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Chapter 1
Introduction
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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
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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)
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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
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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.
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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
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Fig. 1.2 Process for the manufacture of black tea leaf
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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
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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
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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
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(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
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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
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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
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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
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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
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(-)-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)
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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)
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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
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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
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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.,
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+ 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
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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
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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.