Food Chemistry 178 (2015) 164–171

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Evolution of phenolic compounds and metal content of wine during

alcoholic fermentation and storage
Andreas Bimpilas a, Dimitrios Tsimogiannis a, Kalliopi Balta-Brouma b, Theopisti Lymperopoulou b,
Vassiliki Oreopoulou a,⇑
a
Laboratory of Food Chemistry and Technology, Department of Chemical Engineering, National Technical University of Athens (NTUA), 5 Iroon Polytechniou Str., Athens GR
15780, Greece
b
Environment and Quality of Life Center, Department of Chemical Engineering, National Technical University of Athens (NTUA), 5 Iroon Polytechniou Str., Athens GR 15780, Greece

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

Article history: Changes in the principal phenolic compounds and metal content during the vinification process and stor-
Received 2 July 2014 age under modified atmosphere (50% N2, 50% CO2) of Merlot and Syrah wines, from grapes cultivated in
Received in revised form 12 November 2014 Greece, have been investigated. Comparing the variation of metals at maceration process, with the var-
Accepted 19 January 2015
iation of monomeric anthocyanins and flavonols, an inverse relationship was noticed, that can be attrib-
Available online 24 January 2015
uted to complexing reactions of polyphenols with particular trace elements. Cu decreased rapidly,
whereas a similar behavior that could be expected for Fe and Mn was not confirmed. Differences in
Chemical compounds studied in this article:
the profile of anthocyanins and flavonols in the fresh Merlot and Syrah wines are reported. During 1 year
Malvidin-3-O-glucoside (PubChem CID:
443652)
of storage monomeric anthocyanins declined almost tenfold, probably due to polymerization reactions
Petunidin-3-O-glucoside (PubChem CID: and copigmentation. Also, a decrease in flavonol glycosides and increase in the respective aglycones
443651) was observed, attributed to enzymatic hydrolysis. The concentration of total phenols and all metals
Delphinidin-3-O-glucoside (PubChem CID: remained practically constant.
443650) Ó 2015 Elsevier Ltd. All rights reserved.
Quercetin (PubChem CID: 5280343)
Myricetin (PubChem CID: 5281672)
Isorhamnetin-3-O-glucoside (PubChem CID:
44258009)
Syringetin-3-O-glucoside (PubChem CID:
44259492)
Laricitrin-3-O-glucoside (PubChem CID:
44259475)
trans-Caftaric acid (PubChem CID:
13887348)

Keywords:
Wine
Merlot
Syrah
Metal content
Polyphenols
Anthocyanins

1. Introduction
Wine is one of the most widely consumed alcoholic beverages
and its quality control is very important for both producers and
consumers. Among many different organic and inorganic mole-
⇑ Corresponding author. Tel.: +30 2107723166; fax: +30 2107723163.
E-mail address: vasor@chemeng.ntua.gr (V. Oreopoulou).
cules of wine, polyphenols and metals are two categories of partic-
ular interest.
Phenolic compounds of wine contribute to its sensorial proper-
ties such as color, bitterness, astringency and mouthfeel (Boulton,
2001; Vidal et al., 2004). On the other hand, according to several
epidemiological, clinical and in vitro studies, these compounds
reduce the risk of various degenerative diseases (cardiovascular
diseases, cancer, neurodegenerative diseases, diabetes and osteo-
porosis) due to their antioxidant activity (Scalbert, Manach,

http://dx.doi.org/10.1016/j.foodchem.2015.01.090
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 A. Bimpilas et al. / Food Chemistry 178 (2015) 164–171
Morand, Rémésy, & Jiménez, 2005; Stoclet et al., 2004). A high phe-
nolic content in wine is desirable not only due to their beneficial
health effects, but also due to their ability to protect wine against
oxidation. On the other hand, the sensorial properties are affected
by the content of wine in specific phenolic substances. In particu-
lar, the color is mainly affected by the anthocyanin content, while
co-pigmentation reactions of monomeric anthocyanins may result
in color stabilization.
Several studies have reported the phenolic content of Merlot
and Syrah wines (Fanzone et al., 2012; Ivanova-Petropulos et al.,
2015; Puértolas, Álvarez, & Raso, 2011) and in particular some of
them focus on varieties cultivated in Greece (Arnous, Makris, &
Kefalas, 2001; Kallithraka, Tsoutsouras, Tzourou, & Lanaridis,
2006; Makris, Kallithraka, & Mamalos, 2006). These varieties are
used widely in Greek wineries, due to their repute in producing
quality wines. However, an extensive study on the evolution and
complete profile of anthocyanins including minor compounds
(acetates and coumarates) as well as flavonols for varieties culti-
vated in Greece is not available in literature, to the best of our
knowledge. The quantification of acetyl- and coumaroyl-glucosides
of anthocyanins is important as it is related to the activity of
enzymes of the anthocyanin synthesis pathway in grapes and is
suggested to be used to verify the authenticity in red wines
(Fanzone et al., 2012; Ivanova-Petropulos et al., 2015). Moreover,
Figueiredo-González et al. (2012) claimed that the biosynthesis
of flavonols is closely related to that of anthocyanins and their pro-
file could be also used to characterize red grapes varieties.
The presence of metals like Fe, Cu, Mn, and Zn in wine regarding
their properties and the effect on wine quality has been studied
elsewhere (Sebecic, Pavisic-Strache, & Vedrina-Dragojevic, 1998;
Soto Vázquez, Rio Segade, & Fernández Gomez, 2013). It is known
that they act as catalysts of oxidation reactions or as promoters of
some enzymes. They also play an important role on the stability,
color and clarity of wines that affect its organoleptic characteristics.
Natural polyphenols have a great metal complexing capacity.
Bai et al. (2004) indicated that all the transition metals can form
complexes with a particular flavonol (rutin), Esparza, Salinas,
Santamaria, Garcia-Mina, and Fernandez (2005) studied the Zn
and Cu interactions with three selected flavonoids (catechin, quer-
cetin and rutin) and Smyk, Pliszka, and Drabent (2008) investi-
gated the interaction between a Cyanidin 3-glucoside and Cu(II)
ions in a model system. However, data on the changes in metal
content as related to the phenolic content of the wine during the
fermentation and aging are limited and with contradictory results
(Esparza et al., 2004; Soto Vázquez et al., 2013; Zúñiga, Pérez-Roa,
Olea-Azar, Laurie, & Agosin, 2014).
Therefore, the present work was undertaken to study the evolu-
tion of polyphenols, especially anthocyanins and flavonols, and the
metal content, in wines from Merlot and Syrah varieties cultivated
in Attica, Greece. The identification and quantification of anthocy-
anins and flavonols was attempted, while the metal content was
examined throughout the vinification process and changes were
correlated with the polyphenols variations. After the vinification
process the wines were stored under modified atmosphere (50%
N2, 50% CO2), which in a previous experimental work proved to
protect wine against deterioration. Changes in polyphenol and
metal content of wines were studied throughout a storage period
of 1 year.
2. Materials and methods
2.1. Reagents and standards
Standard malvidin-3-O-glucoside was purchased from Extra-
synthèse (Genay, France) and quercetin dihydrate from Sigma–
165
Aldrich (Steimheim, Germany), while the rest standards and
reagents were the following: multi-elemental standard solution
containing 26 elements (Scharlau, Barcelona, Spain), CertiPURÒ
multi-elemental standard solution (Merck, Darmstadt, Germany),
gallic acid (98% (w/w), Acros Organics, Fair Lawn, New Jersey),
Folin–Ciocalteu reagent (Merck, Darmstadt, Germany), methanol
(>99.8% (v/v), Sigma–Aldrich, Steimheim, Germany), hydrochloric
acid (37% (v/v), Sigma–Aldrich, Seelzem, Germany), ammonium
hydroxide (27% (v/v), Sigma–Aldrich, Steinheim, Germany), formic
acid (98–100%, Merck, Darmstadt, Germany), nitric acid (65% (v/v),
Merck, Darmstadt, Germany), sodium carbonate (Mallinckrodt, St.
Louis, Missuri). Water and acetonitrile used for chromatography
analyses were of HPLC and MS grade (Fisher Chemical, Leicester-
shire, UK).
2.2. Vinification process
Monovarietal grapes of the species Vitis vinifera cv. Merlot and
Syrah were harvested from Markopoulo, Attica (Central Greece).
The vinification process was conducted by Allagiannis winery
(Markopoulo, Attica, Greece) as summarized below: After crushing
and destemming, sodium metabisulphite (90 g/tn) and pectinolytic
enzymes (Uvazym couleur, Enartis, San Martino, Italy, 20 g/tn)
were added, followed by the inoculation with Saccharomyces cere-
visiae (200 g/tn). The alcoholic fermentation took place in stainless
steel tanks at a constant temperature of 22 °C and finished after
12 days, whereas the skins were removed after the 7th day. Sam-
ples were withdrawn for analysis every second day. The young
wines were placed for stabilization in plastic barrels at 4 °C for
10 days. Finally samples of both varieties (20 mL) were packaged
in laminated bags (OPP 20 lm/ink/adhesive/PET MET 12 lm/adhe-
sive/PE 75 lm STC) under modified atmosphere (50% N2, 50% CO2).
2.3. Total phenols (TP)
The Folin Ciocalteu method (Waterhouse, 2005) was used for
the quantification of TP after dilution of samples (1:10) in ethanolic
solution 10% (v/v). The results were expressed in gallic acid equiv-
alents (GAE), through construction of a reference curve. All sam-
ples were analyzed in duplicate and the presented results are
mean values.
2.4. Solid phase extraction for analysis of non-anthocyanin phenolic
compounds
Anthocyanin-type pigments cause great interference in the
chromatographic separation and identification of non-anthocyanin
phenolic compounds and especially flavonols. The removal of
anthocyanin pigments was carried out according to a method
described by Nixdorf and Hermosín-Gutiérrez (2010) by means
of solid-phase extraction using a combination of reversed-phase
and cationic-exchanger material, OasisÒ MCX cartridges (Waters,
Milford, Massachusetts, 6 cm3 capacity filled with 500 mg of
absorbent).
Wine (3 mL) was diluted with 3 mL of 0.1 M HCl and added to
the cartridge. In acidic conditions anthocyanins are present as
flavylium ions, so they can be retained by ion exchange on MCX
cartridge, previously conditioned with 5 mL of MeOH and 5 mL of
H2O. Further washing with 5 mL of 0.1 M HCl and 5 mL of H2O
was carried out.
The anthocyanin free fraction (Fr1) was eluted with 3  5 mL of
MeOH. The eluate was dried in a rotary evaporator (40 °C) and re-
dissolved in 3 mL of MeOH. This fraction was used for the identifi-
cation and quantification of all phenolic compounds apart from
anthocyanins.
166 A. Bimpilas et al. / Food Chemistry 178 (2015) 164–171

2.5. Determination of phenolic composition by HPLC–ESI-MS/MS
The identification of wine phenolic compounds was performed
on a Varian 212-LC chromatography system, equipped with a
reversed phase Hypersil C18 column (ODS 5 lm, 250  4.6 mm,
MZ Analysentechnik, Mainz, Germany). The chromatography sys-
tem was coupled to a Varian 500-MS ion trap mass spectrometer
equipped with an electrospray interface. System control and data
acquisition was performed using the Varian Workstation software
(Varian Inc., Palo Alto, California) and coupled to Varian Worksta-
tion data processing software. The wine samples were injected after
filtration (0.2 lm, PVDF syringe filters, Teknokroma, Barcelona,
Spain) on a reversed phase Hypersil C18 column (ODS 5 lm,
250  4.6 mm, MZ Analysentechnik, Mainz, Germany) and the
chromatographic separation of compounds was based on the OIV
method for analysis of anthocyanins in red wines (OIV, 2003)
with specific modifications (Mylona, Bimpilas, Tsimogiannis, &
Oreopoulou, 2013). The solvents consisted of water/acetonitrile/
formic acid (87:3:10 (v/v/v), solvent A; 40:50:10 (v/v/v), solvent
B) and the flow rate was 0.4 mL/min. The linear gradient for solvent
B was: 0 min, 6%; 21.5 min, 30%; 42 min, 50%; 50 min, 60%; 54 min,
60%; 65 min, 6%. For the identification of anthocyanins ESI-MS and
ESI-MS/MS was used, employing the following parameters: positive
ionization mode, drying gas, N2; drying temperature, 350 °C; nebu-
lizer, 65 psi; capillary, 2.5 kV; compound stability, 100%; scan
range, 100–1200 m/z.
Flavonols and phenolic acids were identified in the anthocya-
nin-free fraction (Fr1) of wines, setting positive and negative ioni-
zation mode settings and compound stability 100% and 40%.
2.6. Quantification of phenolic compounds by HPLC-DAD
Equipment consisting of an HP 1100 chromatography apparatus
coupled to an HP 1100 diode array detector (Agilent Technologies,
Santa Clara, California) was employed. Analyses were performed
on the same column and gradient program that had been used
for HPLC–ESI-MS. The injection volume was 20 lL and the detec-
tion of anthocyanins was accomplished at 520 nm, flavonols at
360 nm, hydroxycinnamic acids at 320 nm and flavan-3-ols at
280 nm. The elaboration of the chromatographic data was carried
out on a ChemStation for LC 3D software (Agilent Technologies,
Santa Clara, California). Total monomeric anthocyanin fraction
(MA) was quantified as the sum of concentrations of individual
identified anthocyanins and is expressed in malvidin-3-O-gluco-
side equivalents via reference curve of the respective standard
compound. Similarly, concentration of total flavonols (TF) was
quantified in quercetin equivalents, whereas individual hydroxy-
cinnamic acids and tartaric esters were quantified as caffeic acid
equivalents.
2.7. Determination of metals by ICP
Inductively coupled plasma optical emission (ICP-OES) and
mass spectrometry (ICP-MS) have been used for the determination
of metals in wine, after wet digestion of the samples. Alcohol was
removed by heating 25 mL of wine sample, at 80 °C on a hot plate
until a final volume of 10 mL, which was mixed with 5% nitric acid
(65% w/w) up to its starting volume. Determination and quantifica-
tion of metals was performed on an Agilent 7700 ICP-MS (Agilent
Technologies, Santa Clara, California) and Perkin Elmer Optima
7000 DV, ICP-OES (Perkin Elmer, Waltham, Massachusetts), consid-
ering individual concentrations. The operational conditions of
instruments are reported in literature (Gonzálvez, Llorens,
Cervera, Armenta, & De la Guardia, 2009; Moreno et al., 2008;
Nicolini, Larcher, Pangrazzi, & Bontempo, 2004). Multi-elemental
standard solution containing 26 elements from Scharlau (Barce-
lona, Spain), and CertiPURÒ multi-elemental standard solution
(Merck, Darmstadt, Germany) were used as stock solutions.
3. Results and discussion
3.1. Identification of phenolic compounds
All identified compounds are presented in Table 1, together
with their absorption maxima and mass of the precursor ions
and the most abundant fragments.

Table 1
Identification and quantification of individual phenolic compounds of fresh wines.

Phenolic compound Retention time (min) kmax (nm) [MH]+ and fragments (m/z) Merlot (mg/L) Syrah (mg/L)
Gallic acid 11.6 275 169, 125a n.q.b n.q.b
trans-Caftaric acid 12.3 329 311, 179a 34.2 ± 0.4 35.2 ± 0.3
(+)-Catechin 15.2 285 289a n.q.b n.q.b
trans-Coutaric acid 15.6 316 295, 163a 5.8 ± 0.1 16.7 ± 0.3
Caffeic acid 20.5 324 179, 135a Trace Trace
Delphinidin-3-glucoside 24.1 526, 346, 278 465, 303 20.8 ± 0.3 18.0 ± 0.0
p-Coumaric acid 25.5 310 163, 119a Trace Trace
Myricetin-3-glucuronide 27.1 260, 356 493, 317a 8.0 ± 0.7 7.0 ± 0.4
Myricetin-3-glucoside 27.8 260, 257 479, 317a 29.6 ± 0.3 37.0 ± 0.7
Petunidin-3-glucoside 29.7 526, 350, 276 479, 317 37.3 ± 0.2 31.7 ± 0.4
Quercetin-3-glucuronide 32.5 257, 355 477, 301a 26.0 ± 0.4 23.5 ± 0.4
Peonidin-3-glucoside 33.1 518, 362, 278 463, 301 19.8 ± 0.9 26.2 ± 0.8
Quercetin-3-glucoside 33.2 258, 354 463, 301a 18.3 ± 0.7 35.1 ± 1.1
Laricitrin-3-glucoside 34.5 258, 357 493, 331a 9.7 ± 0.2 12.8 ± 0.4
Malvidin-3-glucoside 35.1 530, 350, 278 493, 331 280.6 ± 4.6 256.5 ± 1.5
Myricetin 39.0 255, 374 317a 12.4 ± 0.5 14.1 ± 0.7
Isorhamnetin-3-glucoside 40.2 255, 356 477, 315a 8.1 ± 1.0 16.5 ± 1.1
Syringetin-3-glucoside 41.0 256, 358 507, 345a 9.9 ± 0.7 11.5 ± 0.9
Petunidin-3-acetyl glucoside 41.5 528, 278 521, 317 18.3 ± 0.7 15.0 ± 1.8
Peonidin-3-acetyl glucoside 48.5 526, 372, 282 505, 301 26.0 ± 0.2 25.4 ± 1.6
Quercetin 49.4 256, 372 301a 34.4 ± 0.4 33.8 ± 0.6
Malvidin-3-acetyl glucoside 50.3 530, 350, 278 535, 331 122.8 ± 1.6 111.7 ± 1.8
Petunidin-3-coumaroyl glucoside 54.9 532, 282 625, 317 19.8 ± 0.7 16.4 ± 0.2
Peonidin-3-coumaroyl glucoside 59.4 522, 282 609, 301 15.7 ± 0.7 13.8 ± 0.4
Malvidin-3-coumaroyl glucoside 60.9 536, 284 639, 331 60.8 ± 3.0 45.7 ± 0.2
a
[MH] and fragment.
b
Not quantified.
 A. Bimpilas et al. / Food Chemistry 178 (2015) 164–171
In both varieties, malvidin was the main anthocyanidin. It was
detected as malvidin-3-O-glucoside (mv-3-glc), malvidin-3-O-
(600 -acetyl)glucoside (mv-3-acglc), and malvidin-3-O-(600 -p-couma-
royl)glucoside (mv-3-coumglc). Moreover, seven other anthocyanins
were determined in lower quantities: delphinidin-3-O-glucoside
(dp-3-glc), petunidin-3-O-glucoside (pt-3-glc), peonidin-3-O-glu-
coside (pn-3-glc), petunidin-3-O-(600 -acetyl)glucoside (pt-3-acglc),
peonidin-3-O-(600 -acetyl)glucoside (pn-3-acglc), petunidin-3-O-
(600 -p-coumaroyl)glucoside (pt-3-coumglc) and pn-3-O-(600 -p-cou-
maroyl)glucoside (pn-3-coumglc).
Quercetin (Q) and myricetin (M) were the predominant
flavonols, and were detected as 3-O-glucuronides (3-glcr),
3-O-glucosides (3-glc) as well as aglycones. Moreover laricitrin-
3-O-glucoside (L-3-glc), isorhamnetin-3-O-glucoside (I-3-glc) and
syringetin-3-O-glucoside (S-3-glc) were detected and to the best
of our knowledge this is the first time that these compounds are
identified in Greek wines.
trans-Caftaric acid was the main hydroxycinnamic acid for both
Merlot and Syrah, followed by trans-coutaric acid, while caffeic and
coumaric acid were found in traces in both varieties. Other pheno-
lic compounds that were identified in both wines were gallic acid
and procyanidin monomers, i.e. catechin and epicatechin.
3.2. Variation of phenolic compounds and metal content during
fermentation
The highest concentration of monomeric anthocyanins (MA)
was observed at the beginning of maceration (1647 mg/L for Mer-
lot and 1388 mg/L for Syrah), as indicated in Fig. 1. Anthocyanins
are found in grape skins, they are water soluble and their extrac-
tion is imminent after crushing, during the maceration process.
Flavonols contribute to the bitter taste of red wine and to stabiliz-
ing and increasing wine color through copigmentation with antho-
cyanins. Although flavonols are predominantly synthesized in the
Fig. 1. Variation in the concentrations of monomeric anthocyanins (MA, expressed as mv-3-glc), total flavonols (TF, expressed as quercetin), total phenols (TP, expressed as
gallic acid equivalents) and trace elements, during the first stages of vinification of Merlot and Syrah.
167
grape skin, they are also located in the flesh of the berry
(Figueiredo-González et al., 2012). The solubility of flavonols in
water is lower than anthocyanins, and therefore their extraction
was enhanced by the increase of ethanol during the alcoholic fer-
mentation. This was particularly observed for Syrah, where the
highest concentration of TF was reached after 10 days (200 mg/L).
On the contrary, the highest TF concentration in Merlot was
noticed on the 2nd day of the maceration process (185 mg/L). By
quantification of the individual flavonols, the faster extraction of
flavonol glycosides was noticed, as they are more water soluble
than the respective aglycones (data not shown). In fact, Q-3-glyco-
sides and M-3-glycosides amounted to 130 mg/L after 2 days of
fermentation in Merlot and 165 mg/L after 6 days in Syrah. The
extraction of Q and M aglycones was slower and reached a maxi-
mum after 6 days in Merlot (34 mg/L) and after 13 days in Syrah
(26 mg/L). The quick extraction of anthocyanins and flavonol gly-
cosides should be also attributed to the use of pectinolytic
enzymes during maceration.
Considering the TP fraction, the highest concentration for
Merlot was found on 2nd day (2528 mg/L), but for Syrah on 8th
day (2066 mg/L). Ortega-Regules, Ros-García, Bautista-Ortín,
López-Roca, and Gómez-Plaza (2008) indicated the thicker walls
and the largest amount of cell wall material in the pulp of cultivar
Syrah, versus Merlot, as the reason for the slower extractability of
polyphenols.
The metal content was also determined during the fermentation
of both varieties. The results indicated that the concentration of Fe,
Cu, and Mn (to a lesser extent) showed significant variation, as
shown in Fig. 1. On the contrary the concentrations of Ni, Pb, Cr,
Cd, Ba, Zn and Al, were practically constant. A high concentration
of Cu was found in the musts of both varieties that can be attrib-
uted to the use of copper fungicides, for the protection of the cul-
tivations from fungi. However, at the early stages of fermentation,
a remarkable decrease in the concentration of Cu was noticed for
168 A. Bimpilas et al. / Food Chemistry 178 (2015) 164–171

both cultivars, which remained constant later. Comparing the con-
centration of Cu with MA, TF, and TP, it is observed that as MA, TF,
and TP concentrations increase, the concentration of Cu decreases.
Similar variations proving this inverse relationship are reported by
Esparza et al. (2004). Flavonoids can chelate metals, such as Cu and
Fe. The ability to form complexes is mainly related to hydroxyl
functional groups linked in ortho-positions on the B ring. Therefore
petunidin and quercetin can form complexes, while the methoxy
group on the B ring of malvidin lowers its ability to form com-
plexes with metal ions (Nicolini et al., 2004). Moreover, complexes
formed by the reaction of metals with flavonoid glucosides at dif-
ferent stoichiometric ratios are reported by Bai et al. (2004),
Esparza et al. (2005). Several compounds possessing ortho-hydro-
xyl groups are present in both varieties, i.e. quercetin, myricetin,
petunidin, delphinidin, either glycosylated or not as indicated in
Table 1. Thus the extraction and the consequent increase of poly-
phenols concentration during the maceration process, leads to
complexing reactions and precipitation of Cu for both cultivars.
Similar behavior would be expected for the concentration of Fe
and Mn; on the contrary a slight increase for both metals has been
observed that can be attributed to the presence of oxides of these
metals in yeast and to the use of stainless steel tanks (Bai et al.,
2004; Castiñeira, Brandt, Jakubowski, & Andersson, 2004).
3.3. Quantification of phenolic compounds and general characteristics
of the fresh wines
The quantification of individual phenolic compounds of the
fresh wines, at the end of fermentation is also presented in Table 1.
In both varieties, malvidin was the main anthocyanidin amounting
to almost 80% of MA. A difference was detected in the concentra-
tion of peonidin (especially pn-3-glc) that was higher in Syrah.
Apart from peonidin, all other anthocyanins were found in higher
concentrations in Merlot, whereas in both varieties pt-3-glc was
the second more abundant unacylated anthocyanin. These results
are partially in agreement with Makris et al. (2006) who examined
wines from Merlot and Syrah grapes cultivated in several Greek
regions, Puértolas et al. (2011) who examined wines from Merlot
and Syrah grapes cultivated in Spain and Ivanova-Petropulos
et al. (2015) who studied the respective varieties in Macedonia.
Nonetheless, differences are observed in the concentration and rel-
ative proportions of anthocyanins for grape cultivars grown in dif-
ferent regions and consequently the anthocyanic profile seems to
be affected both by cultivar and geographical origin (Ivanova-
Petropulos et al., 2015; Makris et al., 2006).
Quercetin and myricetin were the predominant flavonols,
amounting to 83% of TF in Merlot and 78% in Syrah. Comparing
the two cultivars, Merlot presented Q-3-glcr in higher concentra-
tion than the respective glucoside. These results are in agreement
with Rodríguez Montealegre, Romero Peces, Chacón Vozmediano,
Martínez Gascueña, and García Romero (2006) who indicated the
predominance of Q-3-glcr at the skins of Merlot grape. Fanzone
cultivar. Also, Rodríguez Montealegre et al. (2006) found a similar
content of trans-caftaric acid in Merlot and Syrah grape skins, but a
twofold higher content of trans-coutaric acid in Syrah.
Table 2 presents the general characteristics of fresh wines, the
TP content as determined by the Folin–Ciocalteu method, and the
content of phenolic sub-groups determined as the sum of the
respective individual phenolics presented in Table 1. Comparing
the two cultivars, the higher concentration of alcohol observed in
Merlot should be noted since alcohol enhances the extractability
of phenolic compounds from berries (Cheynier, 2006). The TP con-
tent of Merlot is higher than Syrah, contrary to what Arnous et al.
(2001) have observed for wines from northern Greece. Also, Merlot
wine presented a higher MA content, while Syrah a higher TF con-
tent. The higher TF content in Syrah wine compared to Merlot was
observed again by Puértolas et al. (2011), Castillo-Muñoz, Gómez-
Alonso, García-Romero, and Hermosín-Gutiérrez (2007). On the
contrary, Makris et al. (2006) reported similar TF content for Mer-
lot and Syrah wines from several Greek geographic regions, and
higher MA content in Syrah.
Acetylated anthocyanins amounted to 27% for both varieties,
whereas coumaroylated anthocyanins amounted to 15% for Merlot
and 14% for Syrah (see Table 2). The ratio of acetylglucosides to
coumaroylglucosides that has been proposed to varietal character-
ization was equal to 2.0 and 1.7, for Syrah and Merlot wines,
respectively. Puértolas et al. (2011) reported similar values, i.e.,
2.1 and 1.6 for Syrah and Merlot, respectively, while Ivanova-
Petropulos et al. (2015) found values of 2.6–4.8 for Syrah and
2.3–3.8 for Merlot, and Fanzone et al. (2012) 2.0 and 2.9 for Syrah
and Merlot, respectively. These values indicate that the acetylglu-
cosides to coumaroylglucosides ratio varies a lot, depending also
on geographical origin of the variety.
Glucosylated flavonols predominated in both varieties and
amounted to 41% and 51% in Merlot and Syrah, respectively.
According to Fanzone et al. (2012), 3-glucosides are the main flavo-
nol derivatives synthesised in Merlot and Syrah grapes, contrary to
other varieties where glucuronides or galactosides predominate.
Therefore, our results, regarding both the predominance of glu-
cosylated form and the specific flavonol glycoside profile of each
variety, agree with previous works and support further the sugges-
tion to use the flavonol profile for cultivar differentiation of fresh
wines, to the extent it will be proved valid for a larger number of
wines.
The metal content of the fresh wines, at the end of alcoholic fer-
mentation, is presented in Table 3. Maximum levels allowed,
according to OIV (2011), and mean concentrations reported in
Greek wines are also presented. As can be concluded all concentra-
Table 2
Analysis of fresh wines before storage, quantification of total phenols [GAE], total
monomeric anthocyanins [mv-3-glc eq.], total flavonols [Q eq.], and their sub-groups.
Parameter Merlot Syrah

et al. (2012) also observed a higher concentration of Q-3-glcr than Alcoholic strengtha 14.3° 12.2°
Q-3-glc in Merlot wine but not in Syrah. On the other hand, M-3- Density (20 °C)a 0.9943 g/mL 0.9964 g/mL
Total acidity [tartaric acid eq.]a 5.4 g/L 5.8 g/L
glc was detected in higher concentration than the respective glucu-
Volatile acidity [acetic acid eq.]a 0.345 g/L 0.195 g/L
ronide in both varieties, as also observed by Rodríguez Total sulfitesa 73 mg/L 67 mg/L
Montealegre et al. (2006), Fanzone et al. (2012). pHa 3.74 3.76
trans-Caftaric acid was the predominant hydroxycinnamic acid Total phenols [GAE] 2143 ± 45 mg/L 1954 ± 21 mg/L
Monomeric anthocyanins [mv-3-glc eq.] 622 ± 14 mg/L 560 ± 13 mg/L
for both Merlot and Syrah, amounting to 35 mg/L in both fresh
% Glycosylated anthocyanins 58 ± 2% 59 ± 3%
wines. trans-Coutaric acid concentration in fresh Syrah wine was % Acetylated anthocyanins 27 ± 1% 27 ± 2%
threefold higher (16.8 mg/L) than the respective concentration in % Coumaroylated anthocyanins 15 ± 2% 14 ± 1%
Merlot, while caffeic and coumaric acid were found in traces in Total flavonols [Q eq.] 161 ± 5 mg/L 193 ± 6 mg/L
both varieties. These observations are in agreement with Makris % Glucosylated flavonols 41 ± 1% 51 ± 1%
% Glucuronated flavonols 24 ± 1% 19 ± 0%
et al. (2006), Puértolas et al. (2011), and support their suggestion
% Flavonols aglycones 35 ± 2% 30 ± 1%
that trans-caftaric and trans-coutaric acid could be ‘marker com-
a
pounds’ for the differentiation of young wines in function of the Determined by the vinification company.
 A. Bimpilas et al. / Food Chemistry 178 (2015) 164–171 169

tions are within the allowable limits. Cu was reduced from the ini-
tial concentration of 1394 to 413 lg/L and of 1249 to 340 lg/L, in
Merlot and Syrah wine, respectively. The Fe content, although
slightly increased during fermentation as discussed in Section
3.2, was far below the allowable limits. The observed differences
in metal content between the two varieties can be attributed to
grape variety, cultivation practices, and soil composition (Soto
Vázquez et al., 2013), while the measured values are within the
values reported for several Greek wines (Galani-Nikolakaki,
Kallithrakas-Kontos, & Katsanos, 2002; Tariba, 2011).

3.4. Variations in phenolic content during storage of wine

One month after the initiation of the vinification process, the TP

concentration in Merlot was 2143 mg/L and in Syrah was 1954 mg/L,
whereas the concentrations of monomeric anthocyanins amounted
to 622 and 560 mg/L, respectively. As can be seen in Fig. 2, the TP
concentration in both varieties remained practically constant,
throughout 11 months storage period. This is attributed to the fact
that reactions between phenolic substances of wine are mainly
polymerization reactions (anthocyanins–procyanidins) rather than
degradation or oxidation reactions so they have minor effect on the
total number of hydroxyl groups that are measured by the TP
assay.
In accordance to the concentration of TP, the concentration of
MA, at the beginning of the storage period, is slightly higher for
variety Merlot. During storage for 11 months, an almost tenfold
decrease in MA concentration was observed for both varieties.
Color and taste changes taking place during wine aging have long
been ascribed to conversion of grape anthocyanins to polymeric Fig. 2. Variation in the concentrations of monomeric anthocyanins (MA, expressed
as mv-3-glc), total flavonols (TF, expressed as quercetin), total phenols (TP,
pigments through addition reactions with flavanols, i.e., catechin expressed as gallic acid equivalents), during storage of Merlot and Syrah wines.
oligomers. Three mechanisms have been postulated: The first one
involves nucleophilic addition of flavanols (in C8 or C6) onto the
C4 position of the anthocyanin flavylium ion yielding 4-flavanyl-
anthocyanins, which are also referred to as anthocyanin–flavanol we concluded that the consistency of modified atmosphere during
(A–F) or anthocyanin–tannin (A–T) adducts. The second mecha- storage influences the rate of polymerization reactions of anthocy-
nism is based on nucleophilic addition onto the carbonium ion anins with the other wine phenolics, and a higher rate is observed
formed in acid solutions from flavan 3,4-diols or by cleavage of under 50% N2 and 50% CO2, compared to 100% N2 and 100% CO2, for
procyanidins. Anthocyanins, either in the flavene form or in the Merlot and Syrah varieties (unpublished data). The higher rate is
hemiketal form, are added onto the carbonium ion, leading to desirable as it enhances the color stabilization of the product.
F–A adducts. The third mechanism proposed, involves nucleophilic The initial concentration of TF, after the end of cold stabiliza-
addition of the flavanol onto protonated acetaldehyde, followed by tion, in cultivar Merlot was 161 mg/L and Syrah 193 mg/L,
protonation and dehydration of the resulting adduct and nucleo- expressed as quercetin equivalents. In both varieties the TF content
philic addition of a second flavonoid onto the carbocation thus declined slightly during storage as shown in Fig. 2. The decrease of
formed. The resulting products are anthocyanin–flavonol adducts flavonols is attributed to the fact that they can be oxidized through
in which the flavonoid units are linked in C6 or C8 position through coupled reactions and/or they act as co-pigments with anthocya-
a methylmethine bond. These phenomena result in reducing the nins in co-pigmentation processes. Similar observations have been
fraction of monomeric anthocyanins in wine (Cheynier, 2006; made by Figueiredo-González, Regueiro, Cancho-Grande, and
Figueiredo-González, Cancho-Grande, & Simal-Gándara, 2013; Simal-Gándara (2014) studying Garnacha Tintorera-based sweet
Figueiredo-González et al., 2014). In a previous experimental work wines from Spain.

Table 3
Metals detected in fresh wines [lg/L].

Element Syrah Merlot Maximum levels Detection limits Mean concentration in greek wines
Fe 795 ± 15 627 ± 13 20,000 20a 4700–12,000
Cu 340 ± 5 413 ± 5 1000 5a 200–600
Mn 649 ± 7 928 ± 8 – 10a n.d.-10,000
Zn 652 ± 5 978 ± 7 5000 40a 300–3100
Ni 5 ± 0.05 10 ± 0.05 100 0.3b n.d.-2300
Pb 25 ± 0.10 21 ± 0.10 150 0.01b 18–420
Cr 20 ± 0.10 15 ± 0.10 100 0.02b n.d.-1600
Cd 7 ± 0.02 8 ± 0.02 10 0.01b n.d.-6
Ba 278 ± 5.0 269 ± 4.0 – 0.4b
Al 175 ± 3.0 155 ± 2.0 10,000 0.4b 360–950
a
Quantification with ICP-OES.
b
Quantification with ICP-MS.
170 A. Bimpilas et al. / Food Chemistry 178 (2015) 164–171
Fig. 3. Percentage of glycosylated flavonols versus aglycones during storage of Merlot and Syrah cultivars.
While the TF content declined during storage the quantification
of individual flavonols indicated that concentrations of myricetin
and quercetin aglycones increased slightly. Fig. 3 presents the per-
centage of glycosylated flavonols versus aglycones during storage.
A constant decrease of glycosylated forms and increase of agly-
cones was observed that should be attributed to hydrolysis of fla-
vonol glycosides catalyzed by enzymes, like b-glucosidase. Some S.
cerevisiae yeasts possess a gene coding for b-glucosidase. Mateo
and Di Stefano (1997) demonstrated the hydrolysis of grape glyco-
sides by crude extracts of Saccharomyces strains. This activity was
suggested to be related to the major exo-b-glucanase of S. cerevisi-
ae (Gil, Manzanares, Genovés, Vallés, & González-Candelas, 2005).
4. Conclusions
The extraction of phenolic compounds, in particular anthocya-
nins and flavonols, was accompanied by a rapid decrease of the
Cu content in the early stages of vinification, probably due to com-
plex reactions with polyphenols. The quantification of the individ-
ual anthocyanins and flavonols in fresh Merlot and Syrah wines
from Attica, Greece, revealed differences between the two varie-
ties, while some minor compounds were identified for the first
time in the Greek varieties.
During storage, monomeric anthocyanins decline rapidly as
they participate in copigmentation and mainly polymerization
reactions, while a small decrease in total flavonols is also observed.
Additionally, glycosylated flavonols decrease, while the respective
aglycones increase, indicating the enzymatic hydrolysis of the for-
mer. However, the reactions of the above constituents do not affect
the total polyphenol content, which remains constant.
Acknowledgements
The authors would like to thank the managers of Allagiannis
winery, Mrs. Maria Allagianni and Mr. Vassilis Allagiannis, who
provided the raw materials for this research and cooperated for
all the technical details of the fermentation process.
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