CA1087446A - Tea - Google Patents

Abstract

ABSTRACT
Cold water-extractable tea leaf is obtained when sub-stantially unfermented green tea leaf is treated with tannase.
The treated leaves can be processed further by conventional means to obtain colong or black tea.

Description

cQ 364a ~37~
The pre~ent invention relates to a process for obtaining cold water extractable tea leaf and to ~he improved product~ thereby obtained. In particular, the invention relates to a method of treating fresh green tea leaf with the enzyme tanna~e to produce dry tea leaf~ green, blacX or oolong, which i~ cold w~ter extractable, and which ha~ a full comple-ment of ~ea flavour~
Black tea iB usually prepared by subjecting freshly picked tea leaves to a ~eries of processing conditions includ-ing the withering and rolling of freshly harve~ted leaves,foll~wed by a fermentation ~tep (enzymatic oxidation) during which much of the characteri~tic colourr flavour and aroma ~f black tea are developed~ The ermentation i~ ~topped a~ter a ~luitable period of time by "firing" (drying at temperature~
rangin~ from about 65 C to about 100C~ the tea to inactivate - the enzymes causing the fermentation and to complete tha cievelopment of the flavour and colou:r of the tea productO ~he extent of fermentation varie3, in ~ommercial practice, from black to variou~ gradation~ between green and bla~k. Par~ially ~enmented tea~ are known a~ "oolong" tea~. Green tea~ are made by :firing gre2n teal bsfore fermentation ha~ taken plaoe. The gr~en, oolong, and black teas of commerce each provide a beverage ha~ing di~tinctive flavour and colour characteristics.
When conventional teas ~re extracted with cold water ~r ~hort periods of time (le~ than 15 minutes), the tea ~everage produced has a low concentration o$ extractable tea ~-~olid~, a very light colour and almo3t no tea-like taste.
Conventionally, water at temperatuxe~ of about 100 C i~ u~ually employed to obtain a ~ati~factory bever~ge from tea leave~;

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-Various procedure~ are knownfor making cold watersoluble instant tea powders by ~olubilising tea cream obtained from black tea extract~, as de~cribed in U.S. P~tent No. ,7

2,831,772; U.S. Patent No. 2,863,775 and U.S. Patent No.

3,136,539. While each of the~e proces6es are succe~sful to varying degrees in producing a cold water soluble instant tea powder, each has disadvantages. Most fail to provide a tea pow~er which, on recon3titution, give~ a beverage having a natural flavour and colour. Furthermore, none of the~e prior art proce~se~ provide a tea powder which will provide the "milk reaction" provided by natural tea. Naturally brewed tea, when milk i~ added, takes on a bright pinkish-reddish colour which is pleasing to the eye. The instant tea powders produced by the prior art technique~ de~cribed above, when reconstituted a~ a hot beverage, react badly to the addition of mi~k, taking on a dull unplea~ant colourationO It i8 al~o known from the U~S. Patent ~o. 3,812,266, that fresh green tea ho~ogenate, when given a pre-converl~ion t~nna~e treatment, will yield an instant tea powder which i8 cold water soluble.
It h~s now been found that tea leaf products which are capable of producing a high quality tea beverage upon brewing with either cold or hot wat~r~ and which have a ~ub~tantially reduced level of cold water insoluble solids are obtained by contacting comminuted fre~h green tea with tanna~e under condition~ which are at least initially ~s~entially anaerobic an~ thereafter firing and drying the leaf to a moi~ture content of below 5%. In the ca~e of oolong and black tea, the proces~ of the invention in~lude3 the ~tep~ of converting the fresh ~re~n t~a after tannase tr~abment to -- . . .

- 3010ng or black tea by the txaditional method~ o:E fermentation, using the natural tea enzymes, followed by fixing~, The tea leaf products of the invention have the unique property of producing high quality ~ea beverages when brewed with cold water. ~n particular, black tea beverages produced by either cold water or hot water extraction of black tea leaves produced by our new proce~s have the excellent flavour of good quality commercial black teas, a highly ac-cept~ble bright red colour, good clarity when cold and a "milk-reaction" characteristic of conventionally brewed tea.
Moreover, the cold wa~er extractable tea leave~ of the invention can be used tv produce a high quality instant tea product by the simple process o~ extracting the tea leaves with boiling water, clarifying the extract, and drying it.
The instant tea producad by thi3 method give3 a higher yield of cold water ~oluble tea ~olid~ than in~tant t~a produced by conventional method~.
: The term "fresh ~reen tea", as used herein and in the appended cl~im~, include~ fre~hly gathered tea flu~h; namely, the entire shoot tips of the tea plant, Camellia ~ine~is, (L) O. Xunt~e, including both 8tem8 and attached leaVeBO ~he term "fre~h green tea" in~ludes any tea plant material contain- :
ing the naturally occurring enzyme~ in an active form in the ::
ti8~1Ue3 of the fre~hly gathered tea. ~he term "fre~h green . . .
tea 1ush" include~ freahly harvested tea 1ush~ withered tea flu~h, and fre~hly harvested tea flush that ha3 been frozen to pre~erve the enzyme 8y~3tem~ therein. In each in~tan~e, the : fre~h green teæ ~çscribed above has under~one ~ubatantially no ~ fesment~tion to the bla~k tea ~tate~

cQ 364a ~7~4L6 In accord~ncs with a preferred embodiment of the invention, frPsh green tea flush i8 comminuted to en~ur~ that a major portion of the ~oluble tea~lu~h constitutents are brought into contact with one anoth~r to orm a green tea ~ystem capable of undergoing conver~ion to bla~k tea. Com-minution may be carried out in any convenient manner which will not destroy the activity of the tea enzymes, the latter being required for the subsequent step of converting the green tea to black; Suitable comm~ution techniques includa passing fresh green tea through closely spaced rollers or forcing fre~h green tea through a meat grinding machine. The full benefit from the comminution process i9 realised When it i~ carried out in such a way a~ to minimise the amount of aeration of the tea lea~ prior to tanna~e treatment. Thi~ i8 de~irable so as to ~inimi~e the amount of tea fçrmentation, which is an aerobic pro~ess, from taking plac~ until the pre-conver~ion tannase treabment described below has been initiated.
In one preferred embodiment, potassium hydroxide i8 ~ ~:
added a~ a 7% aqueou~ olution, in the amount of 9 mg ~OH per gram dry weight of tea material, and i8 mixed by rolling the tea. It has been noted that the pro~ess of the present inven-tion brings about a slight reduction in the pH of the resulting tea beverage. While it is not a necessary ~tep, it may be de~irable to add sufficient alkali to restore the pH of the tea to its normal lavel. This p~ adjusbment ~tep may be accompli~h-ed either before or after the tanna~e treatment ~tep.
The comminuted fresh green tea is next given a pre-~onversion tanna~e treatment by contacting it with a tannase ~nzyme preparationO Th~ tanna~e ~nzyme i~ preferably added to th~ co~minut~d gre~n tea in the amount of about 8 enzyme units, , .

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mea~ured hy th~ modified :ribuchi et al methodO de~cribed infra, per gram dry weight of green tea ti~sue. MiXing i8 en~ured by further rolling of the gre~n tea, It i~ preferred that the comminuted green tea flush now be maintained under essentially anaerobic conditions in contact with ~he tanna~e for about 15 minutes to one hour at 25 C, ~o as to enable the tannase treatment to take place before an appreciable amount of tea ermentation ha~ taken place. Maintenance of essentially anaerobic conditions may be accompli~hed by placing the com-minuted tea flush in a closed ve~sel and ~parging the systemwith nitrogen ga~ to di~place the air present in the vessel, or, more simply, by keeping the comminuted leaf in a large mass such that the ~3urfa~e area exposed to air is minimised.
Following the~e procedures will maximise the cold water extract-ability of the products. Indeed~ most of the cold waterextra~tability i8 obtained without the requirement of an anaerobic condition during the pre-fermentation tannase treat- :
ment, a8 long as the tannase i~ added to the commlnuted fre~h tea flu~h prior to tea ferment~tion, virtually all the bene~it 20 i8 lo~t if the tannase i8 added after tha tea fermentation is ~omplete.
~ he anaerobic pre-conversion tanna~e treatment step may be conduct~d u~ing a range of enzyme concentration~, ~emperature~, and treabment time~. An enzyme level ranging from 2 to 20 or more ~anna~e units per gram dry weight of green tea, as measured by the Ikbu~hi et al methodO infra, may be :
usedO ~ level a~ low as 2 unit~ per gram of total green tea ~olid~ may be used, but the yield and quali~.y of the product - i~ les~ th~n optimum~ A level of tannase about 8 en~yme units may be used, but no su~3tantial additional benefit i~ obtained.
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cQ 364a . .
The temperature at whîch the pr~ conversion tannase treatment i8 carried out may be varied over a wide range.
Favoura~e yields are obtained at temperatures ranging from 25 C to 550CD At the higher limits of the range, that i8, at 55 CO a le8s desirable conver~ion of green tea ~olids to black i~ obtained. This result i~ probably due to inactivation of ~he natural tea enzyme~ at the~e higher temperature~.
The range of time during which the pre-conversion tannase treatment is conducted under anaerobic conditions has some bearing on the yield of cold water extractable tea solids and the lightnes~ of the colour obtained. Unless the comminuted tea leaf i~ contacted with the tannase under anaerobic condi-tions for a few minutes, a less than optimum product, which gives a light coloured beverage, is obtainedO Additional treatment, say up to about 15 minute~, provid~ improved results.
~avourable cold w~ter extractability and colour characteristic~
are obtaine~ after a 15 minute pre -conver~ion tannase treat-ment under anaerobic conditions. Additional times up to 120 minutes or more have been u~ed but without substantially improving the yield or colour of the product.
Following ~ompletion of the pre-conversion tannase tre~tmentO the tea conver~ion process i3 efected by adjusting the temperature of the con~ninuted green tea to about 32C, and contacting the green tea wit~ air at high relative humidity.
A sati~factory conversion i8 generally effected in about 90 minute~. The fermented tea flush i~ fired by plàcing it in a forced air oven at about 80C for about 30 minutes to r~duce the moisture content of the tea material to below about 5%.
~?iring o~ the fermonted tea leaf ~8 important for the full ~ .
, : , ' ' cQ 364a ~ ~7 ``~evelopment of blaek tea flavour~
The enzyme tannase which i9 u~ed in accordance with the present inventi~n is kn~wn to hydroly3e the e~ter linkages as well as the depside linkages of tannic acid between gallic acid and glucose. It also attack~ gallic acid methyl ester.
one source of the enzyme i8 as a~ elaboration product of the growth of certain molds belonging to the genus Asper~illu8 or Penicillium. For example~ AsPergillus flavus grown on a medium containing tannic acid as a sole carbon source provide~
tannase in sub~tantial amoun~. Two specific strains of mic~oorganisms known to produce substantial quantities of tannase are Asper~illus o~yzae~ ATCC No. 9362 and AsPerqillus niger, ATCC ~o. 16888. one suitable preparation of tannase enzyme i8 available commercially from the ~nzyme Development Corporation. Thi~ preparation i~ obtained with a strain o~

A~PerqLillu~ oryzae ' ' Tannase suitable for use :in the present invention i8 ..
available commercially as a powder, and it is supplied at varying levels of activity, depending upon its source and the method o recovery u~ed. ~he commercial preparati~n described above, has about 20,000 enzyme unit~ per gram of p~wder, as determined by the modified ~ibuchi et al method de~cribed below.
While the exact mechanism by which the invention works i8 not kno~n, it is believed th~t the proce~ses described above cause the tanna~e enzymes to enter the ti~sue~ o~ the green tea thereby causing the hydrolysi~ of the ester bonds which link gallic a~id and tea ~atechins to provide a higher yield of cold ~ater extractable tea solids~ It appears that this effect is ~hieved becau~e there i~ a relea~e of juices , cQ 364a 7~
hen the green tea leaf material~ are ~queezed during the rolling process. When the ~queesing pres~ure i~ released, these juices are reab~orbed causing the tannase enzyme that was added to the green tea leaf material to be ab~orbed ints the tea leaf tissue~. The enzyme th~n proceeds to de-esterify the tea catechin~. After this pre-conver3ion tannase treat-ment i~ completed, the normal tea manufacturing proce~s, for either green,oolong or black tea, may be carried out.
The process of the present invention may also be carried out with the enzyme preparations fixed onto solid ~upports ~uch a~ glas~ or polymeric materials to allow removal from the ~ystem and reuse of enzymes. Enzyme preparations of thi~ nature are termed "immobilised" or "insolubilised" in the art. Techniques for accompli~hing the fixation of enzymes on insoluble matrices are disclo~ed in US Pat~nt ~o~ 3,519,538, 3,536,587, and 3,55S,945. Additional discus~ions of these techniques may be ~ound in 0.R. Za~orsky, C~R~Co ~andbook of Immobili8ed Enzym~, 197-~9 the Chernical Rubber Co., Cleveland, ohio. In general, enzyme insolubili~ation proces~es re~uire the fixation of the enzyme to an insoluble matrix by covalent bonding, by absorption~ or by occlusion. The fixed enzyme preparation~ may be recov~red for reus~ and provide a method ~r completely removing the enzyme from the tea materials undergoing treatment~ In addition, the in~olubilised enzymes are conveniently provided in particulate ~onm which~ in turn, m~y be packed into col~mn~ for use in continuous proce~e~.
The proce~ using immobilised en~ymes makes u~e of an aqueou~ extract of green tea leaf prepared at le~ than 50C to retain enzyme~ in their active formO The eætract i8 giv~n pre-~onYersion t~nna~e treat~e~t by contacting the - .

~7446 cQ 364a ``extract solid~ with immobilised tannas~ ~ixed on a suitable ~upport, 3uch a~ by pas~ing the tea extract through a column packed with the enzyme fixed on glas~ particle~. The tea conver~ion proces~ i8 then effected by contacting the tannase S treated extract with the green tea flush residue, drying them together, and ~iring the tea to form a cold water extractable blac~ tea leaf product.
The cold water extractable tea leaf can also be u~ed to prepare a superior in~tant tea produce by the simple pro-cedure of extracting the tea leaf, clarifying the extract and thereafter drying it. By contrast~ instant tea i8 u~ually produced by extracting regular blaok tea, solubilising the tea cr~am~ clarifying the tea cream and then drying it. By using the cold water extra~table tea leaf of the invention, a higher yi~ld of instant tea solids i~ obtained becau~e the :
tannase treabment i8 applied before any "permaaent"
in~olubilisation of the tea leaf solid~ has occurred as t~e ~e~ult of the more orthodox black tea manufacturing proce~s.
For exampla, a~ not~d above, the u~ual method of producinq an in~tant tea starts with the extraction of regular black tea. In the manufacture of regular black tea, fermenta~ion and firing re~ult in the formation of polyphenolic oxidation ~ .
product~ which are "permanently" in~olubilised by attachment to the insoluble matter of tha tea leaf materialO This ~: :
re~ult~ in the los~ of ~ome of the extractable tea solids~ :
. By contrast~ the pr~fe~mentation tannase treat- . :
ment o~ the pr~sent invention result~ in the ormation of polyphenoli~ oxidation product~ that are le~s prone to become "perman~ntly~ in~olubili3ed by at a~hm~nt to the in~oluble --10-- .. .....
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cQ 364a 37~
~a leaf mat~rial~ This mean~ that a higher economic yield of cold water ~oluble instant tea ~olids i~ obtained by ~tart-ing with the t~a leaf of the pre~ent invention rather than with regular black tea. Moreovex, because the tea leaf of the present invention i~ fired at the ~nd of the enzymic conversion process~ the instant tea product has a high quality black tea flavour.
A method that can be u~ed for the determination of tannase activity of enzyme preparations i5 a modification of the method described by Sadaaki Iibuchi, Yasuji Minoda, and Koichi Yamada in Agricultural and Biological Chemistry, Volv 31, p 513 (1967). Thi~ method determine~ the amount o~
tannic acid which i~ hydrolysed in the presence of a mea~ured amount of the tannaQe enzyme under a ~pecified set of conditions 15 by m~a~uring the decr2ase in absorbancy of the ~ubstrate at a wavelength of 310 nm using a re~ording ultraviol~t ~pectophoto-meter. The solution u~ed for this assay i~ a 0.004% tannic acid solution in 0.02M acetate buffer (pH 5.0). The tannic acid used i8 Analytical Reagent Grade Tannic Acid, Catalogue 20 No. 1764r Mallinckrodt Chemical Work~, St. Louis, Missouri~
3O0 ml of this solution is placed in a 1 ~m cuvet which is held at 30 C in the temperature controlled ~ample chamber of a Cary* Model 14 recording ultraviolet spectrophotometex;
the devi¢~ is ~old by Cary In~truments, Monrovia, California.
~5 Finally, 0.1 ml of enzyme ~olution i8 added and the absorbance change at a wavelength of 310 mm is followed continuously.
The concentration of the enzyme solution i8 adju~ted until an absorbanc~ ~hange of about ~.01 ~bsorbance unit~/minute i8 obserb~d. one minute of tannase enzyme activity measured by * trad~ mark ~Q364a ~7~
` hi~ method i8 defined as that activity which will give a change of 1 ab~orbance unit per minute at 310 nm (30&, 1 cm cell) using a 0.~04% tannic acid 301ution in OJ02M acetate buffer (p~ 500~ with a starting absorbance of about 0.7.
Three tannic acid supplies were te~ted in this a~ay:
~amely, (a) Tannic acid N.F. - Catalogue ~o. 1750 and (b) Tannic acid Rnalytical ~eagent - Catalogue ~o. 176~ fr~m Mallinckrodt Chemical Works. St. Louis, Mi~ouri; and (cj Tannic acid ~.F, - catalogue No. 1198, Allied Chemical Corpor-ation, Morristown, ~ew Jerse~. All three samples of tannic aci~ give the same re~ults when used in the assay d~cribed above. ~he commercial tannase enzyme obtained from the Enzyme Development Corporation, New York, N~Y~, was found by this method to have about 2n, ooo unit~ of activity per gram.
Data her~in which des~ribed the colour and haze of tea beverage products are given in terms of results obtained by conducting standard te~t~ u~ing a Hunter D-25* colourimeter -~.
made by ~unter Re~earch Associates of Fairfax, Virginia. The h~ze value~ ~rs determined by reflectance, and the ~igures - ;
gi~en indicate the amount of re~lected light~ The l~wer the haze numbar, the ~learer the tea solution. The tri-stimulus ~oloux determination i8 based on measurement~ o~ transmitted light. The 1 value i~ the lightnes~ factor: A value of 100 indicates no colour, whereas a value of 0 indicates absolute darkne~. The a value i9 a measure of the red-blue range of colour~, and the b valu~ is a mea~ure of the gr~en-yellow line.
When tea ~olution~ are analy~ed at ~e~erage ~trength, ie 0.35%
tea solids~ in a 5 cm cell, th~ pref~rred L value i8 in ~he r~nge of 20 to 3S~ ~he preerred ~ value, is at lea~t 5 un~t~ -trade maxk .. ., - -. . . ~
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cQ 364a 79~
~reatex than th~ L value, and the preferred b value i8 between 15 and 25.
he invention i~ illu3trated by the following Exampl~R
~ PLE 1 Fresh green tea was obtained from a tea farm near Charleston, South Carolina, and wa~ u~ed fre~h, or wa~ ~tored frozen at -40C for u~e at a~later date.
Product ~.s A 300 g sample of fre~h green tea ~aving a total solids content of about 25%, was withered for 18 hour~ at 25 C
to 35% 901id8, and wa~ then ~liced in a noodle slicer (model "Reginan*, m2~nufactur~d by Giunta Brother~, Philadelphia, Penna.) such that the leaf pre~ent was ~liced into strip~
about 2 mm wide. The withered, ~liced green tea ~a5 pa~ed bet~een a pair of roll~rs sat with a ~pacing of 0.63 mm. This rolling operation wa~ repeated four time~ in ~ucce~sion to fully comminute the green tea. The comminuted green tea wa~ mixed with 0"7 g o~ pota~;sium hydroxide in 10 mls of water for 2 mirlut2~ in a E~obar~ eccentric paddle mixer (model ~-50, Hobart Manufacturing Company, Troy, ohio). ~ext, the green tea mat-erial wa~ pas$~d ~i ce through the roller to ensure even dis-tribution of potassium hydroxide throughout the green t~a mass.
The gr~en t~a material wa8 again pla~ed in the ~obart mixer, 600 units of tannase enzym~ dissolved in 10 ml of wat~r wa8 added, and mixed. The pr~paration wa mixed for 2 minute~.
Aft~r mixing, ~he green tea material wa8 ag~in pu~ through ~he -:
roll~rs twice. The co~ninu ed gr~e~ t~a m~terial containing -~
tanna~Q ~nzyme ~a8 then held under ~sentially anaerobic con-* tra~e mar~

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cQ 364a 7~
lition.~ in a covered beaker for 30 minutes at 25C. After the holding period, the green t~a material waæ spread on a tray ~o as to allGw air pa~age to all green tea particles, and it was allowed to ferment for 90 minutes at 32 & and at a relative humidity higher than 90%. Following this, the con-verted tea flush material wa~ *ired by pa~sing air at 80-85 C
through it for 30 minutes reducing its moisture content to le~s ~han 5%. The black tea produced by thi~ process was then sieved, and the fines: namely, the material pa~ing through a US Standard 40 me~h screen, which was only about ~% of the black tea pruduced~ were discarded. The finished black tea product (Product A) wa~ then placed in an aluminium oil lined paper pouh to age to 90 days.
_oduct B: -The procedure used in preparing Product A was repeat-ed on an additional 300 g o~ ~re~h green tea using the same conditions except that the potas~ m hydroxide and tannase : additions, and the anaerobic holding pariod were omittedO Thi~
procedur~ i~ es~sntially the staRdard pro~es~ for black tea ` 20 manufacture.
Product C:
This i~ a blend of teas, who~e origin would include India, Sri Lanka, and Ea~t Africa, which may be Pound commerci-ally ~vailable black tea product~0 ie, ïOo8e tea or tea bag~
Bo~h Product~ A and ~ were judged to be sound black : tea products in that the hot tea be~erages made from Product~ ~, B, anld C ware found to be ~imilar in ~erm~ of extracted ~olid~
colour, and ~a~te.
. .
~xtraction yield ~ udies for Products A~ Bd and C :
w~re caxried out over a range of extra~tion temperatures~

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cQ 364a 7~
~or each mea~urement, 2.25 g of leaf wa~ placed in a beaker with 200 ml of deionised water at the desired temperature.
After stirring for 3 minute~ with an overhead ~tirrer opera-ting a~250 RPM, the spent leaf was separated from the ex-tract by pas~ing the extract through one layer of Miracloth,*
and the solids in the beverage were detexmined by taking the extract to dryne~ and weighing the residue. The results of this ~tudy (~u~marised in Table 1) ~how clearly that Product A
(tannase treated) had a good cold water extractability while Product B (not tannase treated) was poorly extractable with cold water. Finally, Product C (commercial tea, no tannase treabment~ had the poorest cold water extractability of the three product~ tested. The extraction yield at 30 & for Produ~t A almost equal~ that of B and C at near boiling extraction ~onditions of 95 C.

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aQ 364a 7~6 ~X~PLE II
The procedure used in preparing Product A was repeated on 5 additional 300 g b~tches of fresh green tea using the same condition~ except that tannase wa~ used in the amount of 0, 2, 4, 8 and 16 units per g dry weight of green tea to give Products D, E, F, G and H respeetively.
The cold water extractahility of the~e products using 20~
~ater was determined in the manner described in Example 1.
For further evaluation, beverage strength extracts were prepared by placing 2~25 g of leaf in 200 ml of distilled water at 20C, stirring for 3 minutes with an overhead stirrer at ~50 rpm, and filtering the slurry through a medium ~intered - glass funnel. Th~ colour of the beverage was then determined in terms of L, a, b and haze values~
The result~ of these tests are ~ummarised in ~able 2 and sho~ that a satisfactory product can be made using 4 to 16 units of tannase activity per gram of green tea.
Products F, G, and H hava increased extraction yie~d and greatly improved colour.

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cQ 364a 37~

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cQ 364a EXAMP~E III ~74 The procedure u~ed in preparing Product ~, a~
de~cribed in Example I~ was u~ed in preparing Product~
J, K and L, where the only change in procedure was the amount of pota-~sium hydroxide added to the comminuted green tea.
Potassium hydroxide in the amount of 0, 0.35, 1.05 and 1.40 g was dissolved in 10 ml of water and wa used to prepare Product~ Io J, K and L respectively. The cold water extrac-tion yield at 20C wa8 determined for ~11 4 products as des-cribed in Example I. The pH of each product was determined by extracting 2 g of the leaf with 50 ml boiling deionized water, and allowing it to cool to room temperature (23C).
All of the Product~ were organolepti~ally evaluated as bever-age~. The ta~te of the resultant beverages varied somewhat between the different sample6, but they were quite ac~eptable in ever~ ~ase. The re~ults, given in Table 3, indicate that the extractable solids remain almo~t the same even though the pH of the re~ulting beverages i8 affected by the amount of KOH :
used in the process.

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cQ 364a 37~

EFFECT OF AMOUNT OF POTASSIUM HYDROXIDE
ON EXTRACTION YIELD OF BLACK TEA
_ KOH Extraction Product Added (g) PH Yield (~) ...._ I 0 4.30 22.8 J 0.35 4,55 24.5 ; A 0.7 4.80 24.8 ,' `: .
~ K 1.05 5.05 24.0 .
L 1.40 5.40 25.5 ~' ~

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:; ~ : , . : - -cQ 364a E ~ PLE IV
The procedure u~ed .in the preparation of Product A
waq repeated with three further batches of leaf, The only change made to the pro~edure was the temperature used ~or the tannase treatment. Temperatures of 35t 45 and 55C were u~ed in the preparation of product~ M, M and t respectively.
Extraction yield~ were determined at 20C as de~cribed in Example I for the ~hree products. The result~ are given in Table 4 and they indi~te that the optimum tannase treatment 0 wa8 achi~ved in the temperature range 25 to 35C, while partial suc~es~ wa~ obtained at 45 C, EFFECT OF TEMPERATURE OF TA~NASE_TREATM~T
UPO~ E~TRACTION YIELD
15 Product Temperature (C)Extraction Yield at 20C (%
A 25 24.8 M 35 22.4 ~S 21.4 ~.
16.0 EX~MP~E V
The ~procedure u~ed in th2 preparation of Product A
wa~ repeated on a further 300 g bat~h of tea leaf except that : after comminuting the green leaf9 tannase was added with no addition o~ ~0~, and then after the 30 minute anaerobic hold 2S period, 0.7 g KOH in 10 ml water W~8 added~ This procedure ~ ntially the same as used in the preparation of Product A except that the KO~ addition ~tep and the tannase trea~ment period have been reversed in orderO ~he produc~ mad~ by thi~ -modified proc2ss wa~ identical to Product A in extractability _ 21 -cQ 364a , ~- beverage colour and clarity.

EXAMPLE VI
.
I90 gms of fresh green tea flush was frozen to -20F, mixed with solid CO2, and cryogenically milled in a hammer mill such that all particles passe~ a .035" herringbone sieve. The milled flush was then dispersed in 600 mls of distilled water, and the slurry was brought to 22~C. Tannase enzyme was added in the proportion of 10 units per gram of (dry weight basis), and the slurry was stirred for one houx. At the end of the one hour period, the p~ of the slurry was adjusted to 5.6 with 10~ KOH in water solution, and the slurry was aerated with continuous stirring for 90 minutes. After fermentation, the slurry was then placed in a rotary evaporator, and water was removed under ~7" Hg vacuum, while the slurry was held at 40C. When the slurry was dry enough, such that it began to tumble inside the rotary evaporator, the vacuum `~
was dropped back to 19" Hg, and the temperature of the leaf slurry was allowed to rise to 95C over a period of 20 minutes, and held at 95C for 10 minutes. The vacuum was broken three times, to allow leaf which had become stuck to the sides of the flask to be s~aped off and to allow manual mixing of the contents. After 10 minutes at 95C, the leaf was dry enough such that it could be ; removed and set thinly on an enamel tray. The tray was heated to 70C, and leaf dried to 3% moisture. The leaf was sieved, and all material passing a US standard 40 mesh screen was discarded.
The black tea leaf produced, Product P, was cold water ~20C) extractable and gave a tea beverage with good colour, taste, and clarity. The iced tea beverage produced by cold water extrac-tion of product P was judged to be far superior to a beverage pro-duced by aninstant tea made by the homogenate process without the 3G benefit of a firing treatment. This instant tea was made in the same way as Product P except that, instead of drying the slurry after conversion, an instant tea was prepared directly from the homogenate as described in US Patent No. 3,812,266 cQ 364a -37~6 3 instant tea was made, there~ore, without the firing step normally associated with the manufacture of black tea le~f by dry-ing the hot water extractable tea solids separate from the remainder of the tea flush material. Firing of fermented tea flush is known to play an important role in the formation of black tea flavour and it may well be that firing of the whole fermented tea flush accounts for the good black tea flavour of the cold water extractable black tea produced by this new green tea homogenate conversion process.
EXAMPLE VII
The procedure used in the preparation of Product P, Example VI, was repeated except that immobilized tannase was used in place of soluble tannase. This required separating the slurry into extract and leaf residue prior to tannase treatment. This was carried out following a 30 minute hold period at 50C to in-crease extracted solids content of the water fraction. The clear extract was then passed over a column of tannase immobilized on glass beads until no further pH change was detected. The tannase treated extract was added back to the leaf residue, which still had active tea enzymes, to reform the homogenat slurry. The remain-ing procedures adopted in the preparation of Product P were fol-lowed to produce a tea laaf, Product Q, which was almost indisting-uishable from Product P.
EXAMPLE VIII

.
280 yrams of fresh tea leaf was placed in an eccentric paddle Hobart mixer and macerated for 4-1/2 minutes. The leaf was transferred to a Waring~ Blender along with 500 milliliters -distilled water, and was further macerated for 3-1/2 minutes. The resultant slurry was sparged with nitrogen gas for 2 minutes to remove all disolved oxygen. Tannase enzyme was added to the slurry . . .~ , . . - .
in the proportion of 10 units per gram dry weighk of leaf, and the slurry was stirred for one hour. The pH was adjusted to 5.6 with 7% ~OH solution and aeration was then carried out with continuous stirring, for 90 minutes. The slurry was then placed in a Buchi*
rotary evaporator, and processed in the same manner as described in * trade mark .. .. . . .... . . . .
~ ., - , , . ~ , : -cQ 364a ~ .nple VI for Product P. The black tea leaf prepared, Product R, was judged to be very similar to Products P and Q, having good cold water extractability and giving a beverage with good colour, clar-ity, and taste.
EXAMPLE IX
The procedure described in Example I for the preparation of Product A was repeated except that the fermentation period was shortened to 5 minutes under ambient conditions. This resulted in a tea, Product S, with o~long-like character. As a control, the process for preparing Product B was repeated with the same short-ening of the fermentation step. The control, Product T, and Product S were extracted with 20C water as described in Example I. Product S (tannase treated) had an extraction yield of 19.4~ while Product T
gave a yield of 13.9%. Further, Product S had fuller flavour than Pxoduct T, and it was characterised as a very acceptable oblong tea.
EXAMPLE X
. .
The procedure used in the preparation of Product A was repeated with three further batches of green tea. The only change in the procedure for the first two batches was the l~ngth of the pre-fermentation anaerobic hold period: anaerobic hold times of 15 and 0 minutes were used for the preparation of Products U and V~ respectively. Product W was prepared to demonstrate the effect of a post-fermentation tannase treatment on the cold water extract-ability of the finished black tea product. The following procedure was used to produce Product W. A 300 g batch of green tea was withered, comminutedt and 0.7 g of potassium hydroxide in 10 ml. of -water was added as described for Product A in Example I~ No tannase was added and no anaerobic hold period was used. Thè comminuted green tea was fermented under the conditions used in the ~ -prepartion of Praduct A. After fermentation, 8 units of tannase enzyme per gram dry weight of fermented tea leaf material were added, and the tea flush was passed 2 times through rollers with 0O62 mm spacing to ensure thorough mixing of enzyme and fermented tea leaf material. The tannase treatment was allowed to continu~

for 30 minutes under anaexobic conditions after which the fermented, _ 24 _ ~7~ cQ 364a _~ aase-treated, tea flush was dried by the same method as used for Product A.
~ Extraction yields for Products U,V and W were determined at 20C as described in Example I. The results obtained (Table 5) indicate that a pre-fermentation tannase treatment is required in order to obtain the desired cold water extractability and that an anaerobic hold period of about 15 to 30 minutes is required to maximize the cold water e~tractability of the finished black tea leaf product.

Table 5. Effective Point of Addition of Tannase on Extraction Yield at 20C of 4 Black Teas . .
Tannase Anaerobic Extraction Product Point of Addltion Hold Period (min) at 20C (%) A Pre-fermentation30 24.8 U Pre-fermentation15 24.2 V Pre-fermentation 0 22.9 W Post-fermentation30 13.0 EXAMPLE XI
300 gms of fresh green tea leaf were placed in a micro wave oven (Litton* model 403.000, Litton~Industries, Minneapolis, Minnesota) for 2 minutes, during which time the leaf was heated sufficiently to inactivate the leaf enzymes before any fermen-tation could take place, The leaf was then passed twice through a Hobart meat grinder, whose front plate had holes 3 mm in diameter.
.... .... ..
Half of the commlnuted leaf was then treated with tannase (at a level of 8 units per gram dry weight), dissolved in 10 ml of water and passed once again through the meat grinder. The tannase treat~d portion and the untr~ated portion were then held at 20C for 30 minutes. Both batchas were then fired at 85C until the moisture content was below 5%. The green tea without the tannase treatment was Product X, and the tannase treated green tea was Produc~ Y.
Extraction yields for the Products X and Y were deter- .
mined at 20DC as described in Example I. Product X (no tannase) * trade mark _ 25 -'~ ' , .

cQ 364a 374~

an extraction yield of 15.5~ while Product Y (tanasse treated) had an extraction yield of 20.2~. Product Y had fuller flavour than Produet X when both were brewed up at 20C as described in Example II, and Product Y was found to produce a good quality iced green tea beverage.
EXA~PLE XII
A pilot scale method for preparing cold water extractable black tea was used in this example. F~esh green tea flush was withered overnight to a moisture content of about 70%. An S pound batch of the leaf material was comminuted by passing it three times through rollers set at a gap of 0.014 - 0.020 inches. The comminu-ted tea flush was placed in a Model VL 401 Hobart mixer, and tan-nase, in the proportion of 8 enzyme units per gram dry weight of tea flush, was added as a water solution and mixed thoroughly. To ensure complete mixing, the tea was passed once through the rollers.
The mixture was held at ambient temperatures for 30 minutes under essentially anaerobic conditions. The leaf was again placed in the ~obart mixer and potassium hydroxide, in the proportions of 0.9 parts by weight of potassium hydroxicle per 100 parts of the dry tea flush, was added as an aqueous solution. After one additional ; rolling, the tea flush was passed once through an Osborn* 4 inch CTC machine (Davidson, Calcutta, India) and fermented for 2.75 hoùrs. The flush was then fired at 180F and the dry black tea was sieved through a 10 mesh screen.
An instant tea was prepared from this cold water extract-` able black tea by extracting twice with ten volumes of boiling water. The combined extracts were concentrated to about 6~ solids, and 1% calcium chloride dihydrate ~on a solids basis) were added.
The extract was then cooled to 50F and clarified by centrifuging for ten minutes at 6800 x g and thereafter freeze-dried.

When reconstituted as a beverage, the instant tea, Product Zl~ had excellent clarity and the colour of freshly brewed black tea. The instant tea product dissolved rapidly in either hot or * trade mark _ 26 -. . - . "

cQ 364a 7~
C~ d water, and it had a most satisfying black tea flavour when served either as hot or as iced tea.
A control black tea was prepared from the same withered tea flush. A 4 pound batch of this withered tea flush was passed three times through the rollers and once through the Os~orn CTC
machine. The comminuted leaf was fermented at 80F for 3.25 hours, and then it was fired at 180F and sieved through a 10 mesh screen.
An instant tea was prepared from this black tea by ex-tracting it twice with ten parts boiling water. The extract was treated for 30 minutes with tannase enzyme added in the proportion of 20 units per gram of tea solids. (This is equivalent to 7 units per gram of starting tea leaf). After the tannase treatment, the pH was ad~usted to 5.0 with 2N potassium hydroxide. The extract was concentrated to about 6~ solids, and 1% ealcium chloride dihydrate (on a solids basis) was added. After clarification by ~ eentrifuging for 10 minutes at 50F and 6800 x g, the extract was -I freeze dried. The instant tea, Product Z2~ when reconstituted, also gave a good quality tea beverage that had a satisfactory black tea clarity, eolour, and flavour when used as either a hot or ieed tea product.
A comparison of the extraetion yield, polishing loss, and process yield for these two blaek tea produets (Table 6) shows that the eold water extraetable black tea leaf process gives a fired blaek tea whieh yields more teas solids (Produet Zl) upon extrae-tion with hot water than the control, or regular, fired black tea.
The lower polishing loss also adds to the proeess yield whieh is signifieantly higher for the process used to prepare Produet Zl-. .

Instant Teas made from Black Tea using Tannase TannaseExtraction Polishing Proeess ~, Tea Sample TreatmentYield (%) Loss (%)_ Yield Produet Zl~ Prior to fermlentation 37.3 7.5 34.0 Produet Z2 After extraetion 31.7 14.4 27.4 '. :. : ' ; - _ 27 _

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a dry leaf tea extractable in cold water, wherein comminuted fresh green tea leaf is contacted with tannase under conditions which are at least initially essentially anaerobic, and the tannase-treated leaf is fired and dried to a moisture content of below 5%.

2. A process as claimed in claim 1, wherein the fresh green tea leaf is contacted with tannase under essentially anaerobic conditions throughout the contacting period, and the unfermented tannase-treated leaf is fired and dried to produce a dry green leaf tea.

3. A process as claimed in claim 1, wherein the fresh green tea leaf is contacted with tannase, the tannase-treated leaf is fermented, and the fermented tannase-treated leaf is fired and dried to produce a dry leaf tea of black or oolong character.

4. A process as claimed in claim 1, wherein the tannase treatment is conducted for from 15 minutes to one hour at a temperature in the range 25 to 55°C using an enzyme level in the range of 2 to 20 tannase units per gram dry weight of fresh green tea leaf.

5. A dry leaf tea prepared by a process as claimed in any one of claims 1 to 3.

6. A dry leaf tea prepared by a process as claimed in Claim 4.

7. A process for the preparation of an instant tea powder, wherein a dry leaf tea as claimed in Claim 6, is extracted with hot water, and the extract is clarified and dried to yield an instant tea powder.

8. An instant tea powder prepared by a process as claimed in Claim 7.