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(21)    Application Number: KE/P/2007/ 000594

(22)    Filing Date: 17/04/2007

(30) Priority data:00480/KOL/2005  07/1212005  IN

(51)    IntCL7, A 23F 3/06,3/14

(73)    Owner:DEPARTMENT OF BIOTECHNOLOGY of Block-2, C.G.O.Complex, Lodi Road, New Delhi, 110 003, India and BOSE INSTITUTE of 93/1 APC Road, Kolkata, 700 0009, India

(72) Inventor:    Dr. Swati SEN-MANDI

(74)    Agent/address for correspondence: Ndungu Njoroge & Kwach Advocates, P.O. Box41546-00100,Nairobi


(57) Abstract: A process for enhancing flavour in leaves of field grown tea plants comprising: growing tea plants in field and exposing the said tea plants to artificial U.V. radiation.


This invention relates to a process for enhancing flavour in tea using U.V. radiation.


Till date enhancement of quality in black tea has been attempted mainly by mixing tea leaf from different clones; the proportion and number of different clones being used for mixing in each batch of manufactured tea is the trade-secret for each tea brand. While some attempts have also been made for enhancing tea quality by exposing leaf•pieces to UV radiation there is no report on means I process of enhancing flavour producing compounds in tea leaf of different tea clones. - Da~eeling 1ea has •alwaysbeen appreciated•foritsnigh flavoar/ arom-a-compared to Assam tea. While several studies•have been conducted to look for soil factors as well as flavour causing biochemicals in tea leaf, the role of UV radiation in enhancing aroma producing compounds in leaf on .whole plants/isolated leaf segments have never been reported or successfully undertaken commercially.

UV radiation is generally known to cause cell molecular damage in plants/microbes and has never been considered as a factor for enhancing flavour in tea.


An object of this invention is to propose a process for enhancing flavour in tea using U.V. radiation.

Another object of this invention is to propose a process for enhancing flavour in tea using U.V. radiation for commercial use.

Further, object of this invention is to propose a process for value addition in commercial tea using UV radiation.

Still further object of this invention is to propose a process for enhancing flavour in tea using U.V. radiation with insignificant expenditure for large scale production of tea with high aroma.


Fig 1: shows flavonid analysis in high (H) and low (L) altitude grown tea plants of the same clone by HPLC scan recorded at 285nm using DMSO:MeOH solvent system at 0-55min retention time in hypersil ODS2 column a= T78(L), a'=T78(H),b=AV2(L), b'=AV2(H),c=CP1(L),c'=CP1(H).

Fig 2: shows spectrophotometric assay of enzyme phenylalanine ammonia lyase of tea clones, mentioned in Fig 1.

Fig 3: shows thin layer chromatographic separation of flavonoid glycoside in tea clones growing in high & low altitude; the high attitude clones posses higher content of flavonoid glycosides.

Fig 4: shows spectrophotometric assay of UV absorbing compounds in tea clones (A=absorbance at 305nm).

Fig 5: shows spectrophotometric assay of Beta D Glucosidase enzyme activity of tea clones (1=T78(H), 2=T78(L), 3=AV2(H), 4=AV2(L), 5=CP1(L), 6=TV2(L), 7=P312(H), 8=B157(H) ).

Fig 6: shows RAPD analysis of Tea clones using primer designed from 13-D .glucosidase gene-sequeAce-M=1 OObp-DNA -Ladder (Geneir, -

Fig 7: shows general lay-out of the cultivation site.

• Water sprinkler

C Untreated control plants

-• UV lamp stand (L-shaped) with erect arm 0.75 mt from ground and ttie lamp suspending at right angles to it

•    Tea plants

•    shade plants.

Fig 8.: Representative experimental plot.


According to this invention there is provided a process for enhancing flavour in tea using U.V. radiation.

Plants use sunlight for photosynthesis and as a consequence are exposed to UV radiation that constitutes the harmful component of sunlight; among. the more harmful components of sunlight is UV-8 radiation (280nm-315nm) that cause oxidative stress in living cells. Being exposed to this necessary evil, plants have evolved cell protective/repair mechanisms (QY producing antioxidants) that equip them to grow and flourish under spontaneous UV radiation. Such antioxidants namely the flavonoid group of compounds are known to produce colour/flavour in plants.

In plants the enzyme 13-D glucosidase is know to cleave flavonoid glycoside into aroma producing aglycon moiety. Earlier studies in the laboratory relating to oxidative stress and associated cell protection in tea plants growing in high altttude (at -450011 above sea level) and in low altitude (at -350 ft above sea level) have shown that flavonoid glycosides and 13-D glucosidase are high in the high altitude grown tea clones than in the same clones growing in low altitude both sttes being at the same latitude is 27". HPLC assay of flavonoid glycoside
and enzyme assay of ~D Glucosidase activity have shown markedly high values in extracts of leaves of tea clones growing in high altitude compared to leaves of tea clones growing in low altitude, both at the same lamude viz at -27•N.

In view of the fact that places in high altitude are subject to higher W radiation compared to places in low altitude, particularly in the subtropical region, of the northern hemisphere and since these compounds viz. flavonoids I flavonoid glycosides and the enzyme J3 -D glucosidase are known to be signaled for enhanced production by UV radiation, a process is being developed that could use UV radiation to enhance aroma producing compounds in tea leaf. Although production of favonoid group of compounds (viz. flavonoid glucoside) and ~D glucosidase have been studied in other plants, the effect of UV radiation in enhancing flavour I aroma in tea leaf has never been reported.

In  addition  to  enhancing  aroma  compounds  in  field  grown  tea  plants  UV

. -in:adiation-coula alse be -used-to -enhance aroma trr phlc"kea rea le-al auniig the- - process of tea manufacturing at the "fermentation" step when cut (macerated) tea leaves are left under conditions that allow enzyme activity in the leaf pieces. During this period the enzyme could act to produce the aroma producing aglycon moiety that would enhance flavour in made tea.

It is seen that total flavonoids (Fig1 and Table1), one enzyme activity in the flavonoid. producing pathway viz. phenylalanine ammonia lyase (Fig 2) and also flavonoid glycosides content (Fig 3) activity of B.D glucosidase (Fig 4) are higher in high altitude grown tea gardens; the values are much lower in_tea plants of same varieties grown in low altitude lea gardens. [II is pertinent to mention here that average UV fluence at -840011 above msl at -•21•N latitude is found 1~ be 4.6 MED/h and at Siliguri -492ft above msl it is 2.80 MED/h]. Corroborating the above data, (in fig 1-3 and table 1), total UV absorbing compounds (Fig 4), comprising mostly of the flavonoid group of compounds was also found to be higher in the high attitude grown plants than in the low altitude grown plants belonging to the same clones. Assay of LI-D glucosidase enzyme that cleaves flavonoid glycosides to release the flavour producing moiety also shows higher activity in the high altitude grown tea clones than in the same tea clones growing in low altitude (Fig 5).

In PCR based genome analysis using primers designed from LI-D glucosidase gene sequence lor sequence tagged sites (STS) analysis no significant difference was found belween DNA exlracted from the tea plants (clones) grown

-    - ~n- high -aKitude and from -plants •ofihe- same --varielies (clones)'growing- rn-lov/ altitude (Fig 6). Such experimental data provide evidence to suggest that the difference in aromatic compounds in high and low altitude grown tea clones is largely due to UV signalling of cell molecular events in lea leal. II is pertinent to mention here that constitutive levels of UV absorbing compounds and the readiness of plants to accumulate them have been correlated with UV tolerance also in other studies (Mazza el al 2000); such response was found. to be most rapid for UVB, often showing increase of these compounds within hours (Strid  and Perra, 1992) of UV signalling. Such studies were conducted on soybean plants in the context of cell protection and not for production of flavour related compounds as has been realized in tea plants ( reported in our study).

In this report artificial UV lamps that emit longer wave length radiation of UV viz. UVA 240-280 nm and NBUVB at 311 nm was used in an attempt to simulate near-natural UV radiation to develop a process for enhancing flavonoid compounds in tea plants.

Table: Baseline 810 Peak Integration Report

Name of Tea variety /clones    Total Peak Area(Microvolt-sec)
T78(H)    115321747.3
T78(L)    50419160.0
AV2(H)    68752569.5
--•-•••--AV2{t)- -  --    - "332'01!922.0
CP1 (H)    63778939.3
CP1(L)    29532734.0



Tea plants were grown in the field at altitude -154 ft from msl and at 22•N latijude.

Diagrammatic representation of the plots (SITE PLAN is given in Fig 7 and 8). UV radiation at the experimental site under natural ambient UV in low a~ttude + artificial UV lamps is given below.

Table 2: UV ftuence on experimental plot due to natural UV radiation at 154 ft above msl + artificial UV lamps (in MED/h):

Date:    Time    UV fluence in (MED/h)        Weather condijion   
-OHHo2007--    ---  - --- --  -    -(nirtural-+lNiamps)-    --------------   
(IN ONE                   
    10:35    1.50        Clear sky (some times   
DAY).                hazy)  with  slow wind   
    13:00    1.79           
                from West to East.   
    16:30    0.83           
IMED- 5.83 X 10.~.watt/em
Experimental Material:

Plants of 2 tea clones TV-1 and TV-25 that are known conventionally to be of high and low flavour, respectively, grown in a tea cultivation field at Kharagpur, were used. In this cultivation site, agronomic care viz watering, weeding etc were routinely conducted uniformly in all the plots.

Experimental Set-up:

W~hin the tea cultivation area experimental plots each of 11 It x Bit containing-plants at plant to plant distance of 2.511 and row to row of 3.511 were subjected to add~ional UV radiation from suspended UV lamp (vide SITE PLAN-Fig 7 and B). All plants in the plots were about 2.511 height from the soil.

Over both the selected plots UV lamps were suspended from L-shaped stand

-    -with-the loRgef arm fixed -to the-soij. -The-shorter "llrm was lms-pended •arlight-angle to the stand and at a height of 2.51! above the leaf canopy. The field set-up is represented in Fig 7 and B. The UV lamps used were UVA (40W) emitting wavelength 240-2BO nm and narrow band (NB) NBUVB (20W) emitting

wavelength 311 nm. The UV lights were switched "on" eve_ry morning at 7am (close to sunrise) and sw~ched off every evening at 6pm (close to sunset). It is pertinent to mention here that in other plants (viz soybean plants) most of .the phenylpropanoid (flavonoid group) compounds have been shown to be induced by UVB of wavelengths S315 nm. (Mazza 2000)

Tea leaves, 2 and a bud, (conventional unit collected for tea manufacturing ) were colleted for flavonoid assay from the treated tea plants at 11 day intervals.

Untreated control leaf material for flavonoid assay were collected from plants far away from experimental plots.

Table 3: Spectrophotometric data showing total flavonoid in treated/untreated Samples:

Variety    Treatment    Optical Density at 285nm   
TV1    UVA+UVB (treated)    2.1298 ± 0.238   
    Control (Untreated)    1.2770 ± 0.208   
TV25    UVA + UVB (treated)    1.8907 ± 0.099   
    Control (Untreated)    1.5640 ± 0.285   

-conclusion~ Tnis-is prefimiiiary fiela<fafa~ ---- -- - --- -

Further fi!"ld experiments with better arrangement for UV fluence on the tea plants and also studies on effect of UV on isolated leaf is under way.


1.    A process for enhancing flavour in leaves of field grown tea plants comprising:  growing tea plants in field  and  exposing the said tea plants to artificial U.V. rad.iation.

2.    The process as claimed in claim 1, wherein said U.V. radiation uses the production of flavonoid compounds viz flavonoid glycosides and 13-d glucosidase in the tea leaves which interact to enhance aromatic compounds in tea leaves.

3.    The process as claimed in claim 1 wherein the artificial U.V lamp used hard wave length radiation of UVA 240-280nm & NBUVB 311nm.  


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