FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)


PROCESS FOR PREPARATION OF THEAFLAVIN-ENHANCED
TEA PRODUCTS
HINDUSTAN UNILEVER LIMITED, a company incorporated under
the Indian Companies Act, 1913 and having its registered office
at 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India

The following specification particularly describes the invention and the manner in which it is to be performed


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PROCESS FOR THE PREPARATION OF THEAFLAVIN-ENHANCED TEA PRODUCTS
Technical Field of the Invention
The invention relates to the field of processing of tea. It 5 particularly relates to a process for preparation of theaflavin-enhanced tea product.
Background of the Invention
Any discussion of the prior art throughout the specification 10 should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Theaflavins are compounds characterised by the presence of
15 the benzotropolone ring and are formed when tea leaf is
enzymatically oxidized, or fermented to produce black tea.
Theaflavins are unique to black tea and oolong tea with
theaflavin (TF1), theaflavin-3-monogallate (TF2),
theaflavin-3'-monogallate (TF3) and theaflavin-3-3'-
20 digallate (TF4) being the four major species present. In tea
leaves, epicatechin (EC) and epicatechin gallate (ECG) are
simple catechins, while epigallocatechin (EGC) and
epigallocatechin gallate (EGCG) are gallocatechins which
participate in the formation of the four theaflavin species.
25 The oxidative biotransformation of catechins, like other
biotransformations, is complex and involves multitude of
reaction pathways resulting into dimeric theaflavins and
higher molecular weight compounds known as thearubigins.
30 Theaflavins are known to have antioxidant, antimicrobial and anti-inflammatory properties. Theaflavins have been reported as being effective against various diseases including
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cancer, cardiovascular and cerebro-vascular diseases, diabetes, and hypercholesterolemia. However, normal levels of theaflavins present in tea are 1-2 % on dry weight basis, depending on factors such as geographical location of tea 5 and variety of tea. It is therefore desirable to obtain tea products with enhanced level of theaflavin. In the past, researchers have tried to enhance theaflavin in tea products by various approaches.
10 Cloughley and Ellis (J. Sci. Food Agric. 31 (1980) 924-934) have described a method of theaflavin-enhancement of tea by carrying out fermentation step at a low pH. However, tea obtained by this method may have residual acidity which can cause curdling of milk when added while preparing a milked
15 tea beverage.
US6113965 (2000, Lipton) discloses a method for producing theaflavin by separating theaflavin from a slurry oxidative fermentation product of green leaf tea that has been treated 20 with tannase prior to slurry oxidative fermentation. It also teaches methods for making theaflavin-rich extracts and cold water soluble tea powders and products are provided.
However, enhancement of theaflavin obtainable by the above 25 methods is limited and there is a continuing need for tea products having further enhanced levels of theaflavin.
In addition, the methods reported in prior art have been found to be expensive and therefore there is a further need 30 for a cost-effective method of preparing theaflavin-enhanced tea products.
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It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. 5
One of the objects of the present invention is to provide a process for preparing a theaflavin-enhanced tea product comprising higher levels of total theaflavin as compared to prior art methods. 10
Another object of the present invention is to provide a process for preparing a theaflavin-enhanced tea product in a cost-effective manner.
15 The present inventors have surprisingly found that presence of exogenous epicatechin during enzymatic fermentation of a tea-source leads to a theaflavin-enhanced tea product.
Summary of the Invention
20 According to the present invention there is provided a process for preparing a theaflavin-enhanced tea product including a step of subjecting a tea-source to enzymatic fermentation in the presence of added exogenous epicatechin.
25 Preferably, the enzymatic fermentation according to the present invention is by means of endogenous enzymes in the tea-source.
According to one of the preferred aspects of the invention,
30 the amount of said epicatechin is 35 to 750, more preferably
75 to 520, most preferably 120-260 mg per gram dry weight of
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soluble solids in said tea-source. According to a preferred aspect, epicatechin is added prior to the fermentation of the tea-source.
5 The tea-source is preferably treated with tannase prior to or during the fermentation.
According to a preferred aspect, the fermentation is in the presence of added water. The amount of added water is 10 preferably 0.1-500 gram per gram of soluble solids in the tea source.
Definitions
The term "tea-source" as used herein refers to any material 15 obtained from the plant Camellia sinensis or Camellia assamica or derived therefrom after processing such a plant material.
The term "tea product" as used herein means any product 20 derived from a tea-source. It includes, but is not limited to black tea, oolong tea, instant tea and green tea. It also includes slurry comprising mixture of a tea product with water.
25 The term "theaflavin-enhanced" as used herein refers to a tea product with enhanced level of total theaflavins including TF1, TF2, TF3 and TF4 as compared to the tea source. In particular, the tea product will usually have a higher amount of theaflavins per gram dry weight of soluble
30 solids in the tea product than the amount of theaflavins per gram dry weight of soluble solids in the tea source.
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The term "tea leaves" as used herein includes tea leaves,
buds and other parts of the plant Camellia sinensis or
Camellia assamica. 5
The term "dhool" as used herein means macerated tea leaves.
Tea leaves may be macerated by commonly used methods such as
rolling, crush tear curl (CTC), blending using a kitchen
blender etc. 10
The term enzymatic fermentation as used herein means
oxidative biotransformations mediated by enzymes.
Fermentation can be by means of enzymes endogenous in the
tea-source and capable of enzymatic fermentation- Non-15 limiting examples of such a tea-source include tea leaves
and dhool. Fermentation can also be by means of exogenous
enzyme and corresponding co-substrate.
20
25
The term "substantially deactivated endogenous enzymes" refers to a tea-source subjected to a step of deactivation of enzymes endogenously present within the tea-source. Typically, deactivation is thermal i.e. by subjecting to a step of exposure to a temperature greater than 100°C, also known as firing. Examples of tea-sources with substantially deactivated endogenous enzymes include instant tea and green tea.
The term "soluble solids in a tea-source" as used herein means the amount of solids obtained by (a) contacting a tea-30 source with water at 90° C for 1 hour, (b) separating the liquor from insoluble solids by centrifuge/filtration

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(Whatman 541 filter) and (c) evaporating water from the liquor to obtain soluble solids.
The term "dry weight basis" as used herein refers to a ratio 5 or weight % of a composition expressed on moisture-free basis. The term "fresh weight basis" as used herein refers to a composition expressed as % wt of a material including moisture. Typically tea leaves contain about 7 0% moisture and about 30% solids. Of the total solids, soluble solids
10 are usually about 40% by weight (on dry weight basis). Thus -12% soluble solids on fresh weight basis is equivalent to -40% soluble solids on dry weight basis. It should be noted that typical figures given above are representative and actual figures may vary somewhat depending on geographical
15 origin, species of tea and humidity.
The term "slurry fermentation" as used herein means fermentation of a tea-source where the ratio of added water to the tea-source is greater than 2:1 on fresh weight basis 20 or 6.7:1 on dry weight basis and 17:1 on soluble solids basis.
The term "solid state fermentation" as used herein means enzymatic fermentation of a tea-source where the ratio of 25 added water to the tea-source is less than 2:1 on fresh weight basis or 6.7:1 on dry weight basis and 17:1 on soluble solids basis. Typically the ratio of added water to the tea-source is less than 2:1 on soluble solids basis.
30 The term "exogenous epicatechin" as used herein refers to epicatechin that is externally added to the tea-source and

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excludes any epicatechin that may be present in the tea-source.
Enzyme activity is expressed as activity units or simply 5 units. Tannase activity units are quoted from supplier's data. For exogenous enzymes like polyphenol oxidase (PPO) and Peroxidase (POD), the enzyme activity can be determined as follows
10 Assay for polyphenol oxidase (PPO)
PPO is assayed spectrophotometerically using (+)-catechin as the substrate. The assay is performed with a reaction volume of 3 mL containing 3 mM catechin in 0.1 M phosphate citrate buffer pH 5.5. A 150 jlL aliquot of a freshly
15 prepared 60 mM catechin stock solution in water (70% v/v) -ethanol (30% v/v) mixture is used to obtain a catechin concentration of 3 mM in the reaction mixture. Enzyme solution of appropriate dilution is added to initiate reaction and the mixture is incubated at 40°C for 10-20 min.
20 The reaction is stopped using 200 JJ.L of a solution comprised of acetonitrile (60% v/v), acetic acid (10% v/v) and water (30% v/v) . The absorbance at 4 00 nm is then recorded and the activity calculated based on the relationship: activity of 1 unit = absorbance increase of 0.001.
25
Assay for peroxidise (POD)
The assay for POD is identical to that for PPO except that the reaction mixture also contains 7.3 mM of hydrogen peroxide. The requisite amount of hydrogen peroxide is
30 delivered using a 1.5% w/v stock solution. The reaction


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mixture is incubated for 10-20 min at 30°C. The reaction is then terminated as described above and the absorbance at 400 nm is determined. The activity was again calculated based on the relationship: activity of 1 unit = absorbance 5 increase of 0.001.
Detailed Description of the Invention
According to the present invention there is provided a process for preparing a theaflavin-enhanced tea product 10 including a step of subjecting a tea-source to enzymatic fermentation in the presence of added exogenous epicatechin.
Preferably, the enzymatic fermentation according to the present invention is by means of endogenous enzymes in the 15 tea-source capable of carrying out the fermentation.
Commercially available exogenous epicatechin (e.g. from Sigma, Herbs-Tech etc) can be added. Other sources of exogenous epicatechin that can be added according to the 20 present invention include epicatechin-rich extracts of other plant materials such as cocoa, grape juice etc.
The process according to the present invention can be in the presence of other exogenous catechins (including epicatechin
25 gallate, epigallactocatechin and epigallactocatechin gallate) added along with exogenous epicatechin. Preferably, the ratio of exogenous epicatechin to total exogenous catechins is greater than the ratio of endogenous epicatechin to total endogenous catechins in the tea-source.
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Preferred amount of exogenous epicatechin that is added is selected such the molar ratio of total epigallocatechin to total epicatechin is from 1:1 to 1:16, preferably from 1:1.5 to 1:10, more preferably from 1:2 to 1:9. 5
According to one of the preferred aspect of the invention, the amount of the added epicatechin is 35 to 750, preferably 75 to 520, more preferably 120 to 260 mg per gram dry weight of soluble solids in the tea-source. 10
According to a preferred aspect, when the tea source is tea leaves, epicatechin is added to the tea leaves after plucking. According to an alternative preferred aspect, the epicatechin is added to the tea leaves prior to plucking. 15
It is preferred that the tea source is substantially unfermented, i.e., it has not been previously subjected to a step of enzymatic fermentation. In particular, it is preferred that the weight ratio of total catechins to 20 theaflavins in said tea-source is greater than 10, preferably greater than 20, more preferably from 50 to 10000. Alternatively or additionally, the tea source preferably has no more than 10 mg, more preferably 5 mg, most preferably 2 mg theaf lavin per g of dry weight of 25 soluble solids. It is particularly preferred that the tea-source is substantially devoid of theaflavins.
The process according to the present invention preferably includes a step of treating the tea-source with tannase 30 prior to or during said fermentation.


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According to a preferred aspect, the step of treating the tea-source with tannase is prior to the fermentation. The treatment with tannase is preferably carried out in an atmosphere of nitrogen (to prevent fermentation) at a 5 suitable temperature and for a suitable time. Suitable conditions can be determined by experiment. According to an alternate preferred aspect, the step of treating the tea-source with tannase is during fermentation. It is preferred that the amount of the tannase is 0.0 8 to 8, more preferably 10 0.2 to 4, most preferably 0.4 to 2 mg per gram dry weight of the soluble solids in the tea-source.
According to a preferred aspect, the fermentation is in the presence of added water. The amount of added water is
15 preferably 0.1 to 500 gram per gram of soluble solids in the tea source, in one of the preferred aspect, the amount of added water is 0.1 to 17, more preferably 0.1 to 10, most preferably 0.1 to 2 gram per gram of soluble solids in the tea-source leading to solid state fermentation. According to
20 an alternate preferred aspect, the amount of added water is 17 to 500, more preferably 20 to 400, most preferably 25 to 200 gram per gram of soluble solids in the tea-source, leading to slurry fermentation.
25 According to a preferred aspect, the tea-source is tea leaves including endogenous enzymes capable of enzymatic fermentation and the process includes the steps of:
(a) withering of the tea leaves;
(b) macerating the withered leaves;


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fe) subjecting the macerated tea leaves to
enzymatic fermentation in presence of added
exogenous epicatechin; and
(d) firing, preferably to a temperature of about
5 120°C, to obtain a theaflavin-enhanced black
tea.
The process listed above preferably includes a step of treating the tea-source with tannase prior to or during the 10 step (c), i.e. enzymatic fermentation.
According to another preferred aspect, the tea-source is tea
leaves including endogenous enzymes capable of enzymatic
fermentation and said process includes the steps of:
15 (a) withering of said tea leaves;
(b) macerating of the withered leaves and adding
to the macerated tea-leaves 17 to 500 gram
water per gram of soluble solids to prepare a
slurry;
20 (c) subjecting the slurry to enzymatic fermentation
in presence of added exogenous epicatechin to obtain theaflavin-enhanced slurry.
The process listed above preferably includes a step of 25 treating the tea-source with tannase prior to or during the step (c), i.e. enzymatic fermentation.
Theaflavin-enhanced slurry can be preferably dried to reduce
moisture content to less than 5% to obtain theaflavin-
30 enhanced black tea. In another preferred aspect, insoluble
solids are separated from the theaflavin-enhanced slurry to


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obtain theaflavin-rich solution. The theaflavin-rich solution may be preferably dried to get theaflavin-enhanced tea product, which may, for example, be added to black tea, green tea, oolong tea, or instant tea. According to an 5 alternate preferred aspect, the theaflavin-rich solution can be coated on black tea, green tea or oolong tea and dried to obtain theaflavin-enhanced tea.
According to another preferred aspect, the enzymatic 10 fermentation is by means of an exogenous enzyme and a co-substrate. The fermentation by means of an exogenous enzyme and a co-substrate is preferred when the tea-source has substantially deactivated endogenous enzymes. Examples of tea-source with substantially deactivated endogenous enzymes 15 are green tea or instant green tea. Slurry fermentation is preferred when the tea-source has substantially deactivated endogenous enzymes.
A suitable combination of an exogenous enzyme and
20 corresponding co-substrate is chosen such that the
combination is capable of the fermentation of the tea-
source .
According to a preferred aspect, the exogenous enzyme is 25 polyphenol oxidase and the corresponding co-substrate is oxygen. According to a further preferred aspect, the amount of said polyphenol oxidase is 1.5*104 to 1. 5*107, more preferably 3.5*10^ to 1.5x10s, most preferably 7.5*104 to 7. 5*105 units per gram dry weight of soluble solids in said 30 tea-source. Polyphenol oxidase can be commercially obtained,


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e.g. from Worthington or can be extracted and purified from suitable plant material.
According to another preferred aspect, the exogenous enzyme 5 is peroxidase and the co-substrate is hydrogen peroxide. Peroxidase can be obtained from commercial sources such as Sigma or can be extracted and purified from suitable plant material. According to a further preferred aspect, the amount of said peroxidase is 1.5><104 to 1.5><107, more 10 preferably 3.5*104 to 1.5*105, most preferably 7.5*104 to 7.5*105 units per gram dry weight of soluble solids in said tea-source.
It is preferred that the amount of said hydrogen peroxide is 15 7 to 700 mg, more preferably 15 to 300 mg, most preferably 35 to 100 mg per gram dry weight of soluble solids in said tea-source.
According to a preferred aspect, the tea-source is green tea 20 or instant green tea and the process includes the steps of:
(a) adding 17 to 500 gram water per gram of
soluble solids to the tea-source to prepare a
slurry;
(b) subjecting the slurry to enzymatic
25 fermentation by adding an exogenous enzyme and
a co-substrate, in presence of added exogenous epicatechin, to obtain theaflavin-enhanced slurry.
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The process listed above preferably includes a step of treating the tea-source with tannase prior to or during the step (b), i.e. enzymatic fermentation.
5 Theaflavin-enhanced slurry can be preferably dried to reduce the moisture content to less than 5% (by weight) to obtain theaflavin-enhanced black tea. In another preferred aspect, insoluble solids are separated from the theaflavin-enhanced slurry to obtain theaflavin-rich solution. The theaflavin-
10 rich solution may be preferably dried to get theaflavin-enhanced tea product, which may, for example, be added to black tea, green tea, oolong tea, or instant tea. According to an alternate preferred aspect, the theaflavin-rich solution can be coated on black tea and dried to obtain
15 theaflavin-enhanced black tea.
According to another preferred aspect, the theaflavin-enhanced slurry obtained in the above process is coated on black tea to obtain theaflavin-enhanced black tea. 20
According to another preferred aspect, the theaflavin-enhanced slurry is coated on instant tea to obtain theaflavin-enhanced instant tea.
25
Examples
The invention will now be described with reference to specific examples. The following examples are presented by way of illustration only and they do not limit the scope of 30 the invention in any way.


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Materials and Methods
Table 1 gives a list of materials used in examples.
Table 1: Materials used

Method of analysis of theaflavins
Extraction of theaflavins
10 The total theaflavin level was determined using an aqueous methanolic extract.
The samples from solid state fermentation were extracted using 70% methanol extraction at ^80°C for 10 min at the 15 high aqueous methanolic solution to dhool ratio of 16.7:1 on fresh weight basis. A sample of the extract was diluted 1:1 using a solution comprised of 90% v/v water, 10% v/v


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acetonitrile, 0.5 g/L ascorbic acid and 0.5 g/L ethylenediamine tetraacetic acid (EDTA) to prevent oxidation of theaflavins and catechins.
5 For the slurry fermentation system this extract was prepared by adding requisite amount of 100% methanol to the aqueous slurry to obtain the composition 70% methanol and 30% water. The amount .of methanol added was 2.33 fold the volume of water present therefore the extraction is performed at
10 aqueous methanolic solution to dhool ratio of 16.7:1 on fresh weight basis or -55.7:1 on dry weight basis. At this high ratio of 70% methanol to dhool quantitative extraction of theaflavin is obtained at ~80°C with extraction time of 10 min.
15
Analysis of theaflavins
The samples were stored at -80°C and theaflavins were analysed using high performance liquid chromatography (HPLC) with diode array detection.
20
The theaflavins were analysed by HPLC (Shimadzu) using an octadecylsilica (C18) column (Nova-pak ex. Waters, 3.9 mm i .d.x 150 mm) with detection at 380 nm, column temperature of 40CC, injection volume of 20 }UL and flow rate of 1
25 mL/min. The mobile phases for theaflavin analysis were 2%
(v/v) acetic acid in water (mobile phase A) and
acetonitirile (mobile phase B) . A gradient from 8% B to 69
% B over 50 min was used to separate the theaflavins
following which the column was equilibrated with 8% buffer A
30 for 5 min. Commercially available high purity theaflavins were used as standards for quantification.


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Solid state fermentation
The solid state fermentation was performed at room temperature (~25°C) by spreading of 5 g of dhool on a flat 5 surface exposed to atmosphere. The fermentation time was 90-120 min. When solid state fermentation was performed with tannase, the tannase dosage was 1.67 mg per gram soluble solids in the tea-source. Water containing the requisite amount of epicatechin, or epicatechin and tannase was added 10 to the dhool at the ratio of 1.7:1 (on soluble solids basis).
The amounts of epicatechin and tannase added in solid state fermentation are given in Table 2 for various examples.
Table 2: Examples of solid state fermentation

Example Epicatechin (rag per gram soluble solids in dhool) Tannase (mg per gram soluble solids in dhool)
Comparative example A 0 0
1 35 0
2 118 0
3 258 0

Comparative example Bl 0 0
Comparative example B2 0 1.67
4 35 1.67
5 118 1.67
6 258 1.67
7 518 1.67
Examples 1-7 are examples of processes according to the 20 present invention whilst Comparative examples A, Bl and B2 are outside the scope of invention.
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Slurry Fermentation
The slurry fermentation was performed in shake-flasks using an orbital shaker at 190-200 rpm at 30°C. 5 g (fresh weight) 5 of dhool was used for the fermentation experiment with the water to soluble solids ratio of 41.7:1 (or 5:1 on fresh weight basis). The required amount of epicatechin was added to the slurry prior to fermentation. The fermentation time was 90 min. When slurry fermentation was performed with 10 added tannase (activity of 50,000 tannase activity units per gram, ex. Kikkoman) , tannase was dosed at 0.83 mg/g soluble solids basis using a stock solution of 1 g/L.
The amount of epicatechin and tannase added in slurry 15 fermentation are given in Table 3 for various examples.
Table 3: Examples of slurry fermentation

Example Epicatechin