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Antioxidant Properties of Tea Extracts as Affected by Processing
Article No. fs980491
Lebensm.-Wiss. u.-Technol., 31, 694]698 Ž1998 .
Antioxidant Properties of Tea Extracts as Affected by Processing L. Manzocco*, M. Anese and M. C. Nicoli
L. Manzocco, M. C. Nicoli: Dipartimento di Scienze degli Alimenti, Universita ` degli Studi di Udine, via Marangoni 97, 33100 Udine ŽItaly. M. Anese: Istituto di Produzioni e Preparazioni Alimentari, Universita ` degli Studi di Bari, via Napoli 25, 71100 Foggia ŽItaly. (Recei¨ ed May 25, 1998; accepted September 8, 1998)
The changes in the antioxidant properties of green and black tea extracts as a consequence of processing and storage conditions were e¨ aluated through measurements of chain-breaking acti¨ ity, oxygen sca¨ enging capacity and redox potential. Pasteurisation and storage as well as forced oxygenation in both tea extracts caused an increase in the optical density and a decrease in the reducing power of the be¨ erages; both were accompanied by unexpected increases in the chain-breaking acti¨ ity of the be¨ erages. Simple model systems containing catechin at a concentration similar to that found in the tea extracts were also considered to simulate the effects of enzymatic browning in tea. Possible explanations for the chain-breaking acti¨ ity gain obser¨ ed in the tea extracts as a consequence of processing were proposed.
q 1998 Academic Press Keywords: green and black tea; antioxidant activity; polyphenols; DPPH? ; processing; storage
Introduction Recent experimental studies have recognised that tea Ž Camellia sinensis. exhibits a significant health protecting activity due to its high flavanoid content. Flavanoids are the most abundant compounds in fresh tea leaves and extracts Ž1]3.. Flavanoids have long been recognised to have strong antioxidant activity, they are therefore considered to be responsible for the anticarcinogenic and antimutagenic properties of tea, as well as its protective action against cardiovascular diseases Ž4]6.. The antimutagenic effects of tea extracts are thought to be due to the action of polyphenols in inhibiting carcinogen-metabolising enzymes; these compounds would also be able to scavenge reactive intermediates of the carcinogenic process, thus preventing their interaction with DNA Ž7]9.. It is well known that tea manufacturing processes can greatly affect the oxidation of tea polyphenols. While green tea is produced from fresh leaves, preventing the oxidation of flavanoids, black tea manufacture results in a high degree of enzymatic aerobic oxidation of flavanoids Ž1, 3, 10.. Despite the different flavanol content of green and black tea, both possess considerable antioxidant properties. However, recent findings demonstrated that green tea extracts exhibit stronger * To whom correspondence should be addressed.
antioxidant activity than those of black tea Ž8, 11.. While many authors have tried to relate the antioxidant properties of various tea extracts to the degree of enzymatic oxidation of polyphenols in tea leaves, there is no information available concerning the outcome of these species during tea beverage production and storage. It is well known that many food antioxidants can be significantly lost as a consequence of sterilisation, pasteurisation, dehydration, as well as during prolonged storage Ž12.. Processing and storage are not always responsible for a depletion in the antioxidant properties of foods. In some cases, these factors can induce the formation of compounds with novel antioxidant properties, which can maintain or even enhance the overall antioxidant potential of foods Ž13]15.. Recent studies carried out on various red wines containing polyphenols showed that prolonged air exposure caused a progressive increase in the chain-breaking activity Ž16.. These changes were attributed to the partial oxidation of the wine polyphenols to form macromolecular compounds which still maintain a remarkable radical scavenging activity. As previously described, tea flavanoids are very reactive species which can easily undergo enzymatic and chemical reactions, which may be responsible for changes in the antioxidant properties of the product. Since it is likely that chemical modifications of the
0023-6438r98r070694 q 05 $30.00r0 q 1998 Academic Press
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lwtrvol. 31 Ž1998 . No. 7 & 8
flavanoids may take place during the processing of tea beverages and storage, this study was designed to evaluate the changes in the antioxidant properties of green and black tea extracts as a consequence of pasteurisation and storage. Simple model systems containing catechin which is the most abundant flavanol in tea leaves were also considered. The antioxidant properties were evaluated through the measurement of the chainbreaking activity, the oxygen scavenging capacity and the redox potential.
Materials and Methods Sample preparation Green and black dried tea leaves Ž Camellia sinensis. were purchased from a local market. Tea extracts were obtained by solid-liquid extraction using deionized water at 100 8C Žtea leaf-water ratio was 1:100 wrw.. The extraction time was 5 min. The beverages were then rapidly filtered through Whatman No.4 filter paper. In order to minimise the contact of the beverages with atmospheric oxygen, extraction and filtration were carried out under nitrogen flow Ž5 mLrmin.. The samples were then immediately cooled under cold running water. Volumes of 50 mL of green and black tea extracts were bottled in screw capped flasks in the presence of air and subjected to pasteurisation at 105 8C for 20 min in a laboratory autoclave ŽFVS 13, Fedegari, Italy.. The beverages were then stored at 25 8C for up to 30 d. Additional samples of freshly prepared green and black
tea extracts, not subjected to pasteurisation, were placed in a thin layer in a Petri dish and maintained in contact with atmospheric oxygen whilst stirring at room temperature for up to 24 h. In order to simulate the enzymatic oxidation of polyphenols, which takes place during black tea production, a volume of 50 mL of 2.4 = 10y3 molrL Žq. catechin ŽC-1251, Lot 100H0586, Sigma, St. Luis, Mo, U.S.A.. solution was added to 100 units of mushroom tyrosinase ŽT-7755, Lot 48F9610, Sigma, St. Luis, Mo, USA. and maintained in ambient air whilst stirring for at least 2 h. The measurement of the antioxidant activity of the enzymatically browned solution was carried out after the enzymatic reaction reached a steady state which was detected through optical density measurements at 390 nm. Analytical determinations Total solid content. Total solid content determinations were carried out according to AOAC methods Ž17.. The measurements were made in triplicate. The coefficient of variation, expressed as the percentage ratio between the standard deviation and the mean value, did not exceed 5%. Total polyphenol content. The analysis was carried according to the Folin-Ciocalteu methodology. The results were expressed as gallic acid equivalents. Measurements were made in duplicate and the difference in results between two determinations carried out on the same sample did not exceed 2%.
Fig. 1 Percentage of residual DPPH? as a function of time for increasing concentration of a black tea extract
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Optical density. Measurement was carried out at 390 nm using a Uvikon 860 spectrophotometer ŽKontron Instruments, Milano, Italy.. This wavelength is in the spectral region of maximum absorption. The tea extracts were diluted Ž1:10 v:v. with distilled water to ensure that absorbance values were on scale. Measurements were carried out in triplicate. Coefficients of variation were less than 5%. Chain-breaking acti¨ ity. The chain-breaking activity was measured according to the methodology described by Brand-Williams et al. Ž18.. The bleaching rate of a stable free radical, 2,2-diphenyl-1-picrylhydrazyl ŽDPPH? . at a characteristic wavelength in the presence of the sample was monitored. In its radical form, DPPH? absorbs at 515 nm, but upon reduction by an antioxidant or a radical species its absorption disappears. A volume of 3.0 mL of 6.1 = 10y5 M DPPH? methanol solution was used. The reaction was started by the addition of 10 m L of samples. The bleaching of DPPH? was followed at 515 nm ŽUvikon 860, Kontron Instruments, Milano, Italy. at 25 8C for at least 60 min. In all cases the DPPH? bleaching rate was proportional to the sample concentration added to the medium. In view of the difficulty of achieving a plausible kinetic model to determine the order of the different reactions involved, the radical scavenging activity is generally expressed in terms of an efficient concentration ŽEC 50 ., that is, the amount of antioxidant necessary to decrease the initial DPPH? concentration by 50% Ž18]19.. The EC 50 value is obtained by plotting the percentage of residual DPPH? at the steady state as a function of the antioxidant concentration. This method is difficult to apply when the chain-breaking activity has to be evaluated in complex mixtures whose steady state is often uncertain, as in the case of foods ŽFigure 1.. In such cases, that is, in the presence of phenolic compounds, the overall DPPH? bleaching rate follows complex multistep, generally parallel or consecutive, reactions Ž18, 20]21.. Thus, different kinetic models, whose determination coefficients Ž r 2 . and P values are shown in Table 1, were considered to obtain a suitable indicator of the chain-breaking activity. Under the experimental conditions used, all the DPPH? bleaching curves obtained are described by fourth order reaction kinetics. The following equation was chosen in order to obtain the reaction rate, k: 1 3
A
y
1 A30
Table 1 Coefficients of determination Ž r 2 . and P values obtained from increasing reaction order regression of the DPPH? bleaching curves Reaction order Zero First Second Third Fourth Fifth
r2
P
0.82 0.90 0.96 0.98 0.99 0.96
10y5 10y5 10y5 10y5 10y8 10y6
extracts was measured according to the methodology described by Anese et al. Ž22.. A YSI mod. 53 oxygen monitor ŽYellow Springs Instruments Co. Inc., Yellow Springs, Ohio, U.S.A.. equipped with an oxygen electrode was used. The airtight reaction vessel was filled with 3.0 mL of tea beverage conditioned at 25 8C. The oxygen consumption was recorded using a Bromma recorder ŽLKB Producter, Bromma, Sweden.. Distilled water was used as a control. The rate of oxygen uptake was calculated from the initial linear portion of the curves obtained. A good linearity was found for at least 10 min. The oxygen consumption activity was expressed as m moles oxygen consumed miny1 rg d.m., considering that the oxygen concentration in air-saturated water at 25 8C is 237 m molrL Ž23.. Measurements were made, at least in triplicate, and the coefficients of variation of the determinations performed on the same sample were less than 10%. Redox potential. Measurements were made with a platinum indicating electrode and a AgrAgCl, Cly sat reference electrode connected with a voltmeter ŽCrison, mod. 2002, Alella, Spain.. Calibration was performed against a redox standard solution ŽE s 468 mV at 25 8C.. Electrodes were inserted into a 50 mL 3-neck flask containing a volume of 20 mL of sample. Prior to analysis, oxygen was removed from the system by continuous nitrogen flushing for a period of 10 min. Millivolt values were recorded for at least 5 min, until a stable potential was reached. A stable potential was arbitrarily defined as a change of less than 1 mV in a 5 min period. The analyses were carried out in duplicate and the difference in results between two determinations performed on the same sample did not exceed 3%.
Results and Discussion
s y3k t
where A 0 is the initial optical density and A is the optical density at increasing time, t. The chain-breaking activity was expressed as krmg d.m ŽyO.D.y 3 r minrmg d.m. ., assuming that all the dry matter of the tea extract possesses antioxidant properties. The data are expressed as the mean of at least three repetitions and the coefficients of variation were less than 10%. Oxygen uptake. The oxygen consumption of the tea
In Table 2 the phenol content and the chain-breaking activity, expressed both on total dry matter and on total polyphenol content, of the green and black tea extracts used are reported. The analyses were performed immediately after extraction. Green tea extracts showed higher phenol content and chain-breaking activity than those obtained from black tea leaves. These results are in agreement with observations previously carried out by Yen and Chen Ž8. who analysed several tea extracts having different ‘fermentation’ degrees. The authors
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Table 2 Values of total phenol content and chain-breaking activity of green and black tea extracts GAE a Green tea Black tea a
Chain-breaking activity ŽyO.D.y3rminrmg d.m. . ŽyO.D.y3rminrmg polyphenol .
Žmg Ly1 .
Sample
953.84 801.16
5.60 1.95
11.15 5.36
Total phenols are expressed as gallic acid equivalents ŽGAE.
Table 3 Values of optical density at 390 nm and chain-breaking activity of a 2.4 = 10y3 molrL catechin solution before and after the addition of 100 units of mushroom tyrosinase Sample
Chain-breaking activity O. D. 390 nm ŽyO.D.y3rminrmg.
Catechin 0.083 Catechin q tyrosinase 0.640
3.18 0.98
attributed the loss in antioxidant properties to the enzymatic oxidation of polyphenols, which is the basis of black tea manufacture. The enzymatic oxidation of catechins in tea leaves leads to the formation of a large number of compounds, generally known as teaflavins and thearubigins. Very little is known about the chemical structure of these compounds and their antioxidant properties Ž1.. To confirm the role of enzymatic browning in reducing the original antioxidant activity of tea leaves, an aqueous solution of catechin at a concentration similar to that found in green tea extract was prepared. The catechin was rapidly oxidised by the addition of appropriate units of tyrosinase. The effect of the enzymatic oxidation of catechin on the optical density and on the chain-breaking activity is shown in Table 3. The results acquired clearly demonstrate that the enzymatic browning caused a marked decrease in the radical scavenging properties of catechin. To evaluate the influence of some processing conditions which are commonly used in the industrial production of tea beverages, samples of green and black tea extracts were bottled in the presence of air, pasteurised at 105 8C for 20 min and then stored at 25 8C.
Table 4 shows the chain-breaking activity, the oxygen scavenging activity, the redox potential, and the optical density at 390 nm measured before and after pasteurisation, and also at different storage times of the beverage samples. It is interesting to observe that in both cases pasteurisation, in addition to the subsequent storage, caused an unexpected increase in the chain-breaking activity of the tea extracts. However, the gain observed for the green tea beverage was one order of magnitude higher when compared to that of the black tea. The differences in radical scavenging properties initially found between the green and the black tea samples appear to be enhanced as a consequence of the thermal treatment and the storage that followed. The oxygen scavenging properties gradually decrease during pasteurisation and storage. The reduction in the oxygen scavenging ability of tea extracts was correlated with a progressive increase in the redox potential value for the black tea extracts. These results indicate that the gain in the radical scavenging activity is associated with a corresponding decrease in the reducing power of the tea extracts. As can be observed in Table 4, the changes in the antioxidant properties were accompanied by an increase in the optical density at 390 nm for both green and black teas. It is known that processing and storage can promote a progressive polymerisation of phenolic compounds to form brown-coloured macromolecular products. In some cases the oxidation of polyphenols leads to the formation of stable intermediates which can still exhibit strong antioxidant activity Ž24.. Thus, the modifications in chain-breaking activity and oxygen uptake
Table 4 Changes in chain-breaking, oxygen scavenging properties, redox potential and optical density at 390 nm of green and black tea extracts as a consequence of pasteurisation and storage at 25 8C Samples
Chain-breaking activity ŽyO.D.y3rminrmg d.m. .
Oxygen uptake Ž m mol O 2rminrg d.m. .
E ŽmV. vs AgrAgCl, Cly sat
O.D. 390 nm
Green tea Fresh Pasteurised 7 d storage 30 d storage
5.60 24.24 33.83 105.63
184.6 166.6 159.5 149.0
n.d. n.d. n.d. n.d.
0.131 0.199 0.207 0.214
Black tea Fresh Pasteurised 7 d storage 30 d storage
1.95 5.37 9.73 12.86
184.3 184.0 159.5 149.0
180 220 360 360
0.160 0.186 0.187 0.235
n.d.s not determined
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detected in the tea extracts could be ascribable to the progressive oxidation of polyphenols, a process leading to the formation of macromolecular compounds with stronger radical scavenging power. This may be attributable to the increased resonance delocalisation as well as to the higher stability of the aryloxyl radicals incurred by hydrogen bonding Ž25.. Nonpasteurised green tea extracts maintained in openair while stirring for 24 h at 25 8C were tested for the chain-breaking activity. Green tea samples bottled under a nitrogen atmosphere and maintained at the same storage temperature were kept as control. The forced oxygenation caused a rapid increase in the chainbreaking activity of the tea extracts, whilst no changes were detected for the control samples. The chainbreaking activity was 5.60 and 8.24 for the control and oxygenated samples, respectively. As expected, the oxygen scavenging activity of the oxygenated samples resulted in a value 25% lower than that of the control Ždata not shown.. The results clearly indicate the critical role of oxygen in promoting changes to the chain-breaking activity and the reducing power of the tea extracts during storage. However, these findings apparently contradict those previously observed for the antioxidant properties of green and black tea extracts. While the enzymatic oxidation of the polyphenol fraction caused a decrease in the chain-breaking activity, chemical oxidation was found to give rise to the opposite effect. A possible explanation could be that the enzymatic and chemical oxidation of polyphenols follow different pathways, which lead to the formation of compounds having markedly contrasting radical scavenging capacities. A further hypothesis could be that although both reactions follow the same pathway, the chemical oxidation proceeds much slower than the enzymatic one and the compounds formed during pasteurisation and storage would have an intermediate oxidation status when compared to those formed by enzymatic oxidation. Since the oxidation products of phenolic compounds still retain antioxidant activity Ž25., the increased stability of partially oxidised polyphenols would explain the gain in chain-breaking efficiency found during the processing of the beverages. References 1 GRAHAM, H. N. Green tea composition, consumption, and polyphenol chemistry. Pre¨ enti¨ e medicine, 21, 334]350 Ž1992. 2 LUNDER, T. E. Catechins of green tea: antioxidant activity. In: HUANG, M. T., HO, C. T. AND LEE, C. Y. ŽEds.., Phenolic compounds in food and their effects on health II. Washington DC: ACS Symposium series 507, pp. 114]120 Ž1992. 3 BALENTINE, D. A., WISEMAN, S. A. AND BOUWENS, C. M. The chemistry of tea flavanoids. Critical Re¨ iews in Food Science and Nutrition, 37, 693]704 Ž1997. 4 SHAHIDI, F. AND WANASUNDARA, P.D. Phenolic antioxidants. Critical Re¨ iews in Food Science and Nutrition, 32, 1, 67]103 Ž1992. 5 WISEMAN, S. A., BALENTINE, D. A. AND FREI, B. Antioxidants in tea. Critical Re¨ iews in Food Science and
Nutrition, 37, 705]718 Ž1997. 6 TIJBURG, L. B. M., MATTERN, T., FOLTS, J. D., WEISGERBER, U. M. AND KATAN, M. B. Tea flavanoids and cardiovascular diseases: a review. Critical Re¨ iews in Food Science and Nutrition, 37, 771]785 Ž1997. 7 YANG, C. S. AND WANG, Z. Y. Tea and Cancer. Journal of the National Cancer Institute, 85, 13, 1038]1049 Ž1993. 8 YEN, G. C. AND CHEN, H. Y. Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agriculture and Food Chemistry, 43, 27]32 Ž1995. 9 BU-ABBAS, A., COPELAND, E., CLIFFORD, M. N., WALKER, R. AND IOANNIDES, C. Fractionation of green tea extracts: correlation of antimutagenic effect with flavanol content. Journal of the Science of Food and Agricolture, 75, 453]462 Ž1997. 10 HARBOWY, M. AND BALENTINE, D. A. Tea chemistry. Critical Re¨ iews in Plant Science, 16, 415]480 Ž1997. 11 CAO, G., SOFIC, E. AND PRIOR, R. L. Antioxidant capacity of tea and common vegetables. Journal of Agriculture and Food Chemistry, 44, 3426]3431 Ž1996. 12 JONSSON, L. Thermal degradation of carotenoids and influence on their physiological functions. In FRIEDMAN, M. ŽEd.., Nutritional and Toxicological Consequences of Food Processing. New York: Plenum Press, pp. 75]82 Ž1991. 13 LERICI, C. R., MANZOCCO, L., ANESE, M. AND NICOLI, M. C. Cambiamenti del potere antiossidante in alimenti sottoposti a processi di trasformazione e conservazione. Industrie Alimentari, 36, 977]982 Ž1997. 14 NICOLI, M. C., ANESE, M., MANZOCCO, L. AND LERICI C. R. Antioxidant properties of coffee brews in relation to the roasting degree. Lebensmittel-Wissenschaft und-Technologie, 30, 292]297 Ž1997. 15 NICOLI, M. C., ANESE, M., PARPINEL, M., FRANCESCHI, S. AND LERICI C. R. Loss andror formation of antioxidants during food processing and storage. Cancer Letters, 114, 71]74 Ž1997. 16 TUBARO, F. AND URSINI, F. Misura di attivita ` antiossidante tramite l’ananlisi di una cinetica di competizione. In: CONTE, L., DALLA ROSA, M. AND ZAMORANI, A. ŽEds., Proceeding of the workshop Antiossidanti naturali negli alimenti. Aspetti tecnologici e qualitati¨ i. Udine, 5]6 December 1995. Padova: CLEUP, pp.45]53 Ž1995. 17 AOAC, Official Methods of Analysis, CUNNIF, P. ŽEd.., Association of Official Analytical Chemists International, Arlington, Virginia, U.S.A. Ž1995. 18 BRAND-WILLIAMS, W., CUVELIER, M. E. AND BERSET, C. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenshaft und-Technologie, 28, 5]30 Ž1995. 19 SANCHEZ-MORENO, C., LARRAURI, J. A. AND SAURA-CALIX, F. A procedure to measure the antiradical efficiency of polyphenols. Journal of the Science of Food and Agriculture, 76, 270]276 Ž1998. 20 GORDON, M. H. The mechanism of antioxidant action in vitro. In: HUDSON, B. J. F. ŽEd.., Food antioxidants. London: Elsevier Applied Science, pp. 1]18 Ž1990. 21 CONNORS, K. A. Chemical kinetics: the study of reaction rates in solution. New York: VCH Publishers, pp. 59]76 Ž1990. 22 ANESE, M., PITTIA, P. AND NICOLI, M. C. Oxygen consuming properties of heated glucoserglycine aqueous solutions. Italian Journal of Food Science and Technology, 1, 75]79 Ž1993. 23 CHAPPEL, C. Solubility of oxygen in buffered mitochondrial medium equilibrated with air. Journal of Biochemistry, 90, 255]260 Ž1964. 24 POKORNY, J. Majors factors affecting the autoxidation in lipids. In: CHAN, H. ŽEd.., Autoxidation of unsaturated lipids. London: Academic Press, pp. 141]206 Ž1987. 25 KIKUGAWA, K., KUNUGI, A., KUREKI T. Chemistry and implication of degradation of phenolic antioxidants. In: HUDSON, B. J. F. ŽEd.., Food antioxidants. London: Elsevier Science, pp. 65]98 Ž1990.
698
Lebensm.-Wiss. u.-Technol., 31, 694]698 Ž1998 .
Antioxidant Properties of Tea Extracts as Affected by Processing L. Manzocco*, M. Anese and M. C. Nicoli
L. Manzocco, M. C. Nicoli: Dipartimento di Scienze degli Alimenti, Universita ` degli Studi di Udine, via Marangoni 97, 33100 Udine ŽItaly. M. Anese: Istituto di Produzioni e Preparazioni Alimentari, Universita ` degli Studi di Bari, via Napoli 25, 71100 Foggia ŽItaly. (Recei¨ ed May 25, 1998; accepted September 8, 1998)
The changes in the antioxidant properties of green and black tea extracts as a consequence of processing and storage conditions were e¨ aluated through measurements of chain-breaking acti¨ ity, oxygen sca¨ enging capacity and redox potential. Pasteurisation and storage as well as forced oxygenation in both tea extracts caused an increase in the optical density and a decrease in the reducing power of the be¨ erages; both were accompanied by unexpected increases in the chain-breaking acti¨ ity of the be¨ erages. Simple model systems containing catechin at a concentration similar to that found in the tea extracts were also considered to simulate the effects of enzymatic browning in tea. Possible explanations for the chain-breaking acti¨ ity gain obser¨ ed in the tea extracts as a consequence of processing were proposed.
q 1998 Academic Press Keywords: green and black tea; antioxidant activity; polyphenols; DPPH? ; processing; storage
Introduction Recent experimental studies have recognised that tea Ž Camellia sinensis. exhibits a significant health protecting activity due to its high flavanoid content. Flavanoids are the most abundant compounds in fresh tea leaves and extracts Ž1]3.. Flavanoids have long been recognised to have strong antioxidant activity, they are therefore considered to be responsible for the anticarcinogenic and antimutagenic properties of tea, as well as its protective action against cardiovascular diseases Ž4]6.. The antimutagenic effects of tea extracts are thought to be due to the action of polyphenols in inhibiting carcinogen-metabolising enzymes; these compounds would also be able to scavenge reactive intermediates of the carcinogenic process, thus preventing their interaction with DNA Ž7]9.. It is well known that tea manufacturing processes can greatly affect the oxidation of tea polyphenols. While green tea is produced from fresh leaves, preventing the oxidation of flavanoids, black tea manufacture results in a high degree of enzymatic aerobic oxidation of flavanoids Ž1, 3, 10.. Despite the different flavanol content of green and black tea, both possess considerable antioxidant properties. However, recent findings demonstrated that green tea extracts exhibit stronger * To whom correspondence should be addressed.
antioxidant activity than those of black tea Ž8, 11.. While many authors have tried to relate the antioxidant properties of various tea extracts to the degree of enzymatic oxidation of polyphenols in tea leaves, there is no information available concerning the outcome of these species during tea beverage production and storage. It is well known that many food antioxidants can be significantly lost as a consequence of sterilisation, pasteurisation, dehydration, as well as during prolonged storage Ž12.. Processing and storage are not always responsible for a depletion in the antioxidant properties of foods. In some cases, these factors can induce the formation of compounds with novel antioxidant properties, which can maintain or even enhance the overall antioxidant potential of foods Ž13]15.. Recent studies carried out on various red wines containing polyphenols showed that prolonged air exposure caused a progressive increase in the chain-breaking activity Ž16.. These changes were attributed to the partial oxidation of the wine polyphenols to form macromolecular compounds which still maintain a remarkable radical scavenging activity. As previously described, tea flavanoids are very reactive species which can easily undergo enzymatic and chemical reactions, which may be responsible for changes in the antioxidant properties of the product. Since it is likely that chemical modifications of the
0023-6438r98r070694 q 05 $30.00r0 q 1998 Academic Press
694
lwtrvol. 31 Ž1998 . No. 7 & 8
flavanoids may take place during the processing of tea beverages and storage, this study was designed to evaluate the changes in the antioxidant properties of green and black tea extracts as a consequence of pasteurisation and storage. Simple model systems containing catechin which is the most abundant flavanol in tea leaves were also considered. The antioxidant properties were evaluated through the measurement of the chainbreaking activity, the oxygen scavenging capacity and the redox potential.
Materials and Methods Sample preparation Green and black dried tea leaves Ž Camellia sinensis. were purchased from a local market. Tea extracts were obtained by solid-liquid extraction using deionized water at 100 8C Žtea leaf-water ratio was 1:100 wrw.. The extraction time was 5 min. The beverages were then rapidly filtered through Whatman No.4 filter paper. In order to minimise the contact of the beverages with atmospheric oxygen, extraction and filtration were carried out under nitrogen flow Ž5 mLrmin.. The samples were then immediately cooled under cold running water. Volumes of 50 mL of green and black tea extracts were bottled in screw capped flasks in the presence of air and subjected to pasteurisation at 105 8C for 20 min in a laboratory autoclave ŽFVS 13, Fedegari, Italy.. The beverages were then stored at 25 8C for up to 30 d. Additional samples of freshly prepared green and black
tea extracts, not subjected to pasteurisation, were placed in a thin layer in a Petri dish and maintained in contact with atmospheric oxygen whilst stirring at room temperature for up to 24 h. In order to simulate the enzymatic oxidation of polyphenols, which takes place during black tea production, a volume of 50 mL of 2.4 = 10y3 molrL Žq. catechin ŽC-1251, Lot 100H0586, Sigma, St. Luis, Mo, U.S.A.. solution was added to 100 units of mushroom tyrosinase ŽT-7755, Lot 48F9610, Sigma, St. Luis, Mo, USA. and maintained in ambient air whilst stirring for at least 2 h. The measurement of the antioxidant activity of the enzymatically browned solution was carried out after the enzymatic reaction reached a steady state which was detected through optical density measurements at 390 nm. Analytical determinations Total solid content. Total solid content determinations were carried out according to AOAC methods Ž17.. The measurements were made in triplicate. The coefficient of variation, expressed as the percentage ratio between the standard deviation and the mean value, did not exceed 5%. Total polyphenol content. The analysis was carried according to the Folin-Ciocalteu methodology. The results were expressed as gallic acid equivalents. Measurements were made in duplicate and the difference in results between two determinations carried out on the same sample did not exceed 2%.
Fig. 1 Percentage of residual DPPH? as a function of time for increasing concentration of a black tea extract
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lwtrvol. 31 Ž1998 . No. 7 & 8
Optical density. Measurement was carried out at 390 nm using a Uvikon 860 spectrophotometer ŽKontron Instruments, Milano, Italy.. This wavelength is in the spectral region of maximum absorption. The tea extracts were diluted Ž1:10 v:v. with distilled water to ensure that absorbance values were on scale. Measurements were carried out in triplicate. Coefficients of variation were less than 5%. Chain-breaking acti¨ ity. The chain-breaking activity was measured according to the methodology described by Brand-Williams et al. Ž18.. The bleaching rate of a stable free radical, 2,2-diphenyl-1-picrylhydrazyl ŽDPPH? . at a characteristic wavelength in the presence of the sample was monitored. In its radical form, DPPH? absorbs at 515 nm, but upon reduction by an antioxidant or a radical species its absorption disappears. A volume of 3.0 mL of 6.1 = 10y5 M DPPH? methanol solution was used. The reaction was started by the addition of 10 m L of samples. The bleaching of DPPH? was followed at 515 nm ŽUvikon 860, Kontron Instruments, Milano, Italy. at 25 8C for at least 60 min. In all cases the DPPH? bleaching rate was proportional to the sample concentration added to the medium. In view of the difficulty of achieving a plausible kinetic model to determine the order of the different reactions involved, the radical scavenging activity is generally expressed in terms of an efficient concentration ŽEC 50 ., that is, the amount of antioxidant necessary to decrease the initial DPPH? concentration by 50% Ž18]19.. The EC 50 value is obtained by plotting the percentage of residual DPPH? at the steady state as a function of the antioxidant concentration. This method is difficult to apply when the chain-breaking activity has to be evaluated in complex mixtures whose steady state is often uncertain, as in the case of foods ŽFigure 1.. In such cases, that is, in the presence of phenolic compounds, the overall DPPH? bleaching rate follows complex multistep, generally parallel or consecutive, reactions Ž18, 20]21.. Thus, different kinetic models, whose determination coefficients Ž r 2 . and P values are shown in Table 1, were considered to obtain a suitable indicator of the chain-breaking activity. Under the experimental conditions used, all the DPPH? bleaching curves obtained are described by fourth order reaction kinetics. The following equation was chosen in order to obtain the reaction rate, k: 1 3
A
y
1 A30
Table 1 Coefficients of determination Ž r 2 . and P values obtained from increasing reaction order regression of the DPPH? bleaching curves Reaction order Zero First Second Third Fourth Fifth
r2
P
0.82 0.90 0.96 0.98 0.99 0.96
10y5 10y5 10y5 10y5 10y8 10y6
extracts was measured according to the methodology described by Anese et al. Ž22.. A YSI mod. 53 oxygen monitor ŽYellow Springs Instruments Co. Inc., Yellow Springs, Ohio, U.S.A.. equipped with an oxygen electrode was used. The airtight reaction vessel was filled with 3.0 mL of tea beverage conditioned at 25 8C. The oxygen consumption was recorded using a Bromma recorder ŽLKB Producter, Bromma, Sweden.. Distilled water was used as a control. The rate of oxygen uptake was calculated from the initial linear portion of the curves obtained. A good linearity was found for at least 10 min. The oxygen consumption activity was expressed as m moles oxygen consumed miny1 rg d.m., considering that the oxygen concentration in air-saturated water at 25 8C is 237 m molrL Ž23.. Measurements were made, at least in triplicate, and the coefficients of variation of the determinations performed on the same sample were less than 10%. Redox potential. Measurements were made with a platinum indicating electrode and a AgrAgCl, Cly sat reference electrode connected with a voltmeter ŽCrison, mod. 2002, Alella, Spain.. Calibration was performed against a redox standard solution ŽE s 468 mV at 25 8C.. Electrodes were inserted into a 50 mL 3-neck flask containing a volume of 20 mL of sample. Prior to analysis, oxygen was removed from the system by continuous nitrogen flushing for a period of 10 min. Millivolt values were recorded for at least 5 min, until a stable potential was reached. A stable potential was arbitrarily defined as a change of less than 1 mV in a 5 min period. The analyses were carried out in duplicate and the difference in results between two determinations performed on the same sample did not exceed 3%.
Results and Discussion
s y3k t
where A 0 is the initial optical density and A is the optical density at increasing time, t. The chain-breaking activity was expressed as krmg d.m ŽyO.D.y 3 r minrmg d.m. ., assuming that all the dry matter of the tea extract possesses antioxidant properties. The data are expressed as the mean of at least three repetitions and the coefficients of variation were less than 10%. Oxygen uptake. The oxygen consumption of the tea
In Table 2 the phenol content and the chain-breaking activity, expressed both on total dry matter and on total polyphenol content, of the green and black tea extracts used are reported. The analyses were performed immediately after extraction. Green tea extracts showed higher phenol content and chain-breaking activity than those obtained from black tea leaves. These results are in agreement with observations previously carried out by Yen and Chen Ž8. who analysed several tea extracts having different ‘fermentation’ degrees. The authors
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Table 2 Values of total phenol content and chain-breaking activity of green and black tea extracts GAE a Green tea Black tea a
Chain-breaking activity ŽyO.D.y3rminrmg d.m. . ŽyO.D.y3rminrmg polyphenol .
Žmg Ly1 .
Sample
953.84 801.16
5.60 1.95
11.15 5.36
Total phenols are expressed as gallic acid equivalents ŽGAE.
Table 3 Values of optical density at 390 nm and chain-breaking activity of a 2.4 = 10y3 molrL catechin solution before and after the addition of 100 units of mushroom tyrosinase Sample
Chain-breaking activity O. D. 390 nm ŽyO.D.y3rminrmg.
Catechin 0.083 Catechin q tyrosinase 0.640
3.18 0.98
attributed the loss in antioxidant properties to the enzymatic oxidation of polyphenols, which is the basis of black tea manufacture. The enzymatic oxidation of catechins in tea leaves leads to the formation of a large number of compounds, generally known as teaflavins and thearubigins. Very little is known about the chemical structure of these compounds and their antioxidant properties Ž1.. To confirm the role of enzymatic browning in reducing the original antioxidant activity of tea leaves, an aqueous solution of catechin at a concentration similar to that found in green tea extract was prepared. The catechin was rapidly oxidised by the addition of appropriate units of tyrosinase. The effect of the enzymatic oxidation of catechin on the optical density and on the chain-breaking activity is shown in Table 3. The results acquired clearly demonstrate that the enzymatic browning caused a marked decrease in the radical scavenging properties of catechin. To evaluate the influence of some processing conditions which are commonly used in the industrial production of tea beverages, samples of green and black tea extracts were bottled in the presence of air, pasteurised at 105 8C for 20 min and then stored at 25 8C.
Table 4 shows the chain-breaking activity, the oxygen scavenging activity, the redox potential, and the optical density at 390 nm measured before and after pasteurisation, and also at different storage times of the beverage samples. It is interesting to observe that in both cases pasteurisation, in addition to the subsequent storage, caused an unexpected increase in the chain-breaking activity of the tea extracts. However, the gain observed for the green tea beverage was one order of magnitude higher when compared to that of the black tea. The differences in radical scavenging properties initially found between the green and the black tea samples appear to be enhanced as a consequence of the thermal treatment and the storage that followed. The oxygen scavenging properties gradually decrease during pasteurisation and storage. The reduction in the oxygen scavenging ability of tea extracts was correlated with a progressive increase in the redox potential value for the black tea extracts. These results indicate that the gain in the radical scavenging activity is associated with a corresponding decrease in the reducing power of the tea extracts. As can be observed in Table 4, the changes in the antioxidant properties were accompanied by an increase in the optical density at 390 nm for both green and black teas. It is known that processing and storage can promote a progressive polymerisation of phenolic compounds to form brown-coloured macromolecular products. In some cases the oxidation of polyphenols leads to the formation of stable intermediates which can still exhibit strong antioxidant activity Ž24.. Thus, the modifications in chain-breaking activity and oxygen uptake
Table 4 Changes in chain-breaking, oxygen scavenging properties, redox potential and optical density at 390 nm of green and black tea extracts as a consequence of pasteurisation and storage at 25 8C Samples
Chain-breaking activity ŽyO.D.y3rminrmg d.m. .
Oxygen uptake Ž m mol O 2rminrg d.m. .
E ŽmV. vs AgrAgCl, Cly sat
O.D. 390 nm
Green tea Fresh Pasteurised 7 d storage 30 d storage
5.60 24.24 33.83 105.63
184.6 166.6 159.5 149.0
n.d. n.d. n.d. n.d.
0.131 0.199 0.207 0.214
Black tea Fresh Pasteurised 7 d storage 30 d storage
1.95 5.37 9.73 12.86
184.3 184.0 159.5 149.0
180 220 360 360
0.160 0.186 0.187 0.235
n.d.s not determined
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detected in the tea extracts could be ascribable to the progressive oxidation of polyphenols, a process leading to the formation of macromolecular compounds with stronger radical scavenging power. This may be attributable to the increased resonance delocalisation as well as to the higher stability of the aryloxyl radicals incurred by hydrogen bonding Ž25.. Nonpasteurised green tea extracts maintained in openair while stirring for 24 h at 25 8C were tested for the chain-breaking activity. Green tea samples bottled under a nitrogen atmosphere and maintained at the same storage temperature were kept as control. The forced oxygenation caused a rapid increase in the chainbreaking activity of the tea extracts, whilst no changes were detected for the control samples. The chainbreaking activity was 5.60 and 8.24 for the control and oxygenated samples, respectively. As expected, the oxygen scavenging activity of the oxygenated samples resulted in a value 25% lower than that of the control Ždata not shown.. The results clearly indicate the critical role of oxygen in promoting changes to the chain-breaking activity and the reducing power of the tea extracts during storage. However, these findings apparently contradict those previously observed for the antioxidant properties of green and black tea extracts. While the enzymatic oxidation of the polyphenol fraction caused a decrease in the chain-breaking activity, chemical oxidation was found to give rise to the opposite effect. A possible explanation could be that the enzymatic and chemical oxidation of polyphenols follow different pathways, which lead to the formation of compounds having markedly contrasting radical scavenging capacities. A further hypothesis could be that although both reactions follow the same pathway, the chemical oxidation proceeds much slower than the enzymatic one and the compounds formed during pasteurisation and storage would have an intermediate oxidation status when compared to those formed by enzymatic oxidation. Since the oxidation products of phenolic compounds still retain antioxidant activity Ž25., the increased stability of partially oxidised polyphenols would explain the gain in chain-breaking efficiency found during the processing of the beverages. References 1 GRAHAM, H. N. Green tea composition, consumption, and polyphenol chemistry. Pre¨ enti¨ e medicine, 21, 334]350 Ž1992. 2 LUNDER, T. E. Catechins of green tea: antioxidant activity. In: HUANG, M. T., HO, C. T. AND LEE, C. Y. ŽEds.., Phenolic compounds in food and their effects on health II. Washington DC: ACS Symposium series 507, pp. 114]120 Ž1992. 3 BALENTINE, D. A., WISEMAN, S. A. AND BOUWENS, C. M. The chemistry of tea flavanoids. Critical Re¨ iews in Food Science and Nutrition, 37, 693]704 Ž1997. 4 SHAHIDI, F. AND WANASUNDARA, P.D. Phenolic antioxidants. Critical Re¨ iews in Food Science and Nutrition, 32, 1, 67]103 Ž1992. 5 WISEMAN, S. A., BALENTINE, D. A. AND FREI, B. Antioxidants in tea. Critical Re¨ iews in Food Science and
Nutrition, 37, 705]718 Ž1997. 6 TIJBURG, L. B. M., MATTERN, T., FOLTS, J. D., WEISGERBER, U. M. AND KATAN, M. B. Tea flavanoids and cardiovascular diseases: a review. Critical Re¨ iews in Food Science and Nutrition, 37, 771]785 Ž1997. 7 YANG, C. S. AND WANG, Z. Y. Tea and Cancer. Journal of the National Cancer Institute, 85, 13, 1038]1049 Ž1993. 8 YEN, G. C. AND CHEN, H. Y. Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agriculture and Food Chemistry, 43, 27]32 Ž1995. 9 BU-ABBAS, A., COPELAND, E., CLIFFORD, M. N., WALKER, R. AND IOANNIDES, C. Fractionation of green tea extracts: correlation of antimutagenic effect with flavanol content. Journal of the Science of Food and Agricolture, 75, 453]462 Ž1997. 10 HARBOWY, M. AND BALENTINE, D. A. Tea chemistry. Critical Re¨ iews in Plant Science, 16, 415]480 Ž1997. 11 CAO, G., SOFIC, E. AND PRIOR, R. L. Antioxidant capacity of tea and common vegetables. Journal of Agriculture and Food Chemistry, 44, 3426]3431 Ž1996. 12 JONSSON, L. Thermal degradation of carotenoids and influence on their physiological functions. In FRIEDMAN, M. ŽEd.., Nutritional and Toxicological Consequences of Food Processing. New York: Plenum Press, pp. 75]82 Ž1991. 13 LERICI, C. R., MANZOCCO, L., ANESE, M. AND NICOLI, M. C. Cambiamenti del potere antiossidante in alimenti sottoposti a processi di trasformazione e conservazione. Industrie Alimentari, 36, 977]982 Ž1997. 14 NICOLI, M. C., ANESE, M., MANZOCCO, L. AND LERICI C. R. Antioxidant properties of coffee brews in relation to the roasting degree. Lebensmittel-Wissenschaft und-Technologie, 30, 292]297 Ž1997. 15 NICOLI, M. C., ANESE, M., PARPINEL, M., FRANCESCHI, S. AND LERICI C. R. Loss andror formation of antioxidants during food processing and storage. Cancer Letters, 114, 71]74 Ž1997. 16 TUBARO, F. AND URSINI, F. Misura di attivita ` antiossidante tramite l’ananlisi di una cinetica di competizione. In: CONTE, L., DALLA ROSA, M. AND ZAMORANI, A. ŽEds., Proceeding of the workshop Antiossidanti naturali negli alimenti. Aspetti tecnologici e qualitati¨ i. Udine, 5]6 December 1995. Padova: CLEUP, pp.45]53 Ž1995. 17 AOAC, Official Methods of Analysis, CUNNIF, P. ŽEd.., Association of Official Analytical Chemists International, Arlington, Virginia, U.S.A. Ž1995. 18 BRAND-WILLIAMS, W., CUVELIER, M. E. AND BERSET, C. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenshaft und-Technologie, 28, 5]30 Ž1995. 19 SANCHEZ-MORENO, C., LARRAURI, J. A. AND SAURA-CALIX, F. A procedure to measure the antiradical efficiency of polyphenols. Journal of the Science of Food and Agriculture, 76, 270]276 Ž1998. 20 GORDON, M. H. The mechanism of antioxidant action in vitro. In: HUDSON, B. J. F. ŽEd.., Food antioxidants. London: Elsevier Applied Science, pp. 1]18 Ž1990. 21 CONNORS, K. A. Chemical kinetics: the study of reaction rates in solution. New York: VCH Publishers, pp. 59]76 Ž1990. 22 ANESE, M., PITTIA, P. AND NICOLI, M. C. Oxygen consuming properties of heated glucoserglycine aqueous solutions. Italian Journal of Food Science and Technology, 1, 75]79 Ž1993. 23 CHAPPEL, C. Solubility of oxygen in buffered mitochondrial medium equilibrated with air. Journal of Biochemistry, 90, 255]260 Ž1964. 24 POKORNY, J. Majors factors affecting the autoxidation in lipids. In: CHAN, H. ŽEd.., Autoxidation of unsaturated lipids. London: Academic Press, pp. 141]206 Ž1987. 25 KIKUGAWA, K., KUNUGI, A., KUREKI T. Chemistry and implication of degradation of phenolic antioxidants. In: HUDSON, B. J. F. ŽEd.., Food antioxidants. London: Elsevier Science, pp. 65]98 Ž1990.
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