Journal of Food Composition and Analysis 22 (2009) 709–713

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Article

Tyrosinase immobilized reactor as a fast tool for polyphenolic index of tea

Anna Maria Girelli *, Enrico Mattei, Domenica Papaleo
Dipartimento di Chimica – Sapienza Università di Roma, P.le A. Moro 5 - 00185 Roma, Italy

A R T I C L E I N F O A B S T R A C T

Article history: A new approach for the determination of polyphenolic index of black and green teas has been developed
Received 26 May 2008 employing a bioreactor with mushroom tyrosinase immobilized on aminopropyl-controlled pore glass
Received in revised form 16 March 2009 (AMP-CPG). Initially a linear correlation (r2 = 0.997) of catechin content between the Folin-Ciocalteu (FC)
Accepted 5 April 2009
and the enzymatic assays is obtained. Our method appears to be more specific and rapid. Successively
the inhibition of the enzymatic oxidation of catechin by some tea components like gallic acid (GA),
Keywords: epigallocatechin (EGC), epigallocatechingallate (EGCG) and epicatechin (EC) is investigated. Finally
Immobilized tyrosinase
when tea samples are analyzed by using the new method it appears that green tea is the strongest
Enzymatic bioreactor assay
Tea
inhibitor followed by black tea and flavored tea. The level of polyphenols, which is correlated to the
Polyphenolic index extent of inhibition, is reported as epigallocatechin equivalents and the results are compared with those
Flavonoids obtained using the FC assay and the vanillin index. The differences in phenol content found by applying
Antioxidant capacity of green tea the three methods are discussed in terms of the different specificities of the analytical basis.
Food analysis ß 2009 Elsevier Inc. All rights reserved.
Food composition

1. Introduction properties. More recently analytical techniques are used to isolate,

Recently the recognition of the antioxidant properties of
polyphenols, greatly abundant in our diet, has reached a sparked
interest since their probable role in the prevention of various
diseases associated with oxidative stress, such as cancer, cardio-
vascular and neurodegenerative diseases. In particular it has been
demonstrated that considerable antioxidant properties in vivo and
in vitro have been found in wine, coffee and tea, the most important
beverages in the world (Gardner et al., 2007; Manach et al., 2004;
Rechner et al., 2002; Richelle et al., 2001). The total phenolic content
of these commodities is therefore an important parameter for the
evaluation of their antioxidant properties and quality.
The major phenolics present in the teas are comprised of
flavonoids, in green tea ranging from flavan-3-ols and flavonols to
their complex oxidation products, in black tea mainly theaflavins
and thearubigins. The main flavan-3-ols are epicatechin and its
gallate derivatives, while the main flavonols in tea are conjugates
of quercetin and kaempferol with lower levels of myricetin and the
conjugating moiety varying from mono- to di- and tri-glycosides.
However, the composition of tea varies, besides its form (green,
oolong or black), with crop season, the age of leaf, climate and
horticultural practices.
Conventional methods for the determination of total poly-
phenols content are based on either their oxidation or reduction
identify and determine individual polyphenolic compounds by
HPLC (Friedman et al., 2006; Neilson et al., 2006; Saito et al., 2006;
Yang et al., 2007), but spectrophotometric methods are still the
most widely used, due to their simplicity for the determination of
total phenolic compounds. The leading spectrophotometric
method is Folin-Ciocalteu (FC) assay which is based on a non
specific phenol oxidation reaction by the two strong inorganic
oxidant (phosphotungstic and phosphomolibdic acids) (Laken-
brink et al., 2000; Stevanato et al., 2004) Another important
spectrophotometric method is based by the colorimetric reaction
with vanillin on the estimate of free C6 and C8 atoms of flavanols.
The goal of the present study is to determine the whole profile
of phenolic compounds in some traditional commercial teas by the
evaluation of inhibition of tea components on the catechin
oxidation to catechinquinone catalyzed by a tyrosinase immobi-
lized bioreactor. This work may also be a contribution to the few
existing data on the polyphenol content of tea infusions in ordinary
hot water (Arts et al., 2000; Chandra and De Mejia, 2004; Del Rio
et al., 2004; Liang et al., 2003; Liang and Xu, 2003), which can be
suitable for further use in epidemiological studies.
2. Materials and methods
2.1. Apparatus and chemicals

Catechin, epigallocatechin (EGC), epigallocatechingallate

* Corresponding author. Fax: +39 06490631. (EGCG), gallic acid, vanillin, Folin-Ciocalteu phenol reagent and
E-mail address: annamaria.girelli@uniroma1.it (A.M. Girelli). ethanol were supplied by Sigma (Milan, Italy). For buffers

0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2009.04.006
710 A.M. Girelli et al. / Journal of Food Composition and Analysis 22 (2009) 709–713
solutions, phosphoric acid (Carlo Erba, Milan, Italy), potassium
dihydrogenphosphate and dipotassium hydrogenphosphate epta-
hydrate (Sigma, Milan, Italy) of analytical grade were used. The
water was purified by Milli-Q Ultra-pure system (Millipore,
Bedford, MA, USA).
Chromatographic analysis was performed with a Kontron
system (Milan, Italy), consisting of a model 422 pump and a
UV–visible 432 detector, complete with a Rheodyne 7125 injector
with a 20 mL sample loop (Rheodyne, Berkeley, CA, USA). The
absorbance values of the effluent monitored at 440 nm (corre-
sponding to catechinquinone absorption), were registered by an
integrator system constituting a PC equipped with an Intel
processor, Pentium III 800 MHz CPU, and Agilent ChemStation
software for LC version A.08.03(847) running under MS Windows
NT 4.00.31 OS.
Spectra of peaks were obtained with a Thermo Quest Spectra
Series HPLC (Thermoquest Inc., San Josè, CA, USA) equipped with a
Spectra Series UV 6000 photodiode array detector (Thermoquest
Inc.) using the Chrom Quest software.
Spectrophotometric determinations were performed on a
Kontron Uvikon 920 UV–vis spectrophotometer with cuvettes of
1 cm length.
2.2. Sample collection and preparation
2.2.1. Tea
Three green teas from Camelia sinensis (Chinese, bioagricoltural,
green tea mixture), three fruit-flavored green teas (apple flavored,
rooibos–elder flavored and an infusion of mate containing green
teas), six classic blends of black tea from different brands (English
Breakfast, Ceylon, Prince of Wales, Earl Grey, blackcurrant flavored,
red fruit flavored), all in teabag form, were purchased from a local
supermarket. To prepare the tea, 0.5 g of tea was infused for 5 min
with 100 mL of boiled water. The infusion was successively filtered
on Acrodisk 0.45 mm cartridge (Millipore, Bedford, Mass., USA) and
successively several aliquots of the filtrate were frozen for further
use.
2.3. Immobilized tyrosinase bioreactor
Tyrosinase was immobilized on aminopropyl-controlled pore
glass with the in situ technique as previously described (Girelli
et al., 2007a). Briefly a stainless steel column (50 mm  2.1 mm ID)
was dry-packed with 70 mg of AMP-CPG (505 Å mean pore
diameter) and tyrosinase was immobilized by covalent bond
through glutaraldehyde as cross-linking agent. Reduction of the
Shiff’s base formed between enzyme amino groups and glutar-
aldehyde was made using a solution of cyanoborohydride
according to our previous study (Girelli et al., 2007a).
2.4. Assay of teas by tyrosinase immobilized bioreactor
The polyhenolic index of the tea samples was determined by
their inhibition power on the catechin oxidation to catechin-
quinone, and the results were expressed as epigallocatechin
equivalents. The extent of tea inhibition was determined by
subtracting from the peak area of catechinquinone, enzymati-
cally formed by the injection of 20 mL of a catechin ethanolic
solution (84 mg/L), the peak area of the same enzymatically
formed product obtained from a mixture of catechin and
tea infusion. This latter mixture was prepared by adding
50 mL of tea infusion diluted fivefold at pH 3 (to prevent
autoxidation) to 100 mL of catechin (210 mg/L) (Mochizuki et al.,
2002; Roginsky and Alegria, 2005) and a volume of ethanol to
reach total volume of 250 mL (final catechin concentration was
84 mg/L).
Data reported were obtained by triplicate analysis. For the
calibration curve, the mixture was prepared employing epigallo-
catechin ranging between 32 and 500 mg/L instead of tea infusion
and plotting on the y-axis the response as
Response ¼ Area0  Areast
where Area0 was relative to the catechinquinone formed by the
oxidation of the catechin (84 mg/L) alone and Areast was relative to
the catechinquinone formed by the oxidation of the catechin
(84 mg/L) in presence of EGC. The calibration curve equation was
y ¼ 140:3x þ 6833 ðr 2 ¼ 0:994Þ:
2.5. Vanillin index determination
The concentration of polyphenols reactive to vanillin in a highly
acidic environmental was determined according to the method
described by Vacca et al. (2003): 5 mL of tea infusion and 3 mL of
vanillin 4% (w/v) in methanol were placed in an amber flask
maintained in a cold water bath for 3 min. Then 1.5 mL of H2SO4
were added and the mixture was incubated for 15 min. Finally
absorbance at 500 nm was spectrophotometrically read against a
blank prepared in the same way of the sample but containing
water instead of tea infusion. Each sample analysis was repeated
twice.
2.6. Folin-Ciocalteu assay
The level of total phenols was determined using Folin-Ciocalteu
reagent (Folin-Ciocalteu, 1992; Singleton et al., 1999) at 784 nm,
and the results were expressed as catechin equivalents. Standard
solutions of catechin in the concentration range between 0.05 and
2 g/L were used to prepare the calibration curve. Each sample
analysis was repeated twice.
3. Results
Preliminary measurements were performed in order to select
the reference substrate for tyrosinase bioreactor. The chromato-
grams obtained by the injection of three flavanols commonly
present in teas: catechin, epigallocatechin (EGC) and epigalloca-
techingallate (EGCG) on the immobilized enzyme reactor (IMER),
show that the quinones of catechin and of EGCG were fully
separated by their substrate whereas that of EGC was eluted with
the void volume together with the not reacted substrate (Fig. 1).
The individuation of the species, confirmed by the spectra
registered with photodiode array detector, showed that the
retention times for catechinquinone and epigallocatechingallate
quinone were 1.8 min and 10 min, respectively. In addition
taking into account also that catechin is the flavanol present in the
teas at the lowest percentage (2 and 5% for green and black teas,
respectively) (Khokhar and Magnusdottir, 2002), this compound
was chosen as reference substrate. In this way, if the sample can be
diluted, the eventual contribution of tea catechin component to the
catechinquinone area may be considered negligible.
Successively the response of the IMER to the oxidation of
catechin was performed by injecting a different amount of catechin
in the chromatographic system and determining the catechinqui-
none peak areas at 440 nm. The area values were compared, with
the absorbance values obtained with the same solutions by Folin-
Ciocalteu assay (Fig. 2). The good linearity (correlation coefficient
of 0.997) indicated that the bioreactor was sensitive to the catechin
amount.
In order to ascertain the inhibition of the enzymatic oxidation of
catechin by flavanols present in the teas, stock solutions of gallic
acid, EGC and EGCG at the amount generally present in green teas,
 A.M. Girelli et al. / Journal of Food Composition and Analysis 22 (2009) 709–713
Fig. 1. Eluted peaks from IMER: (A) catechin quinone (C-Q), (B) epigallocatechin quinone (EGC-Q), (C) epigallocatechingallate quinone (EGCG-Q).
68, 1000 and 1200 mg/L, respectively, were investigated. The
inhibition extent, reported in Table 1, indicates that the system is
sensible to the components in the teas. In particular, it appeared
that when the 3-OH group is free in the flavon-3-ol skeleton (EGC)
a higher tyrosinase inhibitory strength is obtained respect to that
of EGCG which can give steric hindrance for the galloyl moiety at
the 3-position.
A preliminary spectrophotometric study focused on the
determination of the color intensity and color tonality, as the
sum and the ratio, respectively, of the absorbance values at 420 nm
(corresponding to yellow pigments of condensed compounds) and
520 nm (corresponding to red pigments of antocyans), and the
Fig. 2. Correlation between the experimental signals obtained with Folin method
and the chromatographic areas by IMER.
711
amounts of total phenols by FC method and flavanols with free C6
and C8 positions by vanillin index was performed on different black
and green tea samples commonly consumed in Italy. Data reported
in Table 2 confirm the higher amount of yellow colored species as
theaflavins and thearubigins in black teas, and higher amount of
flavanols with free C6 and C8 positions and flavonoids in green teas
(USDA, 2006).
A systematic study on the same tea samples was successively
performed by injecting 20 mL aliquots of reaction mixture
containing catechin and tea infusions diluted fivefold at pH 3
(to prevent autoxidation) into the chromatographic system
(Mochizuki et al., 2002; Roginsky and Alegria, 2005) The optimal
bioreactor experimental conditions of flow rate 0.7 mL/min,
temperature 20 8C and pH 6.5, as previously reported (Girelli
et al., 2007b) were chosen. In conjunction the wavelength of
440 nm corresponding to the absorption maximum of catechin-
quinone, enzimatically formed was fixed. The high tea sample
dilutions render negligible the contribution of eventual absorbing
interferents. The data were normalized respect to their own
maximal values and compared with those obtained with FC and
vanillin indices (Figs. 3 and 4). The tea samples were ordered
Table 1
Percent inhibition of gallate, epigallocatechin
(EGC) and epigallocatechin gallate (ECGC) on
catechin oxidation by IMER.
Inhibition %
Gallic acid 66  4
EGC 99  10
EGCG 38  2
712 A.M. Girelli et al. / Journal of Food Composition and Analysis 22 (2009) 709–713

Table 2
Color tonality, color intensity, total phenols and vanillin index of various green and black teas.

Color tonality (A420/A520) Color intensity (A420 + A520) Total phenols (Folin) (g/L eq. catechin) Vanillin index (A500 nm  290.8)

Black teas
Prince of Wales 3.52 1.18 0.62 61.1
English Breakfast 2.96 2.30 0.89 87.2
Ceylon 2.36 2.14 0.60 81.4
Earl Grey 3.52 1.24 0.72 61.1
Flavored (blackcurrant) 3.48 1.15 0.62 63.9
Flavored (red fruits) 4.02 1.30 0.62 49.4

Green teas
Bioagricoltural 3.18 0.99 1.15 157.0
Chinese 2.15 0.52 0.98 154.1
Infuse 3.54 0.53 0.34 81.4
Mixture 3.04 0.68 1.17 162.8
Flavored (apple) 2.74 0.65 1.23 119.2
Flavored (rooibos–elder) 3.03 1.01 0.72 119.2

Table 3
Comparison of flavonoid content obtained in different commercial teas by triplicate IMER analysis (RSD = 3%) and the data collected by USDA.

Teas (Camellia sinensis) mg/L EGC equivalents (=X) X̄ (IMER) (mg/L EGC equivalents) Mean flavonoid content (USDA)
(mg/100 g edible portion)

Green teas brewed Chinese 415  12 380 133

Green teas mixture 379  9
Bioagricoltural 346  10

Flavored green teas brewed Apple 269  11 265 54

Rooibos and elder-tree 268  12
Infuse 257  8

Black teas brewed English Breakfast 374  13 328 120

Ceylon 319  10
Prince of Wales 292  8

Flavored black teas brewed Earl Grey 289  5 282 Not available
Blackcurrant flavored 289  6
Red fruits flavored 269  8

following the decrease of % phenols content obtained by the
enzymatic method.
The better agreement of the IMER data sequence with the trend
of vanillin index (r2 is 0.871 and 0.933 respectively for black and
green teas) rather than FC method values (r2 is 0.682 and 0.863
respectively for black and green teas) is shown. This result may
probably be ascribed to the poor specificity of Folin-Ciocalteu
method, which can give an overestimated value of polyphenol
content since this assay can detect substances like protein, ascorbic
acid or sugar (Singleton et al., 1999). For example, the greater
content of total polyphenols for apple-flavored antioxidant
capacity green tea determined by FC method may be ascribed to
the positive interference of ascorbic acid, originated with the fruit
flavor (Arain, 1997), since it is known (Szeto et al., 2002) that tea
contains little or no ascorbic acid.
In order to define a quantitative index of the teas, a calibration
curve was performed using as reference a substance which must

Fig. 3. Histogram of % data obtained by IMER, Folin-Ciocalteu and vanillin assays on Fig. 4. Histogram of % data obtained by IMER, Folin-Ciocalteu and vanillin assays on
green teas. black teas.
 A.M. Girelli et al. / Journal of Food Composition and Analysis 22 (2009) 709–713
act as a strong inhibitor of catechin enzymatic oxidation but not
interfere chromatographically with the catechinquinone peak.
Epigallocatechin, as seen in Fig. 1 and data reported in Table 1,
appeared to be the most suitable compound for this purpose.
Comparing the flavonoid content expressed as EGC equivalents
with the data collected by United States Department of Agriculture
(USDA) (Table 3), it is evident that the polyphenol content in green
teas was higher by 16% than that of black teas in agreement with
the 10% value determined by the USDA flavonoids database. On the
contrary, the flavonoid content in fruit-flavored green teas
determined by IMER assay was slightly different from that of
USDA reported data; the different nature and amount of flavors
added to the tea base composition influences the analytical
response, making the comparison difficult.
4. Conclusions
The proposed enzymatic bioreactor assay appeared to be
sensitive, rapid and simple, making it an attractive monitoring tool
for food technologies and in particular for the fast screening of
polyphenolic index of complex commodities as tea beverages. In this
regards, green teas showed the highest polyphenolic index value,
followed by the black tea and finally by flavored green teas as reported
about this topic for other countries (USDA, 2006). In particular,
Chinese green tea was the beverage with the best polyphenolic
content and consequently with the high antioxidant capacity.
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