Caffeine in Tea Plants [Camellia Sinensis (L) O. Kuntze] : in Situ Lowering by Bacillus Li Chen~Fonnis (Weigmann) Chester

Indi an Journal of 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 li chen~fonnis (Weigmann) Chester

S Ramarethinam & N Rajalakshmi * Research and Development Centre, United Nil giri Tea Estates Co Ltd ., 3. Savithri Shanmugham Road, Race Course, Coimbatore 64 1 0 18, Ind ia

Received 20 March 2003; revised 3 1 Ma rch 2004

Tea plants (Calll ellia sill ell sis) contain 5-6% caffeine th at is responsible fo r th e stimulating effect of the beverage. As th e to lerance to caffein e varies among indi vid uals, low caffein e tea would be an ideal altern ati ve. While assessing the potenti al of a few selected bacteri a-Bacillus lichell i{o nllis. B. sub/ilis and B. jim illS. to multiply o n nutrient medium supplemented with g lucose (5 % ) and tea leaf extract (2 %), it was observed th at only B. lichelli{o rlllis could proliferate on thi s medium. Hence. B. lichelli{o nllis was used for further studies. Tea pl ants were sprayed with a suspe nsio n of B. lichelli{orlllis at a dilution o f 5 x 108 CFU/ml cont aining 0. 1% Tween 80 as surfactant. III situ lowering of caffe ine from tea leaves was evide nt without affecting the quality of th e oth er tea compo nents. Further, there was no change in the morpho logical and physio logical characteri sti cs as well. It is suggested th at spraying of B. lichell i/imllis may be useful in yielding decaffein ated tea with good fl avour and aroma.

Keywords: Bacillus lichellij'o rlll is. Call1 ellia sill ell sis, Caffe in e- free tea IPC Code: Int C I7 AO I N63/00; C 12N9/56

Tea used as a drink is made from the api cal two the medi cinal value of tea. The terminal bud of tea 4 leaves and a bud of the plant, Camellia sil/ el/ sis (L) O. pl ants contains the maximum caffein e • Withered Kuntze. The leaves contain in addition to the normal leaves have more caffeine ( 18-2 1% )5 in compari son to constituents, hi gh level of polyphenols (30-40%) and fresh leaves probabl y due to degradati on of purine caffeine (5-6%). Caffein e (I ,3,7-trimethylxanthine) nucleotides6 during withering. al so present in other beverages (coffee and cocoa), T olerance to caffeine di ffers among in dividuals. stimulates the central nervous system, increases and therefore, low-caffeine tea would be an advantage mental alertness, betters memory and mood, improves for sensiti ve people. Effo rts to produce decaffeinated reasoning powel'l and is o f valu e in the treatment of tea and coffee have intensified in th e past two gout, hypertensive headaches, myocardial in fa rcti on, decades. Decaffeination, in volving th e selective etc. The common effect of caffeine when taken in re moval of caffeine using organi c solvents or super­ hi gh doses is increased metabo li c rate, irritability, criti cal gases, also removes most of th e components sleep di sturbance2 and gastrointestinal aches, in responsible for fl avour and aroma leavin g a bland tea addition to inducti on of hypercholestrolemi a, without any taste and less of its stimulating effects on osteoporosis, ul cers and cancers. Though the intake of the body. Geneti c engineering may hold th e key to caffeine from tea is not deleteri ous to health per se, as making decaffeinated tea w ithout loss of aroma and tea (-20 mg/l OO ml ) contains less caffeine than coffee fl avour. Identification of the gene encodin g the (- 120 mgll 00 ml), chocolates (-35 mg/28 g bar) or enzyme caffeine synthase7 opens up th e possibili ty of cola drinks (-30 mg/l OO ml)3, lu xury consumption is producin g geneti call y engin eered tea plants in wh ich a possibility sin ce caffeine is widely distributed in a th e expression of the gene has been suppressed. Such vari ety of foods. Apart from caffeine, tea also an atte mpt has been successful in coffee pl ants that contains isomeri c dimeth ylxanthines - theophylline have been transformed w ith an anti sense RN A fo r at and theobrom i ne that are analogues of caffe i ne and least one enzyme in the pathway of caffei ne intermedi ates in its synthesis. These also contribute to biosynthesiss. However, gene manipulation may not be a reali sti c alternati ve for tea. which is a perennial ' Corresponde nt author- herb whose producti ve li fe span is extended to 80- 100 Ph one-0422-2220 125: 2220566; Email : tearesdev@vsnl. in years due to pruning effect obtained by plucking the 576 INDIAN J EXP BIOL, JUNE 2004 leaves once a week. Hence, short-term measures to plucked after 24 hr and also on day 5 and 10. Each lower the caffein e content of leaves when they are experiment was repeated thrice. sti ll on the plant would be an easier approach to Tea processing-Tea was manufactured by the obtain caffeine-free tea that is hi gh in flavour and orthodox method that in volves the stages of aroma. In thi s paper, th e effects of spraying the withering, rolling, fermentation and drying, before the bacterium, Bacillus lichen (jO rl'l1 is, on tea plants are green leaves are converted to black tea of commerce. presented. A 2% brew was prepared from processed tea and its colour and strength as well as theafl av in and Materials and Methods thearubigin content were measured 10. Bacterial cultures-Cultures of Bacillus Estimation of caffeill e-Caffeine from fresh tea lichellijorlnis (Weigmann) Chester, B. subtilis and B. leaves or made tea was analyzed finnus, isolated from tea pl antation were identified by spectrophotometrically'l or after separati on by I 2 morphological, physiological and biochemical tests9 HPLC and quantified in rel ati on to standard in coll aboration with the Centre for Advanced Study caffeine. Theophylline and theobromine were also in Botany, University o f Madras and the extracted along w ith caffeine and separated by I 3 Microbiology Department, PSG Institute of Medical HPLC on a Tiger LC System (Waters Corporation Sciences and Research, Coimbatore. They were USA) and quantified using authentic samples, each of maintai ned on nutrient agar medium (peptone, 5 gil ; which was separately passed through HPLC column sodium chl oride, 5 gil ; beef extract, I gil; yeast at different conce ntrati ons. Based on the area of each extract, 2 gil ; agar, 15 gil; pH 6.8) slants at room peak, a standard graph was drawn for each temperature (300±2°C). For experimental purposes, methy lx anthine in relation to its concentrati on. After log phase cultures were transferred to liquid medium separation by HPLC, the peak area for each (nutrient agar medium without agar) and incubated for methylxanthine in tea samples was measured and its 24 hr before spraying in the fields. concentration was calculated from the respecti ve standard graph. Utilisatioll of tea liquor for bacterial growth-The Pigment allalysis-Within 4 hr after plucking fresh capacity of the selected bacteria to utilise tea tea leaves, pigments from these leaves ( lO g) were components for growth was monitored by inoculating extracted in 100 ml aqueous acetone (80% v/v) and the bacteria into nutrient medium supple mented with their absorbance of visible li ght was scanned using a glucose (5%) and green leaf extract (2%) and C hemito UV -Vis spectrophotometer. The amount of incubati ng at 30° ± 2°C in an orbital shaker. Changes chl orophyll s and carotenoids were calculated in the optical density (at 660 nm) of the culture broth according to Mackinney'4. were measured spectrophotometri call y at every 24 hr Estimation of proteills and phellols-The content for a period of 5 days, and used as an index of of total proteins and phenols were estimated bacterial growth . spectrophotometri cally in aqueous extracts of the Tea plants-Tea, Ca ll/ ellia sin ensis (L) O. Kuntze, leaves, following the methods of Lowry et al. ' 5 and growing in plantations of this organization at Chamraj Schanderi 16 respecti vel y. Estate (N il giri Hills) was used for this study. Experimental plots were demarcated in triplicate, each Results and Discussion comprisi ng thirty productive bushes and covering an To assess the suitability of the selected bacteri a for area of 1.25 cents. Plants were twenty years old and field spray, their potential to multi ply utilising tea were hard -pruned two years before the study period. constituents was monitored by growing the cultures They were sprayed with the culture at a dilution of 5 x on medium contai ning glucose (5%) and tea leaf 108 CFU/ml culture broth containing Tween 80 extract. It was observed that only B. lichen(jorillis (0. 1% ) as surfactant. The same solution without could pro li ferate while th e other bacteria were unable spores served as control and was sprayed on another to grow on thi s medium. Hence, they were not Llsed set of thirty bushes in the adjacent plot. Plants were for spray ing on tea plants in th e plantation and all drenched totally using 10 I of spray sol ution per pl ot further experiments were carried out onl y w ith B. through a knapsack sprayer fitted with NMD nozzle Iicll .en (jorll/is. and sprayed at the rate of 1 !lmin. Spraying was The bacterium, B. licli enijormis, was sprayed on performed in the morning (6 - 7 a.m.) . Leaves were 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 (Table 3). The leaves showed a significant decrease in again after 24 hr, 5 and 10 days of treatment. Effects caffeine content in 24 hr. However, by day 5, th e of spraying the bacteria on tea plants were monitored decrease in caffeine content was lesser, and by lO th both morphologically and physiologically. Tea plants day, the decrease was even lesser. Hence, it appears in the plantation did not show any change in their that the microbe should be sprayed on day 6 of a 7- appearance and the overall morphology of the plant day plucking cycle or on day 9 of a 10-day plucking was the same as that of untreated plants. Hence, other cycle, in order to obtain the maximum lowering in parameters such as, pigment content, total phenols caffeine level of tea leaves. It appeared that the and total protein were monitored. In keeping with the lowering in caffeine induced by the bacterium similarity in physical a pp~arance of plants, there was counteracted the increase of caffeine during no variation in the pigment content, both in terms of withering, thereby reducing the overall caffeine level the levels of individual pigments and their relative of the made tea. proportion (Fig. 1, Table 1). It was also observed that Consistency of results obtained prompted further the protein content (Table 1) did not show any work on the fate of caffeine within the cells. significant change indicatin g that infection with B. Methylxanthines were separated by HPLC (Fi g. 2) lichelliforlllis did not produce much change in the into three components, which were identified using biochemistry of treated plants. However, tea plants authentic standards as theophylline, theobromine and showed enhancement in total phenolic content, since caffeine. The level of caffeine was higher th an the bacterium was recognized as a contagion. This theophylline or theobromine, and hence, separation would mean that the liquor quality of tea prepared was done at different dilutions in order to monitor from such leaves would be better than normal tea. changes in the levels of minor components When these tea leaves were used to prepare black tea, (theophylline and theobromine). This exercise proved there was no difference in its physical appearance and to be rewarding as marked variations in the levels of liquor characteristics (Table 2). Spraying of Bacillus theophylline and theobromine were noticed in sp., termed as Tea Beneficial Microbe has been response to spraying the bacterium, B. lichellifo rl11 is. l shown to increase ? the photosynthetic rate by 30% The levels of caffeine and theophylline got lowered and net yield by 29-65%, in addition to improving while theobromine content increased in response to bud density, growth rate, leaf area, thickness and infection. It was found that caffeine content decreased shoot weight. Caffeine content was monitored in terminal leaves Table I-Pigments, protein and phenol content of normal and (which are normally used for tea processing), mother treated tea leaves leaves (lower mature leaves) and in made tea Parameters Normal Treated** Chlorophyll a* 205 184 Chlorophyll b* 74 79 Total carotenoids* 143 148 Total protein s'!' 2.65 2.42 Total phenolS'!' 15 .5 16.4

* Pigment content expressed as mg % dry weight. '!' Expressed as % dry weight. ** B. lichelliforlllis suspension sprayed on tea leaves.

Table 2- Characteri sti cs 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 1.85 400 500 600 700 Day 10 Control 0.59 11 .34 12.85 1. 83 Wavelength (nm) Day 10 Treated 0.48 12 .62 14.44 1.90 Fig. I- Absorption spectra of acetone extract of normal tea leaves TF - Theatlavins; TR - Thearubi gin s; HPS - Hi ghl y pol ymeri sed (-) and leaves sprayed with culture of Bacililis Iichelll!anllis (----). substances 578 INDIAN J EXP BIOL, JUNE 2004

by about 30-35% confirming the results obtained by three methylxanthines-caffeine, theophylline and the spectrophotometric method. Theophylline theobromine, in response to spraying B. lichen ijo rill is. declined by 16%, while theobromine increased by It was however not clear from these results, as to 60%. It was obvious from these results that there was whether these conversions took place during the an interconversion (as reported earlier's) between the formation of caffeine or during its breakdown. Since

0 0 (-) Normal ~ ~ co ... (---1 Treated ~, CD N ;:I; ~ " '" ~ , II) ("") " 0 0 11 @ 0 ~ @l m ill Z N z 11 ill :: ~ , ; LL LL 0 1 0 0 a:: 1 « ~ 0 - U m N... 1 ... 0 N 0 0 .... ill , N J: ... I I l- , ill I ::> --, 0 Z ~ 11 CD :J I...... J N I E ~ ~ ~ c E Cl. c 0 0 0 ill 0 r-- 0 ~ ~ N 0 0 r-- N ~ ... N N .... _- co (j) co u (j) 0 g c u 0 - c o:i (\) (\) -;r. a - -2 CD 0 -e rJ) 0 .D rJ) 0 g _

0 1 : 0 N ~

0 o 0 ~ - CD N N I ------~. o ~ o m __ I -'----'--'---1 o Time (min)

Fig. 2- HPLC separalio n* of theobromine, Ih eophylline and caffeine iso lated from normal tea leaves (- ) and leaves sprayed wi th cultures of Bacilllls lichelliforlllis (----). " The meth ylxanthines were separnted at different dilutions and the in dividua l runs have been superimposed 10 show the separati on of all three compo unds in a sing le figure. The X-axis depicting Run time in minutes has been ke pi co mmon for all three compound s. The scale on Y I axis is for th eophyll ine and th eobromine whi le the scale o n Y ~ axis is for caffei ne.

Table 3--Caffeine contelll" of leaves and tea made from leaves sprnyed with B. iichelliforl'lis

Sample Two leaves & bud Moth er 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 Treal ed 3. 14 « 2 1%) 2.5 1 « 15% ) 3.78 « 18%) Day 10 Conlrol 3.9 1 3.08 4.76 Day 10 Treated 3. 17 « 19% ) 2.7 1 « 12% ) 4. 14 « 13% )

"Caffeine conlenl ex pressed as % dry weighl o f sample. Va lu es in parelllhesis denole '70 lowering in caffein e conl ent over the control. RAMARETH[NAM & RAJALAKSHM[ : CAFFEINE LOWER[NG BY B. LlCHENIFORMIS 579 the increase in theobromine was significant, it would content of the leaves. It is suggested that spraying of appear to be derived from the conversion of both this bacterium may be useful in yielding decaffeinated caffeine and theophylline, although reports on tea with good fl avour and aroma. interconversion of theophylline to theobromine are rare. But, in relation to th e caffeine content (on a mole References to mole basis), the actual theobromine content was [ Li eberman H R. Wurtman R J, Emde G G & Coviell a [ L. very low, and 60% increase in its level was much The effects of caffeine and aspirin on human mood and perform ance, J Clill Psychopharlllocol, 7 ( 1987) 3 15. lesser than th e actual decrease in caffeine level. 2 Curatolo P W & Robertson D. The health consequences of Earlier studies on coffee, tea and cocoa have shown caffein e, A IIII 1111 Med. 98 (1983) 641. that both theophylline and theobromine are involved in 3 Barone J J & Robert s H R, Caffeine consumption. Food Ch em Toxicol, 34 (1 996) I 19. formation as well as degradation of caffeine. In vivo 4 Cloughley J S, Factors influencing the caffeine content s of and ill vitro studies have shown that theobromine is black tea. The effect of fi eld variables, Food Chelll . 9 ( 1982) sy nthesized from 7-methylxanthine and transformed 269. into caffeine. Caffeine ( I ,3 ,7-trimethylxanthine) 5 Clough ley J B. Factors influenci ng the caffein e contenl of synthesis is considered analogous to th at of uric acid, bl ack tea: Part 2 - The effect of production va ri abl es. Food Chelll , 10 ( 1983) 25. either from purine pool7 via theophylline, or from l 6 Ogutu ga DBA & North cote D H, Bi osynthesis of caffeine nucleic acids '!.2o via th eobromine. It has also been in tea callus ti ssues, Biochelll J. 11 7 ( 1970) 715. observed that the former pathway operates in young 7 Kato M, Mizuno K. Crozier A. Fujimura T & Ashihara H leaves whi le the latter path is predo minant in mature 2000. Caffeine sy nth ase gene from tea leaves. Na fllre . .+06 (2000) 956. tea leaves 18. In ripened coffee fruits, bi odegradation of 8 Sti les J I. Moisyad i I & Neupane K R, Purified protein s. caffeine takes pl ace forming theophylline and 21 recombinant DNA sequences and processes for producing theobromine , whi ch get converted into xanthine, uric caffeine free beverages, US Palelll 1/ 6075184. (2000) acid , all antoin and all antoic acid before getting lost to Uni versit y of Hawaii . the atmosphere as ammonia and carbon di oxide. While 9 Sneath P H A. Endospore forming Gram positi ve rod s and theophylline is in volved o nl y in the breakdown path of cocci. [n Bergev', I/wlI1.wl of SVSl elllalic baCleriologv. vo l. 2. ed ited by P H A Sneat h, N S Mair. M E Sharp & J G Holt caffein e, theobro mine is associated with both the (Williams & Wil li ams. Baltimore) 1986. 11 04. synthesis and degradation paths. 10 Robcrts E A H & Smith R F. Spectrophotometri c determinati on of th eaflavins and thearubi gin s in black tea In other studies in volvi ng use of mi croorgani sms to liquors in assessment s of quality in teas. Th e All al.".I·I. 86 degrade caffein e, conversio n of caffeine to ( 196 1) 94. th eobro mine by cultures of th e bacteria Pseudolllollas II ewton J M. Spectrophotometri c determin at ion of ca ffcine aemgilloso and P. put ida have been recorded22 due to in coffee products: Coll aborati vc stu dy. J Assoc OfT All al the presence of the enzymes xanthine dehydrogenase Ch elll , 62 ( 1979) 705. 2 12 Ashoor S H. Seperich G J, Mont e W C & Welt y J. HPLC and uri case .1 in these organi sms. In these studies, the determin ati on of caffeine in decaffein ated coiTec. te;] and microbes have been grown in culture flasks in the beverage prod ucts. J AI.loc Ofr All al Chelll . 66 ( 1983) 606. laboratory using caffein e as a so urce of nitrogen. 13 Franconi L C. Hawk G L. Sandmann S J & Han ey W 13 . Serraria lIIorcescclIs isolated from soil in a coffee Determin ati on of th eoph yll ine in plasma ultrafilt rate by plantation has been shown to utili ze caffeine, but not reversed phase hi gh-pressure liquid chromatograp hy. AI/{t! Ch elll , 48 ( 1976) 372. theophylline, as a source of carbon and nitrogen for 2 1.+ Mackinncy G. Absorpt ion of li ght by chl oroph yll so lut ions. J growth .J. During bi odegradation of caffein e by Bioi Ch elll. 140 ( 1941) 3 15. microbes. theobromine is formed as an intermediate. It t :) Lowry 0 H. Rosebrougl N J. f arr A L & Randall R J. must be emphasized th at th ese stu dies have been Protein measurement wit h the Foli n phcnol reagent. J Bioi conducted using caffeine as a substrate for growth of Cli elll , 193 ( 1951) 265. 16 Schanderi S H. in Me lhods ill food (//w/l·.I·is (Academic Press. microbes under ill Film co ndit i o n ~. The present study New Yo rk) 1970.70';). was an effort to reduce the eaffei ne cO l1lent under ill 17 Han gu Z. l3aoy u H. Zhenghe Z. Min glong W. Ha nzhi T. situ condition". It appeared that B. Iic!J clli{onllis Jiaren L. Shu xia H. Xuhu i G. Changj u X. Wan li M. Anzhi H induced a breakdo wn of caffein e in tea leaves th rough & Guobin F, Effect or tea benefic ial mi crobc on the a pathway involving theobromine, d uring the course of development an d th e yield of tea plaill s. J Tea Sci. 15 ( I lJ95) 15. its penetration and prol i fe rati on in tea leaves. In 18 Ashih ara I I, Gillies F M & Crozier A. Metaboli sm of addition, the quality of tea manufactured from such caffei ne ane! related purine alkaloid s in leaves of lea leaves was improved cl ue to in crease in pheno li c (C{// I/ eiliu sill ells is L ). PlolIl Cell Phvsiol. 38 ( 199 7) 4 13. 580 INDIAN J EXP BIOL, JUNE 2004

19 Anderson L & Gibbs M, The bi osynthesis of caffein e in Pseudomonas pulido strain , Hoppe Seyler's Z Physiol Cheln . coffee plants. 1 Bioi Chem. 237 ( 1962) 194 1. 358 (1977) 807. 20 Suzuk i T & Takahashi E, Metaboli sm of xanthine and 23 Kurtzmann R H & Schwimmer S, Caffeine removal from hypoxanthinc in the tea plant (Thea sinensis L), Biochem 1, growth medi a by mi croorga ni sms, Expen melllia. 127 ( 1971) 146 ( 1975) 79. 481. 2 1 Suzuki T & Waller G R, l3iodcgradation of caffein e: 24 Mazzafera P, Ol sson a & Sand berg G, Degradati on of Form ati on of theophylline and theobromin e in mature Coffea caffein e and related methylxanthines by Serralia lII arcescens arabica fruit s, 1 Sci Food Agric, 35 (1984) 66. iso lated fro m so il under coffee culti vati on. Microb Ecol. 31 22 Blec her R & Lingens F, The metaboli sm of caffeine by ( 1996) 199.