LWT - Food Science and Technology 44 (2011) 1931e1938

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LWT - Food Science and Technology

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Cabernet Sauvignon wines from two different clones, characterization

and evolution during bottle ageing
Vívian Maria Burin a, Léa Luzia Freitas Costa b, Jean Pierre Rosier c, Marilde T. Bordignon-Luiz a, *
a
Departamento de Ciência e Tecnologia de Alimentos CAL/CCA, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, CEP: 88034-001, Itacorubi,
Florianópolis, SC, Brazil
b
Laboratório Central de Saúde Pública e LACEN, Santa Catarina, Brazil
c
Empresa de Pesquisa e Extensão Agropecuária de Santa Catarina (EPAGRI-SC) Estação, Experimental de Videira SC, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Phenolic compounds constitute important quality parameters of wines. Wines produced from different
Received 24 September 2010 clones of the same grape variety show differences in relation to their chemical composition. The aim of
Received in revised form this study was to characterize and differentiate Cabernet Sauvignon wines from two clones in relation to
2 May 2011
their chemical composition and to examine changes in the phenolic composition and antioxidant activity
Accepted 3 May 2011
during wine ageing in the bottle. All wines were produced with Cabernet Sauvignon grapes, clones 685
and 169, from two vineyards, under the same microvinification conditions. The wines were characterized
Keywords:
in relation to phenolic composition and antioxidant activity, as well as monitored over 11 months of
Red wine
Clones
bottle ageing. A significant difference was observed between the chemical compositions of the wines
Bottle ageing produced from clones 169 and 685, clone 169 showing the highest phenolic content while clone 685 had
Phenolic compounds better color characteristics. The wines showed high antioxidant activity. Principal components and
Antioxidant activity cluster analyses demonstrated separation of the wine according to the clone. In relation to wine bottle
ageing, for both clones evaluated was observed a decrease in all phenolic compound, except of quercetin,
and the antioxidant activity of these wines increased during storage.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Many factors influence the chemical composition of wine, such

Phenolic compounds play an important role in grape and wine
quality, contributing to sensory properties such as color, astrin-
gency and bitterness. Polyphenols from wine can be classified into
two groups: non-flavonoid compounds (hydroxycinnamic and
hydroxybenzoic acids and their derivatives and stilbenes) which
participate in oxidation reactions that lead to the browning of the
must and wine, and flavonoid compounds (anthocyanins, flavanols
and flavonols) which contribute to the wine color since anthocya-
nins are the main pigments of red wine. Besides these functions,
the chemical structure of polyphenols, mainly flavonoids and stil-
benes (resveratrol), makes them suitable to act as antioxidants,
trapping and neutralizing free radicals. The phenolic content of
wine has been extensively studied, mainly in relation to providing
beneficial effects on health, since in addition to antioxidant
capacity it also has anti-inflammatory and anticarcinogenic effects,
among others (Frankel, Bosanek, Meyer, Silliman, & Kirk, 1998).
* Corresponding author. Tel.: þ55 48 3721 5376; fax: þ55 48 3721 9943.
E-mail address: bordign@cca.ufsc.br (M.T. Bordignon-Luiz).
as grape variety, degree of grape ripeness at harvest, climatic
conditions, cultural practices and technology, affecting the final
quality of wine. Moreover, studies have shown that different clones
of the same grape variety also have significant differences in rela-
tion to their chemical composition. It has been shown that some
clones have the capacity to produce wines with distinct color,
aromatic profile and phenolic content (Zamuz, Martínez, &
Vilanova, 2007). Clonal selection has allowed significant gains in
viticulture by allowing the exploration of an important source of
genetic variability, resulting in specific characteristics of the grapes
and wines.
The stability of a wine during storage is known to be dependent
on its chemical composition. However, the composition changes
continuously during the storage period mainly due to factors such
as temperature, light, bottle position, oxygen content and storage
time. These changes show varied intensity and complexity and can
affect the aroma, color, and phenolic composition (Hernanz et al.,
2009). Most phenolic compounds are highly unstable, taking part
in reactions that occur during the wine ageing, especially, oxida-
tion, polymerization and copigmentation. During storage, these
reactions would presumably lead to changes in the antioxidant

0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2011.05.001
1932 V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938
capacity of the wine as a consequence of changes in the redox
balance (Monagas, Gómez-Cordovés, & Bartolomé, 2006).
Studies have been conducted to establish the evolution of
different families of phenolic compounds during wine ageing, in
the barrel or the bottle (Cadahía, Simón, Sanz, Poveda, & Colio,
2009; García-Falcón, Pérez-Lamela, Martínez-Carbalho, & Simal-
Gándara, 2007). However, there are few studies in the literature
regarding the behavior of these compounds during the storage of
wines from different clones of the same grape variety, such as
Cabernet Sauvignon. The aim of this study was to characterize and
differentiate Cabernet Sauvignon wines from two clones in relation
to their chemical composition, and to examine changes in the
phenolic composition and antioxidant activity during wine ageing
in the bottle.
2. Material and methods
2.1. Chemical
All chromatographic solvents were HPLC grade and were
purchased from Merck (Darmstadt, Germany). Standards of (þ)cate-
chin, p-coumaric acid and morin (20 ,3,40 ,5,7-penthydroxyflavone),
DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS [2,2-azino-bis(3 ethyl-
benzothiazoline-6-sulphonic acid) and trans-resveratrol were
purchased from SigmaeAldrich (St. Louis, MO, USA), quercetin, ferulic
acid, caffeic acid, FolineCiocalteu were obtained from Fluka (Stein-
heim, Germany). Malvidin, delphinidin and peonidin-3-glucoside
were obtained from Extrasynthese (Genay,France).
2.2. Samples
This study was carried out at two vineyards (A and B) located in
the São Joaquin region of Santa Catarina State (SC), Brazil, at
similar altitudes (1200 m). The soil of this region is of the type
Inceptisol according to U.S.D.A. classifications and the weather of
the São Joaquim region is classified as “Cool, Cool nights and
Humid” according to the Geoviticulture Multicriteria Climatic
Classification System and as “Region I” (<1389 GDD), a “cold
region”, in terms of the Winkler Regions (Gris, Burin, Brighenti,
Vieira, & Bordignon-Luiz, 2010). From each vineyard Cabernet
Sauvignon grapes from two clones, 685 and 169, 2008 vintage,
were evaluated, designated as: A-685; A-169; B-685 and B-169.
The training system used was the V System and the rootstock was
Paulsen 1103 (Vitis berlandieri Planch  Vitis rupestris Scheele). For
all plants, the row and vine spacing were 3.0  1.5 m, respectively.
Twelve plants from each vineyard were previously marked,
randomly, in four central rows. The grapes were harvested at the
stage of technical maturity and had sugar readings of between 19.1
and 23.5  Brix, titratable acidity of 0.61e0.68 g/100 mL and pH
values between 3.69 and 3.98. These parameters were determined
according to OIV (1990).
The wines were all produced under the same microvinification
conditions at the state agricultural research center - Empresa de
Pesquisa e Extensão Agropecuária de Santa Catarina, in Videira, SC,
Brazil. The grapes were separated from the stalks, crushed and
maintained in a 20 L capacity stainless steel vat. The maceration
period was 10 days, with two daily pumping events at 22  C. The
must was separated from the solid parts and transferred to other
stainless steel vats. Prior to initiating alcoholic fermentation,
a commercial sulfiting agent (20 g per 100 kg of must, corre-
sponding to 10 mg/L) comprised of free SO2 (Noxitan, Pascal
Biotech, Paris, France), a Sacharomyces cerevisae strain (20 g per
100 kg; Fermol Rouge, Pascal Biotech, Paris, France) and commer-
cial enzymes with pectinolytic activity (2e4 g/h L; Pectinex SPL/
Ultra, Pascal Biotech, Paris, France) were added to the musts. Malic
acid consumption by lactic bacteria occurred spontaneously within
20e25 days. Once alcohol fermentation had finished, the wines
were chilled to 4  C for 10 days and Noxitan (35 mg/L of free SO2, on
average) was added before bottling. The wines were analyzed
between 6 and 8 months after microvinification. The wine samples
were stored at 5  C, under same conditions prior to analysis. All
analyses were carried out in triplicate with three repetitions for
each wine sample.
2.3. Wine characterization
2.3.1. Spectrophotometric analysis
Wines were analyzed in relation to total polyphenols (TP) (mg/L
gallic acid) using the FolineCiocalteu reagent (Singleton & Rossi,
1965). Non-polymerized polyphenols (NPP) (mg/L catechin) was
assayed by vanillin reaction and polymerized polyphenols (PP)
(mg/L catechin) was calculated as total phenolic (using
FolineCiocalteu reagent and expressed in mg/L catechin) minus
non-polymerized polyphenols (Paronetto, 1977). Color tonality (CT)
and color intensity (CI) were determined by absorbance measure-
ments at 420, 520 and 620 nm (Glories, 1984). Total monomeric
anthocyanin (TMA) content was determined by the pH difference
method and express as mg/L malvidin-3-glucoside (Giusti &
Wrolstad, 2001).
2.3.2. HPLC analysis
Chromatographic analysis was performed using a Shimadzu
(Kyoto, Japan) liquid chromatograph, equipped with a vacuum
degasser (DGU-14A), quaternary pump LC-10AT, UVeVis detector
(SPD-10AV) and an injector (Rheodyne) with a 20 mL loop, with
CLASS-VP software (v. 6.1). The column (4.6 mm  250 mm, 5 mm
particle size) and guard column (4.6 mm  12.5 mm) were C18
reversed-phase (Hichrom, Berkshire, UK). For analysis of poly-
phenols, trans-resveratrol and anthocyanins, wine samples were
filtered through a 0.45 mm PTFE membrane filter modified with
13 mm of diameter (Millipore, Bedford, MA) and directly injected
into the HPLC.
HPLC separation of the phenolic compounds (catechin, quer-
cetin, gallic acid, caffeic acid, ferulic acid and p-coumaric acid) was
carried out according to a method validated in our laboratory (data
not shown), by internal standardization (morin solution, 16.2 mg/
L). The mobile phase consisted of acetic acid in filtered Milli-Q
water adjusted to pH 2.6 as solvent A and acetonitrile: solvent A
(80:20) as solvent B. Phenolic compounds were eluted in three
segments of linear gradients: 0e30% solvent B for 35 min, 30e50%
for 5 min, 50e100% for 15 min and the last 15 min was used to
recondition the column in preparation for a new chromatographic
run (conditioning step). The flow rate was 1.2 mL/min, with
detection at 280 nm. The trans-resveratrol was determined
according to Souto et al. (2001), by external standardization, with
isocratic elution: water:acetonitrile (75:25) adjusted to pH 3 with
phosphoric acid. The flow rate was 1.2 mL/L and detection was at
306 nm.
Anthocyanin analysis was carried out according to García-Falcón
et al. (2007) with modification by external standardization. The
mobile phase consisted of Milli-Q water:formic acid (95:5 v/v) as
solvent A and formic acid in methanol as solvent B. The elution
gradient used was: 20e40% of solvent B for 40 min, 40e100% of B
for 10 min, and 100% of B for 5 min. The flow rate was 0.8 mL/L, with
detection at 520 nm.
Flavonoid and non-flavonoid phenolic compounds were iden-
tified through comparison of their retention times and UVeVis
spectra with those obtained by injection of the standard solution
under the same conditions. Peak area was used for the quantifica-
tion. Limits of detection (LOD) and quantification (LOQ), linearity
 V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938 1933

Table 1
Total phenolic compounds and color parameters of Cabernet Sauvignon wines, clones 169 and 685, vineyards A and B.

A-685 A-169 B-685 B-169

TP* 2114.95a  10.28 2431.66b  29.65 2205.74c  27.96 2569.81d  32.46
PP* 1293.66a  24.87 1458.44b  9.32 1319.56c  17.98 1516.69d  21.36
NPP* 878.71a  19.45 834.34b  6.94 736.03c  11.25 753.01d  17.41
TMA* 185.22a  3.72 164.08b  3.26 209.33c  1.69 167.67d  2.59
CIy 12.03a  0.02 10.89b  0.01 12.73c  0.01 11.72d  0.02
CTy 0.73a  0.01 0.80b  0.01 0.63c  0.01 0.78b  0.01

*mg/L.
y
expressed as index.
Means  SD of three replication for each wine sample. Different letters in the same line represent significant differences (P < 0.05) between samples.
TP, total polyphenols; PP, polymerized polyphenols; NPP, non-polymerized polyphenols; TMA, total monomeic anthocyanins; CI, color intensity; CT, color tonality.

range (R2), recovery (%) and precision (RSD) of the methods were
determined.
2.3.3. Antioxidant activity
The antioxidant activity of the wine was determined by three
methods: DPPH, ABTS and FRAP. The DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical activity was measured through the extinc-
tion of the maximum absorption at 517 nm (Kim, Guo, & Packer,
2002). The ABTS [2,2-azino-bis(3 ethylbenzothiazoline-6-
sulphonic acid)] radical antioxidant activity was determined
according to Re et al. (1999). The FRAP (ferric reducing ability of
plasma) method was carried out according to Arnous, Makris, and
Kefalas (2002), with measurement at 620 nm. Results are
expressed in Trolox equivalent antioxidant activity (mmol TEAC/L
wine).
2.4. Influence of bottle storage of young red wines on their
evolution
The evolution of the main individual phenolic compounds by
HPLC, total phenolic (polymerized and non-polymerized), antho-
cyanins, as well as the antioxidant activity were monitored for
samples of Cabernet Sauvignon, clones 685 and 169, from vineyard
A. All wine samples were submitted to the same conditions after
bottling. The evaluation period for bottle storage time began after
six months of vinification and was conducted for 11 months of
storage.
2.5. Statistical analysis
All analyses were carried out in triplicate and results expressed
as mean values  standard deviation. The STATISTICA v. 6.0 (2001)
(StatSoft Inc., Tulsa, OK, USA) program was used for the analysis of
variance (ANOVA), Tukey test (P < 0.05), correlation analysis,
principal component (PCA) and cluster analysis.
lower CT values, which is in agreement with research performed by
Cliff, King, and Schlosser (2007).
The TP values for different Cabernet Sauvignon wines from
Santa Catarina State, Brazil are in agreement, and can be considered
as high when compared with wines from the same grape variety
produced in other countries. Results from other studies have shown
that TP values for Cabernet Sauvignon wines from China ranged
from 1410 to 2488 m/L (Li, Wang, Li, Li, & Wang, 2009). Roginsky
et al. (2006) assessed red wines from California and found that
Cabernet Sauvignon wines of different vintages had an average TP
value of 1800 mg/L.
3.1.2. Flavonoid and non-flavonoid phenolic compounds
The main individual phenolic compounds present in wines were
determined by liquid chromatography and showed good analytical
validation parameters (Table 2) according to the literature (García-
Falcón et al., 2007).
Flavonoid and non-flavonoid phenolic compounds are widely
used to differentiate wines produced from different grape varieties,
however, there are few studies using the main phenolic compounds
to differentiate between wines produced with different clones of
the same grape variety. Of the compounds determined (Table 3),
catechin and gallic acid showed the highest concentrations in the
wines produced with clone 169, which also contained a higher
concentration of all non-flavonoid compounds studied, regardless
of the vineyard. Wines produced with clone 685 from the two
vineyards studied showed the highest concentrations of mono-
meric anthocyanins, especially malvidin-3-glucoside, which was
the major compound in these wines. Different clones with different
oenological characteristics are typical of several grape varieties, but
the literature contains data that differ regarding the differences in
the chemical composition of clones. Research carried out to
differentiate clones grown at the same place showed that the
Table 2
Validation parameters for determination of compounds in wines.

Compounds Range* R2 LOD* LOQ* rec RSD Rt

3. Results and discussion
Flavonoids
malvidin-3-glucoside 0.9 e 90.0 0.9999 0.07 0.23 94.3 2.2 32.5
3.1. Wine characterization delphinidin-3-glucoside 1.0 e 50.0 0.9978 0.03 0.10 92.3 2.1 19.2
peonidin-3-glucoside 0.2 e 50.0 0.9926 0.01 0.10 90.1 1.9 30.0
3.1.1. Total content of the phenolic families and color parameters catechin 0.3 e 150.0 0.9992 0.04 0.12 93.2 1.3 23.2
Total content of the phenolic families in the wine sample were quercetin 0.3 e 75.0 0.9991 0.05 0.15 93.4 2.5 45.0

determined by spectrophotometric methods (Table 1) and signifi- Non-flavonoids

cant differences could be observed (P < 0.05) between wine trans-resveratrol 0.1 e 25.0 0.9984 0.05 0.10 95.6 1.9 11.3
gallic acid 8.0 e 80.0 0.9981 0.32 0.97 88.4 2.2 10.0
samples from the clones of the Cabernet Sauvignon grapes. In
caffeic acid 0.3 e 75.0 0.9991 0.02 0.06 98.8 1.7 25.6
relation to the content of polyphenolic compounds, such as TP and ferulic acid 0.3 e 30.0 0.9998 0.04 0.05 101.9 2.6 34.7
PP, the clone 169 showed the highest values, independent of the p-coumaric acid 0.3 e 30.0 0.9998 0.08 0.24 102.6 2.5 31.5
vineyard. In contrast, the highest values for TMA content was LOD: limit of detection; LOQ: limit of quantification; Rec: recovery (%); RSD: relative
observed for clone 685 from both vineyards. The higher anthocy- standard deviation (%); Rt: retention time (min).
anins content contributed to these wines having higher CI and * mg/L.
1934 V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938

Table 3
Content of main flavonoid and non-flavonoid phenolic compounds (mg/L) in Cabernet Sauvignon wines, clones 169 and 685, A and B vineyards determined by HPLC.

Compounds A-685 A-169 B-685 B-169

Flavonoids
malvidin3glucoside 35.70a  0.15 32.32b  0.61 42.21c  0.27 31.71d  0.09
delphinidin3glicosídeo 4.27a  0.08 3.76b  0.03 7.1c  0.12 3.26d  0.08
peonidin3glucoside 17.32a  0.20 14.07b  0.18 22.85c  0.45 14.12d  0.15
catechin 17.51a  0.12 50.42b  0.85 9.96c  0.05 39.23d  0.33
quercetin 38.69a  1.11 15.36b  0.96 24.56c  0.12 52.61d  1.25
Non-flavonoids
trans-resveratrol 0.75a  0.02 1.52b  0.04 0.65c  0.02 1.89d  0.03
gallic acid 30.29a  0.06 32.63b  0.71 31.52c  0.33 44.22d  0.12
caffeic acid 1.56a  0.09 5.16b  0.38 1.79c  0.05 1,84d  0.16
ferulic acid 0.08a  0.01 0.12b  0.01 0.05c  0.01 0.14b  0.01
p-coumaric acid 1.47a  0.04 2.50b  0.03 0.79c  0.02 6.80d  0.09

Means  standard deviation over three replications for each wine sample. Different letters in the same line represent significant differences (P < 0.05) between the wine
samples.

anthocyanins composition differs significantly between samples
from clones of the same variety (Forveille, Vercauteren, & Rutledge,
1996) which is in agreement with the results obtained in this study.
The phenolic compounds concentration in the Cabernet Sau-
vignon wines evaluated was found to be high, especially gallic acid
and quercetin, when compared with wines of the same variety from
Spain, which showed values for these compounds of 13.6 and
5.0 mg/L, respectively (Monagas, Suárez, Gómez-Cordovés, &
Bartolomé, 2005). The trans-resveratrol concentration was also
significant in these wines from Santa Catarina State and higher than
in Cabernet Sauvignon wines from the northeast region Brazil, with
values ranging from 0.55 to 0.04 mg/L (Lucena et al., 2010). For
Spanish wines this value was 0.4 mg/L according to studies con-
ducted by Cadahía et al. (2009). Souto et al. (2001) determined trans-
resveratrol in commercial wines from southern Brazil and found for
Cabernet Sauvignon, from different vintages, concentrations
ranging from 0.32 to 3.57 mg/L. These differences in the phenolic
composition of Cabernet Sauvignon from different countries may be
related to different clones of the same variety, vinification process
(which influences the extraction and diffusion of compounds from
the grape to the wine), and factors that affect the berry development
such as soil, geographical origin and climatic conditions.
3.1.3. Correlation between antioxidant activity and phenolic
compounds
The determination of the antioxidant activity of the wines was
carried out using the ABTS and DPPH methods, which are based on
the capture of free radical and FRAP method based on antioxidant
act as antioxidants. The ABTS method showed higher correlation
with the polyphenols than the DPPH and FRAP methods for the
wines, a finding also reported by Burin et al. (2010) and
Rivero-Pérez, González-Sanjosé, Ortega-Herás, and Muñiz (2008).
Catechin was the main phenolic compound, giving the highest
correlation with antioxidant activity for the three methods evalu-
ated, with a Pearson correlation coefficient (R) greater than 0.9,
which was also reported by other researchers who affirm that
catechin is the compound that most contributes to the antioxidant
activity of wine (Arnous et al., 2002; Di Majo, La Guardia,
Giammanco, La Neve, & Giammarnco, 2008). This is consistent
with observations cited in the literature that catechin is the most
important flavanol found in wines of different varieties (Gómez-
Alonso, Garcia-Romero, & Gutierrez, 2007). The concentration of
catechin, total polyphenols (TP) and polymerized phenolic (PP)
present in the wines also showed strong correlations with the
antioxidant capacity of wine (R > 0.8). These results are consistent
with research conducted by Alén-Ruiz, García-Falcón, Pérez-
Lamela, Martínez-Carballo, and Simal-Gándara (2009) who
demonstrated that polymeric forms present in wine are primarily
responsible for the antioxidant activity. These results indicate that
the antioxidant potential of wine is not related to only one
compound, since there is synergy between the antioxidant activi-
ties of different polyphenolic classes.
24 b

ability to reducing to iron. The antioxidant activity found through 22 b b

different methods for Cabernet Sauvignon wines, produced with 20 b

two clones (Fig. 1), showed a significant difference (P < 0.05) 18
between methods, with the exception of sample B-685 which 16
antioxidant activity

showed no difference using the DPPH and FRAP methods. For all
14
wine samples, the highest antioxidant activity was obtained with
(mmol/L)

a c
12 a c a
the ABTS method. Researches carry out by Li et al. (2009) and Burin a c a
10
et al. (2010) also found higher values for antioxidant activity using
the ABTS method. The clone wines showed a significant difference 8

(P < 0.05) in antioxidant capacity, clone 169 from the two vineyards 6
evaluated showing the highest antioxidant activity for the three 4
methods, which is consistent with the higher phenolic content 2
found in these wines. 0
Statistical analysis showed positive correlations between the A685 A169 B685 B169
antioxidant activity determined by the three methods (DPPH, ABTS wines
and FRAP) and each individual phenolic compound determined in
Fig. 1. Antioxidant activity expressed as Trolox equivalents (mmol TEAC/L wine) for
this study. This is in agreement with the results of other studies that Cabernet Sauvignon wines from clones 169 and 685 and vineyards A and B. The results
confirm the antioxidant capacity of wine is dependent on the are expressed as means  standard deviation over three replication for each wine
phenolic composition (Zafrilla et al., 2003), however, there are sample. Clones with different letters for the same sample represent significant
disagreement in the literature regarding the main compounds that differences (P < 0.05) between the methods (L DPPH; ABTS; FRAP).
 V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938
Fig. 2. Principal Components Analysis (PCA) with the results for antioxidant activity
and phenolic compounds for Cabernet Sauvignon wines, clones 169 and 685, A and B
vineyards. QUER, quercetin; GAE, gallic acid; CUM, p-coumaric acid; TP, total popy-
phenol; CAT, catechin; FRAP, ferric reducing ability of plasma; ABTS, 2,2-azino-bis(3
ethylbenzothiazoline-6-sulphonic acid); DPPH, 1,1-diphenyl-2-picrylhydrazyl; CAF,
caffeic acid; RESV, trans-resveratrol; DELF, delphinidin-3-glucoside; MALV, malvidin-3-
glucoside; PEON, peonidin-3-glucoside; TMA, total monomeric anthocyanins.
Anthocyanins are the compounds which showed the lowest
correlation with the antioxidant activity, as also evidenced by
Sánchez-Moreno, Cao, Ou, and Prior (2003) for the total antho-
cyanin content. Other polyphenols have also shown positive
correlations, however with R < 0.50, as also evidenced by Di Majo
et al. (2008) who correlated the individual phenolic compounds
with the antioxidant capacity of wines and obtained R values
of <0.55.
3.1.4. Multivariate analysis
The evaluation of Cabernet Sauvignon wine samples was carried
out by data analysis using multivariate techniques in order to verify
whether the clones 169 and 685 showed different characteristics.
The separation was obtained using principal component analysis
(PCA) (Fig. 2) and cluster analysis (Fig. 3), and were performed
using flavonoid and non-flavonoid phenolic compounds deter-
mined by HPLC, antioxidant activity (ABTS, DPPH and FRAP
methods), total monomeric anthocyanins and total polyphenols.
The wine samples were clearly separated by two functions
(PC1  PC2) (Fig. 2), which explain 81.43% of the total data vari-
ability. It may be noted that the wine samples were separated
according to type of clone considering PC1 which explained most of
the variability of the data. Wines from clone 685 are positively
located in relation to CP1, while wines from clones 169 are nega-
tively located. Regarding the variables analyzed, there was sepa-
ration from PC1, where anthocyanins were located positively and
negatively in relation to polyphenols and antioxidants, respectively.
Wines from clone 685, A and B vineyards, showed positive corre-
lations with all anthocyanins, higher color characteristics being
observed for the wines produced with this clone. However, wines
from clone 169 showed higher positive correlations with antioxi-
dant activity and polyphenols, mainly catechin, p-coumaric and
caffeic acids. These data are in agreement with those described in
Tables 1 and 3.
Cluster analysis was carried out by Ward’s method (Galgano,
Favati, Caruso, Scarpa, & Palma, 2008) (Fig. 3) and graphical
1935
700
600
Distancia euclidiana (%)
500
400
300
200
100
A-685 B-685 A-169 B-169
Fig. 3. Dendogram of cluster analysis using total polyphenols, individual phenolic
content determined by HPLC and antioxidant activity for Cabernet Sauvignon wines
produced with 169 and 685 clones, from vineyards A and B.
representation was presented in the form of a dendrogram, where
the separation criterion was Euclidean distance (%). It was possible
to clearly observe the formation of two homogenous groups. In one
group there are wines produced with clone 169 from the two
vineyards (A and B) and the other group contains the wines of clone
685. These results suggest that the phenolic profile determined in
this study was sufficient to differentiate between the wines
produced with different clones, indicating that each clone showed
particular characteristics which are transferred to the wines.
3.2. Evolution of the phenolic compounds during bottle ageing
Changes in the phenolic content of the wines produced with
clones 169 and 685 from the same vineyard (A) stored in bottles
were monitored over 11 months (Figs. 4 and 5). The non-
polymerized polyphenols decreased while polymerized poly-
phenols increased during the bottle ageing of the wine, which was
also evidenced by Monagas et al. (2006) and Roginsky et al. (2006)
in research on red wines.
Among non-flavonoid compounds, the behaviors of the main
hydroxycinnamic acids (p-coumaric, caffeic and ferulic) were
similar for the two wines, with decreasing concentrations during
wine ageing. This behavior was also observed by other researchers
(Gutiérrez, Lorenzo, & Espinosa, 2005; Monagas et al., 2005), and
probably occurs through the formation of copigments with
anthocyanins. Also, in the wines studied, a reduction of half in the
gallic acid concentration was observed, and the greatest decrease in
the concentration was between the fourth and eighth months of
storage. Contrasting data are presented in the literature in relation
to gallic acid content during wine ageing. Results similar to those
found in this study were reported by García-Falcón et al. (2007) for
red wines and by Hernanz et al. (2009) for white wines. However,
some researchers, such as Gutiérrez et al. (2005) and Monagas et al.
(2006), assessed Cabernet Sauvignon wines and observed an
increase in gallic acid concentration over time. Revilla and
González-Sanjosé (2003) found no significant changes in the
gallic acid concentration after 24 months of ageing.
The flavonoid compounds, catechin and quercetin, showed
different behaviors over time, with a decrease in catechin concen-
tration and an increase in the quercetin content in both wines. The
catechin concentration at the end of 11 months was close to zero.
 (A-685) (A-169)
2400
a 2400
2200
2200
2000
2000

polyphenol content (mg/L)

polyphenol content (mg/L)

1800
1800
1600
1600

1400 1400

1200 1200

1000 1000

800 800

600 600

400 400
0 2 4 6 8 10 12 0 2 4 6 8 10 12

35 35
b
30 30

25 25

non-flavonoids (mg/L)
non-flavonoids (mg/L)

20 20

15 15

5
1.5
4
1.0
3
0.5 2

0.0 1
0
-0.5
0 2 4 6 8 10 12 0 2 4 6 8 10 12

c 60 60

50 50

40
40
flavonoids (mg/L)

flavonoids (mg/L)

30
30

20
20

10
10

0
0 2 4 6 8 10 12 0
0 2 4 6 8 10 12

d 22 22

20 20
antioxidant activity (mmol/L)

antioxidant activity (mmol/L)

18 18

16 16

14 14

12
12

10
10

0 2 4 6 8 10 12
0 2 4 6 8 10 12
T im e (m onth)
T im e (m o n th )

Fig. 4. Evolution of phenolic content and antioxidant activity (DPPH and ABTS methods) during bottle ageing (11 months) of Cabernet Sauvignon wines from clones 169 and 685
grown in the same vineyard (A). The results are expressed as means  standard deviation over three replication for each wine sample. (a) (e,e) total popyphenol; (eBe)
polymerized polyphenols; (e6e) non-polymerized polyphenols. (b) (e,e) gallic acid; (eBe) caffeic acid; (e6e) ferulic acid; (e7e) p-coumaric acid. (c) (e,e) catechin;
(eBe) quercetin. (d) (eBe) ABTS (2,2-azino-bis(3 ethylbenzothiazoline-6-sulphonic acid)); (e,e) DPPH (1,1-diphenyl-2-picrylhydrazyl).
 V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938
(A-685)
180
160
140
120
anthocyanins (mg/L)
100
80
40
20
0 0
0 2 4 6 8 10
Time (month)
Fig. 5. Evolution of the individual anthocyanins during bottle ageing (11 months) in Cabernet Sauvignon wines obtained from grape clones 169 and 685, and produced at the same
vineyard (A). The results are expressed as means  standard deviation over three replication for each wine sample. (eBe) malvidin-3-glucoside; (e9e) delphinidin-3-glucoside;
(e6e) peonidin-3-glucoside; (e,e) total monomeric anthocyanins.
This decrease during storage has also been reported by Gutiérrez
et al. (2005) who evaluated red wines and by Hernanz et al. (2009)
for white wines during bottle ageing (12 months). Research has
shown that derivatives of the flavonol glycosides decreased during
ageing. As a result, an increase in the corresponding aglycones could
occur due to the hydrolysis of glycosides (Gutiérrez et al., 2005;
Zafrilla et al., 2003). Thus, this may explain the increase in the
quercetin concentration observed in our study. García-Falcón et al.
(2007) evaluated the quercetin content in wines immediately after
malolactic fermentation for three months, and also after one year of
bottle ageing, and it was possible to quantify quercetin in the wine
after one year of bottling. Cadahía et al. (2009) observed this
behavior for myricetin, which belongs to the same class as quercetin,
and the content increased during the 12 month evaluation period,
with a consequent reduction in glycosylated derivatives.
The antioxidant activity, determined by the DPPH and ABTS
methods showed a significant increase during bottle ageing. The
behaviour was similar in both methods probability due to their being
methods that work with the same antioxidant mechanism mediated
by electron transfer. The information in the literature regarding the
antioxidant activity evolution during wine ageing is still very con-
flicting. Studies by Zafrilla et al. (2003) showed no evidence of
changes in antioxidant activity using these methods, during seven
months of storage for red wines. De Beer, Joubert, Gelderblom, and
Manley (2005) affirm that antioxidant activity decreases over
time. However, there are reports in the literature which are in
agreement with our findings, demonstrating that antioxidant
activity increases during wine storage (Kallithraka, Salacha, &
Tzourou, 2009) because the compounds present in these wines
have greater ability to capture free radicals compared with young
wines. Characteristically, this means that the number of active-OH
groups, responsible for antioxidant activity, remain unchanged
under anaerobic conditions, which may have contributed to the
antioxidant activity of the wines not decreasing over time.
As expected, we observed a decrease in the individual antho-
cyanin concentrations in the wine samples (Fig. 5), and this
decrease was also evidenced for total monomeric anthocyanins
content during the time assessed. Gutiérrez et al. (2005) also
observed a decrease (68%) in the monomeric anthocyanin
concentration of Cabernet Sauvignon wine after 9 months of bottle
ageing. This decrease is consistent with the involvement of these
1937
(A-169)
180
160
140
120
anthocyanins (mg/L)
100
80
20
12 0 2 4 6 8 10 12
Tim e (m onth)
compounds in numerous condensation reactions during the
storage period, as well as in hydrolytic reactions. Bakker, Picinelli,
and Bridle (1993) investigated the anthocyanin evolution in
a model wine solution, in order to avoid interference from
constituents of the medium, and observed a logarithmic decrease
in the monomeric anthocyanins concentration during storage. The
fastest changes in the color composition occurred during the first
year of storage, where a color modification from bright red, typical
of young wines, to brown red occurred. These changes are mainly
caused by the disappearance of monomeric pigments and the
appearance of more stable oligomeric forms. Gutiérrez et al. (2005)
affirm that the main reaction involving monomeric anthocyanins,
which results in a decrease in the concentration, is the formation of
polymeric pigments by condensation with other phenolic
compounds, mediated and accelerated by acetaldehyde. However,
there is also the formation of new pigments derived from antho-
cyanins, called pyranoanthocyanins, originated from the reaction
between monomeric anthocyanins, particularly malvidin-3-
glucoside, and compounds present in the wines, especially
hydroxycinnamic acids (Rentzsch, Schwarz, & Winterhalter, 2007).
4. Conclusions
The wines showed different characteristics according to the
clones from which they were produced. Clone 169 showed the
presence of polyphenols and clone 685 had a predominance of
anthocyanins regardless of the vineyard. These results were
confirmed by multivariate analysis. The results of this study show
that the determination of the main phenolic compounds may be
used as a tool for wine classification and for discrimination
between different clones. The wines presented high antioxidant
activity for the three methods evaluated and the best results were
obtained with the ABTS method. Positive correlations were found
for all phenolic compounds and antioxidant activity, catechin
showing the highest correlation followed by total polyphenols and
polymerized polyphenols.
During bottle ageing a decrease in the concentration of total and
non-polymerized polyphenols in the wines was observed, with
a consequent increase in the polymerized polyphenol content.
Flavonoid and non-flavonoid phenolic compounds also decreased
over the ageing period evaluated, with the exception of quercetin
1938 V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938
which increased in concentration. The antioxidant capacity of
wines increased during ageing according to the two methods
evaluated. The evolution of the different phenolic compounds was
very similar for the wines produced with different clones, indi-
cating that the changes were mostly due to the ageing process.
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