Enzymatic treatment of black tea leaf

A process for the enzymatic treatment of black tea leaf is disclosed. In particular, black tea leaf is wetted with water containing tannase and one or more cell-wall-digesting enzymes, such as cellulase, pectinase, papain, or hemicellulase, prior to extraction. The enzyme-moistened tea leaf is incubated in a closed system at room temperature for a few hours. The enzyme-treated tea is then neutralized with a suitable food grade base and the enzymes are inactivated by heating. The resulting treated tea can be extracted and processed in the usual manner or dried for use in tea bags in the usual manner. As a result, a higher yield of tea is obtained and the resulting instant tea has better solubility in cold water than conventional teas. The bag tea products an infusion which does not form haze at cold temperatures.

TECHNICAL FIELD 
This invention relates to the enzymatic treatment of black leaf tea. 
"Teas", loosely defined, can be made from a variety of botanical materials 
by extraction with water. Of particular importance is Camellia sinensis, 
or "true" tea, which accounts for a large proportion of the world's tea 
consumption. Two- to three-leaf shoots of the tea plant are harvested for 
processing. 
Tea processing technology is aimed at modifying the chemical composition of 
the raw tea leaf material in order to produce new flavor and aroma 
compounds, responsible for the taste, color and aroma of the manufactured 
teas that are so highly valued by the consumer. Green tea leaf has a 
bitter taste and grassy odor. This is transformed into an aromatic and 
flavorful manufactured tea by biochemical processes which are well known. 
In the production of conventional black tea, the tea leaves undergo the 
following treatment processes: (1) withering, (2) rolling and sorting, (3) 
fermentation, (4) firing, and (5) final sorting of the dried tea. These 
steps are described in excellent detail by Bokuchava et al., "Biochemistry 
and Technology of Tea Manufacture", CRC Critical Reviews and Science and 
Nutrition, 12 (4) pp. 309-370 (July 1980), which is incorporated herein by 
reference. The fermentation process for conversion of green teas to black 
tea is well known to involve a variety of enzymes, including those used 
herein. This role of enzymes is well summarized in two articles, Sanderson 
and Coggon, ACS Symposium Series, 47, 12-26 (1977), and Roberts, J. Sci. 
Food Agric., 3, 193 (1952). 
Manufactured teas on the world market can be subdivided into four groups 
with respect to the processing employed. This classification is based on 
the activity of tea enzymes in the course of raw tea treatment. Enzyme 
action throughout the entire process, including withering and fermenting 
stages, yields fermented teas which include all brands and grades of black 
tea, including instant tea made from black tea. Arresting the enzyme 
activity at an early stage of tea processing by steaming or roasting the 
raw tea provides unfermented or green tea, which has a specific taste and 
aroma. Partial (10% to 15%) action of enzymes in processing yields a 
yellow tea with a flavor and aroma different from the flavor and aroma of 
green tea and/or heavily processed teas. Partial but greater (20% to 30%) 
action of the enzymes yields red, or oolong tea, which has a rich, strong 
aroma and flavor, and a reddish color. 
Unfortunately, prior art tea processing techniques involve only partial 
conversion of tea aroma and flavor precursors to tea aroma and flavor 
compounds. Further, prior art techniques are able to extract only a 
portion of these aroma and flavor compounds from the black fermented tea 
leaf. Still further, many of these materials, once extracted, are prone to 
polymerization, precipitation and insolubility problems, further reducing 
the overall quality and overall yield of the tea. 
U.S. Pat. No. 4,051,264, issued in 1977 to Sanderson et al., teaches the 
conversion of nonfermented (green) tea to black tea by the use of tannase. 
However, the patentees state that it is important to apply the tannase 
treatment before any "permanent" insolubilization of the tea leaf solids 
has occurred as a result of the more orthodox black tea manufacturing 
process. 
It is an object of this invention to provide an enzymatic process for the 
treatment of black tea leaf which produces a high fidelity product in 
greater yield. 
It is another object of this invention to provide a process for enzymatic 
treatment of black tea leaf which produces a product having greater cold 
water solubility than conventional black tea extracts, 
These and other objects of the invention will be evident from the following 
disclosure. 
DISCLOSURE OF THE INVENTION 
The process of this invention involves pre-treatment or conditioning of 
black tea leaf with an enzyme solution containing tannase in conjunction 
with cell-wall-digesting enzymes such as cellulase, pectinase, and 
hemicellulase, after conventional fermentation but prior to extraction. 
In accordance with this invention it has been found that clarified tea 
infusions can be obtained from black tea which has been treated with 
tannase in conjunction with the cell-wall enzymes of this invention, such 
as cellulase, pectinase or hemicellulase prior to extraction. While not 
intending to be limited by theory, the likely mechanism by which this 
process works is that upon imbibition of enzyme solution by black tea leaf 
and the swelling of black tea leaf tissues, the enzymes are absorbed into 
or absorbed onto the tissues, causing the release of immobilized teal 
solids from tea leaf material and hydrolysis of released tannins to 
provide a higher yield of cold-water soluble tea solids. The cell-wall 
enzymes are specific for hydrolysis of major cell-wall components, such as 
cellulose, pectin, hemicellulose, lipids and protein. 
In a preferred process process of this invention, black tea leaf is mixed 
with an enzyme solution at a weight ratio of from 1:4 to 2.5:1. The enzyme 
solution contains tannase and one or more cell-wall-lysis enzymes. 
Preferably, the enzyme solution contains from 0.5 to 32 units of tannase 
per gram of black tea, and from 5 to 20 units of cellulase, pectinase, or 
hemicellulase, or a mixture thereof, per gram of black tea. The wetted tea 
is incubated, preferably in a closed system at a temperature of from about 
20.degree. to about 65.degree. C. for from 1/2 to 20 hours. The resulting 
enzyme-treated black tea is neutralized with an edible base to the desired 
final pH. The enzymes are inactivated by heating to a temperature greater 
than about 65.degree. C., and the tea is then ready for conventional 
packaging or extraction. 
STARTING MATERIALS 
The starting tea material of this invention is black tea leaf. While tea 
particle size is not critical to the practice of this invention, most 
imported black tea has already been finely comminuted in processing, and a 
fairly fine grind is preferred to provide maximum surface area for wetting 
of the tea. Ground black tea can be used as received, and typically has a 
particle size distribution providing roughly 45% retained on a 20 mesh 
screen and essentially none through a 48 mesh screen. This is preferably 
further ground by any convenient means so that about 15% are retained on a 
20 mesh screen and about 5% are through a 48 mesh screen. (All percentages 
are measured by screening the tea on metal sieves on a Ro-Tap shaker, made 
by Tyler Industrial Products, for 3 minutes.) 
Enzyme Solution 
By "cell-wall-digesting enzyme" herein is meant 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, such as pancreatic lipase or castor-oil lipase. 
In the practice of this process, the black tea leaf is preferably only 
wetted with enzyme solution. No free-flowing "tea juice" is created. Since 
tea absorbs about 2.5 times its weight in water, the tea is preferably 
wetted with enzyme solution in a solution:tea ratio of from about 1:4 to 
about 2.5:1. Enzymes can be used at any convenient level. Higher levels of 
enzyme permit shorter incubation times, but are also more expensive. 
The enzyme tannase which is used in this invention is known to hydrolyze 
the ester linkages of tannic acid between gallic acid and glucose. It also 
attacks gallic acid methyl ester. The enzyme is an elaboration product of 
the growth of certain molds belonging to the genera Aspergillus and 
Penicillium. Aspergillus flavus grown on a medium containing tannic acid 
as a sole carbon source provides tannase in substantial amounts. Two other 
specific strains of microorganisms known to produce substantial quantities 
of tannase are Aspergillus oryzae, ATCC No. 9362, and Aspergillus niger, 
ATCC No. 16888. One suitable preparation of tannase enzyme is available 
commercially from the Enzyme Development Corporation. Yet another is 
available from Novo Industri A/S, Bagsvaerd, Denmark. The other 
cell-wall-digesting enzymes, such as cellulase, papain, pectinase, and 
hemicellulase can be obtained from similar commercial enzyme sources. 
Examples of measurement of enzyme activity are given below. 
One unit of tannase activity in the practice of this invention is defined 
as the amount of enzyme which is able to hydrolyze one micromole of ester 
bond in tannic acid in 1 minute under the following conditions: 
Substrate: tannic acid (0.35%, weight/volume in 0.1 m. phosphate buffer, pH 
5.5). 
Temperature: 30.degree. C. 
Enzyme concentration: approx. 4-6 units/ml. (estimated) 
Reaction time: 10 minutes 
This method is described by Iibuchi, S., Minoda, Y. and Yamada, K., 
"Studies on Tannin Acyl Hydrolase of Micro-organisms, Part 2. A new method 
determining the enzyme activity using the change of UV absorption", 
Agricultural and Biological Chemistry, Vol. 31 (5), pp. 513-18 (1967). 
The procedure was as follows: To four parts of substrate (0.350 w/v% of 
tannic acid dissolved in 0.05M citrate buffer, pH 5.5), one part of the 
enzyme solution was added. After t minutes reaction at 30.degree. C., 0.1 
part of the mixture was added to ten parts of 90% ethanol. 
The optical density of the ethanol solution at 310 m.mu. was measured. 
Tannase activity (unit/ml) was given by following equation. 
##EQU1## 
Where E.sub.t1 and E.sub.t2 mean the optical density of the ethanol 
solution at 310 m.mu. prepared after t.sub.1 and t.sub.2 minutes reaction, 
and one unit of the enzyme means the amount of the enzyme which is able to 
hydrolyze one micro mole of the ester bond in tannic acid in one minute. 
One unit of cellulase activity is defined as the amount of enzyme required 
for producing 1 micromole of reducing carbohydrate per minute under the 
following conditions: 
Substrate: avicell microcrystalline wood cellulose, 10 gram/liter. 
pH: 4.8 
Temp.: 50.degree. C. 
Reaction time: 20 minutes 
Enzyme concentration: 0.01-0.03 units/ml. (estimated) 
Pectinase activity is expressed in polygalacturonase units (pgu). One pgu 
is defined as the amount of enzyme required to release 1 micromole of 
reducing group from pectic acid per minute. The reducing group is 
determined by adding excess iodine solution and back titrating with sodium 
thiosulfate solution. The method is described by Colowick, S. P. and 
Kaplan, and O, Methods in Enzymology, Vol. 1, pp. 162-64 (1955). 
To 99 ml. of 0.5% pectic acid, previously adjusted to pH 4.0 and 
25.degree., 1 ml. of enzyme soluton is added. The time is noted, and 5-ml. 
aliquots are removed at various times (depending on enzyme activity) and 
added to 0.9 ml. of the Na.sub.2 CO.sub.3 solution in a glass-stoppered 
flask. Now 5 ml. of iodine solution is added, and after thorough mixing 
the mixture is allowed to stand for exactly 20 minutes; then 2 ml. H.sub.2 
SO.sub.4 is added and the excess of iodine titrated with the Na.sub.2 
S.sub.2 O.sub.3 soluton. A calibration curve is prepared with the 
galacturonic acid monohydrate solution. Under these conditions 1 meq. of 
reduced iodine corresponds to 0.513 mM. of aldose liberated. Controls are 
run without enzyme as well as with heated enzyme solution. The activity 
may be expressed as (PG u.)/ml., indicating the millimoles of reducing 
groups liberated per minute per milliliter of enzyme. 
Activities of other cell-wall digesting enzymes can be measured, and 
suitable levels determined, by recourse to standard reference texts in 
enzymology. In general, it is acceptable to have sufficient cell-wall 
digesting enzyme(s) to cause measurable digestion and fermentation of 
cell-wall components in the tea leaf. 
Incubation 
The enzyme-wetted tea is then incubated to cause partial digestion and 
fermentation of the tea. This fermentation and digestion is in addition to 
the fermentation and digestion which occurs in conventional black tea 
processing. Incubation is preferably done in a closed system at 
temperatures of from about 20.degree. C. to about 65.degree. C., for from 
1/2 to about 20 hours. The closed system insures that delicate aroma 
volatiles are neither lost nor damaged. Inert gas blanking of the 
incubating tea is also preferred to avoid oxidative damage to delicate 
flavor and aroma volatiles, but is not required for the enzymatic 
reactions to proceed. The enzymes are active at temperatures ranging from 
well below ambient up to about 65.degree. C., at which temperature the 
enzymes are destroyed. Temperatures below about 20.degree. C. are 
acceptable, but, because they are below ambient, require cooling and 
longer process times and therefore are less preferred. A temperature of 
about 40.degree. C. is optimal. Incubation times from 1/2 hour to 20 hours 
are accceptable, but times greater than about 4 hours produce little 
noticeable change in end results, and therefore shorter times are 
preferred. An optimum incubation time is about 21/2 hours. The tea can be 
gently agitated to assure uniform wetting, but agitation is not required 
during incubation. 
Neutralization 
During the course of incubation, the action of the enzymes upon released 
tea solids causes acidification of the incubating tea leaf. In particular, 
gallic acid and other organic acids are released by enzymatic action upon 
polymeric tea solids, with a consequent reduction in pH to 4.25 to 4.75. 
This pH range is organoleptically unacceptable for tea. Therefore, in the 
practice of this invention it is important to neutralize the tea to a 
typical tea pH in the range of from 4.75 to 5.75, preferably 5.0 to 5.5. 
Neutralization can be accomplished with any food-compatible base, such as 
sodium hydroxide, sodium bicarbonate, potassium hydroxide, potassium 
bicarbonate, etc. The level of base added can range from about 3 mg. to 
about 60 mg. per gram of black tea, depending upon the starting pH and the 
desired final pH of the tea product. By the terms "food-compatible" and 
"edible" herein is meant both toxicological and organoleptic acceptability 
under the conditions of use in this invention. 
Neutralization can be performed at any convenient point in processing after 
incubation. Preferably it is performed immediately after incubation, but 
it can be postponed until after extraction, for example. Then, of course, 
it is the extract which is neutralized. 
Enzyme Inactivation 
Following neutralization of the tea by addition of edible base, the enzymes 
used should be inactivated by denaturing. The enzymes can be denatured by, 
for example, exposure to heat. The heat can be supplied by any 
conventional means, including microwave, irradiation, conductive or 
convective air heat or steam heat. Inactivation of the enzymes can also be 
achieved during the tea leaf drying process, providing the drying oven 
temperature and drying time are sufficient. If hot water or steam are used 
for extraction of the leaf tea, these can be used for inactivation of the 
enzymes, provided the temperatures are high enough and the extraction step 
substantially immediately follows the incubation step. Preferably, for 
good flavor retention, the minimum heat exposure necessary for 
inactivation of the enzymes is used. For example, freeze drying or vacuum 
ove drying is better than regular atmospheric oven drying in terms of 
flavor retention. 
Further Processing 
Following or concurrent with enzyme inactivation, the tea is further 
processed by conventional techniques. It can be dried for sale in bulk or 
in bags, or extracted for production of soluble tea products. The 
processed leaf tea of this invention produces an extract the solids of 
which are substantially completely soluble in cold (4.degree. C.) water, 
as measured by the technique used in Example 1, below, when extracted with 
boiling water to an extract solids concentration of from 0.3% to 10%. 
It will be appreciated that the process of this invention is compatible 
with conventional art techniques for stripping and recycling or 
re-addition of aroma and flavor components, as already used in tea 
processing.

All percentages herein are by weight unless otherwise indicated. The 
following examples illustrate the features of this invention without 
intending to be limitative thereof. 
EXAMPLE 1 
125 gram portions of a black tea blend were mixed with 100 ml. distilled 
water, 100 ml. distilled water containing 500 units of tannase, and 100 
ml. of distilled water containing 500 units of tannase plus 2500 units of 
cellulase, respectively, in closed gallon jars. All three samples were 
incubated at 25.degree. C. for 3 hours. At the end of the 3 hours, 6.5 ml. 
of 2M potassium bicarbonate solution was added to the jar containing 
tannase alone, and 11.5 ml. of 2M potassium bicarbonate solution was added 
to the jar containing tannase plus cellulase. Each of the samples was 
heated in a microwave oven (Sharp Model R-7600, 700 watt, 2450 MHz) for 3 
minutes at full power to stop the enzymatic reactions. 1250 ml. of boiling 
distilled water was added to each jar and each sample was stepped for 10 
minutes in the closed jar. The tea extract was separated from the spent 
tea leaf by filter paper. The tea extract was cooled to 10.degree. C. in 
an ice bath. The cold extract was filtered through a diatomaceous 
earth-coated filter paper (24 cm. diameter). The final extract was then 
freeze dried to tea powder. The results are presented in Table 1. 
TABLE 1 
______________________________________ 
Effect of tannase/cellulase treatment on extraction 
yield in cold water solubility of instant tea. 
% Solids of % Extraction 
% Insoluble in 
Treatments 
Tea Extract.sup.a 
Yield.sup.b Iced Water.sup.c 
______________________________________ 
Control 2.59 22.2 9.0 
Tannase 2.84 24.2 3.0 
Tannase + 
3.30 30.4 0.0 
Cellulase 
______________________________________ 
.sup.a % solids was determined with a Bausch & Lomb refractometer. 
.sup.b % extraction yield = (% solids of tea extract .times. total weight 
of tea extract)/weight of black tea. 
.sup.c insoluble in iced water was measured by filtering out insoluble 
solids in iced water with a filter paper and determining the dry weight o 
insoluble solids after drying to constant weight in an oven. 
In Table 1, extraction yield was increased substantially by tannase plus 
cellulase. The instant tea produced after tannase/cellulase treatment is 
completely soluble in iced water. 
EXAMPLE 2 
Four hundred grams of a black tea blend was mixed well with 400 ml. of 
enzyme solution containing 200 units of tannase and 7500 units of 
pectinase in a glass jar and incubated at 40.degree. C. in a water bath 
for 3 hours with the jar closed. The enzyme-heated tea was loaded into an 
extraction column (Pyrex, 13.times.33 cm). Steam was injected from the top 
of the column onto the tea bed. The temperature in the middle of the tea 
bed reached about 70.degree. C. The steam injection process was repeated 
twice in 10 minutes. Then 3000 ml. of boiling distilled water was 
introduced from the top of the column and the tea extract was pulled by 
vacuum into a receiving tank connected to the bottom of the extraction 
column. The receiving tank was submerged in an iced water bath. The 
maximum bed temperature reached 65.degree. C. at the end of the 
extraction. The boiling distilled water was introduced at a rate of 100 
ml. per min. and the whole extraction process took 40 minutes. Another 
glass tank submerged in liquid nitrogen was connected between the 
receiving tank for tea extract and the vacuum source to recover tea 
volatiles. This tea volatile frost was added to the tea extract before 
drying. The pH of the tea extract was adjusted to 5.2 with 2M potassium 
bicarbonate solution. The extract was dried to powder by vacuum drum 
drying. The instant tea prepared by this process was completely soluble in 
iced water and looked reddish clear in a glass. Its taste was judged by 
expert tasters to be flavorful and brewlike. Two more separate extraction 
runs were performed without enzyme and with 200 units of tannase alone, 
respectively, under the same conditions. The results are summarized in 
Table 2. 
TABLE 2 
______________________________________ 
Effect of tannase/pectinase treatment on extraction 
yield in cold water solubility of instant tea. 
% Solids of % Extraction 
% Insoluble in 
Treatments 
Tea Extract.sup.a 
Yield.sup.b Iced Water.sup.c 
______________________________________ 
No enzymes.sup.a 
3.4 22.1 9.2 
Tannase.sup.b 
4.0 26.1 3.0 
Tannase + 
4.9 33.9 0.0 
pectinase 
______________________________________ 
.sup.a average of three runs. 
.sup.b average of two runs. 
.sup.c average of five runs. 
EXAMPLE 3 
Fifty grams of the black tea blend used in Example 1 was mixed well with 50 
ml. of enzyme solution containing 100 units of tannase and 500 units of 
pectinase. The wetted tea was incubated in a closed jar at 25.degree. C. 
for 3 hours. After incubation, 3.75 ml. of 2M potassium bicarbonate 
solution was added to the tea sample. The tea sample was heated in a 
microwave oven as in Example 1, for 2 minutes. The tea sample was dried at 
85.degree. C. in an oven. A separate sample was prepared without enzyme, 
as a control. 2.25 gm. portions of the black teas prepared by these 
processes were put into "flow through" tea bags. For product evaluation, 
each tea bag was brewed in 250 ml. of boiling water for 3 minutes. Solids 
concentrations were determined with a Carl Zeiss Refractometer in the 
usual manner. Turbidity was measured with a Hach Ratio Turbidimeter, Model 
18900. The turbidimeter can measure 0-200 Nephelometric turbidity units 
(NTU). The correlation between NTU and turbidity of tea in glass is 
described as follows: 
0-50 NTU: crystal clear 
50-100 NTU: clear 
101-200 NTU: hazy 
The results of the evaluation are summarized in Table 3. 
TABLE 3 
______________________________________ 
Brew solids and turbidity of 
enzyme-processed chill-proof bag tea. 
% Brew Turbidity Appearance 
Treatments 
Solids* at 4.degree. C. (NTU)* 
in Glass 
______________________________________ 
Control 0.26 141 Brownish 
& creamy 
Tannase + 0.30 39 Reddish 
pectinase & clear 
______________________________________ 
*Avg. of 4 replicates. 
EXAMPLE 4 
One hundred grams of black tea from commercial tea bags was mixed with 80 
ml. of distilled water with 3,250 units of pectinase and 300 units of 
tannase and incubated in a closed jar at 40.degree. C. in a water bath for 
3 hours. Then 1.2 g. of vitamin C and 0.75 g. of sodium hexametaphosphate 
in 20 ml. of distilled water were added to the tea sample. For 
neutralization 12 ml. of 2M potassium bicarbonate solution was added. The 
tea sample was dried in a vacuum oven at 60.degree. C. The dried tea 
sample was blended with 50 g. of fresh untreated black tea for better 
flavor. The blended sample was put into tea bags at 2.25 g. per bag and 
evaluated as in Example 3. The tea infusion brewed from the tea bags 
prepared by the enzyme process of this invention remained clear and 
reddish in a refrigerator for several days, while the control sample 
turned creamy and brownish within several hours under the same conditions. 
EXAMPLE 5 
The same procedures and materials were used as in Example 2, except that 
the tea extract was not dried to a powder. Instead, the tea extract was 
diluted with distilled water to drinking strength (0.4-0.5% tea solids). 
The diluted tea liquid was bottled in 10 oz. clear glass bottles and 
pasteurized as ready-to-drink iced tea. The resulting ready-to-drink iced 
tea in bottles stayed reddish and crystal clear at 40.degree. for six 
months. It retained a brewlike tea flavor without the typical bitter and 
harsh taste of iced brewed tea.