Effect of tea fungal enzymes on the quality of black tea
G.S.Murugesan,J.Angayarkanni,K.Swaminathan*
Department of Biotechnology,Bharathiar University,Coimbatore-641046,Tamil Nadu,India Received 25October 2001;received in revid form 30January 2002;accepted 30January 2002
Abstract
The cellulolytic enzymes,cellulas,pectinas and xylanas,isolated from the tea fungus (a symbiont of two yeast’s Pichia sp.NRRL Y-4810and Zygosaccharomyces sp.NRRL Y-4882and the bacterium Acetobacter sp.NRRL B-2357)and lacca from Trametes versicolor ,were tried for the improvement of black tea quality.The effects of the enzymes on black tea quality heaflavin (TF),thearubigen (TR),high polymerid substances (HPS),total liquor colour (TLC),total soluble solids (TSS),caffeine (CAF)and dry matter content (DMC),were analyd.Purified cellula amended with Trametes versicolor lacca in the ratio of 3:2(v/v)was found to be most effective in enhancing tea quality.Teas procesd in the conventional manner and fer-mented with commercial Biopectina were maintained as controls.#2002Elvier Science Ltd.All rights rerved.
成绩查询入口Keywords:Tea fungus;Cellulytic enzymes;Theaflavin;Thearubigan;Caffeine
1.Introduction
Tea is the most widely ud stimulant throughout the world.It is generally procesd from the tender shoots as two leaves and a bud of the tea plant (Camellia sinensis ).Variations in the flavour and quality of tea are determined by the interplay of complex metabolic events that occur in the leaves during processing.Dur-ing processing,the enzyme,polyphenoloxida oxidis the polyphenols prent in the tea leaves,resulting in the formation of black tea components.In native leaves,polyphenoloxida is prent in the epidermal cells and the vascular bundles,whereas the Substrate catechin is prent inside the vacuoles of the palisade cells (Bhatia &Ullah,1965).In the conventional method,withered tea leaves are procesd by the CTC process (crush,tear and curl).The partial disruption of tea leaves during the CTC process reduces the amounts of peroxida and polyphenoloxida in the native leaves,thereby sup-pressing the formation of black tea components (Mar-imuthu,Manivel,&Kareem,1997;Senthilkumar,Swaminathan,Marimuthu,&Rajkumar,2000).More-over,the cell wall polysaccharides act as a barrier for the enzyme substrate interaction.To overcome this problem,in the prent study,the cellulolytic enzymes,cellula,pectina and xylana obtained from the tea fungus and the polyphenoloxida,lacca,from Trametes
versicolor were exogenously added during tea fermenta-tion to improve the tea quality.The quality pa
rameters studied were theaflavin,thearubigen,high-polymerid substances,total liquor colour,total soluble solids,caf-feine and dry matter contents.
andreea balan2.Materials and methods 2.1.Tea fungus
The tea fungus (a symbiont of two yeast’s,Pichia sp.NRRL Y-4810and Zygosaccharomyces sp.NRRL Y-4882and a bacterium Acetobacter sp.NRRL B-2357),was obtained from the tribal people of Kolli hills,Tamil Nadu.The fungus was grown in a tea med-ium (Hesltine,1965).2.2.Enzyme production
The tea fungus (1g mat)was inoculated and incu-bated in the minimal medium (Carter &Bull,1969)for 8days on an orbital shaker (120rpm).For cellula,pectina and xylana enzyme production,carbox-ymethylcellulo,pectin and xylan were ud as sub-strates respectively.For lacca production,Trametes versicolor was grown in guaiacol amended minimal medium for four days.After the incubation period,the fungal mat was filtered and the filtrate ud as crude enzyme.
0308-8146/02/$-e front matter #2002Elvier Science Ltd.All rights rerved.P I I :S 0308-8146(02)00157-
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*Corresponding author.
2.3.Enzyme purification and assay
2.3.1.Cellula
The culturefiltrate was mixed with three volumes of chilled ethanol and kept overnight at4 C.The pre-cipitate was collected,dried in a vacuum and dissolved in10ml of0.1M citrate buffer,pH 5.0.The con-centrated enzyme was pasd through a Sephadex G-100column(1.5Â45cm),equilibrated with0.1M citrate buffer,pH5.0and eluted with the same buffer. Fractions of5ml/h were collected and the protein con-tent(Lowry,Robrough,Farr,&Randall,1951)and cellula activity in each fraction were estimated.The cellula activity was determined as the amount of enzyme releasing1m mol of reducing sugar as gluco in 1min(Somogyi&Nelson,1952).The active fractions were collected and pooled.
2.3.2.Pectina
For pectina,the ethanol precipitate was dissolved in 0.1M acetate buffer,pH 5.0,and eluted through a Sephadex G-100column,using the same buffer with a flow rate of5ml/h.In each fraction,the
pectina activity was estimated(Sherwood,1966)and expresd as increa in OD per hour per mg protein.The active fractions were pooled.
2.3.3.Xylana
北京新东方官方网站In xylana purification,the ethanol precipitate was dissolved in0.01M sodium phosphate buffer,pH8.0 and fractionated by Sephadex G-100column chroma-tography,using the same buffer as eluant with aflow rate of5ml/h.The xylana activity was estimated as amount of reducing sugars relead from oat spelt xylan (Somogyi&Nelson,1952).The activity was expresd as amount of enzyme releasing1m mol of reducing sugar as xylo in1min.
2.4.Characteristics of enzymes
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The enzyme ptimum pH,temperature and substrate concentration for maximum enzyme activity(V max and K m),were determined.The molecular weights of the enzymes were estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE;Laemmli,1970),using a Phast Gel gra-dient10–15medium.Pharmacia high molecular weight calibration kit proteins were ud as markers.The methods propod in the Phast system TM paration techniquefile No.110,Pharmacia LKB Biotechnology, Uppsala,Sweden,were followed.
2.5.Tea processing
Tea clone,reprenting‘Assam’(UPASI-3)cultivar, was lected for the study.Tea shoots,consisting of an apical bud and two leaves,were harvested and withered for18h.The withered leaves were made into cut dhool by the CTC(crush,tear and curl)process and four cut-tings were performed.The crude and purified enzymes of cellula,pectina,xylana and biopectina,dilu-ted(1:25v/v)with0.1M acetate buffer of pH5.0,were sprayed individually on cut dhools;cut dhools sprayed with0.1M acetate buffer pH5.0were maintained as controls.The spray volume was25ml for750g of tea leaves for all the treatments.At this volume,the enzyme
Table1
Purification of cellulolytic enzymes from the culturefiltrates of tea fungus
liverpool
Sample Volume
(ml)Activity
(IU/ml)起英文名字
Protein
(mg/ml)
Total
activity
(IU)
Total
protein
(mg)
Specific
activity
(IU/mg)
Yield
(%)
Purification
fold
Cellula
Culturefiltrate2000.55 2.341104680.231001 Ethanol precipitation100.650.78 6.507.800.83 5.91 3.61 Column chromatography Sephadex G100
Fraction350.650.02 3.250.1032.50 2.95141 650.770.02 3.850.1038.50 3.50167 950.820.02 4.100.1041.00 3.73178 Pectina
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Culturefiltrate2000.65 2.14130.004280.301001 Ethanol precipitation100.850.968.509.600.88 6.54 2.93 Column chromatography Sephadex G100
Fraction850.960.03 4.800.1532.00 3.69107 Xylana
Culturefiltrate2000.66 2.10132.004200.311001 Ethanol precipitation100.750.867.508.600.87 5.68 2.81 Column chromatography Sephadex G-100
ious
Fraction950.840.03 4.200.1528 3.1890.3 412G.S.Murugesan et al./Food Chemistry79(2002)411–417
activities were0.80,0.96and0.83IU/ml respectively, for cellula,pectina and xylana.The dhool cut was allowed to ferment for45min at room temperature.The fermented dhool was dried at120 C for20min in an oven and then sifted and graded.The quality para-meters of the black tea,theaflavin,thearubigen,high polymerid substances,caffeine,dry matter contents and total liquor colour,were analyd by the solvent extraction method(Takeo&Osawa,1976);total soluble solids content was estimated by ISS(1973)method.The data on quality parameters were analyd statistically by DMRT(Duncan,1955)and the best treatment for tea quality improvement was identified.In the next t of experiments,the enzyme preparation yielding best quality tea was mixed in various proportions with the polyphenoloxida enzyme,lacca,obtained from the wood rotting fungi Trametes versicolor(Selvam, 2000).Effect of this enzyme mixture on tea quality were analyd.3.Results and discussion
3.1.Purification and properties of cellulolytic enzymes Sephadex G-100column chromatography of cellula enzymes revealed three active fractions.Thefirst frac-tion showed a purification fold of141with a specific activity of32.5IU/mg protein.In the cond fraction, the purification was167-fold a
nd specific activity was 38.5IU/mg protein and in the third fraction,the pur-ification fold and specific activity were178and41.0IU/mg protein,respectively.The enzyme yield(%)was in the range2.95–3.73.In pectina and xylana purifications, the enzymes showed only one fraction on Sephadex G-100chromatography.In pectina fractions,the pur-ification fold was107,specific activity was32.0IU/mg protein and the yield was3.69%.In xylana,the pur-ification fold was90.3,specific activity was28IU/mg protein and the yield was3.18%(Table1and Fig.
1).
The cellula enzymes of tea fungus had an optimum pH of7.0and temperature of50 C.The V max against carboxymethylcellulo was32IU/mg protein and K m was2.50mg/ml.The molecular weights of the three fractions were35,40and42kDa.The optimum pH for pectina activity was6.0and temperature was40 C. V max and K m values were calculated against apple pectin, which were31.0IU/mg protein and7.50mg/ml, respectively.The molecular weight was20kDa.For xylana,the optimum pH was6.0and temperature was 50 C;the V max and K m values against oat spelt xylan were30.0IU/mg protein and2.50mg/ml,respectively and the molecular weight was32kDa(Table2and Fig.2).
3.2.Tea processing
Digestion of cell wall components and relea of coloured phenolic compounds are two important requi-sites in tea manufacture.The enzymes peroxida(PO) and polyphenoloxida(PPO)play an important role in oxidative reactions,governing the distribution of pig-ments responsible for tea quality(Marimuthu,Kumar, Balasubramanian,Rajkumar,&Christie,2000).Poly-phenoloxida oxidis polyphenol bodies to orthoqui-nones.Subquently,orthoquinones,by a process known as dimerisati
on,conden to bis-flavonols and the in turn conden rapidly into theaflavins,which are golden yellow substances(Bhatia,1963;Bhatia& Ullah,1965;Sanderson,1965).Further oxidation results in transformation of theaflavins into thearubigens(red-dish brown pigments),which is an enzyme-independent process.An ideal fermentation result in a proper bal-ance of theaflavins and thearubigens(Sanderson,1965). Theaflavins(TF),which include theaflavin,theaflavin-3-gallate,theaflavin-30-gallate and theaflavin-3–30-digal-late are keys to the colour and taste and account for 2–6%of the dry weight in brewed black tea(Yang, Chung,Yang,Chhabra,&Lee,2000).Thearubigens (TR),such as TR-1,TR-2and TR-3formed during manufacture of black tea,contribute largely towards the total colour(Dix,Fairley,Millin,&Swaine,1981). Thearubigens start forming right from plucking and continue until drying of the tea particles.TR increa lin-early during fermentation and attains their maximum in over-fermented leaves.The role of black tea components in tea liquor was analyd as theaflavins for colour, taste,brightness and briskness and as thearubigens for colour,total depth and strength and caffeine for the stimulation of body and mind(Roberts,1962).
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The tea polyphenols are antioxidants and have the ability to quester metal ions and scavenge reactive oxygen species(Wiman,Balentine,&Frei,1997). Catechins,theaflavins and caffeine are rep
orted to inhi-bit cancer formation in animal models(Yang et al., 2000).The theaflavins in black tea are reported to inhi-bit lung and oesophageal carcinogenesis(Mor et al., 1997;Yang et al.,1997).Yang,Liao,Kim,Yurkow, and Yang(1998)reported that the black tea polyphenol, theaflavin-3–30-digallate,inhibits the growth of Ha-ras-transformed21BES cells.Hence,any increa in tea phenolic compounds will increa the therapeutic value of tea.
In the prent study,the cellulolytic enzymes pro-duced by the tea fungus were ud to increa the maceration of plant tissues so that high amounts of phenolic substrates,catechins and the oxidising enzymes are relead,resulting in incread product formation. Moreover,to enhance the oxidation of phenolic com-pounds,the phenoloxida,lacca,obtained from the wood rot fungi Trametes versicolor(Selvam,2000)was exogenously supplied during tea fermentation.The results revealed that exogenous application of cellula, pectina and xylana incread the black tea compo-nents,theaflavins(TF),thearubigens(TR),caffeine (CAF),high polymerid substances(HPS),total liquor colour(TLC),total soluble solids(TSS)and dry matter
北京培训网Table2
Properties of tea fungal enzymes
Properties Cellula Pectina Xylana Optimum pH7.0 6.0 6.0 Optimum temperature, C504050
V max,IU/mg protein32.0031.0030.00 K m,mg/ml 2.507.50 2.50
Molecular weight,KDa (SDS-PAGE)35,40&4220
45
Fig.2.SDS-PAGE for cellula,pectina and xylana.
414G.S.Murugesan et al./Food Chemistry79(2002)411–417
