Indi an Journal o f Experime ntal Bio logy Vol. 42, June 2004, pp. 575-580

Caffeine in tea plants [Camellia sinensis (L) O. Kuntze] : In situ lowering by Bacillus lichen~fonnis (Weigmann) Chester

S Ramarethinam & N Rajalakshmi*

Research and Development Centre, Un ited Nilgiri Tea Esta tes Co Ltd ., 3. Sav ithri Shanmugham Road, Race Course, Co imbatore 64 1 0 18, Ind ia

Received 20 March 2003; revised 3 1 Ma rch 2004

Tea plants (Calll ellia sillellsis) contain 5-6% caffe ine that is responsible fo r the stimulati ng effect of the beverage. As the to lerance to caffeine varies among indi vid uals, low caffeine tea would be an ideal alternati ve. While assess ing the potenti al o f a few selec ted bacteri a-Bacillus lichell i{onllis. B. sub/ilis and B. jim illS. to multipl y o n nutrient med ium supplemented with g lucose (5 % ) and tea leaf ex tract (2 %), it was observed that only B. lichelli{o rlllis could pro liferate on thi s med ium. Hence. B. lichelli{o nllis was used for furth er studi es . Tea pl ants were sprayed with a suspe nsio n of B. lichelli{orlllis at a diluti on o f 5 x 108 CFU/ml containing 0. 1 % Tween 80 as sur factant. III situ lowering of caffe ine from tea leaves was ev ide nt without affec ting the quality of the o ther tea compo nents. Further, there was no change in the morpho logica l and physio logical charac teri stics as well. It is suggested that spray ing o f B. lichell i/imll is may be useful in yie lding deca ffe inated tea with good fl avour and aroma.

Keywords : Bacillus lichellij'o rlll is. Call1 ellia s illellsis , Caffe ine- free tea

IPC Code: Int C I7 AO I N63/00; C 12N9/56

Tea used as a drink is made fro m the api cal two leaves and a bud o f the plant, Camellia sil/el/ sis (L) O. Kuntze. The leaves contain in additi on to the no rmal constituents, hi gh level of polypheno ls (30-40%) and caffe ine (5-6%). Caffeine (I ,3,7-trimethylxanthine) also present in other beverages (coffee and cocoa), stimul ates the cent ra l nervous sys tem, increases mental a le rtness, bette rs memo ry and mood, improves reasoning powel'l and is o f va lue in the treatment o f gout, hypertensive headaches, myocardial in fa rc tio n, etc. The co mmon effec t of caffeine when taken in hi gh doses is increased metabo lic rate, irritability, sleep di sturbance2 and gastro intestinal aches, in addition to inductio n of hypercho les trolemi a, osteoporos is, ul cers and cancers . Though the intake of caffeine fro m tea is not de lete ri o us to health per se, as tea (-20 mg/l OO ml ) contains less caffeine than coffee (- 120 mgll 00 ml), chocolates (-35 mg/28 g bar) o r cola drinks (-30 mg/l OO ml )3, lu xury consumpti o n is a poss ibility since caffe ine is wide ly distributed in a vari ety of foods. Apart fro m caffeine, tea a lso contains isomeri c d imethylxanthines - theophylline and theobro m i ne that are analogues of caffe i ne and intermedi ates in its synthes is. These a lso contribute to

' Corresponde nt author-Phone-0422-2220 125: 2220566; Email : tearesdev@vsnl. in

the medi cinal value o f tea. The te rminal bud of tea pl ants contains the max imum caffe ine4

• Withered leaves have more caffeine ( 18-2 1 %)5 in compari son to fresh leaves pro babl y due to degradati on of purine nucleotides6 during withering.

T olerance to caffeine di ffers amo ng ind ividuals. and therefore, low-caffeine tea would be an advantage for sensiti ve people . Ef fo rts to produce decaffeinated tea and coffee have intensified in the pas t two decades. Decaffeinati on, in volving the se lect ive re moval of caffeine using organic solvents or super­criti ca l gases, a lso removes most of the co mponents respo nsible for fl avour and aroma leav ing a bland tea without any taste and less of its stimul ating effects on the body . Genetic eng ineering may ho ld the key to making decaffeinated tea w ithout loss of aro ma and fl avour. Identificatio n of the gene encod ing the enzy me caffe ine sy nthase7 opens up the poss ibili ty of producing gene tica ll y eng ineered tea plants in wh ich the ex press ion of the gene has been suppressed. Such an atte mpt has been successful in coffee pl ants that have been transformed w ith an anti sense RN A fo r at least o ne enzy me in the pathway of caffei ne biosy nthes iss. However, gene manipul atio n may not be a rea lis ti c a lte rnati ve fo r tea. which is a perennial herb whose produc ti ve li fe span is ex tended to 80- 100 years due to pruning e ffec t obtained by plucking the

576 INDIAN J EXP BIOL, JUNE 2004

leaves once a week. Hence, short-term measures to lower the caffe ine content of leaves when they are sti ll on the plant would be an eas ier approach to obtain caffeine-free tea that is hi gh in flavour and aroma. In thi s paper, the effects of spray ing the bacterium, Bacillus lichen (jO rl'l1 is, on tea plants are presented.

Materials and Methods Bacterial cultures-Cultures of Bacillus

lichellijorlnis (Weigman n) Chester, B. subtilis and B. fi nnus, isolated from tea pl antation were identifi ed by morphological, physiological and biochemical tests9

in co ll aboration with the Centre for Advanced Study in Botany, University o f Madras and the Microbiology Department, PSG Institute of Medical Sciences and Research, Coimbatore. They were maintai ned on nutri ent agar medium (pepto ne, 5 gil ; sodi um chloride, 5 gil ; beef ex tract, I gil ; yeast extract, 2 gil ; agar, 15 gil ; pH 6.8) slants at room temperature (300±2°C) . For ex perimental purposes, log phase cultures were transferred to liquid med ium (nutrient agar medium without agar) and incubated for 24 hr before spraying in the fields.

Utilisatioll of tea liquor for bacterial growth-The capacity of the selected bacteria to utili se tea components for growth was monitored by inoculating the bacteria into nutrient med ium supple mented with g lucose (5%) and green leaf extract (2%) and

incubati ng at 30° ± 2°C in an orbi ta l shaker. Changes in the optical density (at 660 nm) of the culture broth were measured spectrophotometrically at every 24 hr for a period of 5 days, and used as an index of bacterial growth .

Tea plants-Tea, Call/ellia sinensis (L) O. Kuntze, grow ing in plantations of this organization at Chamraj Estate (N ilg iri Hill s) was used for this study. Experimental plots were demarcated in triplicate, each co mprisi ng thirty productive bushes and covering an area of 1.25 cents . Plants were twenty years old and were hard-pruned two years before the study period. They were sprayed with the culture at a dilution of 5 x 108 CFU/ml culture broth containing Tween 80 (0. 1 %) as surfacta nt. The same soluti on without spores served as control and was sprayed on another set of thirty bushes in the adjacent plot. Plants were drenched totally using 10 I of spray sol ution per plot through a knapsack sprayer fitted with NMD nozzle and sprayed at the rate o f 1 !lmin . Spraying was performed in the morning (6 - 7 a.m.) . Leaves were

plucked after 24 hr and also on day 5 and 10. Each experiment was repeated thrice.

Tea processing-Tea was manufactured by the orthodox method that in volves the stages of withering, rolling, fermentation and drying, before the green leaves are converted to black tea of commerce. A 2% brew was prepared from processed tea and its colour and strength as well as theafl av in and thearubig in content were measured 10.

Estimation of caffeille-Caffei ne from fresh tea leaves or made tea was ana lyzed spectrophotometrically'l or after separati on by HPLC I 2 and quantified in rel ation to standard caffeine. Theophylline and theobromine were also extracted along w ith caffeine and separated by HPLC I 3 on a Tiger LC System (Waters Corporation USA) and quantified using authentic samples, each of which was separately passed through HPLC column at different conce ntrati ons. Based on the area of each peak, a standard graph was drawn for each methy lxan thine in relation to its concentrati on. After separatio n by HPLC, the peak area for each methylxanthine in tea samples was measured and its concentration was calculated from the respecti ve standard graph .

Pigment allalysis-Within 4 hr after plucking fresh tea leaves, pigments from these leaves ( lO g) were extrac ted in 100 ml aqueous acetone (80% v/v) and their absorbance of visible li ght was scan ned using a C hemito UV -Vis spectrophotometer. The amount of chl orophyll s and caroteno ids were calculated according to Mackinney' 4.

Estimation of proteills and phellols-The content of total proteins and pheno ls were estimated spectro photometri cally in aqueous extracts of the leaves, following the methods of Lowry et al. ' 5 and Schanderi 16 respecti ve l y.

Results and Discussion To assess the suitabili ty of the selected bacteri a for

fie ld spray , their potential to multi ply utili sing tea constituents was mo nitored by growing the cultures on med ium contai ning glucose (5 %) and tea leaf extract. It was observed that on ly B. lichen(jorillis could pro li fe rate while the other bacteria were unab le to grow o n thi s medium. Hence, they were not Llsed for spray ing on tea plants in the plan tat ion and a ll further experiments were carried out only w ith B. I icll.en (jorll/is.

The bac terium, B. licli enijormis, was sprayed o n tea plants two days after plucking and allowed to

RAMARETHINAM & RAJALAKSHMI : CAFFEINE LOWERING BY B. L1CHENIFORMIS 577

proliferate on/in the leaves before they were plucked again after 24 hr, 5 and 10 days of treatment. Effects of spraying the bacteria on tea plants were monitored both morphologically and physiologically . Tea plants in the plantation did not show any change in their appearance and the overall morphology of the plant was the same as that of untreated plants. Hence, other parameters such as , pigment content, total phenol s and total protein were monitored. In keeping with the similarity in physical app~arance of plants, there was no variation in the pigment content, both in terms of the levels of individual pigments and their relative proportion (Fig. 1, Table 1). It was also observed that the protein content (Table 1) did not show any significant change indicating that infection with B. lichelliforlllis did not produce much change in the biochemistry of treated plants. However, tea plants showed enhancement in total phenolic content, since the bacterium was recognized as a contagion. This would mean that the liquor quality of tea prepared from such leaves would be better than normal tea. When these tea leaves were used to prepare black tea, there was no difference in its physical appearance and liquor characteristics (Table 2) . Spraying of Bacillus sp. , termed as Tea Beneficial Microbe has been shown to increase l

? the photosynthetic rate by 30% and net yield by 29-65%, in addition to improving bud density, growth rate, leaf area, thickness and shoot weight.

Caffeine content was monitored in terminal leaves (which are normally used for tea processing), mother leaves (lower mature leaves) and in made tea

400 500 600 700

Wavelength (nm)

Fig. I- Absorption spectra of acetone extract of normal tea leaves (-) and leaves sprayed with culture of Bacililis Iichelll!anllis (----).

(Table 3). The leaves showed a significant decrease in caffeine content in 24 hr. However, by day 5, the decrease in caffeine content was lesser, and by lOth day, the decrease was even lesser. Hence, it appears that the microbe should be sprayed on day 6 of a 7-day plucking cycle or on day 9 of a 10-day plucking cycle, in order to obtain the maximum lowering in caffeine level of tea leaves. It appeared that the lowering in caffeine induced by the bacterium counteracted the increase of caffeine during withering, thereby reducing the overall caffeine level of the made tea.

Consistency of results obtained prompted further work on the fate of caffeine within the cells. Methylxanthines were separated by HPLC (Fig. 2) into three components, which were identified using authentic standards as theophylline, theobromine and caffeine. The level of caffeine was higher than theophylline or theobromine, and hence, separation was done at different dilutions in order to monitor changes in the levels of minor components (theophylline and theobromine) . Thi s exercise proved to be rewarding as marked variations in the levels of theophylline and theobromine were noticed in response to spraying the bacterium, B. lichellifo rl11 is. The levels of caffeine and theophylline got lowered while theobromine content increased in response to infection . It was found that caffeine content decreased

Table I-Pigments, protein and phenol content of normal and treated tea leaves

Parameters Normal Treated**

Chlorophyll a* 205 Chlorophyll b* 74 Total carotenoids* 143 Total prote ins'!' 2.65 Total phenolS'!' 15 .5

* Pigment content expressed as mg % dry weight. '!' Expressed as % dry weight.

184 79 148

2.42 16.4

** B. lichelliforlllis suspension sprayed on tea leaves.

Table 2- Charac teri stics of tea liquor made from leaves sprayed with B. Iichellifonllis

Sampl e TF TR HPS Colour

Control 0.22 2.04 7.50 1.80 24 hr Treated 0.1 6 2.22 8. 10 1.90 Day 5 Control 0.17 2.66 6.90 1.75 Day 5 Treated 0.15 2.34 7.90 1.85 Day 10 Control 0.59 11 .34 12.85 1.83 Day 10 Treated 0.48 12.62 14.44 1.90

TF - Theatlavins; TR - Thearubi g ins; HPS - Hi ghl y pol ymeri sed substances

578 INDIAN J EXP BIOL, JUNE 2004

by about 30-35% confirming the results obtained by the spectrophotometric method. Theophylline decl ined by 16%, while theobromine increased by 60%. It was obv ious from these results that there was an interconversion (as reported earlier' s) between the

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Fig. 2- HPLC separalio n* of theobromine, Iheophylline and caffei ne iso la ted from normal tea leaves (- ) and leaves sprayed wi th cu ltures of Bacilllls lichelliforlllis (----) . " The methy lxanthines were separnted at different diluti ons and the in div idua l runs have been superimposed 10 show the separati on of all three compo unds in a s ing le figure. The X-axi s depicting Run time in minutes has been ke pi co mmon for a ll three compound s. The scale on Y I axis is for theophyll ine and theobromi ne whi le the sca le o n Y~ ax is is for caffei ne.

Table 3--Ca ffe ine contelll" o f leaves and tea made from leaves sprnyed with B. iichelliforl'lis

Sample Two leaves & bud Mother leaf Made tea

Conlrol 4. 14 2 .63 4.57 24 hr Treated 3.02 « 27 %) 2.05 « 22% ) 3.49 «240/0) Day 5 Control 3.97 2.95 4.62 Day 5 Trealed 3. 14 « 2 1%) 2 .5 1 « 15% ) 3.78 « 18%) Day 1 0 Conlrol 3.9 1 3.08 4.76 Day 10 Treated 3. 17 « 19% ) 2.7 1 « 12% ) 4. 14 « 13% )

"Caffeine con lenl ex pressed as % dry weighl o f sa mple . Va lues in parelllhes is denole '70 lowe ring in caffeine conlent over the contro l.

RAMARETH[NAM & RAJALAKSHM[ : CAFFEINE LOWER[NG BY B. LlCHENIFORMIS 579

the increase in theobromine was significant, it would appear to be derived from the conversion of both caffeine and theophylline, although reports on interconvers ion of theophy lline to theobromine are rare . But, in re lation to the caffeine content (on a mole to mo le basis) , the actua l theobromine content was very low, and 60% increase in its level was much lesser than the actual decrease in caffeine leve l.

Earlier studies on coffee, tea and cocoa have shown that both theophyll ine and theobromine are involved in formation as well as degradation of caffeine. In vivo and ill vitro studies have shown that theobromine is sy nthes ized from 7-methy lxanthine and transformed into caffeine. Caffe ine ( I ,3 ,7-trimethylxanthine) synthesis is considered analogous to that of uric acid , either from purine poo l7 via theophylline, or from nucleic acids l

'!.2o via theobromine. It has also been

observed that the former pathway operates in young leaves whi le the latte r path is predo minant in mature tea leaves 18. In ripened coffee fruits , biodegradation of caffe ine takes pl ace forming theophylline and theobromine21

, which get converted into xanthine, uric acid, allantoin and allanto ic ac id before getting lost to the atmosphere as ammonia and carbon diox ide. While theophylline is in volved o nly in the breakdown path of caffeine, theobro mine is associated with both the synthesis and degradation paths.

In other studies in volvi ng use of mi croorgani sms to degrade caffe ine, conversio n of caffe ine to theobromine by cultures of the bacteria Pseudolllollas aemgilloso and P. put ida have been recorded22 due to the presence of the enzymes xanthine dehydrogenase and uri case2

.1 in these organi sms. In these studi es, the microbes have been grown in culture flasks in the laboratory using caffeine as a so urce of nitrogen. Serraria lIIo rcescclIs isolated from so il in a coffee plantation has been shown to utili ze caffe ine, bu t not theophylline, as a source of carbon and nitrogen for growth2

.J. During biodegradation of caffe ine by microbes. theobromine is formed as an inte rmediate. It must be emphasized that these stud ies have been conduc ted us ing caffe ine as a substrate for growth of microbes under ill Film condit i o n ~. The present study was an effort to reduce the eaffei ne cOl1lent under i ll situ condition". It appeared that B. Iic!J clli{onllis induced a breakdo wn of caffe ine in tea leaves th rough a pathway invo lving theobromine, d uring the course of its penetration and prol i fe rati on in tea leaves. In addit ion, the qua lity of tea manufactured from such leaves was improved cl ue to increase in pheno lic

content of the leaves. It is suggested that spraying of this bacterium may be useful in yielding decaffeinated tea wi th good fl avour and aroma.

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580 INDIAN J EXP BIOL, JUNE 2004

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