1.2 Traditional method of black tea manufacture

The traditional process of black tea manufacture from fresh green tea leaves is

described by Balentine et al. (1997; 2004). The composition of fresh tea leaves

is given in Table 1.3. The process comprises four major steps: withering, rolling,

fermentation and firing (Fig 1.2). Withering is a process whereby the freshly

plucked tea leaves are stored until the moisture content is reduced to about 55-

72%. Withering causes protein breakdown leading to an increase in free amino

acids, soluble carbohydrate and caffeine, changes in organic acid also takes

place. The withered leaves are crushed by rolling or maceration in order to break

down the leaf cell structure and bring enzymes and the substrate polyphenols

into contact.

23

Table 1.3

Composition of fresh green tea leaves

Components Fresh tea leaves

(% dry weight)

Flavanols 25.0

Flavonols and flavonol glycosides 3.0

Polyphenolic acids and depsides 5.0

Other polyphenols 3.0

Caffeine 3.0

Theobromine 0.2

Amino acids 4.0

Organic acids 0.5

Monosaccharide 4.0

Polysaccharides 13.0

Cellulose 7.0

Protein 15.0

Lignin 6.0

Lipids 3.0

Chlorophyll and other pigments 0.5

Ash 5.0

Volatiles 0.1

Source: Balentine et al., 1998

During fermentation the simple flavanoids in green tea leaves are oxidized

by endogenous tea enzymes, polyphenol oxidase (PPO) and peroxidase (POD)

to produce, the more complex polyphenols that impart a bright red color and the

astringent flavour to black tea. Tea fermentation is truly an enzymatic

polymerization and is not a typical fermentation process as against the

terminology practiced in the tea industry. The role of enzymes in the

fermentation process for conversion of green tea leaves to black tea is well

summarized (Sanderson and Coggon, 1977; Roberts, 1962). Fermented tea is

24

Fig. 1.2 Process for the manufacture of black tea leaf

25

fired (dried) with hot, dry air reducing the moisture content of the leaves to less

than 5%. Firing of tea arrests fermentation by inactivating enzymes and results

in improvement of color and creates the final balance of tea aroma. Following

drying, the tea is sorted and graded to yield a commercial black tea product.

1.3 Ready-to-drink tea

Tea processing has undergone many changes over the last 100 years, from

loose to blended tea, tea packets, tea bags, instant tea, and finally ready-to-drink

(RTD) tea. As consumers look for healthier alternatives to soft drinks, RTD tea

has become a dynamic category in the world market. USA, China, Japan and

Europe are the important markets for RTD tea, which is catching up with other

countries as well.

The tea beverage is generally prepared by brewing tea leaves in freshly

boiled water for a few minutes and perhaps adding milk and sugar. In many

countries, tea is more commonly enjoyed as an iced beverage (iced tea).

However, such a beverage cannot be prepared by infusing traditionally

manufactured tea leaves in cold water since many of the tea compounds

responsible for its organoleptic properties are only sparingly soluble in cold

water. Traditionally, cold tea is prepared by infusion of tea leaves and then taste

enhancers like sugar or lemon juice are added, which is then cooled (>30 min)

before consumption. Many methods have been proposed for manufacturing cold

water infusing tea leaf that offered the convenience of not having to boil water

and wait for it cool down, and the benefit of the fresh brewed tea taste. A more

26

convenient option is to use cold water soluble tea powders for the preparation of

iced tea. However, for many consumers the quality of the final beverage from

instant tea powder is not equal to that prepared from hot infused tea. Besides,

use of powders is perceived to be artificial and therefore unnatural (Goodsall et

al., 2004). The other alternative, instant tea beverages are typically available to

consumers as packaged products in cans, bottles and other sterile containers,

single strength beverage ready for consumption (RTD) or as a concentrate which

is diluted with water to form a drinkable tea beverage (Agbo and Spradlin, 1995).

Since RTD tea offers greater convenience, it is gaining more popularity. The

reference for consumer acceptance is a product that would resemble in its color,

flavor and taste as iced tea made from a hot infused tea.

Industrially, RTD tea is generally prepared by using tea extracts or

reconstituted tea powder with addition of sugar, lemon/peach juice, citric acid and

colorants to modify its flavour, taste and colour. Besides, various additives are

used as stabilising agents. Such type of cold teas, available in the market, does

not ideally meet the consumers' demands who are looking for additive-free foods

of high nutritional value (Todisco et al., 2002).

1.3.1 Problems associated with RTD teas

One of the most relevant problems encountered in the production of natural and

additive-free RTD cold tea is its instability due to development of haze and

formation of tea cream. It gives discoloration and precipitation of complexed

substances, affecting the visual appeal, flavour and colour, besides reduces the

27

shelf stability (Todisco et al., 2002). Since the expected shelf life of RTD tea is

commonly 6 to 12 months under refrigeration, the stability of infusion is of great

importance.

1.3.2 Tea cream

Strong aqueous black tea infusion becomes turbid, changing from clear, deep

red to light brown or orange suspension as it cools down. The coloured

precipitate, whose formation causes turbidity, is known as ‘tea cream’. In simple

words, cold-water insolubles are known as ‘tea cream’ in the art. It is a very

finely divided colloidal precipitate which imparts a distinct opacity to the clear

liquor and comprises tannin complexes that comprise 15-35% of the total tea

solids present in the infusion (Roberts, 1963). Tea catechins and their oxidation

products when interact with caffeine, protein, pectins and metal ions in the

extract form larger complexes that eventually precipitate out (Ekanayake et al.,

2001). Tea cream contains many of the compounds that provide taste and

colour in black tea and its formations cause both loss of taste and colour (Jobstl

et al., 2005).

Bradfield and Penney (1944) were the first to demonstrate a relationship

between tea cream formation and tea quality (strength, pungency and briskness)

in the infusion. The color of the tea cream is determined by the ratio of TRs to

TFs. A high content of TRs results in the production of ‘dull cream’ and ‘bright

cream’ would be associated with high TFs content. Triacetidin is a pink colored

compound and may also play a part in determining the color of the cream

28

(Wickremasinghe and Perera, 1966). Creaming is valued by professional tea

tasters as a contributory indication of tea quality and a visual assessment of

creaming down by a tea taster, is a means whereby it is judged whether the tea

contains sufficiently high amounts of TFs, TRs and caffeine, and whether they

are present in relative proportions for the tea to be considered satisfactory

quality. But it is considered as a negative factor in instant tea processing

industry as it affects the appearance and dispersibility of iced tea beverages

(Roberts, 1963).

Tea cream formation in black tea depends on many factors like

fermentation time, temperature, duration of extraction and water-to-tea ratio

(Liang and Xu, 2003). Concentration, composition, pH and temperature-time

history of the infusion also affect cream formation (Tolstoguzov, 2002). Tea

cream formation is governed by various types of interactions, including

polyphenol–caffeine (with or without lipid) and polyphenol–protein interactions

(Jobstl et al., 2005).

The principal constituents of black tea cream are TRs, TFs and caffeine

(Roberts, 1962) and 97% of the tea cream consists of normal constituents of

black tea (Smith, 1968). The approximate composition of tea cream produced in

Assam tea infusion (1:40) was reported to be 15% TFs, 65% TRs, 14% caffeine,

3% ash, which included potassium (1%) and calcium (0.2%). The remaining 3%

included non-caffeine nitrogenous compounds and other minor constituents

(Smith, 1968). Nagalaksmi et al. (1983) brought out the differences between the

tea creams isolated at ambient as well as at 4°C. Theanine is the major amino

29

acid constituent of the tea infusion but does not take part in cream formation,

proteins to a larger extent and soluble caffeine to a lesser extent seem to play a

significant role in the composition of tea cream isolated at ambient temperature in

comparison to that isolated at 4°C. The polymeric polyphenols do not contribute

significantly to cream formation at ambient temperature, but are the major

constituents of cream isolated at lower temperatures. The nitrogenous

compounds especially caffeine complexes with highly acidic phenolic groups of

tea polyphenols, namely, TFs and TRs via the formation of hydrogen bonds.

These hydrogen bonds are stable at lower temperatures and increasingly

become unstable at higher temperatures.

Liang et al. (2002) reported that caffeine, gallocatechin (GC) and

epigallocatechin gallate (EGCG) are the predominant compounds in green tea

cream, while TRs, GC and TFs in black tea cream. Other tea components like

protein, pectin and calcium also take part in tea cream formation by co-

precipitating with insoluble complexes (Liang et al., 2002). Gallated compounds

offer more hydroxyl groups for hydrogen bonding for tea cream formation. Also

oxidation products of catechins have a stronger creaming capacity than the

unoxidized catechins. Hence, the cream formation is lesser in green tea

compared to oolong and black tea. However, average size of cream particles is

bigger in green than black tea.

1.3.3 Decreaming methods

RTD tea is generally produced from instant tea powder. Decreaming is an

30

important step in the process to meet the cold stability requirements of the

product. Conventional decreaming methods (removal of the precipitating

complexes) include clarification by centrifugation/filtration after adjustments in

temperature, enhancing the solubility by employing chemicals and enzymes, and

other equivalent techniques or combination of these methods. Enzymatic

approaches have overcome some of the disadvantages associated with other

methods, namely, cold water extraction, chill decreaming, chemical stabilization

and chemical solubilization (Rutter and Stainsby, 1975; Clark et al., 1984;

Mishkin, 1962; Tsai, 1987).

1.4 Enzymatic treatments during black tea processing

The role of enzymes in tea processing has been recognized for nearly four

decades and its application to improve the quality of tea. Enzyme treatment

have been given at three stages of black tea processing, i.e. prior to fermentation

of tea, prior to extraction of black tea and to the extract; to improve soluble solids

yield, cold water extractability/solubility and decrease in tea cream formation as

well as to improve the clarity.

Tannase is the most commonly employed enzyme for tea processing.

This enzyme hydrolyses esters of phenolic acids, including the gallated

polyphenols found in tea. Therefore, tea compounds are generally used as

substrate for assessing the activity of tannase. Although tannase can be

obtained from plant, animal and microbial sources, it is mainly produced by the

latter. Aguilar et al. (2007) has summarized the various types of bacteria, yeast

31

and fungi employed in tannase production. Filamentous fungi of the Aspergillus

genus and bacteria of the Bacillus genus have been widely used for producing

tannase. Phenolic compounds such as gallic acid, pyrogallol, methyl gallate and

tannic acid induces tannase synthesis (Bajpai and Patil, 1997). The major

commercial food applications of tannase are in the production processes of

instant tea, acorn liquor and gallic acid. The tannase of some Aspergillus strains

has a molecular weight around 150-350 kDa. Their activity and stability pH are

5.0-6.0 and 3.5-8.0, respectively, whilst optima temperatures ranged from 35ºC

to 40ºC. Tannase is stable for several months at 30ºC (Belmares et al., 2004).

The enzyme is commercialized by many companies with different catalytic units

depending on the product presentation.

Various cell-wall-digesting enzymes have also been employed which react

and modify plant cell wall biopolymers. Cell-wall digesting enzyme is an enzyme

which breaks down one or more tea cell-wall constituents to simpler materials

and thus reduces the structural integrity or increases the permeability of the cell

wall. Plant cell walls are composed primarily of cellulose, but contain lesser

amounts of proteins, hemicellulose, pectins, and lipids. Accordingly, cell-wall

digestive enzymes include cellulase and hemicellulase, proteases such as

papain, pectinase, dextranase, lysozyme and lipases (Tsai, 1987).

1.4.1 Enzymatic preconversion treatment to green tea leaves

The four major catechins (Fig. 1.3) in green tea leaf are epicatechin (EC) and

epigallocatechin (EGC) and the gallated forms of these catechins (bearing a

32

gallic acid (GA) moiety), epicatechin-3-gallate (ECG) and epigallocatechin-3-

gallate (EGCG). During oxidative fermentation of green tea, all these catechins

undergo oxidative biotransformation, through their quinones, into dimeric

compounds known as TFs and higher molecular weight compounds known as

TRs, which are components of black tea (Goodsall et al., 2000). TFs comprise

several well-defined catechin condensation products that are characterized by

their benzotropolone ring (Fig. 1.4). TRs are a group of undefined molecules

with a large variance in molecular weight. TFs and TRs are responsible for the

orange and brown colors of black tea infusions and products as well as making

significant contributions to the astringency and body of the made tea. TRs are

larger in size and darker in color than TFs. The oxidative polymerizations are a

combination of biochemical oxidations mediated by PPO and/or POD enzymes

present in the leaf and chemical reactions of reactive species. The general

reaction catalyzed by tannase (flavanol gallate esterase) is the cleavage of

gallate ester linkages, both on gallated catechins and also from other gallated

compounds within the leaf (Goodsall et al., 2000). Tannase action is also

expected to hydrolyze gallated ester linkages of TFs and TRs releasing gallic

acid in black tea. Galloyl groups are important in cream formation and tannase

has been used extensively for the degallation and solubilization of black tea

cream. EGCG and ECG are the most abundant catechins in fresh tea leaves

and tannase treatment hydrolyzes EGCG to yield. EGC and gallic acid, and

ECG to yield EC and gallic acid by cleaving their ester bonds. Several studies

have been made with tannase treatment on green tea leaf to simplify the mixture

33

(-)-Epicatechin (EC) (-)-Epigallocatechin (EGC)

OH

OH

OH

OH

OH

OH O

OH

OH

OH

OH O

OH

OH

OH

O

OH

C OOH

OH O

OH

OH

OH

OH

O

OH

C OOH

OH O

OH

OH

OH

Fig 1.3 Chemical structures of major catechins

Source: Goodsall et al., 2000

of catechins before the start of the fermentation (Sanderson and Coggon, 1974;

Sanderson et al., 1977; Goodsall et al., 2000; 2004; Balentine et al., 2004).

(-)-Epicatechin-3-gallate (ECG) (-)-Epigallocatechin-3-gallate (EGCG)

34

OOH

OH

O

OHOH

O

OH

OH

OH

OH

OH

O

OH

OH

OH O

OH

OH

OH

OH

OH O

OH

OH

O

OH

C O

OH

OH O

O

OH

OH

O

OH

OH

OH O

OH

OC

OH

OH

OH

OH

OH O

O

OH

OH

O

OH

OH

OH O

OH

OH

O

OH

C O

OC

OH

OH

OH

Fig 1.4 Chemical structures of theaflavin and major theaflavin gallates

Source: Goodsall et al., 2000

Theaflavin (TF1) (EC+EGC)

Theaflavin-3-gallate (TF2a)

(EC+EGCG)

Theaflavin-3’-gallate (TF2b) (ECG+EGC)

Theaflavin-3,3’-digallate (TF3) (ECG+EGCG)

35

1.4.2 Enzymatic treatment of black tea before/during extraction

Enzymatic treatments have been attempted with black tea by treating

withtannase and cell wall digesting enzymes either before (Tsai, 1987) or during

extraction (Lehmberg et al., 1999a;b;c) to improve its quality in terms of stability,

and cold water solubility as well as extraction yield. Cell wall-digesting enzyme

breaks down one or more cell wall constituents to simpler materials and thus

reduces the structural integrity or increase the permeability of the cell wall.

1.4.3 Enzymatic treatment to extract

Tannase has been recommended for hydrolysis of cream to lower molecular

weight compounds, reducing turbidity and increasing cold water solubility

(Sanderson and Coggon, 1974). This treatment to tea extract (Takino, 1976;

Agbo and Spradlin, 1995) converts at least a portion of the insoluble solids of tea

cream to a cold water-soluble form. The enzyme based method eliminates or

reduces the need for inorganic and organic materials normally employed in the

chemical solubilization methods. This approach is rightly referred as ‘Enzymatic

solubilization of cream’ and also as ‘Enzymatic clarification of extract’.

1.4.4 Transformation of tea polyphenols with the action of endo and

exogenous enzymes

PPO and POD are the two natural endogenous enzymes present in fresh tea

leaves responsible for fermentation (oxidation process) in the conversion of

green tea leaves to black tea. During this fermentation process, all catechins

36

undergo enzymatic oxidation and condensation to form dimeric and polymeric

compounds, TFs and TRs (Fig. 1.5A). The role of POD is limited as endogenous

H2O2 produced by PPO is largely consumed by catalase active in tea.

Tannase hydrolyses esters of phenolic acids including the gallated

polyphenols. Tannase treatment to green tea hydrolyses gallated catechins ECG

and EGCG to EC and EGC, respectively, with cleavage of gallic acid from their

ester bonds. This results in producing enhanced levels of TF1, which is the

oxidation and condensation product of EGC and EC. During the subsequent

fermentation process TFs and TRs are formed with a higher proportion of their

ungallated forms (Fig. 1.5 B) compared to untreated sample (Fig 1.5A). An ideal

ratio of EGC(G):EC(G)::3:1 in green tea, facilitates only TF1 production but in

practice a small amount of eTF acid may also be formed if endogenous H2O2

becomes available to activate POD. Although tannase treatment is aimed at

complete degallation, it may not happen. In tannase treated samples, gallic acid

produced could be a measure of extent of degallation.

Addition of H2O2 during oxidative fermentation is beneficial as it can

activate endo-POD that could oxidize gallic acid and EC into eTF acid (Fig 1.5C).

However, if the addition is made during the beginning of fermentation it could be

detrimental to the formation of TF1. It is preferable to add H2O2 after allowing

sufficient time for maximum TF1 formation and subsequent addition could lead to

eTF acid formation utilizing free gallic acid which would otherwise affect the taste

of tea. Such an approach will not interfere either with the formation of beneficial

TF1. Treatment to black tea leaves and extracts with tannase, degallates

37

A Traditional method of conversion of green tea leaves to black teaa

Green tea catechins

Ungallated- EC, EGC

Gallated- ECG, EGCG

PPO/POD*

Aerobic

Catechins

Gallocatechin

quinones

TFs

Ungallated -TF1

Gallated - TF3, gallate

TF3’ gallate

TF3,3’digallate

TRs

Ungallated

Gallated

aTypical catechin composition in green tea - EC:1-3%; EGC:3-6%; ECG:3-6%; EGCG:8-12% (Harbowy and Balentine,1997).

*Role of POD may be limited since catalase active in tea removes peroxides as they form

Usually the oxidation is not complete and some quantity of simple catechins and TFs are present along with TRs. Typical polyp henols

composition in black tea: simple catechins- 15%; TFs-15%; TRs-70% (Collier et al., 1973).

B Tannase preconversion treatment to green tea leavesb

bUsually the degallation and oxidation reactions may not be complete and some quantity of simple catechins and TFs may be

including TF1 are present along with TRs and GA.

TannaseGreen tea catechins

Ungallated - EC, EGC

Gallated - ECG, EGCGAnaerobic

Degallation

EGCG

ECG

EGC + GA

EC + GA

PPO/POD

AerobicTFs+TF1+GA

TRs

Ungallated

Gallated

+ GA

EGC+EC TF1+GA

Fig.1.5 Expected tannase oxidation products of green/black tea catechins during enzymatic conversion

Contd.,

38

+ eTF acid

C Tannase - Peroxide treatment to green tea leavesc

cSome amount of simple catechins, TFs and may be traces of TF1 and GA are present along with TRs and eTF acid

D Tannase treatment to black tea leaves/extractsd

Black tea leaves

Simple catechins

TFs, TRs

Degallation

EGCG

ECG

EGC + GA

EC + GA

Gallated TF TF1

Gallated TRs Ungallated TRs

TF1+ TFs

Ungallated

+TRs

Ungallated

Gallated

+ GA

dSome amount of simple catechins may be present along with TRs, TFs (including traces of TF1) and GA

Green tea catechins

Ungallated - EC, EGC

Gallated - ECG, EGCG

PPO/POD

Aerobic

POD

H2O2Anerobic

Degallation

EGCG

ECG

EGC + GA

EC + GA

TFs+TF1+GA

TRs

Ungallated

Gallated

Tannase

EC+GA eTF acid

Tannase

EGC+EC TF1+GA

Fig.1.5 Expected tannase oxidation products of green/black tea catechins during enzymatic conversion

39

gallated TFs and TRs as well as catechins releasing gallic acid (Fig 1.5D). The

production of gallic acid is a direct measure of hydrolytic activity of tannase and

free gallic acid at elevated levels result in a metallic note affecting the taste of tea

to a greater extent.

1.4.5 Relative merits of various approaches

The tannase treatment is useful at any process stage of its application although

the benefits are more when applied in the pre-fermentation stage as it results in

the formation of polyphenolic compounds that are less prone to become

permanently insoluble, thereby increasing cold water solubility, higher yield,

preventing tea cream formation and higher clarity. However, it may be desirable

to apply enzyme treatment either during extraction or to the extract from the

viewpoint of ease of adoption in the manufacturing process. In the latter

approaches, black tea leaf can be used as produced in the usual manner.

Enzymatic treatment of tea leaves in the solid state is preferable over enzymatic

clarification after the extract is prepared, since a separate enzyme inactivation

step can be avoided. While each of these processes is successful to varying

degree towards improving the quality of RTD beverages either directly or

indirectly, each has inherent disadvantages too. With due considerations to

application and adoption in the manufacturing process, enzymatic treatment to

black tea before extraction may be preferred. The feasibility and economy of

instant tea products are very much dependent on the extract yield. Most of the

work on enzymatic processing of black tea is focused on solids extractability, tea

40

cream solubilization, and clarity but there are no reports on polyphenols

recovery. The future attempts should also aim at improving the extractability of

polyphenols without affecting the quality (polyphenol-to-soluble solids and TF/TR

ratios) of black tea extract in addition to above objectives.

1.5 Extraction of black tea

RTD tea is often made using reconstituted spray dried tea powder. The

extraction efficiency is a critical factor in determining the economics of an instant

tea production process. Besides the extract yield, the quality of the soluble

powder obtained is an equally important factor, which decides the quality of final

converted products. Tea is valued for its colour, strength, briskness and flavour

of the liquor and first three of these could be correlated to the TFs and TRs

content of black tea liquor (Clougley, 1980). Earlier attempts revealed that TFs

content is an important factor in determining black tea quality (Hilton and Ellis,

1972). Liang et al. (2003) analysed the chemical composition, colour differences

of black tea infusions and their relationships with sensory quality assessed by

professional tea tasters as an attempt to develop an objective method of quality

evaluation.

One of the earliest experiments by Natarajan et al. (1962) examined the

brewing behaviour of four different grades of black tea, extraction rates of

different constituents, and effect of water-to-tea ratio, water temperature and

infusion time. Extensive studies were conducted by Spiro and co-workers on

extractability of black tea (Price and Spiro, 1985). All the above studies on

41

extractability of tea were conducted under brewing/infusing conditions close to in-

home infusion preparations. The extraction conditions employed in the instant

tea manufacture are aimed towards the maximum recovery of solids and are

usually harsher compared to the brewing conditions used for tea preparations.

Extraction conditions namely, solvent-solute ratio, extraction time and

temperature greatly influence the extraction process. Long (1977) conducted a

series of bench scale experiments on extraction of black tea and followed it up

with pilot-scale studies on batch-simulated continuous counter-current extraction

with an aim to develop a commercial extraction process (Blogg and Long, 1980).

All these studies were focused on the yield of black tea considering its

importance in the manufacture of instant tea. However, not much research effort

has gone in to the direction of extractability of polyphenols, which contribute to

the organoleptic properties.

1.5.1 Enzymatic extraction of black tea

There are several reports on enzymatic treatment of tea leaves with common

cell-wall digesting enzymes such as pectinases, cellulases, amylases and

proteases prior to extraction to improve the extract yield and also with tannase to

improve the cold water extractability. Tsai (1987) employed an enzyme solution

containing tannase in conjunction with enzymes such as cellulase, pectinase and

hemicellulase with an objective to improve the yield of cold-water soluble solids

from black tea. The likely mechanism by which this process works is that upon

imbibition of enzyme solution by black tea leaves and swelling of leaf tissues, the

42

enzymes are absorbed into or onto the tissues, causing the release of

immobilized tea solids from leaf material and hydrolysis of released tannins to

provide a higher yield of cold-water soluble tea solids. Extraction yield increased

by combined enzymatic treatment compared to tannase treatment alone.

Tannase-pectinase treatment gave greater extraction yield and cold-water

solubility compared to tannase-cellulase treatment. Due to the action of tannase

upon released tea solids, gallic acid and other organic acids are released,

causing a decrease in pH and it may be necessary to adjust the pH,

subsequently to a desired level with any food compatible base. Many

researchers followed this combined enzyme treatment approach with some

variations. Lehmberg et al. (1999b) extracted black tea using a solution

containing a cocktail of enzymes. However, there are no reports to improve the

extractability of polyphenols along with improving the overall extract yield.

1.6 Membrane clarification of tea extracts

There is an increasing demand for foods that are more closely resemble the

original raw materials and have a healthy or natural image, and have fewer

synthetic additives, or have undergone fewer changes during processing

(Fellows, 2009). It is necessary to respond to these pressures from the

consumers. Although, enzymatic approaches have overcome some of the

disadvantages associated with conventional decreaming methods, even

enzymes are not preferred in the production of additive-free natural products. In

43

this perspective, membrane technology is an alternate approach and a mild

physical process, which could overcome most of these disadvantages.

Membrane processing that involves the principles of separation by size

and shape of molecules or particles is a simple procedure. It offers several

advantages over conventional processing methods as they are convenient and

easy to scale-up. Pressure driven membrane processes are often identified by

the range of size of solutes they separate, namely, reverse osmosis (RO) or

hyperfiltration, nanofiltration (NF), ultrafiltration (UF), and microfiltration (MF).

Commercial membrane devices are available in four major types, namely plate

and frame, tubular, spiral-wound and hollow fiber.

Membrane technology for the processing of fruit juices and beverages has

been applied mainly for clarification using UF and MF, and for concentration

using RO. Even though pressure-driven, these processes are attractive and cost

effective, since the absence of phase change and inter-phase mass transfer

necessitates less energy (Raman et al., 1994). Commercially, the major impact

of membranes has been for the clarification of apple juice (Girard and Fukumoto,

2000). MF and UF have been replacing conventional fining and filtration

methods for clarifying apple juices. Advantages of UF and MF over conventional

methods include reduction in enzyme consumption, elimination of fining agents

and their associated problems, and production with a continuous simplified

process (Keefe, 1984; Rösch, 1985).

During enzymatic polymerization, ~10% of the catechins are converted to

TFs, bisflavanols and other oligomers with molecular weights of 500-3000 Da

44

and 75% of them are converted to TRs in black tea. The size of TRs is reported

to be in the range of 700-40000 Da (0.002-0.04 m) (Todisco et al., 2002).

Green tea extracts initially contain high levels of unoxidized flavanols, especially

monomeric catechins such as EC, ECG, EGC and EGCG that impart a desired

taste (astringency) to the beverage. Unfortunately these catechins remaining in

the extract will still be oxidized over time to the less desirable oxidized

polyphenols (Ekanayake et al., 2001). Evans and Bird (2006) suggested that

potentially a physical barrier could be used to separate polyphenols including

TRs from the larger cream aggregates since the majority of black tea cream

particles formed (84.8%) is in the size range of 0.1-1.03 m (Liang and Xu,

2001). However, it may be desirable to employ techniques to prevent/reduce tea

cream formation while retaining natural characteristics. In the last few decades,

some attempts have been made employing membrane technology (pressure

driven) for the clarification of extracts from black and green tea demonstrating its

capability. Attempts made with black tea extracts are described below.

Wickremasinghe (1977) developed a patented method for preparing cold

water soluble tea concentrates and powders by selectively removing high

molecular weight compounds such as chlorophyll, protein, polypeptides and

polysaccharides while retaining the polyphenolic compounds by subjecting the

black tea extract to filtration through a gel (polymerized dextran or

polyacrylamide), porous glass granules or a UF membrane. In the UF process,

extract is prefiltered through glass wool and 35 ml of ethanol (for 25 g black tea)

is added to 200 ml of hot extract (60C) to prevent membrane clogging before UF

45

(20 kDa; 0.1 MPa). The membrane selectively removed high molecular weight

compounds while allowing permeation of caffeine, polyphenols and amino acids.

The pH of the resulting extract (4.8) is adjusted to 5.1 and conventionally

processed to obtain a water soluble tea concentrate or powder.

Todisco et al. (2002) studied the clarification of infusions from commercial

black tea leaves using a 40 kDa ceramic tubular membrane with a focus to

eliminate proteins that interact with soluble tannins and precipitate in the infusion

during storage. The purpose of the work was to integrate between the optimum

infusion until a limiting polyphenol concentration is achieved and UF process to

produce a stable tea with high polyphenols content and reproducible color

quality. Flux and polyphenols rejection were studied over wide range of

operating conditions (70-170 kPa; 0.49-3.20 m/s; 50°C). Low rejection of

polyphenols (~12%) and high flux 150 LMH was observed at the highest flow

velocity and 120 kPa. Polyphenols concentration and color parameters (CIE L,

a, b) remained stable, and no visible haze was observed in the ultrafiltered

product for up to 2 months stored in dark bottles at -4°C. Corresponding

untreated infusions showed a strong reduction of lightness and yellowness

whereas redness increased probably as a consequence of oxidation. There was

a slight decrease in the polyphenols concentration in the direct infusion owing to

the precipitation. However, protein content was not estimated in permeates

which would have established whether proteins were eliminated during UF and

their role in the cream formation.

Evans and Bird (2006) examined UF as a clarifying procedure with two flat

46

sheet polymeric membranes of equal MWCO (30 kDa) made of fluoropolymer

(FP) and regenerated cellulose (RC) in a cross-flow system using reconstituted

spray dried black tea. The permeate quality was analysed in terms of haze and

color (CIE tristimulus values) at 35°C. Haze was characterized by the

absorbance at 900 nm, corresponding to an absorbance minimum of a

centrifuged sample. Color and haze were compared before and after UF (0.1

MPa TMP, 0.44 m/s and 50°C) at similar solids concentrations. Both the

membranes were effective in reducing the haze by at least an order of

magnitude. Lightness and yellowness increased considerably after UF.

Permeate of FP membrane showed greater haze and redness indicating its

potential for transmission of larger molecular weight compounds compared to RC

membrane. At 0.1 MPa TMP and after 30 min of operation, FP and RC

membranes showed a steady flux of 23.0 and 32.1 LMH, rejecting 21% and 27%

of solids, respectively. The membrane was effective in rejecting haze and cream

aggregates, but transmitted lower molecular weight compounds that led to a

relatively low overall rejection of solids. As the TMP increased, both the

membranes rejected more solids.

These researchers also evaluated the solute-membrane fouling

interactions during UF. Being more hydrophobic, FP membrane showed more

fouling tendency than RC membrane with hydrophilic tea components that led to

surface modification of FP membrane resulting in a more hydrophilic surface than

the original membrane. This demonstrated the advantage of using a moderately

hydrophobic membrane for tea liquor filtration in terms of greater flux following

47

multiple fouling and cleaning cycles closer to fluxes similar to those obtained with

hydrophilic materials. Hydrophobic materials generally offer greater chemical

and thermal stability compared to hydrophilic membrane materials and therefore,

preferred for industrial applications.

Evans et al. (2008) in a subsequent study investigated the efficiency of

separation and final product quality using different MWCO RC and FP

membranes (10, 30 and 100 kDa). The FP membranes generally showed lower

fluxes than the RC membranes. FP-10 had the lowest steady state flux of 14

LMH and RC-100 displayed the highest with 32 LMH. All RC membranes

showed similar solids (69-73%) and polyphenols (~90%) transmission. However,

the FP membranes displayed greater variations in their transmission. FP-30

provided the highest solids (73%) as well as polyphenols (~90%) transmission

while FP-10 (65%) and FP-100 (62.5%) gave lower solids transmissions.

Caffeine transmitted through both the types of membranes easily and was thus

found in higher relative concentrations in the permeated solids. The haze

(absorbance @ 900 nm) had been significantly removed by membrane filtration

(<0.002) compared to unfiltered (0.043-0.074) and commercial ice tea (0.025)

samples. Correspondingly, lightness had also increased significantly. According

to these researchers, this would enable using higher solids concentrations of

ultrafiltered solutions in iced tea production. Instead, it would be a good

preposition to improve the clarity without losing much of the original color of tea

liquor. RC-100 gave the reddest and yellowest solution among all the

membranes. The FP membranes were significantly rougher than the RC

48

membranes and increased fouling was present on rougher, more hydrophobic FP

surfaces. The results demonstrated that flux and defined MWCO are not

adequate criteria in themselves to determine membrane selection. Surface

science parameters are important both to the filtration properties of real liquors,

and the resulting fouling and cleaning mechanisms.

Pierre (2008) proposed a process for making a cold water soluble tea

extract with good colour, low or no haze and acceptable yield, without addition of

any chemicals or enzymes. According to the process, the cream fraction

obtained after chill decreaming is solubilized in boiling water (1-15%

concentration) and ultrafiltered (50-200 kDa) at ~45°C. The permeate fraction

upon cooling to room temperature was found to be free from haze, which may be

combined with the decreamed fraction obtained earlier from chill-decreaming

step before or after concentration/drying. In the accompanying example, the

above UF process (100 kDa) greatly reduced the turbidity (0.65 NTU) compared

to mere centrifugation (42.2 NTU) while processing solubilized primary cream

fraction.

Considering the increasing market demand for RTD tea, there is a great

potential for adopting membrane technology in the production process to improve

their stability and decrease the haze developed during refrigerated storage while

retaining most of its natural characteristics. Although the above research works

advanced the application of membrane technology for clarification of tea extracts,

its efficacy has not been completely tested. For instance, none of the above

researchers have studied polyphenols and solids recoveries in the process.

49

Besides, retentate stream is a very rich source of polyphenols and there are no

attempts towards its recovery. It may be desirable to introduce this clarification

technique to the primary extract considering the fact that RTD tea beverages are

generally produced from reconstituted spray dried tea powder. However,

majority of the earlier researchers have used reconstituted tea, which would have

gone through a primary clarification process and may not be a representative

sample for carrying out studies. Besides, most of the researchers relied upon

absorbance/transmittance as a measure of clarity which could be misleading

instead it may be desirable to assess in terms of direct turbidity units. It is also

necessary to measure these quality parameters at uniform strength of samples

for meaningful comparison. The clarification process needs are to be

benchmarked in terms of low turbidity, high retention of polyphenols, high

recovery of solids and storage stability.

1.7 Scope of the present investigation

An exhaustive review of research carried out towards application of enzymes and

membrane technology in the production of RTD black tea beverages forms a

prelude to the present investigation. One of the most relevant problems

encountered in the industrial production of additive-free RTD cold tea is its

instability due to development of haze and formation of tea cream. Conventional

decreaming methods are associated with inherent disadvantages while

membrane technology could overcome some of these disadvantages. Hence,

50

membrane technology has been investigated as a physical method for clarifying

black tea extracts.

RTD tea is often made using reconstituted spray dried tea powder. The

extraction conditions employed are aimed at maximum recovery of tea solids with

due consideration to the economics of the production process. Besides the

extract yield, quality of soluble powder obtained is an equally important factor

which decides the quality of final converted products. However, not much

research effort has gone in to the direction of extractability of polyphenols and

other tea solids. To begin with, the influence of extraction conditions on

polyphenols content and tea cream constituents in black tea extracts was

investigated.

The role of enzymes in tea processing and its application to improve the

quality of tea has been recognized for nearly four decades. There are several

attempts on enzymatic treatment of tea leaves with common cell-wall digesting

enzymes such as pectinases, cellulases, amylases and proteases prior to

extraction to improve the extract yield and also with tannase to improve the cold

water extractability. However, there are no attempts to improve the extractability

of polyphenols along with improving the overall extract yield without affecting the

tea quality. In this study, attempts were made employing enzyme assisted

extraction to enhance the recovery of polyphenols besides ESY, maintaining a

good balance of tea quality, using a cell-wall digesting enzyme (pectinase) and a

tannin hydrolyzing enzyme (tannase).

51

Membrane technology has been explored for the clarification of extracts

from black tea. Although the research carried out in the last three decades

advanced the application of membrane technology for clarification of tea extracts,

the efficacy has not been completely tested. For instance, there are no reports

on polyphenols and solids recoveries in the process. RTD tea beverages are

generally produced from reconstituted spray dried tea powder. Therefore, it is

desirable to introduce this clarification technique to the primary extract.

However, majority of the earlier researchers have used reconstituted tea for

carrying out their studies, which may not be a representative sample as it would

have gone through a primary clarification step in the production process. In the

present investigation, membrane technology was assessed as a clarification

method for black tea extract, obtained under optimized conditions, employing

various MF and UF membranes with a focus on higher yield and greater retention

of polyphenols.

The retentate of the membrane clarification process contained a

substantial amount of polyphenols and hence not to be treated as a reject waste

stream. Attempts were made to evolve a comprehensive membrane process

solution to clarification of tea extracts. A comparative assessment of anti-oxidant

potential of tea solids present in various membrane process streams was carried

out to establish a better utility for the retentate stream as a tea solids conserve.