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(11)    Patent Number:  KE 444   

(45)    Date of grant:  16/06/2011     

(51)    Int.CI.8: A 23F 3/10

(21)Application Number: KElP! 2009/ 000916

(22) Filing Date: 15111/2007

(30) Priority data: 06125217.7  01/12/2006  EP;  07111788.1 05/07/2007 EP and 792/MUM/2007 24/04/2007 IN

(86)  PCT data PCT/EP07 /0623 73 15/11/2007 wo 2008/065006 05/06/2008

(73) Owner: UNILEVER PLC of Unilever House, 100 Victoria Embankment, London, EC4Y ODY, GB formerly of Unilever House, Blackfriars, London, EC4P 4BQ, United Kingdom

(72) Inventor:  MULLICK, Ashim ofHindustan Unilever Limited, Research Centre, 64 Main Road, Whitefield P.O., 560 066, India and PU1REVU, Saroja, Mani of 12-2-823/A/1/3 Brindavan Apartments, Santosh Nagar, Mehdipatnam, Hyderabad 500 028, India.

(74) Agent/address for correspondence: Kaplan & Stratton Advocates, P.O. Box 40111-00100, Nairobi
 
(54) Title: PROCESS FOR THE PREPARATION OF THEAFLA YIN-ENHANCED TEA PRODUCTS.

(57) Abstract:  Provided is 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.

PROCESS FOR THE PREPARATION OF THEAFLAVJN-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        (TFl),        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

cancer, cardiovascular and cerebro-vascular diseases, diabetes, and hypercholesterolemia. However, normal levels of theaflavins present in tea are l-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   thea flavin   obtainable  by   the   above

25    methods is limited and there is a continuing need for tea products having further enhanced levels of theaflavin.

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

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 TFl, 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.
 
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.


The    term    "substantially    deactivated    endogenous    enzymes"

20    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

25    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

(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 70% 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

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 l!L 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 l!L of a solution comprised of acetonitrile (60% v/v), acetic acid (10% v/v) and water (30% v/v). The absorbance at 400 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

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.

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 theaflavin 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.

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 sui table 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  0.08  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;

(c)    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

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 1.5xl0 4 to 1.5xl0 7 , more preferably 3.5xl0 4 to 1.5xl0 6 , most preferably 7.5xl0 4 to 7.5xl0 5 units per gram dry weight of soluble solids in said

30    tea-source.   Polyphenol  oxidase  can  be  commercially  obtained,
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    1.5x10 4     to    1.5x10 7 ,      more 10    preferably   3.5xl0 4     to   1.5x10 6 ,      most   preferably   7.5x10 4     to 7.5xl0 5   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.

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 sol uti on 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.

Materials  and  Methods

Table  1  gives  a  list  of  materials  used  in  examples.

                Table    1:    Materials    used       
                               
        Ingredient    Form  of  ingredient        Source    and   
                                    geographical    origin   
                                    of  ingredient       
                                   
        Tea  leaves    Frozen    (stored    at    SouthIndian    tea   
                               
                -80°C)                    gardens       
                                       
        Dhool    Prepared        from    the           
                frozen    tea    leaves    by           
                immersing   the    leaf    in           
                liquid    nitrogen    and           
                then   blending   it   in   a           
                kitchen  blender.               
                                           
        Epicatechin                        Sigma  Aldrich       
                   
        Tannase  KT-    Stock  solution  of  1  g/1    Kikkoman,  Japan   
    50                                   
5                                           
        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

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 40°C, injection volume of 20 !J.L 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. Corrunercially available high purity theaflavins were used as standards for quantification.

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.

15

Table  2:  Examples  of  solid  state  fermentation

Example            Epicatechin    Tannase    (mg  per
                (mg  per    gram    gram  soluble
                soluble    solids  in    solids    in
                dhool)                dhool)   
Comparative    example    A    0                    0       
1                35                    0       
2                118                    0       
3                258                    0       
                                       
Comparative    example    B1    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, B1 and B2 are outside the scope of invention.

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    Tannase    (mg  per   
                    (mg  per   gram    gram  soluble   
                           
                    soluble  solids  in    solids    in   
                    dhool)    dhool)       
    Comparative    example    C    0    0       
    8                35    0       
    9                118    0       
    10                258    0       
    11                518    0       
    Comparative    example    Dl    0    0       
    Comparative    example    D2    0    0.83       
    12                35    0.83       
                               
    13                118    0.83       
    14                258    0.83       
    15                518    0.83       

Examples    8-15   are   examples   of   processes   according   to   the

20    present invention whilst Comparative example C, Dl and D2 are outside the scope of invention.
Results

Total    theaflavins  were  analyzed  for   tea  products  of  all  the

5    examples and the results are given in Table 4 for solid state fermentation and Table 5 for slurry fermentation.

Table  4:  Total  theaflavins  in  solid  state  fermentation

Example                Total  theaflavins  in
                tea  product
                (weight  % on  dry
                weight  basis)
Comparative    example  A            1.3
1                1.8
2                2.4
3                1.9
               
Comparative  example  Bl    1.9
Comparative    example  B2        3.1
4                4.3
5                4.0
6                4.9
7                5.2
10

It  is  clear  from  the  above  that  tea  products  of  Examples  1-4

prepared    by    a    process    including    step    of    enzymatic

fermentation    in    presence    of    exogenous    epicatechin    have

15    enhanced levels of theaflavins as compared to tea product of Comparative Example A.



Similarly,    tea   products    of   Examples    5-8   prepared   by   a

process    including    step    of    enzymatic    fermentation    in

20    presence of exogenous epicatechin and tannase have enhanced levels of theaflavins as compared to tea products of Comparative Examples Bl and B2.

Table  5:  Total  theaflavins  in  slurry  fermentation

Example                Total  theaflavins  in  tea
                product
                (weight  % on  dry  weight
                basis)
Comparative    example    C    1.9   
8                2.0   
9                2.6   
10                3.1   
11                2.9   
Comparative    example    Dl    1.9   
Comparative    example        D2    3.7   
12                5.1   
13                6.4   
14                6.0   
15                6.4   

It  is  clear  from  the  above  that  tea  products  of  Examples   8-5     11    prepared   by    a    process    including    step    of    enzymatic fermentation    in    presence    of    exogenous    epicatechin    and externally   added   water   have   enhanced   levels   of   theaflavins

as  compared  to  tea  product  of  Comparative  Example  C.


10    Similarly, tea products of Examples 12-15 prepared by a process including step of enzymatic fermentation in presence of exogenous epicatechin, tannase and externally added water have enhanced levels of theaflavins as compared to tea products of Comparative Examples Dl and D2.

15
It is also clear from all the examples above that theaflavin levels increase with the increase in the level of epicatechin up to about 300 mg/g epicatechin per dry weight of dhool. With further increase in epicatechin levels to

20    about 500 mg/g dry weight of dhool, the trend indicates either a plateau or reduction in theaflavin levels. It is therefore envisaged that incorporation of epicatechin at

levels beyond 750 mg per g dry weight of dhool is unlikely to offer further enhancement of theaflavin or be economically viable.


5 Thus, according to the present invention, it has been possible to prepare theaflavin-enhanced tea products using solid-state as well as slurry fermentation by carrying out enzymatic fermentation in presence of exogenous epicatechin, either in presence or absence of tannase.

Claims

1.    A   process    for    preparing    a    theaflavin-enhanced    tea

product    including  a   step  of  subjecting  a   tea-source   to

5    enzymatic fermentation in presence of added exogenous epicatechin.



2. A process as claimed in claim 1 wherein amount of said epicatechin is from 35 to 750 mg per gram dry weight of
10    soluble  solids  in  said  tea-source.


3. A process as claimed in claims 1 or 2 including a step of treating the tea-source with tannase prior to or during said fermentation.

15

4.    A process as claimed in claim 3, wherein amount of said tannase is from 0.08 to 8 mg per gram dry weight of said soluble solids.


20    5. A process as claimed in any one of the preceding claims wherein said fermentation is in the presence of added water.


6.    A  process  as   claimed  in  claim  5  wherein  amount  of  said

25    water is 0.1 to 500 gram per gram dry weight of soluble solids in said tea-source.



7. A process as claimed in any one of the preceding claims wherein said tea-source is tea leaves including

30    endogenous enzymes capable of said enzymatic fermentation and said process includes the steps of:

(a)    withering  of  said  tea  leaves;

(b)    macerating  the  withered  leaves;

(c)    subjecting the macerated tea leaves to

enzymatic    fermentation   in   presence   of   added

5    exogenous  epicatechin;  and

(d)    firing to obtain a theaflavin-enhanced black tea.

8. A process as claimed in any one of the preceding claims wherein said tea-source is tea leaves including endogenous enzymes capable of enzymatic fermentation and said process includes the steps of:

(a)    withering  of  said  tea  leaves;

(b)    macerating the withered leaves and adding to the macerated tea-leaves 17 to 500 gram water per gram of soluble solids to prepare a slurry;

(c)    subjecting the slurry to enzymatic fermentation in presence of added exogenous epicatechin to obtain theaflavin-enhanced slurry.
 


9.    A process as claimed in any one of the preceding claims wherein said fermentation is carried out by adding exogenous enzyme and a co-substrate.

25

10. A process as claimed in claim 9 wherein said tea-source has substantially deactivated endogenous enzymes.

11.    A  process   as   claimed   in   claim   9   or   10   wherein   said

30    exogenous enzyme is polyphenol oxidase and said co-substrate is oxygen.
12.    A process as claimed in claim 9 or 10 wherein said exogenous enzyme is peroxidase and said co-substrate is hydrogen peroxide.

5

13.    A process as claimed in any one of claims 9 to 12 wherein said tea-source is green tea or instant tea and said process includes the steps of:

(a)    adding   17   to    500   gram   water   per   gram   of

10    soluble solids to the tea-source to prepare a slurry;

(b) subjecting the slurry to enzymatic fermentation by adding an exogenous enzyme and a co-substrate, in presence of added

15    exogenous epicatechin, to obtain theaflavin-enhanced slurry.



14.    A  process  as  claimed  in  any  one  of  the  preceding  claims

wherein    the    weight    ratio    of    total    catechins    to

20    theaflavins  in  said  tea-source  is  greater  than  10.

15.    A  process  as  claimed  in  any  one  of  the  preceding  claims

wherein    said    tea-source    has    no    more    than    10    mg

theaflavins  per  g  of  dry  weight  of  soluble  solids.

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