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
Vvian Maria Burin a, La Luzia Freitas Costa b, Jean Pierre Rosier c, Marilde T. Bordignon-Luiz a, *
a

Departamento de Cincia e Tecnologia de Alimentos CAL/CCA, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, CEP: 88034-001, Itacorubi,
Florianpolis, SC, Brazil
b
Laboratrio Central de Sade Pblica e LACEN, Santa Catarina, Brazil
c
Empresa de Pesquisa e Extenso Agropecuria de Santa Catarina (EPAGRI-SC) Estao, 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:
Received 24 September 2010
Received in revised form
2 May 2011
Accepted 3 May 2011

Phenolic compounds constitute important quality parameters of wines. Wines produced from different
clones of the same grape variety show differences in relation to their chemical composition. 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. All wines were produced with Cabernet Sauvignon grapes, clones 685
and 169, from two vineyards, under the same microvinication conditions. The wines were characterized
in relation to phenolic composition and antioxidant activity, as well as monitored over 11 months of
bottle ageing. A signicant difference was observed between the chemical compositions of the wines
produced from clones 169 and 685, clone 169 showing the highest phenolic content while clone 685 had
better color characteristics. The wines showed high antioxidant activity. Principal components and
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.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Red wine
Clones
Bottle ageing
Phenolic compounds
Antioxidant activity

1. Introduction
Phenolic compounds play an important role in grape and wine
quality, contributing to sensory properties such as color, astringency and bitterness. Polyphenols from wine can be classied into
two groups: non-avonoid 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 avonoid compounds (anthocyanins, avanols
and avonols) which contribute to the wine color since anthocyanins are the main pigments of red wine. Besides these functions,
the chemical structure of polyphenols, mainly avonoids and stilbenes (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
benecial effects on health, since in addition to antioxidant
capacity it also has anti-inammatory and anticarcinogenic effects,
among others (Frankel, Bosanek, Meyer, Silliman, & Kirk, 1998).

* Corresponding author. Tel.: 55 48 3721 5376; fax: 55 48 3721 9943.

E-mail address: bordign@cca.ufsc.br (M.T. Bordignon-Luiz).
0023-6438/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2011.05.001

Many factors inuence the chemical composition of wine, such

as grape variety, degree of grape ripeness at harvest, climatic
conditions, cultural practices and technology, affecting the nal
quality of wine. Moreover, studies have shown that different clones
of the same grape variety also have signicant differences in relation to their chemical composition. It has been shown that some
clones have the capacity to produce wines with distinct color,
aromatic prole and phenolic content (Zamuz, Martnez, &
Vilanova, 2007). Clonal selection has allowed signicant gains in
viticulture by allowing the exploration of an important source of
genetic variability, resulting in specic 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, oxidation, polymerization and copigmentation. During storage, these
reactions would presumably lead to changes in the antioxidant

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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, Gmez-Cordovs, & Bartolom, 2006).
Studies have been conducted to establish the evolution of
different families of phenolic compounds during wine ageing, in
the barrel or the bottle (Cadaha, Simn, Sanz, Poveda, & Colio,
2009; Garca-Falcn, Prez-Lamela, Martnez-Carbalho, & SimalGndara, 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 ()catechin, p-coumaric acid and morin (20 ,3,40 ,5,7-penthydroxyavone),
DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS [2,2-azino-bis(3 ethylbenzothiazoline-6-sulphonic acid) and trans-resveratrol were
purchased from SigmaeAldrich (St. Louis, MO, USA), quercetin, ferulic
acid, caffeic acid, FolineCiocalteu were obtained from Fluka (Steinheim, 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 So 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. classications and the weather of
the So Joaquim region is classied as Cool, Cool nights and
Humid according to the Geoviticulture Multicriteria Climatic
Classication 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 microvinication
conditions at the state agricultural research center - Empresa de
Pesquisa e Extenso Agropecuria 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 sulting agent (20 g per 100 kg of must, corresponding 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 commercial 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 nished, 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 microvinication. 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 measurements 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 polyphenols, trans-resveratrol and anthocyanins, wine samples were
ltered through a 0.45 mm PTFE membrane lter modied with
13 mm of diameter (Millipore, Bedford, MA) and directly injected
into the HPLC.
HPLC separation of the phenolic compounds (catechin, quercetin, 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 ltered 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 ow 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 ow rate was 1.2 mL/L and detection was at
306 nm.
Anthocyanin analysis was carried out according to Garca-Falcn
et al. (2007) with modication 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 ow rate was 0.8 mL/L, with
detection at 520 nm.
Flavonoid and non-avonoid phenolic compounds were identied 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 quantication. Limits of detection (LOD) and quantication (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
TP*
PP*
NPP*
TMA*
CIy
CTy

2114.95a
1293.66a
878.71a
185.22a
12.03a
0.73a

A-169







10.28
24.87
19.45
3.72
0.02
0.01

2431.66b
1458.44b
834.34b
164.08b
10.89b
0.80b

B-685







B-169

2205.74c
1319.56c
736.03c
209.33c
12.73c
0.63c

29.65
9.32
6.94
3.26
0.01
0.01








2569.81d
1516.69d
753.01d
167.67d
11.72d
0.78b

27.96
17.98
11.25
1.69
0.01
0.01








32.46
21.36
17.41
2.59
0.02
0.01

*mg/L.
y
expressed as index.
Means  SD of three replication for each wine sample. Different letters in the same line represent signicant 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-2picrylhydrazyl) radical activity was measured through the extinction of the maximum absorption at 517 nm (Kim, Guo, & Packer,
2002). The ABTS [2,2-azino-bis(3 ethylbenzothiazoline-6sulphonic 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. Inuence 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), anthocyanins, 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 vinication 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.
3. Results and discussion
3.1. Wine characterization
3.1.1. Total content of the phenolic families and color parameters
Total content of the phenolic families in the wine sample were
determined by spectrophotometric methods (Table 1) and signicant differences could be observed (P < 0.05) between wine
samples from the clones of the Cabernet Sauvignon grapes. In
relation to the content of polyphenolic compounds, such as TP and
PP, the clone 169 showed the highest values, independent of the
vineyard. In contrast, the highest values for TMA content was
observed for clone 685 from both vineyards. The higher anthocyanins content contributed to these wines having higher CI and

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-avonoid 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 (GarcaFalcn et al., 2007).
Flavonoid and non-avonoid 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-avonoid compounds studied, regardless
of the vineyard. Wines produced with clone 685 from the two
vineyards studied showed the highest concentrations of monomeric 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.
R2

LOD* LOQ* rec

RSD Rt

90.0
50.0
50.0
150.0
75.0

0.9999
0.9978
0.9926
0.9992
0.9991

0.07
0.03
0.01
0.04
0.05

0.23
0.10
0.10
0.12
0.15

94.3
92.3
90.1
93.2
93.4

2.2
2.1
1.9
1.3
2.5

32.5
19.2
30.0
23.2
45.0

25.0
80.0
75.0
30.0
30.0

0.9984
0.9981
0.9991
0.9998
0.9998

0.05
0.32
0.02
0.04
0.08

0.10
0.97
0.06
0.05
0.24

95.6
88.4
98.8
101.9
102.6

1.9
2.2
1.7
2.6
2.5

11.3
10.0
25.6
34.7
31.5

Compounds

Range*

Flavonoids
malvidin-3-glucoside
delphinidin-3-glucoside
peonidin-3-glucoside
catechin
quercetin

0.9
1.0
0.2
0.3
0.3

e
e
e
e
e

Non-avonoids
trans-resveratrol
gallic acid
caffeic acid
ferulic acid
p-coumaric acid

0.1
8.0
0.3
0.3
0.3

e
e
e
e
e

LOD: limit of detection; LOQ: limit of quantication; Rec: recovery (%); RSD: relative
standard deviation (%); Rt: retention time (min).
* mg/L.

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V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938

Table 3
Content of main avonoid and non-avonoid phenolic compounds (mg/L) in Cabernet Sauvignon wines, clones 169 and 685, A and B vineyards determined by HPLC.
Compounds
Flavonoids
malvidin3glucoside
delphinidin3glicosdeo
peonidin3glucoside
catechin
quercetin
Non-avonoids
trans-resveratrol
gallic acid
caffeic acid
ferulic acid
p-coumaric acid

A-685

A-169

B-685

B-169

35.70a
4.27a
17.32a
17.51a
38.69a







0.15
0.08
0.20
0.12
1.11

32.32b
3.76b
14.07b
50.42b
15.36b







0.61
0.03
0.18
0.85
0.96

42.21c
7.1c
22.85c
9.96c
24.56c







0.27
0.12
0.45
0.05
0.12

31.71d  0.09
3.26d  0.08
14.12d  0.15
39.23d  0.33
52.61d  1.25

0.75a
30.29a
1.56a
0.08a
1.47a







0.02
0.06
0.09
0.01
0.04

1.52b
32.63b
5.16b
0.12b
2.50b







0.04
0.71
0.38
0.01
0.03

0.65c
31.52c
1.79c
0.05c
0.79c







0.02
0.33
0.05
0.01
0.02

1.89d  0.03
44.22d  0.12
1,84d  0.16
0.14b  0.01
6.80d  0.09

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

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
ability to reducing to iron. The antioxidant activity found through
different methods for Cabernet Sauvignon wines, produced with
two clones (Fig. 1), showed a signicant difference (P < 0.05)
between methods, with the exception of sample B-685 which
showed no difference using the DPPH and FRAP methods. For all
wine samples, the highest antioxidant activity was obtained with
the ABTS method. Researches carry out by Li et al. (2009) and Burin
et al. (2010) also found higher values for antioxidant activity using
the ABTS method. The clone wines showed a signicant difference
(P < 0.05) in antioxidant capacity, clone 169 from the two vineyards
evaluated showing the highest antioxidant activity for the three
methods, which is consistent with the higher phenolic content
found in these wines.
Statistical analysis showed positive correlations between the
antioxidant activity determined by the three methods (DPPH, ABTS
and FRAP) and each individual phenolic compound determined in
this study. This is in agreement with the results of other studies that
conrm the antioxidant capacity of wine is dependent on the
phenolic composition (Zafrilla et al., 2003), however, there are
disagreement in the literature regarding the main compounds that

act as antioxidants. The ABTS method showed higher correlation

with the polyphenols than the DPPH and FRAP methods for the
wines, a nding also reported by Burin et al. (2010) and
Rivero-Prez, Gonzlez-Sanjos, Ortega-Hers, and Muiz (2008).
Catechin was the main phenolic compound, giving the highest
correlation with antioxidant activity for the three methods evaluated, with a Pearson correlation coefcient (R) greater than 0.9,
which was also reported by other researchers who afrm 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 avanol found in wines of different varieties (GmezAlonso, 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 Aln-Ruiz, Garca-Falcn, PrezLamela, Martnez-Carballo, and Simal-Gndara (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 activities of different polyphenolic classes.

24

b
b

22
20

18
antioxidant activity
(mmol/L)

anthocyanins composition differs signicantly 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 Sauvignon 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, Surez, Gmez-Cordovs, &
Bartolom, 2005). The trans-resveratrol concentration was also
signicant 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 conducted by Cadaha et al. (2009). Souto et al. (2001) determined transresveratrol 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, vinication process
(which inuences 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.

16
14
12

10
8
6
4
2
0
A685

A169

B685

B169

wines
Fig. 1. Antioxidant activity expressed as Trolox equivalents (mmol TEAC/L wine) for
Cabernet Sauvignon wines from clones 169 and 685 and vineyards A and B. The results
are expressed as means  standard deviation over three replication for each wine
sample. Clones with different letters for the same sample represent signicant
ABTS;
FRAP).
differences (P < 0.05) between the methods (L DPPH;

V.M. Burin et al. / LWT - Food Science and Technology 44 (2011) 1931e1938

1935

Distancia euclidiana (%)

700

600

500

400

300

200

100
A-685

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 popyphenol; 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-3glucoside; 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
Snchez-Moreno, Cao, Ou, and Prior (2003) for the total anthocyanin 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 avonoid and non-avonoid phenolic compounds determined 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 variability. 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 negatively located. Regarding the variables analyzed, there was separation 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 correlations 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 antioxidant 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 Wards method (Galgano,
Favati, Caruso, Scarpa, & Palma, 2008) (Fig. 3) and graphical

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 prole determined in
this study was sufcient 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 nonpolymerized polyphenols decreased while polymerized polyphenols 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-avonoid 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
(Gutirrez, 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 Garca-Falcn et al. (2007) for
red wines and by Hernanz et al. (2009) for white wines. However,
some researchers, such as Gutirrez et al. (2005) and Monagas et al.
(2006), assessed Cabernet Sauvignon wines and observed an
increase in gallic acid concentration over time. Revilla and
Gonzlez-Sanjos (2003) found no signicant changes in the
gallic acid concentration after 24 months of ageing.
The avonoid compounds, catechin and quercetin, showed
different behaviors over time, with a decrease in catechin concentration 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

(A-169)
2400

2400

2200

2200

polyphenol content (mg/L)

polyphenol content (mg/L)

2000
1800
1600
1400
1200
1000
800

1600
1400
1200
1000
800

400

400
0

10

12

35

35

30

30

25

25

non-flavonoids (mg/L)

non-flavonoids (mg/L)

1800

600

600

2000

20
15

1.5
1.0

10

12

15
5
4
3
2

0.0

1
0
0

10

12

60

60

50

50

40

flavonoids (mg/L)

flavonoids (mg/L)

20

0.5

-0.5

30

20

10

12

40

30

20

10

10
0
0

10

12

22

22

20

20

antioxidant activity (mmol/L)

antioxidant activity (mmol/L)

18
16
14
12
10

10

12

18
16
14
12
10

T im e (m onth)

10

12

10

12

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)

(A-169)

180

180

160

160

140

140

120

120

anthocyanins (mg/L)

anthocyanins (mg/L)

1937

100
80
40

20

100
80

20

0
0

10

12

10

12

Tim e (m onth)

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 Gutirrez

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 avonol glycosides decreased during
ageing. As a result, an increase in the corresponding aglycones could
occur due to the hydrolysis of glycosides (Gutirrez et al., 2005;
Zafrilla et al., 2003). Thus, this may explain the increase in the
quercetin concentration observed in our study. Garca-Falcn 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. Cadaha 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 signicant 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 conicting. 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) afrm that antioxidant activity decreases over
time. However, there are reports in the literature which are in
agreement with our ndings, 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 anthocyanin concentrations in the wine samples (Fig. 5), and this
decrease was also evidenced for total monomeric anthocyanins
content during the time assessed. Gutirrez 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

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 rst
year of storage, where a color modication 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. Gutirrez et al. (2005)
afrm 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 anthocyanins, called pyranoanthocyanins, originated from the reaction
between monomeric anthocyanins, particularly malvidin-3glucoside, 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
conrmed 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 classication 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-avonoid 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, indicating that the changes were mostly due to the ageing process.
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