Phenolic composition and physicochemical parameters of Carmnre, Cabernet

Sauvignon, Merlot and Cabernet Franc grape seeds (Vitis vinifera L.) during
ripening
Elas Obreque-Slier
a
, Remigio Lpez-Sols
b
, Lorena Castro-Ulloa
a
, Cristian Romero-Daz
a
,
lvaro Pea-Neira
a,
*
a
Department of Agro-Industry and Enology, Faculty of Agronomical Sciences, University of Chile, P.O. Box 1004, Santiago, Chile
b
Program of Cellular and Molecular Biology, Faculty of Medicine-ICBM, University of Chile, Independencia 1027, Santiago, Chile
a r t i c l e i n f o
Article history:
Received 25 October 2011
Received in revised form
30 January 2012
Accepted 6 February 2012
Keywords:
Proanthocyanidins
Anthocyanins
Ripeness
HPLCeDAD
a b s t r a c t
Phenolic composition and some physicochemical parameters of seeds from grape cultivars (Vitis
vinifera L.) Carmnre (CA), Merlot (M), Cabernet Franc (CF) and Cabernet Sauvignon (CS) were
evaluated at four different ripening stages. Compositional differences between cultivars were observed
only in some ripening stages. Nevertheless, signicant differences between CA and CF were observed
throughout all the study period (veraison to harvest) in a number of parameters, including seed
weight, total tannins and polymeric avanol fraction. Each of the four grape varieties showed char-
acteristic sets of colorimetric coordinates (CIElab parameters) at any of the ripening stages. On the
other hand, while M seeds presented a high concentration of both ()-catechin, ()-epicatechin,
epicatechin-3-O-gallate, proanthocyanidin trimers 1, B3 and B4, CA seeds presented characteristic high
concentrations of a series of other proanthocyanidins. CF seeds displayed the highest concentrations of
gallic acid and proanthocyanidin gallates. Altogether, we conclude that Carmnre, Merlot, Cabernet
Franc and Cabernet Sauvignon grape seeds present marked differences in phenolic composition during
ripening.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Phenolic compounds are important secondary metabolites that
are responsible of important properties in foods and drinks, such as
color, aroma, bitterness and astringency (Brossaud, Cheynier, &
Noble, 2001). Seeds are a major source of phenolic compounds in
Vitis vinifera L. grape berries (Kennedy, Troup, et al., 2000; Roby,
Harbertson, Adams, & Mattews, 2004). The synthesis of phenolic
compounds in seeds during ripening depends on edaphic, climatic,
cultural and genetical factors (Downey, Harvey, & Robinson, 2003;
Haselgrove et al., 2000; Kennedy, Troup, et al., 2000; Ollat et al.,
2002). Previous reports have shown a signicant varietal effect in
the evolution and concentration of phenolic compounds in seeds of
different grape berry varieties during ripening (Guendez,
Kallithraka, Makris, & Kefalas, 2005; Guerrero et al., 2009;
Monagas et al., 2003; Rodrguez-Montealegre, Romero-Peces,
Chacn-Vozmediano, Martnez-Gascuea, & Garca-Romero,
2006; Santos-Buelga, Francia-Aricha, & Escribano-Bailn, 1995;
Sun, Ricardo da Silva, & Spranger, 1998).
Most of the grape varieties currently used in wine production
all over the world are derived from Vitis vinifera L. Nowadays, the
varietal patrimony of Vitis vinifera L. comprises over 5000 varie-
ties, which have been grouped in families comprising one or
several members sharing one or several common features. The
Carmenets grape family includes several diversely known varie-
ties, namely, Merlot, Cabernet Franc, Cabernet Sauvignon, Petit
Verdot, Fer Servadou and Carmnre (Reynier, 2002). At one of
this group of grape varieties, Cabernet Sauvignon has been widely
studied because of its world-wide distribution (Guendez et al.,
2005; Guerrero et al., 2009; Kennedy, Matthews, & Waterhouse,
2000; Monagas et al., 2003; Pea-Neira et al., 2004; Roby et al.,
2004 ; Rodrguez-Montealegre et al., 2006; Santos-Buelga et al.,
1995). At the other end, the Carmnre variety, a late-maturing
variety that was devastated from the European vineyards in
1863 due to the phylloxera epidemic, is much less known. In effect,
this variety was rediscovered in Chile in 1994, after years of being
confounded with Merlot and Cabernet Franc, two varieties with
similar ampelographic characteristics. Nowadays, Carmnre is
* Corresponding author. Tel.: 562 9785730; fax: 562 9785796.
E-mail address: apena@uchile.cl (. Pea-Neira).
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doi:10.1016/j.lwt.2012.02.007
LWT - Food Science and Technology 48 (2012) 134e141
cultivated in Chile as single vineyards with a total production area
of 7283 hectares (Servicio Agrcola y Ganadero, 2008). On the
other hand, despite the major commercial importance of the
Carmenets family, comparative information on the phenolic
composition of those varieties is scarce. Studies in that regard have
been limited to only two of those varieties (Cabernet Sauvignon
versus Carmnre) (Fernndez, Kennedy, & Agosin, 2007;
Obreque-Slier et al., 2010). Considering that both evolution and
concentration of phenolic compounds in seeds is dependent upon
the grape variety, the objective of the present study was to eval-
uate the phenolic composition and physicochemical parameters of
grape seeds of the Carmenets varieties Carmnre, Merlot,
Cabernet Franc and Cabernet Sauvignon during ripening, all of
them grown under similar edaphoclimatic and cultural conditions.
Through this study, we expect to gain insights into the varietal
effect in the evolution of phenolic compounds during ripening
among closely related Vitis vinifera L. grape varieties, including the
less known Carmnre.
2. Materials and methods
2.1. Materials and instrumentation
Standards of gallic acid (G-7384), ()-catechin (C-1251),
()-epicatechin (E-1753) and ()-epicatechin-3-O-gallate (E-
3893), as well as phloroglucinol (P-3502) and 0.45-mm pore size
membranes were acquired from Sigma Chemical Company, Saint
Louis, Missouri, USA. Vanillin, triuoroacetic acid, ethyl acetate,
HPLC grade acetonitrile and pro-analysis grade solvents were
purchased from Merck, Darmstadt, Germany. Sep-Pak Plus tC
18
cartridges WAT 036810 and WAT 036800 were obtained from
Waters (Milford, MA, USA).
The HPLC system (Agilent Technologies Santa Clara, CA, USA)
consisted of a photodiode-array detector Model G1315B, a pump
Model Quat G1311A and an autosampler Model ALS G1329A. A
reversed phase Nova Pack C
18
column (4 mm, 3.9 mm ID 300 mm;
Waters Corporation, Milford, MA, USA) was used for HPLCeDAD
analysis of individual phenolic compounds. A reversed phase
LiChro Cart 100 RP-18 column (5 mm, 4 mm ID 250 mm; Agilent
Technologies, Santa Clara, CA, USA) was used in phloroglucinolysis
studies. Absorbances were measured using a Jasco UVeVis spec-
trophotometer Model V-530 (JASCO International Co., Ltd., Tokyo,
Japan).
2.2. Grape samples
Self-rooted grapevine (Vitis vinifera L.) cultivars Carmnre,
Merlot, Cabernet Franc and Cabernet Sauvignon vines, vintage
2008, planted in 2001 and grown in the Maipo Valley at the
Metropolitan Region of Chile were studied. Agronomic, photo-
chemical and technical variables were controlled. Three groups of
120 berries per variety were selected from 4 clusters per plant
among a total of 30 plants. The samples were collected monthly
from February (veraison) to May. The phenolic compounds were
extracted from grape seeds as described in previous reports
(Obreque-Slier et al., 2010; Pea-Neira et al., 2004).
2.3. Spectrophotometric characterization
Total phenolic content was determined by UV-absorptiometry at
280 nm (Glories, 1984) using gallic acid as standard. Total tannin
content was measured by the method of Ribreau-Gayon and
Stonestreet (1966). The CIElab parameters were determined
according to the method of Ayala, Echvarri, and Negueruela (1997).
2.4. Fractionation of proanthocyanidins into monomers, oligomers
and polymers
Grape seed extracts were fractionated by chromatography on
Waters C
18
Sep-Pak cartridges according to the method described
by Sun, Leandro, et al. (1998). Briey, 5 mL of seed extracts were
concentrated to dryness in a rotary evaporator at <30

C. The
residue was dissolved in 20 mL of 67 mmol/L phosphate buffer pH
7.0. The pH of the resulting solution was adjusted to 7.0 with either
NaOH or HCl, as necessary. Two C
18
Sep-Pak cartridges were
assembled (WAT 36800 on top and WAT 36810 at the bottom) and
conditioned sequentially with methanol (10 mL), distilled water
(2 10 mL) and phosphate buffer pH 7.0 (10 mL). Samples were
passed through the cartridges at a ow rate not higher than 2 mL/
min and phenolic acids were then eliminated by elution with 10 mL
of 67 mmol/L phosphate buffer at pH 7.0. The cartridges were dried
with nitrogen gas and eluted sequentially with 25 mL of ethyl
acetate (fraction FI FII containing monomeric and oligomeric
avan-3-ols) and with 15 mL of methanol (fraction FIII containing
polymeric proanthocyanidins). The ethyl acetate eluate was taken
to dryness under vacuum, redissolved in 3 mL of 67 mmol/L
phosphate buffer, pH 7.0, and re-loaded onto the same series of
cartridges that had been conditioned again as described above. The
cartridges were dried by passing a stream of gas nitrogen and
eluted sequentially with 25 mL of diethyl ether (fraction FI con-
taining monomers) and 15 mL of methanol (fraction FII containing
oligomers). Fractions FI, FII and FIII were evaporated to dryness
under vacuum and redissolved in 3 mL of methanol. The total
content of avan-3-ols in each fraction was determined by the
vanillin assay (Sun, Ricardo da Silva, et al., 1998).
2.5. Phloroglucinolysis
Flavanol isolates were characterized by acid-catalysis in the
presence of an excess of phloroglucinol followed by reversed phase
HPLC (phloroglucinolysis) using a previously described method
(Kennedy, Troup, et al., 2000 with some modications by Obreque-
Slier et al., 2010). This assay provides information on mean degree
of polymerization (mDP), percentage of galloylation (%G) and
average molecular weight (aMW) of avanols. Briey, a 3-mL
aliquot of a grape seed extract was concentrated under reduced
pressure, furtherly concentrated to dryness using a rotary evapo-
rator at <30

C and dissolved again in 2.5 mL of methanol (Downey
et al., 2003). One-mL aliquots of each of these proanthocyanidin
solutions were allowed to react with 1 mL of solution C (0.25 g of
ascorbic acid, 1.25 g of phloroglucinol and 215 mL of concentrated
hydrochloride acid in 25 mL of methanol) at 50

C for 20 min. The
reaction was stopped with 1 mL of 200 mmol/L sodiumacetate. The
chromatographic separation by HPLC used a binary-gradient with
mobile phases (owrate 1.0 mL/min) containing 10 mL/L acetic acid
(mobile phase A) and methanol (mobile phase B). Elution was
monitored by UV detection at 280 nm. The elution prole was as
follows: 0e15 min, 100% A; 15e35 min, 95e80% A (linear gradient);
35e61 min, 80e60% A (linear gradient); 61e71 min, 10% A. The
column was nally equilibrated with 100% A for 6 min before the
following chromatographic separation.
2.6. HPLCeDAD chromatography and analysis of individual
phenolic compounds
Grape seed extracts of phenolic compounds were re-extracted
with ethyl ether (3 20 mL) and ethyl acetate (3 20 mL)
(Obreque-Slier et al., 2010; Pea-Neira et al., 2004). The resulting
extracts were evaporated to dryness at 30

C, redissolved in 2 mL of
methanol/water (50:50 mL:mL) and membrane-ltered (0.45 mm
E. Obreque-Slier et al. / LWT - Food Science and Technology 48 (2012) 134e141 135
pore size). Fifty-mL aliquots of the nal solution were subjected to
reversed-phase chromatographic separation at 20

C using a Nova
Pack C
18
column. A photodiode-array detector was set at 280 nm.
Two mobile phases were used: A, water/acetic acid (98:2 mL:mL)
and B, water/acetonitrile/acetic acid (78:20:2 mL:mL:mL). A two-
step gradient was carried out at a constant ow rate of 1.0 mL
per min: 0e55 min, 100e20% A and 55e70 min, 20e10% A. Equil-
ibration times of 15 min were allowed between injections. Each
major peak in the HPLC chromatograms of the extracts was char-
acterized both by retention time and absorption spectrum (from
210 to 360 nm). Identication of specic compounds was achieved
by comparison of UV spectra and retention times against those of
pure standards. Proanthocyanidins, for which standards were
unavailable, were assigned by retention time and spectral param-
eters according to Pea-Neira et al. (2004) and Obreque-Slier et al.
(2010). Quantitative determinations were made by using the
external standard method and commercial standards. All the
qualitative and quantitative analyses of phenolic composition,
including the whole extraction procedure, were performed in
triplicate.
3. Results and discussion
3.1. General analytical parameters
Carmnre (CA), Merlot (M), Cabernet Franc (CF) and Cabernet
Sauvignon (CS) grapes were characterized by a number of analytical
parameters shown in Table 1. The content of total soluble solids
from the four cultivars underwent a signicant increase from rst
to fourth sampling dates. Similar observations had been reported
by other authors (Kennedy, Troup, et al., 2000; Roby et al., 2004).
Note that by the second sampling date, that is, at 45 days after
veraison, all the cultivars had reached technological maturity
(24

Brix). On the other side, a gradual increase in the pH of grapes
was observed between the rst and the last sampling dates for all
cultivars. Also, the titratable acidity was found to decrease to
a minimum value at the last sampling date. Carmnre grapes
showed the lowest titratable acidity values at the end of this study.
Such a decrease in acidity during ripening may make necessary the
addition of organic acids to carry out a normal vinication
(Zoecklein, Fugelsang, Gump, & Nury, 2001). That might be
particularly the case of the Carmnre grapes, which display a late
aromatic and phenolic maturity (Fernndez et al., 2007; Granett,
2004; Reynier, 2002). Finally, the Carmnre grapes presented
the lowest seed weights during ripening, in contrast to the Caber-
net Franc grapes that showed the highest values for this parameter
throughout this period. Probably, that difference may affect the
content of phenolic compounds when expressed on a per berry
basis.
3.2. Phenolic composition of grape seeds during ripening
Table 2 shows the global extractable phenolic composition of
seeds from CA, M, CF and CS grapes during ripening. The content of
total phenols in all the four cultivars was found to decrease grad-
ually between the rst and the last samplings, probably in relation
with oxidative processes (Kennedy, Matthews, et al., 2000). By
contrast, the content of total tannins displayed some peculiarities
in the four varieties. In effect, while CA and Mseeds did not present
any signicant variation in the content of total tannins during
maturation, the CF and CS seeds showed some signicant uctua-
tions with the highest concentrations in the second and third
sampling dates. On the other hand, among the four varieties the CA
seeds presented the highest concentrations and the CF seeds pre-
sented the lowest concentrations of total tannins at the various
maturity stages. The contents of total phenols and total tannins in
this study were similar to those reported in previous works with
these and other varieties grown in various parts of the world
(Downey et al., 2003; Kennedy, Matthews, et al., 2000; Kennedy,
Troup, et al., 2000; Roby et al., 2004). Likewise, the contents of
total phenols and total tannins in the CA and CS grape seeds in this
study were similar to those of a previous report that comprised
a different vintage, plant age and grape growing region (Obreque-
Slier et al., 2010).
3.3. Identication of CIElab parameters
Seed color is a tool commonly used by winemakers to dene the
optimal moment for harvesting during the wine production process
in some product categories (medium and high price). However, this
parameter is estimated visually and there is no enough information
about relations among seed color, different harvest dates and some
chemical parameters of the seeds.
The CIELAB system has been designed to determine chromatic
characteristics on the basis of several colorimetric coordinates
(Prez-Magario & Gonzlez-San Jos, 2003). Table 3 presents the
Table 1
General analytical parameters of Carmnre (CA), Merlot (M), Cabernet Franc (CF) and Cabernet Sauvignon (CS) grapes during ripening.
20-February 20-March 20-April 20-May
Total solids (

Brix) CA 20.2 0.8 a A 24.2 0.5 a B 25.7 0.6 a C 25.7 0.3 a BC

M 21.3 1.0 a A 24.4 1.5 a AB 24.5 1.5 a AB 25.7 1.3 a B
CF 19.2 2.3 a A 24.3 0.6 a B 26.5 0.3 a B 26.4 1.4 a B
CS 18.3 0.5 a A 23.6 0.5 a B 24.5 0.8 a B 24.5 0.4 a B
pH CA 3.4 0.2 a A 3.6 0.0 a AB 3.7 0.0 b B 3.9 0.2 a B
M 3.3 0.1 a A 3.5 0.1 a A 3.3 0.1 a A 4.0 0.2 a B
CF 3.2 0.1 a A 3.6 0.0 a B 3.5 0.0 a B 4.2 0.1 a C
CS 3.3 0.0 a A 3.5 0.1 a AB 3.4 0.1 a A 3.7 0.1 a B
Total acidity (g/L sulfuric acid) CA 3.6 0.2 a A 3.6 0.2 a A 2.9 0.0 a A 2.7 0.0 a A
M 3.7 0.3 a A 4.6 0.3 b A 4.1 0.6 a A 3.8 0.6 b A
CF 4.7 0.7 b A 4.5 0.7 b AB 3.6 0.3 a AB 3.3 0.3 ab B
CS 4.0 0.1 ab A 4.4 0.1 ab B 4.0 0.1 a B 3.4 0.1 b C
Seed weight (g/100 berries) CA 6.2 1.1 a A 5.4 1.2 a A 4.9 0.9 a A 5.4 0.1 a A
M 7.4 0.4 a AB 8.0 0.4 bc B 6.4 0.7 ab A 8.1 0.6 bc B
CF 10.7 0.9 b A 9.4 1.2 c A 9.7 0.7 c A 9.7 1.1 c A
CS 7.9 0.2 a A 7.0 0.4 ab A 7.8 1.4 bc A 7.8 0.3 b A
Figures represent mean standard deviation (triplicates). Values with different small letters in the single columns are statistically different between cultivars and different
capital letters in the single rows are statistically different between sampling dates (Tuckey test, p < 0.05).
E. Obreque-Slier et al. / LWT - Food Science and Technology 48 (2012) 134e141 136
values of clarity (L*), chroma (C*), hue (H*), a* and b* in seed
extracts of the four varieties at different stages of maturity. The L*
coordinate varies between 100 (perfect white) and cero (black)
(Gil-Muoz, Gmez-Plaza, Martnez, & Lpez-Roca, 1997). In this
study, all the four varieties presented their highest L* values in the
second sampling date but, compared to the other varieties, CA
showed a signicantly higher L* value in the last sampling date. The
C* parameter indicates the contribution of a* (red color) and b*
(yellow color) to total color (Gil-Muoz et al., 1997). The four
varieties in this study showed the highest C* values in the rst
sampling date followed by an abrupt decrease in the second
sampling date. However, compared to the other varieties, the CF
seed extracts showed signicantly higher chroma values in the
second and third sampling dates.
The H parameter indicates the tone or hue and varies between
violet (H 315

) and rose (H 0

) (Prez-Magario & Gonzlez-

San Jos, 2003). Throughout ripening, the CA and CS seed extracts
displayed statistically invariant H values. By contrast, the CF and M
seed extracts presented signicant increases in the H parameter
during the second and third sampling dates. Compared to the other
varieties, the CS seed extracts exhibited lower hue values, although
such a difference was statistically signicant only in the last
sampling date.
The a* parameter goes from red () to green colors () (Prez-
Magario & Gonzlez-San Jos, 2003). The a* values of the seed
extracts of CA, M and CF displayed a continuous decrease to
a minimumin the third sampling date. In three of the four sampling
dates, the seed extracts of the CS variety showed the highest a*
values. Finally, the b* values, that vary from yellow () to blue
colors () (Prez-Magario & Gonzlez-San Jos, 2003), were found
to experience abrupt but different decreases at the beginning of
maturation (second sampling date) in the four varieties. Thus,
compared to the other varieties, the CF seed extracts presented the
highest values in the second and third sampling dates whereas the
CS seed extracts showed the lowest b* values in the last three
sampling dates.
Taken together, these observations indicate that close to verai-
son the four varieties displayed fairly similar L, C and H values. By
this time, signicant differences among some cultivars, particularly
CS, did occur in the a* and b* values. By contrast, as maturity of the
four grape varieties progressed, signicant differences among the
cultivars became evident at any of the grape ripening stages under
study with respect to some, but not all, the colorimetric coordi-
nates. Thus, for instance, at harvest, CS was clearly different from
the other Carmenets varieties both in C*, H*, a* and b*. Likewise,
each of the four cultivars displayed signicant changes in most of
the colorimetric coordinates as early as the second sampling date. A
major exception to this observation was the steadiness of the H
value throughout all the ripening period under study in the CA and
CS grape varieties.
Table 3
CIELAB color space values.
20-February 20-March 20-April 20-May
L CA 73.3 8.6 a A 96.4 0.2 a B 96.0 0.9 a B 94.9 0.6 b B
M 79.1 3.1 a A 95.4 0.6 a B 94.7 2.2 a B 93.3 2.0 ab B
CF 74.2 1.6 a A 95.3 1.0 a B 94.9 0.5 a B 91.2 0.8 a C
CS 70.1 4.9 a A 96.1 0.4 a B 91.5 4.9 a B 91.3 1.4 a B
C CA 55.9 10.4 a A 12.7 2.6 a B 15.1 1.4 a B 11.1 1.2 a B
M 60.7 3.3 a A 14.8 1.4 ab B 18.4 0.7 b B 11.9 3.3 a B
CF 47.7 6.5 a A 18.9 2.8 b B 23.7 1.7 c B 10.3 2.7 a B
CS 67.3 8.1 a A 12.1 1.0 a B 16.1 0.7 ab B 7.3 0.8 a B
H CA 85.4 5.7 a A 88.1 3.7 a A 94.5 1.6 a A 85.8 3.6 a A
M 84.7 0.8 a A 92.0 1.4 a B 96.2 1.6 a B 84.0 2.7 a A
CF 79.3 6.3 a A 91.9 1.6 a B 95.9 0.8 a B 79.9 4.8 ab C
CS 78.0 2.1 a A 87.5 0.3 a A 69.1 36.0 a A 71.2 5.2 b A
a* CA 5.1 1.6 a A 0.3 0.1 a B 1.2 0.3 a C 0.8 0.6 a B
M 5.6 0.5 a A 0.5 0.4 a C 2.0 0.5 a C 1.2 0.3 ab B
CF 8.7 2.8 a A 0.6 0.4 a C 2.5 0.5 a C 1.6 0.5 ab B
CS 14.5 0.7 b A 0.5 0.1 a C 4.7 1.1 b B 2.4 0.7 b B
b* CA 55.5 10.1 ab A 12.6 2.7 a B 15.1 1.5 a B 11.1 1.2 a B
M 60.5 3.4 ab A 14.8 1.4 ab B 18.3 0.7 ab B 11.9 3.3 a B
CF 46.9 4.5 a A 18.8 0.4 b B 23.6 1.7 b B 10.1 2.8 a C
CS 65.5 8.2 b A 12.0 1.0 a B 13.2 5.1 a B 6.8 0.7 b B
Figures represent mean standard deviation (triplicates). Values with different small letters in the single columns are statistically different between cultivars and different
capital letters in the single rows are statistically different between sampling dates (Tuckey test, p < 0.05).
Table 2
Global extractable phenolic composition of Carmnre (CA), Merlot (M), Cabernet Franc (CF) and Cabernet Sauvignon (CS) grapes during ripening.
20-February 20-March 20-April 20-May
Total phenols (mg EAG/g seed) CA 28.5 3.4 a A 26.3 3.7 a AB 22.9 3.3 a AB 15.1 1.1 a B
M 25.0 0.3 a BC 26.2 0.8 a C 22.2 2.0 a B 13.5 0.6 ab A
CF 24.2 1.3 a A 27.0 1.3 a A 19.4 0.7 a B 14.1 2.9 ab C
CS 23.8 1.3 a A 23.2 1.1 a A 15.3 5.2 a B 10.2 0.2 b B
Total tannins (mg EC/g seed) CA 14.5 3.9 b A 13.6 2.8 b A 16.4 3.9 b A 16.2 0.4 c A
M 10.7 4.5 ab A 8.4 0.8 a A 11.7 1.2 ab A 11.0 2.2 b A
CF 4.9 0.5 a A 7.7 1.5 a AB 7.9 1.3 a AB 6.6 1.0 a B
CS 7.2 0.5 ab A 11.9 1.0 ab B 12.0 2.6 ab B 8.2 1.1 ab AB
Figures represent mean standard deviation (triplicates). Values with different small letters in the single columns are statistically different between cultivars and different
capital letters in the single rows are statistically different between sampling dates (Tuckey test, p < 0.05). EAG, equivalent gallic acid; EC, equivalent ()-catechin.
E. Obreque-Slier et al. / LWT - Food Science and Technology 48 (2012) 134e141 137
3.4. Extractable low molecular weight phenolic compounds in grape
seeds during ripening
Table 4 shows contents of 13 avonoids and non-avonoids
compounds in seed extracts of the CA, CS, CF and M grape varieties
during ripening. HPLCeDAD analysis showed three avanol mono-
mers [()-catechin (C), ()-epicatechin (EC) and ()-epicatechin-3-
O-gallate (ECG)], ve procyanidin dimers [catechin-(4a/8)-epi-
catechin (B4), epicatechin-(4b/8)-epicatechin (B2), catechin-
(4a/8)-catechin (B3), epicatechin-(4b/8)-catechin (B1) and epi-
catechin-(4b/6)-epicatechin (B5)], one dimer esteried with gallic
acid [catechin-(4a/8)-epicatechin-3-O-gallate (B4G)], one pro-
cyanidin trimer [epicatechin-(4bf8)-epicatechin-(4bf8)-catechin
(C1)], several uncharacterized proanthocyanidins (Ps) and
Table 4
Extractable low molecular weight phenolic compounds (mg/K) of Carmnre (CA), Merlot (M), Cabernet Franc (CF) and Cabernet Sauvignon (CS) seeds during ripening.
20-February 20-March 20-April 20-May
GA CA 143.3 23.1 cb A 107.0 10.0 b B 106.7 30.6 a B 46.7 12.1 b C
M 153.3 5.8 b A 126.7 25.2 ab AB 116.7 5.8 a B 80.0 14.1 ab C
CF 216.7 20.8 a A 150.0 14.1 a B 126.7 5.8 a BC 110.0 10.0 a C
CS 110.0 0.0 c A 73.3 5.8 c B 95.0 7.1 a C 50.0 0.0 b D
B3 CA 203.3 5.8 bc A 150.0 10.0 ab B 143.3 11.5 ab BC 110.0 17.3 a C
M 230.0 17.3 a A 210.0 10.0 a A 210.0 20.0 a A 120.0 0.0 a B
CF 166.7 30.6 ab A 145.0 23.6 ab A 113.3 19.3 b A 93.3 25.2 ab A
CS 140.0 0.0 c A 90.0 0.0 b B 80.0 0.0 b C 60.0 0.0 b D
B1 CA 136.7 28.9 a A 123.3 15.3 a A 106.7 15.3 a A 96.7 23.1 a A
M 126.7 11.5 a A 116.7 5.8 a A 110.0 10.0 a AB 90.0 5.0 a a
CF 106.7 15.3 a A 90.0 28.3 a A 56.7 10.8 b B 56.7 10.8 b B
CS 113.3 5.8 a A 83.3 5.8 a A 65.0 7.1 b A 50.0 0.9 b A
C CA 2700.0 100.8 a A 1823.3 292.4 a B 1780.0 210.8 a B 1153.3 241.2 a C
M 2923.3 342.7 a A 2363.3 300.9 a AB 2260.0 341.8 a C 1645.0 176.8 a B
CF 1910.0 250.3 b A 1825.0 139.0 a A 1103.3 88.1 b B 923.3 78.1 b B
CS 1333.3 58.6 c A 760.0 26.5 b B 620.0 14.1 c C 440.0 36.1 b D
C1 CA 73.3 5.8 ab A 43.3 4.8 b B 50.0 10.0 a AB ND
M 76.7 5.8 a A 66.7 5.8 a A 66.7 5.8 a A ND
CF 60.0 10.0 b A 50.0 4.1 ab B 33.3 5.3 b C ND
CS 66.7 2.8 ab A 40.0 0.0 b A 30.0 0.0 b C ND
B4 CA 193.3 43.7 b A 150.0 36.1 b A 166.7 41.6 b A 130.0 26.1 b A
M 313.3 5.8 a A 276.7 32.1 a A 273.3 20.8 a A 205.0 35.4 a B
CF 203.3 58.6 b A 190.0 49.0 b AB 153.3 35.7 b AB 130.0 32.9 b B
CS 101.0 3.5 c A 112.0 3.2 c B 103.0 3.1 c AB 83.3 5.8 c C
B2 CA 316.7 23.1 a A 336.7 11.5 a A 326.7 85.0 a A 256.7 66.6 a A
M 280.0 17.3 ab A 336.7 49.3 a AB 366.7 15.3 a B 240.0 42.4 a A
CF 250.0 30.0 b A 285.0 63.6 ab A 280.0 72.1 a A 213.3 35.1 a A
CS 270.0 0.0 ab A 226.7 5.8 b A 250.0 0.0 a A 170.0 10.0 a A
EC CA 1136.7 73.7 b A 806.7 132.8 ab B 720.0 156.2 ab B 576.7 123.4 ab B
M 1416.7 126.6 b A 1200.0 240.2 a AB 983.3 105.0 a BC 755.0 148.5 a C
CF 1343.3 213.9 b A 1055.0 231.3 a AB 780.0 146.6 ab B 626.7 149.9 ab B
CS 680.0 20.0 a A 443.3 32.1 b B 330.0 14.1 b C 270.0 10.0 b D
B4G CA 110.0 36.1 a A 53.3 13.1 a AB 40.0 6.5 a AB 16.7 2.8 a B
M 81.3 3.8 ab A 43.0 2.6 ab B 32.0 7.3 a C 12.0 2.6 ab D
CF 76.7 11.5 ab A 55.0 7.1 a B 9.6 5.8 b C 9.2 1.8 b C
CS 36.7 5.8 b A 20.0 0.0 b B 4.0 0.1 b C 10.0 0.0 b D
ECG CA 80.0 13.5 a A 80.0 10.0 a A 76.7 15.3 a A 53.3 5.3 a B
M 70.0 0.0 ab B 86.7 5.8 a A 86.7 20.8 a AB 55.0 11.2 a C
CF 43.3 5.8 c B 55.0 11.2 b AB 66.7 6.2 a A 43.3 5.8 ab B
CS 56.7 5.8 bc A 33.3 11.5 b B 50.0 0.0 b AB 36.7 5.8 b B
B5 CA 1240.0 49.5 a A 510.0 55.1 a B 356.7 31.2 a C 216.7 41.5 a D
M 743.3 90.7 b A 410.0 36.1 b B 253.3 40.4 b C 175.0 35.4 a C
CF 540.0 124.9 b A 345.0 35.4 b B 180.0 35.6 b C 133.3 30.6 a a
CS 250.0 17.3 c A 90.0 20.0 c B 70.0 14.1 c BC 56.7 11.5 b b
Ps CA 444.6 147.4 a A 204.9 135.0 a A 229.7 92.1 a A 169.6 88.1 a A
M 331.3 10.62 ab A 238.0 26.2 a B 223.8 26.2 a B 147.1 13.3 a C
CF 432.7 47.24 ab A 281.2 1.0 a B 220.1 64.6 a B 187.2 31.3 a B
CS 231.0 21.5 b A 91.8 43.9 a B 92.7 5.3 a B 73.1 6.3 a B
GPs CA 154.7 37.7 a A 44.0 9.7 b B 54.1 7.1 a B 30.8 6.9 a B
M 164.3 6.5 a A 53.0 5.1 b B 55.0 10.2 a B 28.9 3.0 a C
CF 120.6 13.4 a A 80.9 12.3 a B 61.1 3.9 a C 34.4 4.8 a D
CS 146.7 35.9 a A 52.7 8.1 b B 31.4 3.0 b C 10.2 1.6 b D
Figures represent mean standard deviation (triplicates). Values with different small letters in the single columns are statistically different between cultivars and different
capital letters in the single rows are statistically different between sampling dates (Tuckey test, p < 0.05). GA, gallic acid; B3, catechin-(4a/8)-catechin; B1, epicatechin-
(4b/8)-catechin; C, ()-catechin; C1, epicatechin-(4bf8)-epicatechin-(4bf8)-catechin; B4, catechin-(4a/8)-epicatechin; B2, epicatechin-(4b/8)-epicatechin; EC, ()-epi-
catechin; B4G, catechin-(4a/8)-epicatechin-3-O-gallate; ECG, ()-epicatechin-3-O-gallate; B5, epicatechin-(4b/6)-epicatechin; Ps, proanthocyanidins; GPs, proantho-
cyanidin gallates.
E. Obreque-Slier et al. / LWT - Food Science and Technology 48 (2012) 134e141 138
proanthocyanidin gallates (GPs), and only one non-avonoid
compound (GA, gallic acid) (Fig. 1). Identity of all those compounds
have been previously reported by different authors (Guerrero et al.,
2009; Obreque-Slier et al., 2010; Pea-Neira et al., 2004;
Rodrguez-Montealegre et al., 2006). During ripening, the concen-
tration of each of these compounds in the seeds of all the grape
varieties under studyexperienceda continuous anddrastic decrease.
This behavior has been previously observed in studies with other
varieties in different parts of the world (Kennedy, Troup, et al., 2000;
Obreque-Slier et al., 2010; Rodrguez-Montealegre et al., 2006).
Those changes have been associated with both oxidative processes
under aqueous conditions and the differential chemical evolution of
these compounds during ripening (Kennedy, Troup, et al., 2000). In
agreement with observations made by other authors working with
other varieties (Guendez et al., 2005; Obreque-Slier et al., 2010;
Rodrguez-Montealegre et al., 2006), C and EC were the most
abundant avonoid compounds. Regarding both compounds and all
the varieties under study, the M seeds showed the highest concen-
trations and the CS seeds the lowest concentrations in the different
sampling dates. With regard to the other identied compounds, the
CA seeds displayed the highest concentrations of B1, B4G and B2 at
least in two sampling dates. Similarly, the M seeds showed the
highest concentrations of B3, T, B4 and ECG in some of the sampling
dates whereas the CF seeds exhibited the highest concentrations of
AG in three sampling dates. It is interesting to note that the CS seeds
showed the lowest concentrations of most of the compounds iden-
tied during the study. A similar observation was made by
comparing CS and M grape seeds (Guendez et al., 2005; Rodrguez-
Montealegre et al., 2006).
3.5. Distribution of extractable proanthocyanidins according to
polymerization degree in grape seeds during ripening
Fig. 2 shows the mono-, oligo- and polymeric fractions of avan-
3-ols in grape seeds at three sampling dates during maturation
(February, March and April). In this assay, data corresponding to
harvest (May) was not included due to insufcient sample size. As
shown in Fig. 2, monomeric avan-3-ols was the less abundant
extractable fraction in seeds from CA, CS, CF and M grapes
throughout the whole study period whereas the polymeric avan-
3-ols represented the most abundant fraction, except in the rst
sampling date of the M seeds. These observations are in full
agreement with a number of studies dealing with other grape
varieties grown in different parts of the world (Jordo, Ricardo da
Silva, & Laureano, 2001; Monagas et al., 2003; Obreque-Slier
et al., 2010). During maturation, the monomeric fraction in grape
Fig. 1. Representative HPLCeDAD chromatogram of an aqueous grape seed extract from Vitis vinifera.
Fig. 2. Flavan-3-ols content in the monomeric, oligomeric and polymeric fractions of
Carmnre, Merlot, Cabernet Franc and Cabernet Sauvignon seeds during ripening.
E. Obreque-Slier et al. / LWT - Food Science and Technology 48 (2012) 134e141 139
seeds of the CA, M and CF varieties, but not that of CS, exhibited
a decreasing tendency between the rst and third sampling dates.
Some signicant differences in this fraction among different grape
varieties did occur at different stages of maturation but toward the
last sampling date the grape seeds of all the varieties exhibited
a similar content of monomeric avan-3-ols.
As to the oligomeric fraction of the grape seeds, no differences
were usually observed among the four varieties at any of the
maturity stages. However, signicant decreases in the contents of
oligomeric avan-3-ols in the M and CF grape seeds were observed
in the last sampling date. With regard to the polymeric fraction, no
major differences among the four varieties were observed at any of
the maturity stages. However, this fraction increased steeply by the
second sampling date and then was partially reduced at the third
sampling date. At this nal stage of maturation the contents of
polymeric avan-3-ols in seeds of the four grape varieties were still
signicantly over those respectively observed in the rst sampling
date.
Altogether, in our study the relative contents of the different
fractions of extractable monomeric, oligomeric and polymeric
proanthocyanidins were found to be similar to those of previous
reports referred to seeds of other grape varieties grown in different
parts of the world (Obreque-Slier et al., 2010). However, at the same
time, the concentrations of avanol fractions observed in this study
are lower than those reported by Sun, Ricardo da Silva, et al. (1998)
and higher than those reported by Monagas et al. (2003). In our
view, such differences may be associated to differences in the
methods used for the extraction of phenolic compounds fromseeds
(Obreque-Slier et al., 2010).
3.6. Phloroglucinolysis
Several studies have reported mDP values in the range from 2.3
to 15.1, which would depend on the analysis method and a varietal
effect (Downey et al., 2003; Kennedy, Matthews, et al., 2000;
Monagas et al., 2003; Moreno et al., 2008; Prieur, Rigaud, Cheynier,
&Moutounet, 1994). The mDP values for the seedextracts of the four
varieties under study presented a tendency to increase from
February to April, with values in the range from1.5 to 3.8 (Fig. 3). In
this assay, data corresponding to May (harvest) was not included
either due to insufcient sample size. The CS grape seeds displayed
the highest mPDvalues in all sampling dates. As to the %G, the seeds
fromCA, CS andCF grapes exhibited constant values during ripening
in contrast with the M seeds that showed a gradual decrease in this
parameter. With regard to aMW, seeds of the CF and CS varieties
presented their highest values in the last sampling date in such
a way that by then the aMW of CS became comparatively higher
thanthose of CAand M. Altogether, boththe %Gvalues and the aMW
of this study are highly coincident with reports from various labo-
ratories working with other grape varieties grown in different parts
of the world (Monagas et al., 2003; Sun, Leandro, et al., 1998).
4. Conclusion
Physicochemical differences do occur between grape seeds of
different varieties at some particular stages of maturation. Among
those differences we can mention total phenols, clarity (between
CA and CS), chroma (between CA and CF), hue (between M and CS)
and the monomeric fraction (between CF and CS). Also, signicant
differences between CA and CF do occur in various parameters
(such as seed weight, total tannins and the polymeric fraction of
avanols) at all the stages of maturation. On the other hand, several
low molecular weight phenolic compounds undergo a gradual
decrease during ripening. While the M seeds present a high
concentration of avanol monomers [()-catechin, ()-epicatechin
and epicatechin-3-O-gallate] and some proanthocyanidins [trimer
1, B3 and B4], the CA seeds display characteristic high concentra-
tions of other proanthocyanidins and the CF seeds showthe highest
concentrations of gallic acid and proanthocyanidin gallates.
Acknowledgments
This study was partially supported by grant 1110832 from Fon-
decyt-Chile and Program U-Inicia VID 2011, grant U-Inicia 11/05
University of Chile.
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