Note: Descriptions are shown in the official language in which they were submitted.
Method of Maximizing Epigallocatechin Gallate Content in Tea
FIELD OF THE INVENTION
[0001] This invention relates generally to novel methods of maximizing the
extraction of
catechins from brewed tea. More specifically, this invention relates to the
maximization of
epigallocatechin gallate (EGCG) levels in tea made from Camellia sinensis.
BACKGROUND
[0002] The current invention has application to the field of tea brewing and
consumption. Tea is
produced from the Camellia sinensis plant, a species of evergreen shrub or
small tree. Two
major varieties of C. sinensis are grown: C. sinensis var. sinensis, and C.
sinensis var. assamica.
The former is used to produce Chinese teas, while the latter is used to
produce Indian Assam
teas. Forms of tea produced from these varieties of C. sinensis include green,
white, yellow,
black, oolong, pu-erh and matcha tea. Most of the teas are produced with the
leaves exclusively,
although some forms of tea also include the twigs and stems.
[0003] Some forms of tea do not contain any C. sinensis, including many herbal
and fruit
infusions. This invention does not pertain to teas that do not contain C.
sinensis materials. For
the purpose of this disclosure, the term "tea" includes only those beverages
produced with
material from C. sinensis.
[0004] For the purpose of this invention, the term "brewed tea" refers to a
tea-based beverage
that results from allowing the C. sinensis plant material to steep in water in
order to extract the
flavor and chemical compounds from the tea. In general, tea is brewed or
steeped using hot
water, most typically boiling water or water that is close to the boiling
point. Some tea is also
"sun tea" that is allowed to brew at ambient temperatures in sunlight. Tea can
also be brewed
using cold water over a longer period of time, optionally in a refrigerated
environment. Any
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method of combining tea leaves with water to produce a beverage is considered
to be "brewed
tea" for the purposes of this disclosure. The time that the leaves are allowed
to remain in the
water can vary from a few seconds up to many hours, depending on the
techniques used and the
desired strength of the beverage.
[0005] C. sinensis is native to East Asia, Southeast Asia, and the Indian
Subcontinent. Today, it
is cultivated around the world in tropical and subtropical regions. The plant
is usually kept
trimmed to less than 2 metres in height in order to facilitate picking. Tea
leaves are typically
harvested several times during the harvesting season, which begins in March
and carries through
to November in the Northern Hemisphere. In countries south of the equator, the
harvesting
season runs October to May. Picking tea typically involves picking a terminal
bud and two
young leaves. Picking can be done mechanically or by hand.
[0006] After picking, the leaves of C. sinensis begin to wilt and oxidize
unless they are heated
immediately. Typically, the enzymatic oxidation reaction in the leaves that
causes darkening and
release of tannins is halted by heating, often simultaneously as the leaves
are dried. Tea is
conventionally dried for ease of packaging, shipping and storage.
[0007] All teas produced from C. sinensis contain a number of chemical
compounds including
caffeine, theobromine, theophylline, and polyphenols. Among the polyphenols
are flavonoids
and catechins. Flavanols (also called flavan-3-ols) are derivatives of flavans
that use the 2-
pheny1-3,4-dihydro-2H-chromen-3-ol skeleton. The compounds from tea that fall
into this group
include catechin (C), epicatechin gallate (ECG), epigallocatechin (EGC),
epigallocatechin gallate
(EGCG), epicatechin (EC), and catechin gallate (CG). C and EC are epimers,
while EGC and
gallocatechins contain an additional phenolic hydroxyl group when compared to
C and EC.
EGCG and EGC are the most abundant catechins in green tea (Williamson et al.
(2011) Moi.
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Nutr. Food. Res. 55:864-873).
100081 The catechins contained in tea have recently been of increasing
interest due to their
strong antioxidant effect, as described in, for example, Zhao B. et.
al.(1989), Cell Biophys. 14,
175, which outlines the scavenging effects of the catechins on active oxygen
radicals, and Huang
M. T. et al., (1992) Carcinogenesis 13, 947, which outlines the inhibition of
tumor initiation and
promotion in mouse skin as a result of topical application of catechins.
100091 In addition, research is increasingly demonstrating a cancer chemo-
preventive effect of
catechins. Singh et al. (2014) Biochem Pharmacol. 82(12): 1807-1821 review a
broad range of
studies showing that EGCG performs as a powerful antioxidant, preventing
oxidative damage in
healthy cells and acting as an antiangiogenic and antitumor agent. In
addition, Singh mentions
the benefits of EGCG in early studies in diabetes, Parkinson's disease,
Alzheimer's disease,
stroke and obesity.
[0010] Given the increasing interest in tea catechins, and EGCG in particular,
there is a need for
methods of maximizing the EGCG content of tea in order to take full advantage
of its beneficial
properties.
SUMMARY OF THE INVENTION
100111 Provided is a method of maximizing EGCG content from tea. In one
embodiment, the
content of EGCG is maximized by harvesting tea leaf and bud material from C.
sinensis,
minimizing the withering time of said leaf and bud material, treating the leaf
and bud material
with an acid treatment, infusing the treated material in water, and macerating
the treated, infused
material to produce a final tea product with higher EGCG content.
[0012] The acid treatment may comprise ascorbic and citric acid and may be
obtained from
citrus fruits or other sources of ascorbic and/or citric acid.
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[0013] Optionally, the treated tea leaf and bud material may be subjected to a
carefully
controlled freezing step in order to maintain shelf life and stability of the
EGCG levels within the
product. The infusion and maceration may optionally occur after the freezing
step.
DETAILED DESCRIPTION
[0014] The following is a detailed description of an embodiment of technology
and methods
enabling improved catechin levels in tea produced from C. sinensis, and in
particularly,
improved EGCG levels in tea produced from C. sinensis.
[0015] Following the picking of tea leaves, the leaves begin to wilt and
oxidize. This is
desirable in tea because it causes the leaves to turn darker as they produce
tannins, which create
the characteristic flavor of tea. The withering process is carefully
controlled by tea producers by
monitoring of the humidity, temperature, and air flow around the leaves. It is
desirable for the
leaves to wither evenly. The darkening, and tannin production, are typically
stopped during the
drying of the tea, by heating. The ending of the withering process is
typically governed by the
amount of water loss, usually measured by percent, and usually a moisture
reduction of one third
to one half is normal. Further moisture reduction occurs later in the
conventional tea processing
cycle.
[0016] In the present application, it is recommended to allow the leaves to
wither for a period of
fewer than 16 hours. The withering time can optionally be eliminated entirely
by processing
using a mobile in-field unit to begin processing the leaves immediately.
Alternatively, withering
can be carried out for a short time, such as a few minutes, 5 minutes, 10
minutes, 15 minutes, 30
minutes, 45 minutes, or up to an hour. Withering can also be carried out for a
longer time,
including 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8
hours, 9 hours, 10 hours,
11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or any period of time up to
16 hours. Withering
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can be achieved using standard techniques known in the art of tea production.
The working
examples described later in this application use a period of 7 hours for
withering. This period
was chosen as the average time frame for the leaves to proceed from picking to
processing in an
average tea harvesting operation.
100171 The withering process reduces the level of catechins in tea, as
demonstrated by Chen.
(Chen, C. (1984) Theory in Tea Processing, 51-52, 205-217, Shanghai: Shanghai
Science and
Technology Press.) As seen in Table 1, below, the catechins in white tea drop
significantly
during the withering process.
Table 1: The variation of catechins (in mg) of white tea during processing
Catechin Fresh Withering 8 Withering 16 Withering 32
Leaves hours hours hours
EGC 36.70 24.54 19.62 8.61
GC 23.74 16.56 11.42 4.91
EC + C 24.32 20.76 15.20 10.51
EGCG 122.56 90.08 77.02 55.49
ECG 40.62 31.89 26.31 20.21
Total 247.94 183.83 149.57 109.73
[0018] During the withering process, oxidation begins to occur inside the tea
leaves.
Chlorophyll in the tea leaves is degrading, while flavor compounds begin to
accumulate. The
cell walls of the individual leaf cells begin to break down, which initiates
the activity of
polyphenol oxidase and peroxidase. These compounds cause oxidation of the cell
contents.
Some components of the leaves will degrade into volatile components. During
oxidation, the
catechins, which are located in the leaf cell vacuoles, are converted into
flavonoids ¨ specifically
theaflavins and thearubigins. These compounds provide the tea with taste and
colour. However,
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this process also reduces the level of catechins because they are being
converted into other
compounds. Therefore, it is permissible to allow some oxidation for the
purpose of flavor, but
ideally, the time from picking to freezing should be minimized in order to
prevent oxidation,
thereby retaining the maximum level of catechins while not completely
compromising flavor
profiles.
[0019] Oxidation in standard tea production requires careful control of
humidity and oxygen.
Oxidation occurs best at a temperature range of 26.7 to 29.4 '14C (80 to 85
F), and is slowed
almost completely at a range of 60 to 65.5 C (140 to 150 F) (Basu & Ullah
(1978) Two and A
Bud 25(1): 7-11). For the purposes of this invention, no oxidation, or very
limited oxidation is
desirable because more of the catechins are left intact. When oxidation is
allowed to continue in
the leaves, many of the catechins will have been converted, leaving far fewer
catechins in the
final tea. Thus, unlike in conventional tea processing, the leaves used to
produce tea according
to this disclosure are not subjected to a specific oxidation step.
[0020] The oxidation process in standard tea production is typically stopped
by heating the
leaves. This is also referred to as fixing or kill-green. To stop oxidation,
the leaves must be
heated to a minimum of 65.5 C (150 F). Numerous methods can be used to heat
the leaves,
including steaming, pan firing, boiling, heated tumbling, heated drying in
ovens or similar units,
and sun drying, which results in fixing as a result of dehydration rather than
heating. Because of
the intent to maximize catechins, the disclosed invention does not use a
heated fixing step. The
intent of the invention is to take the leaves from the picking step to the
freezing step as quickly
as possible by minimizing any withering and also minimizing any oxidation. The
tea leaves in
the present invention are subjected to freezing as soon as possible after
picking. Unlike in
conventional tea processing, in the present disclosure, the combination of the
acid treatment and
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subsequent freezing process acts as a fixing step.
[0021] In general, tea leaves are dried as a final step in their processing.
Drying is a convenient
and relatively simple way to keep tea leaves shelf-stable and the process also
makes the leaves
easier to transport and store. Drying can also potentially enhance the flavor
of the tea. Drying is
typically achieved using a commercial dryer such as a heater or fluidized bed
dryer, an oven, or
sun drying. Less common methods include charcoal firing or drying on a heated
floor. Drying
typically aims to reduce moisture levels to 2-3%, slowing oxidative processes
almost completely
and making the leaves shelf stable. Unlike conventional tea processing, in the
present disclosure,
prolonged drying is not a desirable step because it will reduce catechin
levels.
[0022] Tea leaves are conventionally dried to approximately one quarter of
their original weight.
Typically, 10 grams of leaves/buds are reduced to 2.5 grams, which is used for
a typical 250 mL
serving of brewed tea. Often, tea is dried to a point that it contains one to
six percent moisture
content. Contrary to traditional tea drying methods, the present disclosure
aims to avoid such
drying. The present disclosure aims to produce a tea from leaves that retain
50 to 100% of their
original moisture content at the time of picking. This is achieved by the
minimal withering time
and subsequent treatment of the leaf and bud material. Shorter withering times
result in higher
levels of EGCG and other catechins.
[0023] Ascorbic acid has previously been demonstrated to stabilize catechins
in green tea (Chen
et al. (1998) J. Agric. Food Chem. 46(7): 2512-2516). Chen showed that while
catechins are
typically unstable and degraded quickly, the addition of acid would increase
their stability. Chen
demonstrated that adding ascorbic acid to catechins in a sodium phosphate
buffer improved their
stability for up to 20 hours.
[0024] By using a lemon juice treatment on the harvested and withered tea
leaves, thereby
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providing an acid treatment, the instant invention demonstrates a significant
increase in catechin
levels. In addition, applicants have demonstrated that the maceration of a
lemon-treated sample
of tea leaves, coupled with a second infusion, results in 79 times the level
of EGCG than a
typically-processed tea sample. This increase is surprisingly high in
comparison with typically
processed teas.
[0025] The following examples illustrate the present invention in more detail
and are illustrative
of how the invention described herein could be implemented in tea. The initial
steps of Example
were conducted in North Carolina and the analysis was subsequently conducted
in Quebec,
Canada. Testing was conducted in October and November of 2016.
[0026] Example 1: Preparation of Samples
[00271 The invention described herein was practiced using C. sinensis var.
sinensis leaf obtained
from a nursery in North Carolina. Two leaves and a bud were picked from three-
year old plants.
The total amount picked was between 70 and 80 grams of plant material. The
leaves were
allowed to wither naturally on a bamboo rack for seven hours.
[0028] The gathered, withered plant material was divided into 6 separate
samples. One sample
gave inconclusive results, and another sample was not ultimately tested, so
only four samples
will be reviewed in this example.
[0029] The four samples were subjected to different treatments as shown in
Table 2, below.
Table 2: Sample Treatments
# Name Treatment Methodology
1 Steamed 10 grams of plant material was steamed in a bamboo steaming
rack with a
lid over 95 C boiling water for 40 seconds, then vacuum sealed in plastic
and rapidly frozen in controlled conditions at -27 C using dry ice.
2 Processed 10 grams of plant material was steamed in a bamboo steaming
rack with a
lid over 95 C boiling water for 45 seconds, then left to dry on the
bamboo rack to wither for 30 minutes at a temperature of 65 C, and
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vacuum-sealed. Post-drying weight was 2.6 grams.
3 Lemon- 10 grams of plant material was bathed in approximately 6
tablespoons of
treated freshly-squeezed lemon juice for 10 seconds. The bathing step
was
accomplished using a glass jar, and the leaves were stirred within the
lemon juice and were removed from the bath using a strainer. The sample
was then vacuum-sealed and rapidly frozen in controlled conditions at -
27 C using dry ice.
4 Natural 10 grams of
plant material was vacuum-sealed and rapidly frozen in
controlled conditions at -27 C using dry ice.
[0030] Samples 1, 3 and 4 were transported on dry ice and shipped to a testing
facility in
Quebec, Canada. Sample 2 was shipped to the same testing facility but, because
it was not
frozen, it was treated in the same manner as conventional finished tea and was
therefore
transported at ambient temperature.
100311 Example 2: Infusion Testing
[0032] The treated leaves described in Example 1 were subjected to two
infusions. Each sample
was infused in 250 mL of 95 C (203 F) water for a duration of four minutes.
Then the liquid
was drained and a second 250 mL of 95 C (203 F) water was added to the same
leaves for a
second infusion. Samples 1, 2 and 4 had the liquid drained for testing. Sample
3 was subjected
to a further step of blending for 2 minutes in a blender following the second,
6-minute infusion,
and then the final liquid was filtered prior to testing.
[0033] The results for each of the four samples are provided in Tables 3
through 6, below.
Table 3: Results for Sample 1: Steamed
Analysis Method Infusion Result
1 4 072 ug/lOg
tea
Epigallocatechin (EGC) UPLC-MS
2 5 826
pig/lOg tea
Catechin (C) UPLC-MS 1 673 ug/lOg tea
2 1 288 mg/lOg
tea
1 2 580 mg/lOg
tea
Epicatechin (EC) UPLC-MS
2 3 037 vigil
Og tea
Epigallocatechin gallate (EGCG) UPLC-MS 1 6 170
pig/10g tea
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2 9 387 g/lOg tea
1 3 180 g/lOg tea
Epicatechin gallate (ECG) UPLC-MS
2 3 256 g/10g tea
1 560 g/lOg tea
Catechin gallate (CG) UPLC-MS
2 927 g/log tea
1 40 mg/lOg tea
Caffeine HPLC-UV
2 17 mg/lOg tea
USDA 1 5 131 TEAC '
H-ORAC
Spectrofluorimetry 2 6 806 TEAC '
USDA 1 513 mo1/250 mL tea 2
H-ORAC
Spectrofluorimetry 2 513 mo1/250 mL tea 2
I TEAC: Trolox Equivalent Antioxidants Capacity. Results expressed in mol
TE/100 g of
sample.
2 Units to compared Camellia sinensis results
Table 4: Results for Sample 2: Processed
Analysis Method Infusion Result
1 3034 g/2.6 g tea
Epigallocatechin (EGC) UPLC-MS
2 383 g/2.6 g tea
1 1278 42.6 g tea
Catechin (C) UPLC-MS
2 1596 g/2.6 g tea
1 3541 g/2.6 g tea
Epicatechin (EC) UPLC-MS
2 2760 g/2.6 g tea
1 5318 g/2.6 g tea
Epigallocatechin gallate (EGCG) UPLC-MS
2 1201 g/2.6 g tea
1 3363 g/2.6 g tea
Epicatechin gallate (ECG) UPLC-MS
2 2979 g/2.6 g tea
1 905 g/2.6 g tea
Catechin gallate (CG) UPLC-MS
2 1160 g/2.6 g tea
1 45 mg/2.6 g tea
Caffeine HPLC-UV
2 15 mg/2.6 g tea
USDA 1 27 000 TEAC
H-ORAC
Spectrofluorimetry 2 26 000 TEAC
USDA 1 703 ilmo1/250 mL tea 2
H-ORAC
Spectrofluorimetry 2 675 mo1/250 mL tea 2
I TEAC: Trolox Equivalent Antioxidants Capacity. Results expressed in mol
TE/100 g of
sample.
2 Units to compared Camellia sinensis results
Table 5: Results for Sample 3: Lemon Treated
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Analysis Method Infusion Result
Epigallocatechin (EGC) UPLC-MS 1 14 711 ug/10 g tea
2 60 911 g/10 g tea
Catechin (C) UPLC-MS 1 294 ug/10 g tea
2 2 243 ug/10 g tea
Epicatechin (EC) UPLC-MS 1 4 322 ug/10 g tea
2 15 281 ug/10 g tea
Epigallocatechin gallate (EGCG) UPLC-MS 1 20 604 lig/10 g tea
2 95 156 ug/10 g tea
Epicatechin gallate (ECG) UPLC-MS 1 4 798 pig/10 g tea
2 19 852 ug/10 g tea
Catechin gallate (CG) UPLC-MS 1 ND'
2 ND
1
Caffeine HPLC-UV 42 mg/10 g tea
2 16 mg/10 g tea
H-ORAC USDA 1 8 000 TEAC
Spectrofluorimetry 2 37 415 TEAC 2
H-ORAC USDA 1 800 pmol/250 mL tea 3
Spectrofluorimetry 2 3 741 mol/250 mL tea 3
ND: Not Detected
2 TEAC: Trolox Equivalent Antioxidants Capacity. Results expressed in mai
TE/100 g of
sample.
3 Units to compared Camellia sinensis results
Table 6: Results for Sample 4: Natural
Analysis Method Infusion Result
Epigallocatechin (EGC) UPLC-MS 1 3 7501.1g/10g tea
2 4 119 ilg/lOg tea
Catechin (C) UPLC-MS 1 673 pg/lOg tea
2 1 514 ug/lOg tea
Epicatechin (EC) UPLC-MS 1 2 590 Rg/lOg tea
2 3 356 tig/lOg tea
Epigallocatechin gallate (EGCG) UPLC-MS 1 6 235 ug/lOg tea
2 8 197 ug/lOg tea
Epicatechin gallate (ECG) UPLC-MS 1 2 882 ug/lOg tea
2 4 155 ug/lOg tea
Catechin gallate (CG) UPLC-MS 1 583 ug/lOg tea
2 1 225 vig/lOg tea
Caffeine HPLC-UV 1 35 mg/lOg tea
2 21 mg/lOg tea
H-ORAC USDA 1 5 500 TEAC 1
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Spectrofluorimetry 2 8 100 TEAC 1
H ORAC USDA 1 547 mmo1/250 mL tea 2
- Spectrofluorimetry 2 810 ttmo1/250 mL tea 2
TEAC: Trolox Equivalent Antioxidants Capacity. Results expressed in 1.tmol
TE/100 g of
sample.
2 Units to compared Camellia sinensis results
[0034] Reviewing the results from the tables presented above, it can easily be
seen that all the
frozen samples of tea produced higher levels of EGCG than Sample 2, which was
dried and
processed in the manner of typical tea processing. Furthermore, the dried
sample exhibited a
significant drop in EGCG level in the second infusion, resulting in less than
25% of the amount
of EGCG recovered in the first infusion. Additionally, the frozen samples all
had higher EGCG
levels than the dried sample, and unlike the processed sample where the values
dropped upon the
second infusion, the values for ECGC were significantly higher on the second
infusion for all
three of the frozen samples.
[0035] The natural leaf sample EGCG levels were 221% higher than the processed
sample. The
steamed leaf sample EGCG levels were 238% higher than the processed sample,
and 108%
higher than the natural leaf sample. The lemon-treated sample had overall EGCG
levels that
were 1,776% higher than the processed sample, and 802% higher than the natural
leaf sample.
Notably, the EGCG levels on the lemon-treated sample, which were already
superior to the other
tested samples, nearly quadrupled following the maceration after the second
infusion. The
sample resulted in EGCG levels that were 7,923% higher following the second
infusion and
maceration than the second infusion of the processed sample, and 1160% more
EGCG on the
second infusion in comparison to the natural leaf sample.
[0036] Although various representative embodiments of this invention have been
described
above with a certain degree of particularity, those skilled in the art could
make numerous
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alterations to the disclosed embodiments without departing from the spirit or
scope of the
inventive subject matter set forth in the specification and claims. In some
instances, in
methodologies directly or indirectly set forth herein, various steps and
operations are described
in one possible order of operation, but those skilled in the art will
recognize that steps and
operations may be rearranged, replaced, or eliminated without necessarily
departing from the
spirit and scope of the present invention. It is intended that all matter
contained in the above
description or shown in the accompanying drawings shall be interpreted as
illustrative only and
not limiting. Changes in detail or structure may be made without departing
from the spirit of the
invention as defined in the appended claims.
100371 Although the present invention has been described with reference to the
embodiments
outlined above, various alternatives, modifications, variations, improvements
and/or substantial
equivalents, whether known or that are or may be presently foreseen, may
become apparent to
those having at least ordinary skill in the art. Listing the steps of a method
in a certain order
does not constitute any limitation on the order of the steps of the method.
Accordingly, the
embodiments of the invention set forth above are intended to be illustrative,
not limiting.
Persons skilled in the art will recognize that changes may be made in form and
detail without
departing from the spirit and scope of the invention. Therefore, the invention
is intended to
embrace all known or earlier developed alternatives, modifications,
variations, improvements,
and/or substantial equivalents.
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I. A method of increasing the content of epigallocatechin gallate in brewed
tea, comprising
a) harvesting tea leaf and bud material from C. sinensis;
b) allowing said tea leaf and bud material to wither for fewer than 16
hours;
c) treating said tea leaf and bud material with an acid treatment;
d) infusing said treated tea leaf and bud material in water; and
e) macerating said treated tea leaf and bud material in the water to
produce a
brewed, macerated tea beverage.
2. The method of claim 1 wherein said acid treatment is a result of
treatment in the
juice of one or more fruits originating from the Citrus genus.
3. The method of claim 2 wherein said one or more fruits are selected from
the
group consisting of:
a) lemon;
b) orange;
c) lime;
d) grapefruit; and
e) pomelo
4. The method of claim 1 wherein said infusion in water is conducted with
water that
has a temperature between 0 and 100 degrees Celsius.
5. The method of claim 1 wherein said water infusion step is conducted for
up to 20
hours.
6. The method of claim 1 wherein said treated tea leaf and bud material is
subjected
to rapid freezing following the acid treatment to preserve the stability of
the leaf and bud
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material, and further wherein in the infusing and macerating steps occur at a
later time
using the frozen tea leaf and bud material.
7. The
method of claim 1 wherein said maceration step is accomplished using a tool
selected from the group consisting of a blender, food processor, hand mixer,
knife or
other chopping instrument.
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