Food Chemistry 132 (2012) 112–124

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Food Chemistry
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Aroma potential of Brancellao grapes from different cluster positions

R. Noguerol-Pato a, C. González-Barreiro a, B. Cancho-Grande a, J.L. Santiago b, M.C. Martínez b,⇑,1,
J. Simal-Gándara a,⇑,2
a
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, University of Vigo, Ourense Campus, E-32004 Ourense, Spain
b
Misión Biológica de Galicia, Consejo Superior de Investigaciones Científicas (CSIC), Carballeira 8, E-36143 Salcedo (Pontevedra), Spain

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

Article history: In this study the presence of aroma compounds in grapes of Brancellao (Vitis vinifera L.) was investigated
Received 13 June 2011 in order to obtain its aroma potential fingerprint. It is well known that differences exist in aromatic com-
Received in revised form 11 August 2011 pounds amongst grapevine varieties at ripening stages. Within the framework of an increasingly compet-
Accepted 12 October 2011
itive market, the chance of obtaining different wines from vines of the same variety grown at the same
Available online 19 October 2011
vineyard is becoming of increasing importance. This can be done through the managing of the vineyard,
but also some wineries have assayed the separation of the tip and shoulder berries of the clusters of a
Keywords:
specific variety with this objective. In this work it is evaluated that, in the final stages of maturation, dif-
Vitis vinifera L. cv. Brancellao
Aroma compounds
ferences exist in the probable alcoholic degree, total acidity of the must, as well as in the aromatic com-
Grapes position of skin and flesh of berries coming from the tips and shoulders of the clusters. Gas
Tips chromatography coupled to mass spectrometry (GC–MS) was used to determine the aromatic composi-
Shoulders tion, in the skin and flesh of each sample, either tip or shoulder berries from the clusters. The obtained
Gas chromatography–mass spectrometry results showed that there was not variability for the probable alcoholic degree and total acidity between
(GC–MS) the shoulders and tips, whereas there was variability for their aromatic composition. For the berries from
Odour activity value (OAV) the tips of the clusters most of volatiles were found in the flesh (except aldehydes) and spicy and floral
nuances (with the only exception of b-ionone) were in higher proportions. For the berries from the shoul-
ders of the clusters, most of volatiles were found in the skin (monoterpenes, norisoprenoids, aldehydes,
and C6 alcohols), where the flesh was slightly richer in aromatic alcohols, volatile phenols and pantolac-
tone; b-ionone and herbaceous nuances were in higher proportions. These results are promising for those
wineries that are considering the chance of separating berries from tips and shoulders of the clusters for
the elaboration of different quality wines.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction pounds directly involved in aroma flavour, playing a key role in

Currently, the tendency of the world wine-making market is to
reward the production of wines that have particular and differen-
tiated characteristics, especially those due to aromatic composi-
tion. A relationship between wine character and berry/must
volatile composition has been recently recognised (Rocha, Coutin-
ho, Barros, Delgadillo, & Coimbra, 2007). Therefore, the aroma
composition of berry destined for winemaking may provide useful
information for predicting wine quality (Salinas, Zalacaín, Pardo, &
Alonso, 2004).
Aroma compounds arising from berry grapevine metabolism
are mainly terpenes, norisoprenoids, benzene compounds and C6
alcohols. These compounds in grapevine are present in free and
bound forms (especially glycosidic). Free forms are volatile com-
⇑ Corresponding authors.
E-mail addresses: carmenmartinez@mbg.cesga.es (M.C. Martínez), jsimal@uvi-
go.es (J. Simal-Gándara).
1
Corresponding author for selection of plant material and experimental design.
2
Corresponding author for aroma compounds determination and data discussion.
the quality and the peculiar aroma of wines. Bound glycoside
forms, which are odourless (Günata, Bayonove, Baumes, & Cordon-
nier, 1985; Sánchez-Palomo, Díaz-Maroto, González Viñas,
Soriano-Pérez, & Pérez-Coello, 2007), can be transformed in
volatile compounds by hydrolysis (Fenoll, Manso, Hellín, Ruiz, &
Flores, 2009; Günata, Bayonove, Baumes, & Cordonnier, 1986;
Hellín, Manso, Flores, & Fenoll, 2010). The distribution of free and
bound glycoside forms in the berry is not uniform (Fenoll et al.,
2009; Wilson, Strauss, & Williams, 1986). Skins have been found
to contain more than half the volatile compounds present in grape
berries (García, Chacón, Martínez, & Izquierdo, 2003).
Aroma compounds vary depending not only on cultivar, but also
on certain cultural and climate-related factors (Bureau, Razungles,
& Baumes, 2000; Jackson & Lombard, 1993; Sánchez-Palomo et al.,
2007; Zoecklein, Wolf, Marcy, & Jasinski, 1998). It is well known
that even within the same vine not all clusters have the same char-
acteristics and differences in some parameters could be observed.
In this sense, Kasimatis, Lider, and Kliewer (1975) and Tarter and
Keuter (2005) found significant differences in the °Brix analysed

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.10.042
 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124
in berries from the shoulders, the tips and the centre of one cluster
when they studied Thompson Seedless and Cabernet Sauvignon
varieties, respectively. Tarter and Keuter (2008) also observed dif-
ferences in the weight of the cluster analysing Cabernet Sauvignon,
Merlot and Chardonnay grapes.
One of the factors most influencing the aroma characteristic of a
variety is the stage of ripening of the berries because both free and
glycosylated forms of varietal compounds are accumulated in the
berry during ripening (Bayonove, 2003; Sánchez-Palomo et al.,
2007). To follow berry ripening, classical parameters based on
the percentage of soluble solids, sugar, acidity, pH, and colour are
used (Coelho, Rocha, Barros, Delgadillo, & Coimbra, 2007). How-
ever, in order to trace more specifically the varietal characteristics
to achieve a better product quality, analysis of phenolics (Hellín
et al., 2010; Kennedy, Matthews, & Waterhouse, 2000; Pérez-Mag-
ariño & González-San José, 2005), carotenoids (Razungles, Bayono-
ve, Cordonnier, & Sapis, 1988) and volatile compounds (Coelho
et al., 2007; Fenoll et al., 2009; Gambuti et al., 2007; García et al.,
2003; Kalua & Boss, 2009; Salinas et al., 2004; Sánchez-Palomo
et al., 2007) have been used.
Vitis vinifera L. cv. Brancellao is a traditional black berry grape-
vine from Galicia (NW Spain). It is known also as Serradelo and is
cultivated in Portugal under the name Alvarelhao (Gago et al.,
2009). Today the use of Brancellao for red wine-making is included
in the regulations of three of the five Denominations of Origin
(Appellations of Controlled Origin) existent in Galicia (Rías Baixas,
Valdeorras, Ribeiro, Ribeira Sacra and Monterrei), but it is usually
used as a complement in winemaking. The Brancellao cultivar
could be a possible alternative to the Mencía cultivar for the elab-
oration of quality red wines due to its maturation characteristics
and phenolic composition (Cortés & Díaz, 2011).
The aim of this work is to study in detail the varietal volatile
composition of Brancellao berries. Free and bound glycoside mon-
oterpenoids, norisoprenoids, aromatic alcohols, volatile phenols, C6
alcohols and aldehydes from the skin and flesh were separately
determined at different stages during ripening. Two positions of
grapes within the cluster (tips and shoulders) were assessed to
contribute to the characterisation of this cultivar. All this knowl-
edge about the grape volatile composition offers a means of evalu-
ating the aroma potential of the Brancellao berries taking into
account their position within the cluster as well as the period of
time in which the maximum aroma potential of the berries is
exhibited.
2. Materials and methods
2.1. Chemicals
Solvents (residue analysis grade) used were dichloromethane,
ethanol, ethyl acetate, hexane, methanol and water, purchased
from Panreac (Barcelona, Spain). Other reagents such as sodium
sulphate anhydrous (USP reagent) PA-ACS-ISO, di-sodium hydro-
gen phosphate anhydrous (USP) purissimum-CODEX, sodium di-
hydrogen phosphate 1-hydrate (USP, BP) PRS-CODEX, tri-sodium
citrate 2-hydrate PA-ACS, bromothymol blue and 0.1 N NaOH were
also purchased from Panreac.
The sorbent materials used for solid-phase extraction (SPE) pro-
cedures were Strata-X cartridges (6 ml size), 33 lm polymeric re-
versed phase, 500 and 200 mg from Phenomenex (Torrance, CA,
USA). Small apparatus such as an Ultrasons-H ultrasound bath (JP
Selecta, Barcelona, Spain), a Reax Top vortex (Heidolph, Schwa-
bach, Germany), a Rotina 35R centrifuge (Hettich Zentrifugen,
Tuttlingen, Germany), a Clifton shaking bath (Afora, Spain), a Turbo
Vap LV evaporator (Caliper Life Sciences, Hopkinton, MA, USA), and
a Visiprep™ 24 vacuum manifold (Supelco, USA) were also used.
113
2.2. Samples
Grapes (V. vinifera L.) from Brancellao variety were collected in
the 2009 harvest in Pontevedra, Spain, from an experimental vine-
yard: Salcedo, Pontevedra (NW Spain), latitude 42° 250 N, longitude
8° 380 W, 35 m of elevation. This vineyard is property of Misión
Biológica de Galicia-CSIC. The plot itself has a reference weather
station. The average annual temperature the last 50 years was
14.11 °C and average annual rainfall 1686.68 mm. The extent of
the plot is 1.4 ha and the soil is of sandy loam texture (70.1% sand,
16.1% silt and 13.8% clay) containing 7.3% organic matter. Varietal
identity has been confirmed in previous works by ampelography
and analysis of microsatellite molecular markers (Simple Sequence
Repeats, SSRs) (Gago et al., 2009). The plants of Brancellao are
grown since 1993 and all vines were subjected to identical pruning
and cultivation practices.
Upon véraison a clearing of the clusters was performed, leaving
the same number in all vines. The objective of this practice was to
avoid differences in the parameters studied due to a greater or les-
ser load on the vine (number of clusters per vine).
Three samplings were conducted between September and Octo-
ber 2009, with an interval of 10 days apart. The first one was held
in a third of the vines, taking separate portions from tips and shoul-
ders of the clusters (Fig. 1), to collect an amount of about 3 kg of
sample in each case. In order to see how it would be the natural
evolution of the parameters evaluated without interference intro-
duced by taking portions of clusters (first sampling), the second
sampling was performed on different vines to those used in the
first. The same approach was applied in the third sampling.
Of the 3 kg sample collected from the shoulders of the clusters
for each sampling, a small portion was separated intended for anal-
ysis of must basic parameters (probable alcohol degree and total
acidity). The remainder was used in the determination of volatile
compounds. We proceed similarly with the tips of the clusters. In
all cases the samples were properly separated and labelled in plas-
tic bags; finally, the samples were kept frozen at 80 °C until their
analysis.
2.3. Sample preparation
2.3.1. Probable alcohol degree and total acidity of the must
In each portion of clusters samples, once thawed at room tem-
perature, the berries were picked off and dried with a paper towel
to remove the excess of water from the outside. They, were then
squeezed in a mortar. The resulting must was split: 10 ml to deter-
mine total acidity and 5 ml to determine the probable alcoholic de-
gree. These operations were performed in triplicate for each
sample.
Fig. 1. Diagram of a cluster, specifying the tip and shoulder position within the
cluster.
114 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124
The probable alcohol of each sample was obtained by using a
refractometer. For this purpose, a few drops of juice were placed
on the refractometer to obtain the sugar concentration (°Brix),
and then using a conversion table, the corresponding probable
alcohol degree (% v/v) was calculated.
Total acidity of the must was determined using the volumetric
method with colour pattern (OJ, 1991).
2.3.2. Volatile compounds
The aroma content in flesh and skin was separately analysed.
The berries of each sample were peeled and destemmed by hand.
Skins were centrifuged (4000 rpm, 5 °C, 20 min) to separate from
must and frozen to 80 °C until analysis. Fleshes were collected
in a beaker, subsequently crushed and homogenised with a mixer
(Orbegorzo, Model BV 7000) for 20 s. The triturate was centrifuged
twice at 4000 rpm for 20 min at 5 °C to separate the must from so-
lid phase, followed by a vacuum filtration through 1.2 lm glass fi-
bre filter. The must obtained was mixed with 80 mg/l of SO2 and
frozen to 80 °C until analysis.
2.3.2.1. Isolation of free and glycosidically-bound volatile compounds
from must. Initially, 100 ml of must containing 40 ll of surrogate
standard (4-nonanol at 150 mg/l in ethanol) was loaded onto a
500 mg Strata-X cartridge previously activated and conditioned
with methanol (17 ml) and water (20 ml at pH 4.4) in sequence.
After elution the cartridge was washed with 20 ml of water at pH
4.4 and dried by passing N2 for 45 min. The free volatile com-
pounds were eluted with dichloromethane (10 ml). The bound
compounds were eluted with 25 ml of ethyl acetate:methanol
(9:1, v/v) and this eluate was frozen at 18 °C until the hydrolysis
procedure. The dichloromethane eluate with free compounds was
dried over anhydrous Na2SO4, concentrated to <1 ml under N2
stream, enriched with 20 ll of 2-octanol (25 mg/l in ethanol) as
internal standard and adjusted to a volume of 1 ml with dichloro-
methane prior to gas chromatographic analysis.
2.3.2.2. Isolation of free and glycosidically-bound volatile compounds
from skin. Free aroma compounds from skins were extracted fol-
lowing the method proposed by Ibarz, Ferreira, Hernández-Orte,
Loscos, and Cacho (2006) with some modifications. Twenty
grammes of skins were suspended first in 95 ml of a buffer solution
(0.1 M Na2HPO4/NaH2PO4, pH 7, 13% ethanol), spiked with 40 ll of
surrogate standard (4-nonanol at 150 mg/l in ethanol) and allowed
to macerate for 6 h with agitation at ambient temperature. This
solution was centrifuged at 4000 rpm for 20 min at 5 °C, and the
supernatant was filtrated through 1.2 lm glass fibre filter. A sec-
ond maceration with the skins was carried out in the same condi-
tions to guarantee the complete extraction of the aroma
compounds. The pellet was washed with buffer solution and cen-
trifuged. The liquid phases were assembled until 250 ml of skin ex-
tract was produced. Then the macerate was introduced onto the
SPE cartridge following the procedure described previously in
Section 2.3.2.1.
2.3.2.3. Enzymatic hydrolysis of glycosidically-bound fraction. As
mentioned in the previous Section 2.3.2.1, the precursors were
eluted with 25 ml of an ethyl acetate:methanol mixture (9:1, v/
v). This eluate was evaporated to dryness under a gentle N2 stream
at 35 °C. The enzymatic hydrolysis was adapted from that reported
by Schneider, Razungles, Augier, and Baumes (2001). The dry ex-
tract was reconstituted in 8.2 ml of a 0.1 M citrate/0.2 M phosphate
buffer solution pH 5, and then 800 ll of a 120 mg/ml solution in
the citrate/phosphate buffer solution of AR 2000 pectinase enzyme
preparation (DMS Food Specialties B.V., Delft, The Netherlands)
was added. Enzymatic hydrolysis was carried out at 40 °C for
16 h. A surrogate standard consisting of 40 ll of 4-nonanol
(150 mg/l in ethanol) was then added. This mixture was eluted
through a 200 mg Strata-X cartridge previously activated and con-
ditioned with 7.5 ml of methanol and 10 ml of water at pH 5.0, and
the free compounds released with 10 ml of dichloromethane. The
dichloromethane eluate was dried over anhydrous Na2SO4, concen-
trated to <1 ml under a N2 stream, enriched with 20 ll of 2-octanol
(25 mg/l in ethanol) as an internal standard and adjusted to a vol-
ume of 1 ml with dichloromethane prior to gas chromatographic
analysis.
2.4. GC–MS chromatographic conditions
Volatile compounds were separated and identified on a Trace
GC instrument equipped with a PolarisQ ion trap mass selective
detector (ITMS) that was furnished with an AS 2000 automatic
injector from Thermo Finnigan (Rodano, Italy) and interfaced to a
PC computer running the software Xcalibur 1.4 from Thermo Sci-
entific. Chromatographic separations were done on a HP-Innovax
fused-silica capillary column (60 m  0.25 mm ID, 0.25 lm film
thickness). The carrier gas, helium, was circulated at 1 ml/min in
the constant flow mode. A split/splitless injector was used in the
splitless mode (split time: 0.75 min). The injected volume was
2 ll and the injector temperature 250 °C. The oven temperature
programme was as follows: 45 °C for 2 min; 2 °C/min ramp to
225 °C and held for 15 min. The transfer line temperature was
250 °C and the ion-trap manifold temperature 200 °C. The ion en-
ergy for electron impact (EI) was set constantly at 70 eV.
Identification of the volatile compounds was achieved by com-
paring the GC retention times and mass spectra over the mass
range 35–300 amu for the samples with those for pure standards
analysed under the same conditions (González-Rodríguez, Noguer-
ol-Pato, González-Barreiro, Cancho-Grande, & Simal-Gándara,
2011; González-Álvarez, González-Barreiro, Cancho-Grande, & Si-
mal-Gándara, 2011; Noguerol-Pato, González-Barreiro, Cancho-
Grande, & Simal-Gándara, 2009; Noguerol-Pato, González-Rodrí-
guez, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2011).
Mass detection was performed in the selected ion recording (SIR)
mode for quantitation and 2-octanol was used as an internal
standard.
2.5. Odour activity values (OAVs)
The contribution of each volatile compound to grape aroma was
evaluated qualitatively via its associated descriptor and quantita-
tively via its OAV. OAVs were calculated by using the equation
OAV = c/t, where c is the total concentration of each compound in
the grape samples and t is the odour threshold value of the com-
pound in water (Hellín et al., 2010); threshold values were taken
from information available in the literature (Table 1).
Grouping volatile aroma compounds with similar descriptors
into odorant series allows the sensory profile of a wine to be estab-
lished (Franco, Peinado, Medina, & Moreno, 2004; Moyano, Zea,
Moreno, & Medina, 2002), and, consequently, the sensory profile
of the grape. We used floral, herbaceous and spicy odour series
for our grapes.
2.6. Statistical treatment
A two-tailed t-student test (at 95.0% confidence level) was per-
formed for probable alcohol degree and total acidity in must. Con-
centration values of free and bound volatile compounds were
compared for berry skin and flesh by One-Way ANOVA (at 95.0%
confidence level). This was performed with the statistical software
package Statistica version 8.0 (StatSoft, Inc., Tulsa, OK, USA).
 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124 115

Table 1
Classification of volatiles in odorant series according to their main odour descriptors.

Volatile compound Odour descriptor Odour thresholda (lg/L) Odorant series OAVb,*
Tips Shoulders
Monoterpenes
(+/)-Linalool Orange flowers 6c Floral 8.6 5.5
()-Terpinen-4-ol Iris 5d Floral – –
a-Terpineol Anise 330e Floral 0.034 0.026
(±)-b-Citronellol Citrus 40f Floral 0.097 0.089
Nerol Orange flowers 300f Floral 0.071 0.030
Geraniol Geranium 40e Floral 1.8 1.1
Geranic acid Green 40 Herbaceous 8.9 6.2
trans, trans-Farnesol Anise 20f Floral 0.46 0.36
C13-norisoprenoids
b-Ionone Violet 0.007c Floral 271 372
Aldehydes
Hexanal Grass 5g Herbaceous 334 674
Heptanal Green 3g Herbaceous 0.16 0.18
trans-2-Hexenal Grass 17c Herbaceous 121 80
Benzaldehyde Almond 350g Spicy 0.036 0.014
Alcohols
1-Hexanol Grass 2500g Herbaceous 0.76 0.50
trans-3-Hexen-1-ol Green 1000h Herbaceous 0.017 0.015
cis-3-Hexen-1-ol Green 70c Herbaceous 0.56 0.42
trans-2-Hexen-1-ol Green 100i Herbaceous 12 13
Aromatic alcohols
Benzyl alcohol Walnut 10000g Spicy 0.30 0.23
2-Phenylethanol Rose 1100g Floral 0.37 0.22
Volatile phenols
4-Ethtylguaiacol Phenolic 50j Spicy 0.014 0.0061
Eugenol Clove 6c Spicy 2.6 1.8
4-Ethylphenol Phenolic 440k Spicy 0.0025 0.0017
4-Vinylguaiacol Phenolic 3g Spicy 19 5.6
Syringol Phenolic 570l Spicy 0.0066 0.0061
Vanillin Vanilla 20m Spicy 2.1 2.1
Acetovanillone Vanilla 1000l Spicy 0.089 0.077
Miscellaneous
Hexyl acetate Ripe fruit 2n Floral 0.10 –
Pantolactone Licorice 2200h Spicy 0.0041 0.0058
a
Odour thresholds were taken from the literature, except for geranic acid (not found) which was assumed to be the same as geraniol.
b
OAV of each compound was calculated by adding the OAVs for that compound in free and bound forms for both flesh and skin (free-flesh; free-skin; bound-flesh; bound-
skin) in the 3rd sampling.
c
Buttery, Teranishi, Ling, and Turnbaugh (1990).
d
Zea, Moyano, Moreno, Cortés, and Medina (2001).
e
Takeoka, Flath, Mon, Teranishi, and Guentert (1990).
f
Ohloff (1978).
g
Buttery, Turnbaugh, and Ling (1988).
h
Moyano et al. (2002).
i
Larsen, Poll, and Olsen (1992).
j
Swan and Burtles (1978).
k
Boidron, Chatonnet, and Pons (1988).
l
López, Aznar, Cacho, and Ferreira (2002).
m
Rychlik, Schieberle, and Grosh (1998).
n
Flath, Black, Guadagni, McFadden, and Schultz (1967).
*
In bold, compounds with OAV > 1. In italics, compounds with OAV > 0.2.

3. Results and discussion
3.1. General
Table 2 shows the probable alcohol degree and total acidity
determined in berries coming from the tips and the shoulders of
Brancellao clusters, at three different samplings. Significant differ-
ences (p < 0.05) between tips and shoulders in the probable alcohol
degree were observed for the first sampling. No significant differ-
ences were found in the last sampling, which contradicts the work
of Kasimatis et al. (1975) and Tarter and Keuter (2005) who found
higher °Brix in berries from the shoulders than tips, although these
authors already warned of the need to conduct further studies in
this regard with different varieties. On the other hand, total acidity
of the must (expressed as g tartaric acid/l) showed significant dif-
ferences in the third sampling. In this case, the highest value was
found in the shoulders of the clusters.
3.2. Evolution of the concentration in time of the different chemical
classes of volatile compounds
Free monoterpenes in berry flesh increased around 40% both in
tips and shoulders of the clusters in the 3rd sampling with regards
to the 1st sampling (Table 3). This increase was mainly conditioned
by a 60% increase in geraniol (Table 3). Although in relative terms
(taking into account the subtotal amount of monoterpenes) the in-
crease was similar in both cluster positions, in absolute terms (for
each compound individually) the tips showed a higher level of free
monoterpenes in the 3rd sampling (Table 3). Free monoterpenes
were associated primarily with the skins of the berries (Table 3)
116 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124
Table 2
Changes of probable alcohol (% v/v) and total acidity (g tartaric acid/l) for the three samplings of Brancellao berries located at two different positions within the cluster (tips and
shoulders).
Parameter 1st Sampling
Tips Shoulders
Probable alcohol (% v/v) 9.37 ± 0.06 10.70 ± 0.20*
Total acidity (g tartaric acid/l) 7.53 ± 0.11 7.95 ± 0.33 ns
ns: not significant.
*
t-Student test (p 6 0.05), n = 3.
as other researchers reported for other grape varieties in early
studies (García et al., 2003; Wilson et al., 1986). These compounds
were found at higher levels in the 3rd sampling with regards to the
1st sampling for both cluster positions. In the tips, the increase was
basically resulting of the increase of geranic acid (Table 3) through-
out the three samplings. In the shoulders, no significant increases
were individually found for any free monoterpene, but geranic acid
showed a trend to increase, which led to 39% higher total monoter-
penes in the 3rd sampling (Table 3). Bound monoterpenes reached
higher values than free forms in the flesh of berries throughout all
the maturation period. These results are in agreement with those
found for other grape varieties (Carballeira Lois, Cortés Diéguez,
Gil de la Peña, & Fernández Gómez, 2001; Fenoll et al., 2009; Hellín
et al., 2010; Park, Morrison, Adams, & Noble, 1991). Linalool was
the bound monoterpene with the highest increase in all situations
(Table 4). Free and bound forms of terpenols accumulate in ripen-
ing grapes from véraison onwards, and some works suggest that
they accumulate continuously through to harvest. It is generally
agreed, however, that these compounds reach peak levels just prior
to the hexose sugar peak being attained. Vineyard conditions, such
as temperature and water relations, may be responsible for the
contradictions in results (Hornsey, 2007). In this work the screen-
ing of geraniol and linalool during ripening could be used to define
the evolution profile of the varietal volatile compounds; this find-
ing is in accordance with the study of Coelho et al., 2007.
Free norisoprenoids were reduced in the 3rd sampling in tips
and shoulders, both in the skin and flesh of berries (Table 3). b-Io-
none was the compound at highest level within the group and, in
quantitative terms, the skin is richer in this compound according
to Table 3. However, the changes in the concentrations of bound
norisoprenoids were negligible (Table 4). Similar behaviour was
observed for b-ionone in Monatrell grapes (Salinas et al., 2004),
this compound fell as ripening progressed.
Free aldehydes were mainly in the skin of berries. This agreed
with the study of García et al. (2003) about changes in volatile
compounds during ripening in grapes of Airén, Macabeo and Char-
donnay white varieties. Two compounds, hexanal and trans-2-hex-
enal, were found at higher concentrations. In the berry flesh, these
compounds decreased during ripening in the tips and increased in
the shoulders of the clusters. In the skin, significant differences
throughout samplings were not found. With regards to the differ-
ences at the 3rd sampling, the berries of the shoulders were richer
in aldehydes than those from the tips, with the only exception of
trans-2-hexenal in the skin of the berries (Table 3). For bound alde-
hydes, the only significant changes were found in the berries from
the tips: for hexanal in the flesh (with a decrease of a 55%), and for
trans-2-hexenal (with a significant increase, 116%) in the skin
(Table 4).
The highest level of free C6 alcohols was for hexanol and trans-
2-hexenol. In the flesh of berries, these compounds were relatively
constant in the tips during ripening, but they, respectively, de-
creased by 33% in the tips and a 47% in the shoulders. In the skin
of berries, the hexanol decreased in the shoulders, whereas trans-
2-hexenol decreased in the tips. The berries from the tips showed
the highest concentration of C6 alcohols in the flesh, whereas in the
2nd Sampling 3rd Sampling
Tips Shoulders Tips Shoulders
9.63 ± 0.15 10.03 ± 0.15 ns 10.03 ± 0.15 9.97 ± 0.21 ns
6.15 ± 0.15 5.85 ± 0.08 ns 5.40 ± 0.08 5.93 ± 0.15*
skin the berries from the shoulders were those with the highest
concentration (Table 3). In the case of bound C6 alcohols, the most
quantitatively important was hexanol, and no significant differ-
ences were observed in any case, representing the bound C6 alco-
hols about 6% of total volatiles in the flesh of berries and 7.5% in
the skins (Table 4).
With regards to free aromatic alcohols, a transfer from the skin
(since they decreased until the 3rd sampling) to the flesh (where
their levels were increased) could be possible according to Table 3.
In general terms, the bound aromatic alcohols decreased in the
skin and increased in the flesh, as in their free form. Benzyl alcohol
was the compound at the highest level and remained quite con-
stant with ripening time; the changes in this group (Table 4) were
controlled by the behaviour of 2-phenylethanol. On the contrary,
the evolution of these compounds in Airén, Chardonnay and Mac-
abeo varieties (García et al., 2003) was somewhat erratic, but the
tendency was for a slight decrease in the musts and a slight in-
crease in the skins until approximately the middle of the ripening
period, from which time the concentration in skin also started to
fall.
A lower free volatile phenols concentration was found in flesh
as opposed to the skin. Moreover, in the flesh, the concentration
of these compounds decreased in the 3rd sampling, but increased
in the skin, both in tips and shoulders positions (Table 3). Bound
volatile phenols were mainly found in the flesh of berries. In the
tips, their concentration was increased from the 1st sampling in
most of them, whereas in the shoulders most remained quite con-
stants, with the exception of vanillin (80% increase), ruling the
trend in this group (Table 4).
By comparing the time-evolution of Brancellao berries at two
different positions within the cluster, it seems that the tips are
ahead in developing variety-linked aroma compounds.
3.3. Changes of chemical classes of volatile compounds in grapes with
bunch position
As can be seen in Fig. 2a about the concentration of different
chemical classes of compounds studied in 3rd sampling, the bound
monoterpenes were dominant in the flesh, whereas in the skin the
free form was more abundant. Berries coming from the tips of the
clusters concentrated most of these compounds in the flesh, but in
the shoulders they were mainly in the skin. In general, the tips
showed a larger content of monoterpenes than the shoulders, but
mainly in bound form.
In any position within the cluster the berry skin had a larger
content in norisoprenoids than the flesh (Fig. 2b), and in the skin
they were practically only found in free form. In the flesh, more
than the half of the content in norisoprenoids was in the bound
form. Overall, both positions showed a similar content in norisopr-
enoids, although the highest proportion of bound norisoprenoids
was detected in the tips of the clusters.
The aldehydes (Fig. 2c) were mainly concentrated in the skin in
the free form and there were no significant differences between
both positions (tips and shoulders). There were more free
 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124 117

Table 3
Evolution of free volatile compounds (ng/g of berry) in flesh and skin from Brancellao berries located at two different positions within the cluster (tips and shoulders).

Tips % D31B Shoulders % D31B % DabC

1st Sampling 2nd Sampling 3rd Sampling 1st Sampling 2nd Sampling 3rd Sampling
FleshA
Monoterpenes
(±)-Linalool 0.91 ± 0.11a 0.67 ± 0.048 b 0.76 ± 0.0048 a,b – 0.78 ± 0.0056 x 0.55 ± 0.021 y 0.48 ± 0.010 z 38 ; 58 ;
()-Terpinen-4-ol 0.10 ± 0.0053 b 0.12 ± 0.016 b 0.15 ± 0.0022a 53 " 0.10 ± 0.0083y 0.097 ± 0.0019y 0.12 ± 0.0059x 26 " 26 ;
a-Terpineol 0.45 ± 0.067 a 0.56 ± 0.068 a 0.61 ± 0.030 a – 0.052 ± 0.0055 x 0.058 ± 0.035 x 0.057 ± 0.014x – 970 ;
(±)-b-Citronellol 0.64 ± 0.29 a 0.66 ± 0.066 a 0.62 ± 0.19 a – 0.082 ± 0.00039x 0.098 ± 0.032 x 0.11 ± 0.012 x – –
Nerol 0.97 ± 0.016 b 1.28 ± 0.065 a 1.4 ± 0.12 a 41 "
Geraniol 19 ± 2.1 b 23 ± 0.42 b 31 ± 1.2 a 60 " 9.6 ± 0.42 y 9.4 ± 0.37 y 15 ± 1.7 x 58 " 102 ;
Geranic acid 9.9 ± 0.96 a 9.2 ± 0.46 a 11 ± 0.27 a – 5.1 ± 0.35 x 5.4 ± 0.086 x 5.7 ± 0.53 x – 94 ;
trans, trans-Farnesol
SubTOTAL 32 35 45 41 " 16 16 22 39 "
% 0.93 1.2 1.8 0.86 0.95 1.2

C13-Norisoprenoids
TheaspiraneA
TheaspiraneB 0.28 ± 0.0097 a 0.13 ± 0.0066 b 0.084 ± 0.0025c 70 ; 0.29 ± 0.0033 x 0.13 ± 0.018 y 0.12 ± 0.025 y 57 ; –
b-Damascenone
b-Ionone 0.62 ± 0.059 a 0.32 ± 0.039 b 0.24 ± 0.0037 b 61 ; 0.28 ± 0.018 x 0.18 ± 0.00028y 0.20 ± 0.014 y 28 ; –
SubTOTAL 0.90 0.45 0.32 64 ; 0.57 0.31 0.32 43 ;
% 0.026 0.015 0.013 0.031 0.019 0.018

Aldehydes
Hexanal 468 ± 4.2 a 153 ± 5.7 b 53 ± 0.80 c 89 ; 354 ± 21 y 477 ± 19 x 512 ± 43 x 44 " 90 "
Heptanal
trans-2-Hexenal 588 ± 8.6 a 297 ± 2.9 b 105 ± 3.3 c 82 ; 545 ± 17 y 625 ± 13 x 620 ± 32 x 14 " 83 "
Benzaldehyde 2.8 ± 0.58 b 8.5 ± 1.2 a 3.8 ± 1.4 b – 1.5 ± 0.012 x 0.86 ± 0.29 y 0.82 ± 0.22 y 46 ; –
SubTOTAL 1059 458 162 85 ; 901 1103 1132 25 "
% 31 16 6.6 49 67 64

Alcohols C6
1-Hexanol 1157 ± 16 a,b 1196 ± 10 a 1094 ± 31 b – 406 ± 22 x 227 ± 1.0 y 270 ± 11 y 33 ; 305 ;
trans-3-Hexen-1-ol 10 ± 0.0050 b 10 ± 0.7 a,b 12 ± 0.023 a 14 " 3.9 ± 0.080 x 2.6 ± 0.082 y 3.0 ± 0.25 y 23 ; 290 ;
cis-3-Hexen-1-ol 40 ± 0.80 a 21 ± 1.5 b 16 ± 0.078 c 60 ; 29 ± 0.69 x 11 ± 0.13 y 12 ± 0.39 y 60 ; 39 ;
trans-2-Hexen-1-ol 939 ± 14 b 1027 ± 7.2 a 883 ± 42 b – 330 ± 13 x 161 ± 2.0 y 176 ± 9.3 y 47 ; 403 ;
SubTOTAL 2148 2255 2006 7; 769 402 461 40 ;
% 62 76 81 42 24 26

Aromatic alcohols
Benzyl alcohol 156 ± 6.2 a 147 ± 2.4 b 183 ± 5.3 a – 103 ± 4.0 y 93 ± 5.2 y 134 ± 2.9 x 29 " 37 ;
2-Phenylethanol 41 ± 2.1 b 36 ± 2.6 b 49 ± 0.27 a 20 " 17 ± 0.25 y 14 ± 0.46 z 19 ± 0.63 x 10 " 158 ;
SubTOTAL 198 184 233 18 " 121 108 153 27 "
% 5.7 6.2 9.4 6.6 6.5 8.6

Volatile phenols
4-Ethylguaiacol
Eugenol 1.8 ± 0.073 a 1.4 ± 0.044 b 1.5 ± 0.048 b 16 ; 0.64 ± 0.20 x 0.71 ± 0.26 x 0.64 ± 0.49 x – –
4-Ethyl-phenol
4-Vinylguaiacol 0.19 ± 0.028 b 0.26 ± 0.0094a 0.21 ± 0.022 a,b – 0.28 ± 0.0045 x 0.29 ± 0.013 x 0.29 ± 0.018 x – –
Syringol
Vanillin 7.6 ± 0.11 a 9.0 ± 1.1 a 7.4 ± 0.071 a – 14 ± 1.7 x 12 ± 0.4 x 7.2 ± 0.97 y 49 ; –
Acetovainillone 5.0 ± 0.039 a 3.1 ± 0.28 c 3.9 ± 0.024 b 22 ; 1.2 ± 0.16 x 1.2 ± 0.01 x 1.2 ± 0.094 x – 229 ;
SubTOTAL 15 14 13 11 ; 16 14 9.3 42 ;
% 0.42 0.47 0.53 0.88 0.87 0.52

Miscellaneous
Hexyl acetate 0.084 ± 0.013 b 0.13 ± 0.058 a,b 0.20 ± 0.0079a 142 " –
Pantolactone 2.8 ± 0.28 a 2.0 ± 0.10 b 2.5 ± 0.11 a,b – 3.0 ± 0.42 x 2.8 ± 0.13 x 3.1 ± 0.75 x – 19 "
SubTOTAL 2.9 2.2 2.7 7; 3.0 2.8 3.1 1.4 "
% 0.083 0.074 0.11 0.17 0.17 0.17

SkinA
Monoterpenes
(±)-Linalool 2.0 ± 0.036 a 1.8 ± 0.0097 b 2.0 ± 0.0055 a – 0.46 ± 0.052 x 0.40 ± 0.019 x 0.48 ± 0.072x – 309 ;
()-Terpinen-4-ol
a-Terpineol 0.30 ± 0.017 a 0.27 ± 0.0033 b 0.28 ± 0.013a,b – 0.31 ± 0.013 x 0.32 ± 0.11 x 0.48 ± 0.26 x – 43 "
(±)-b-Citronellol 0.96 ± 0.021 a 0.97 ± 0.22 a 0.85 ± 0.027 a – 1.8 ± 0.23 x 2.5 ± 0.085 x 1.9 ± 0.37 x – –
Nerol 1.5 ± 0.064 a 1.3 ± 0.19 a 1.5 ± 0.098 a – 1.9 ± 0.0048 x 2.2 ± 0.23 x 2.5 ± 0.49 x – –
Geraniol 5.6 ± 0.076 a 7.1 ± 0.99 a 6.6 ± 1.6 a – 5.7 ± 1.1 x 10.2 ± 2.8 x 5.9 ± 1.7 x – –
Geranic acid 78 ± 7.8 b 121 ± 11 a 134 ± 5.8 a 72 " 87 ± 34 x 154 ± 3.6 x 124 ± 33 x – –
trans, trans-Farnesol –

(continued on next page)

118 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124

Table 3 (continued)

Tips % D31B Shoulders % D31B % DabC

1st Sampling 2nd Sampling 3rd Sampling 1st Sampling 2nd Sampling 3rd Sampling

SubTOTAL 88 133 145 65 " 98 169 135 39 "

% 2.1 2.6 3.4 1.3 1.9 2.3

C13-norisoprenoids
TheaspiraneA –
TheaspiraneB –
b-Damascenone –
b-Ionone 1.9 ± 0.052 a 1.9 ± 0.21 a 1.7 ± 0.038 a – 2.7 ± 0.19 x 2.8 ± 0.033 x 2.4 ± 0.32 x – –
SubTOTAL 1.9 1.9 1.7 12 ; 2.7 2.8 2.4 11 ;
% 0.046 0.039 0.039 0.037 0.030 0.041

Aldehydes
Hexanal 1279 ± 64 b 1859 ± 173 a 1592 ± 14 a,b – 2372 ± 767 x 3164 ± 299 x 2837 ± 298 x – 44 "
Heptanal 0.60 ± 0.0037 a 0.42 ± 0.072 b 0.49 ± 0.016a,b – 0.68 ± 0.056xy 0.82 ± 0.072 x 0.55 ± 0.049y – –
trans-2-Hexenal 1188 ± 42 b 1760 ± 242 a 1539 ± 7.2 a,b – 1951 ± 683 xy 2804 ± 288 x 710 ± 80 y – 117 ;
Benzaldehyde 4.2 ± 0.27 a,b 3.9 ± 0.39 b 4.8 ± 0.14 a – 7.0 ± 0.36 x 5.8 ± 0.61 x 3.1 ± 0.23 y 56 ; 55 ;
SubTOTAL 2472 3624 3136 27 " 4330 5975 3551 18 ;
% 60 72 74 59 66 61

Alcohols C6
1-Hexanol 734 ± 51 a 550 ± 52 b 635 ± 18 a,b – 1626 ± 168 x 1565 ± 73 x 876 ± 29 y 46 ; –
trans-3-Hexen-1-ol 8.5 ± 1.0 a 6.6 ± 0.63 a 3.4 ± 0.021 b 60 ; 7.9 ± 0.67 x 7.7 ± 0.91 x 11 ± 3.3 x – –
cis-3-Hexen-1-ol 20 ± 0.30 a 11.4 ± 1.6 b 5.6 ± 0.18 c 72 ; 15 ± 3.3 x 9.4 ± 2.0 xy 5.1 ± 0.92 y 65 ; –
trans-2-Hexen-1-ol 718 ± 52 a 576 ± 98 a 245 ± 3.1 b 66 ; 1092 ± 58 x 1158 ± 60 x 1103 ± 314 x – 78 "
SubTOTAL 1480 1144 889 40 ; 2741 2740 1995 14 "
% 36 23 21 37 30 34

Aromatic alcohols
Benzyl alcohol 40 ± 0.64 b 70 ± 12 a 32 ± 0.034 b – 64 ± 1.2 x 81 ± 21 x 42 ± 3.2 x – 23 "
2-Phenylethanol 27 ± 0.25 b 31 ± 0.20 a 24 ± 2.4 b – 55 ± 6.3 xy 63 ± 1.7 x 48 ± 1.1 y – 51 "
SubTOTAL 67 101 56 17 ; 119 144 90 24 ;
% 1.6 2.0 1.3 1.6 1.6 1.5

Volatile phenols
4-Ethylguaiacol
Eugenol 1.9 ± 0.035 a 1.7 ± 0.11 a 1.9 ± 0.061 a – 2.2 ± 0.21 x 2.2 ± 0.053 x 2.5 ± 0.44 x – –
4-Ethyl-phenol
4-Vinylguaiacol
Syringol 1.2 ± 0.0083 a,b 1.1 ± 0.10 b 1.3 ± 0.023 a – 1.2 ± 0.0033 y 1.2 ± 0.0057 y 1.9 ± 0.31 x 56 " –
Vanillin 12 ± 0.047 b 16 ± 1.4 a 16 ± 0.77 a 33 " 12 ± 0.73 z 14.6 ± 0.36 y 24 ± 0.52 x 93 " 33 "
Acetovainillone 7.0 ± 0.0018 c 12 ± 1.1 b 15 ± 0.39 a 117 " 26 ± 0.25 y 28.6 ± 1.1 y 37 ± 0.85 x 41 " 59 "
SubTOTAL 22 31 34 56 " 42 47 65 55 "
% 0.53 0.62 0.80 0.57 0.50 1.1

Miscellaneous
Hexyl acetate
Pantolactone
SubTOTAL
%
A
Values are mean ± standard error (n = 2). Different letters within rows indicate statistical differences according to ANOVA (p < 0.05): a, b, c for tips positions and x, y, z for
shoulders positions.
B
Increase percentage from the 1st sampling to the 3rd sampling calculated as (((concentration of 3rd sampling – concentration of 1st sampling)/concentration 1st
sampling) * 100).
C
Increase percentage between the tips and shoulders berries at 3rd sampling calculated as (((concentration of tips berries at 3rd sampling – concentration of shoulders
berries at 3rd sampling)/concentration of shoulders berries at 3rd sampling) * 100).

aldehydes in the berry flesh of the shoulders compared to the tips,
where they were mainly found in the bound form.
The C6 alcohols (Fig. 2d) were mainly present in the free form. In
the tips they were mainly found in the berry flesh, whereas in the
shoulders the skin had a concentration four times larger than in the
flesh. In general, the berries from the tips had a higher content of
C6 alcohols than those from the shoulders.
On the contrary, aromatic alcohols (Fig. 2e) were mainly de-
tected in the bound form. The berry skin rarely contained aromatic
alcohols, as the flesh is the compartment where most of these com-
pounds can be found. Considering the different positions of the
berries within the cluster, the tip was richer in these compounds.
Regarding volatile phenols (Fig. 2f), the most remarkable were
their higher content in the bound form in the flesh, especially in
the tips of the clusters. The skin was richer in free volatile phenols.
On the whole, the berries from the tips had a larger proportion of
these compounds, mainly in the bound form.
In the miscellaneous group (Fig. 2g), the main compound was
pantolactone, and it only appeared in the flesh of the berries. The
berries from the shoulders were richer in this compound than
 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124 119

Table 4
Evolution of bound volatile compounds (ng/g of berry) released from flesh and skin from Brancellao berries located at two different positions within the cluster (tips and
shoulders).

Tips % D31B Shoulders % D31B % DabC

1st Sampling 2nd Sampling 3rd Sampling 1st Sampling 2nd Sampling 3rd Sampling
FleshA
Monoterpenes
(±)-Linalool 16 ± 1.6 b 18 ± 0.19 b 37 ± 0.75 a 127 " 6.2 ± 0.031 z 12 ± 0.11 y 13 ± 0.42 x 114 " 176 ;
()-Terpinen-4-ol –
a-Terpineol 6.0 ± 0.55 b 6.0 ± 0.15 b 7.9 ± 0.13 a 31 " 3.9 ± 0.035 xy 4.4 ± 0.25 x 3.7 ± 0.19 y – –
(±)-b-Citronellol 1.9 ± 0.022 b 2.3 ± 0.11 a 2.4 ± 0.035 a 30 " 0.79 ± 0.088 x 0.73 ± 0.015 x 0.84 ± 0.063 x – 187 ;
Nerol 4.5 ± 0.28 c 5.9 ± 0.30 b 6.8 ± 0.0016 a 53 " 3.5 ± 0.22 y 4.4 ± 0.057 x 3.8 ± 0.24 xy – 79 ;
Geraniol 20 ± 1.9 b 24 ± 0.79 a,b 29 ± 4.3 a 45 " 16 ± 1.2 x 18 ± 1.9 x 19 ± 4.6 x – –
Geranic acid 198 ± 3.3 a 187 ± 10.3 a 201 ± 5.5 a – 56 ± 0.29 x 54 ± 2.0 x 52 ± 4.0 x – 289 ;
trans, trans-Farnesol 8.1 ± 0.45 b 9.5 ± 0.37 a 9.2 ± 0.23 a,b – 6.3 ± 0.42 x 7.8 ± 0.32 x 7.3 ± 0.84 x – –
SubTOTAL 255 254 293 15 " 93 101 99 7"
% 7.4 6.3 7.0 3.8 3.6 3.8

C13-norisoprenoids
TheaspiraneA 0.79 ± 0.025 a 0.78 ± 0.0064 a 0.79 ± 0.0020 a – 0.28 ± 0.0024 a 0.30 ± 0.019 a 0.23 ± 0.067 a – 236 ;
TheaspiraneB 0.32 ± 0.029 a 0.29 ± 0.029 a 0.29 ± 0.021 a – 0.32 ± 0.0021 a 0.34 ± 0.027 a 0.23 ± 0.081 a – –
b-Damascenone –
b-Ionone –
SubTOTAL 1.1 1.1 1.1 3; 0.61 0.64 0.46 25 ;
% 0.032 0.027 0.026 0.025 0.023 0.018

Aldehydes
Hexanal 14 ± 1.8 a 7.0 ± 0.50 b 6.2 ± 0.032 b 55 ; 5.1 ± 0.77 x 6.4 ± 2.1 x 4.1 ± 0.60 x – 51 ;
Heptanal –
trans-2-Hexenal 420 ± 41 a 445 ± 46 a 409 ± 37 a – 19 ± 1.3 y 26 ± 3.6 x 21 ± 1.4 xy – 1844 ;
Benzaldehyde 4.1 ± 1.1 a 3.4 ± 0.4 a 3.6 ± 0.058 a – 1.6 ± 0.27 x 1.8 ± 0.38 x 0.69 ± 0.84 x – 418 ;
SubTOTAL 439 456 420 4; 25 35 26 1.7 "
% 13 11 10 1.0 1.2 1.0

Alcohols C6
1-Hexanol 131 ± 3.2 a 148 ± 15.5 a 162 ± 8.0 a – 92 ± 3.1 y 113 ± 3.9 x 97 ± 6.9 y – 67 ;
trans-3-Hexen-1-ol 1.2 ± 0.017 b 1.7 ± 0.21 a 1.7 ± 0.012 a 39 " 1.0 ± 0.021 y 1.5 ± 0.028 x 1.0 ± 0.0009 y – 68 ;
cis-3-Hexen-1-ol 23 ± 3.0 a 23 ± 1.5 a 18 ± 0.57 a – 22 ± 1.2 y 25 ± 0.26 x 13 ± 0.22 z 41 ; 37 ;
trans-2-Hexen-1-ol 31 ± 2.4 b 39 ± 2.1 a 32 ± 0.83 b – 23 ± 1.2 y 37 ± 0.43 x 18 ± 1.4 z 22 ; 83 ;
SubTOTAL 187 213 214 15 " 138 176 129 5;
% 5.4 5.3 5.1 5.6 6.2 5.0

Aromatic alcohols
Benzyl alcohol 2209 ± 21 a 2682 ± 321 a 2775 ± 104 a – 1931 ± 30 x 2262 ± 15 x 2122 ± 366 x – –
2-Phenylethanol 248 ± 8.2 b 272 ± 18 b 329 ± 8.9 a 32 " 197 ± 2.0 x 202 ± 3.0 x 168 ± 48 x – 95 ;
SubTOTAL 2458 2955 3104 26 " 2129 2465 2291 7"
% 71 74 74 87 87 87
Volatile phenols
4-Ethylguaiacol 0.71 ± 0.036 a 0.57 ± 0.022 b 0.70 ± 0.0044 a – 0.35 ± 0.0044 x 0.24 ± 0.029 x 0.30 ± 0.081 x – 132 ;
Eugenol 8.3 ± 0.34 b 8.0 ± 0.34 b 11 ± 0.047 a 32 " 8.4 ± 0.33 x 6.8 ± 0.069 x 6.3 ± 1.3 x – 74 ;
4-Ethyl-phenol 0.94 ± 0.034 b 0.66 ± 0.0027 c 1.1 ± 0.016 a 19 " 0.84 ± 0.016 x 0.71 ± 0.034 x 0.76 ± 0.13 x – –
4-Vinylguaiacol 24 ± 3.7 b 48 ± 4.5 a 57 ± 1.5 a 140 " 11 ± 0.89 x 15 ± 1.1 x 16 ± 4.8 x – 247 ;
Syringol 1.6 ± 0.19 a 1.5 ± 0.029 a 1.9 ± 0.043 a – 0.84 ± 0.068 x 0.81 ± 0.033 x 1.0 ± 0.11 x – 87 ;
Vanillin 15 ± 0.33 b 15 ± 0.33 b 19 ± 0.013 a 23 " 5.4 ± 0.39 y 6.4 ± 0.31 y 9.7 ± 0.59 x 80 " 92 ;
Acetovainillone 51 ± 2.3 c 60 ± 1.6 b 68 ± 0.14 a 35 " 36 ± 0.63 x 33 ± 1.0 y 36 ± 0.019 x – 88 ;
SubTOTAL 101 133 158 57 " 62 63 71 13 "
% 2.9 3.3 3.8 2.5 2.2 2.7

Miscellaneous
Hexyl acetate –
Pantolactone 5.5 ± 0.30 a 6.8 ± 0.79 a 6.4 ± 0.20 a – 7.7 ± 0.11 x 5.2 ± 3.5 y 9.6 ± 0.33 y 25 " 33 "
SubTOTAL 5.5 6.8 6.4 17 " 7.7 5.2 9.6 25 "
% 0.16 0.17 0.15 0.31 0.18 0.37
SkinA
Monoterpenes
(+/-)-Linalool 3.4 ± 0.096 c 5.6 ± 0.24 b 12 ± 0.74 a 259 " 7.7 ± 0.77 z 15 ± 0.049 y 19 ± 0.020 x 147 " 36 "
(-)-Terpinen-4-ol
a-Terpineol 1.5 ± 0.042 b 2.4 ± 0.27 a 2.5 ± 0.073 a 61 " 3.2 ± 0.47 y 3.5 ± 0.097 xy 4.4 ± 0.44 x 38 " 44 "
(±)-b-Citronellol 0.50 ± 0.034 x 0.74 ± 0.078 x 0.65 ± 0.045 x –
Nerol 0.91 ± 0.10 c 9.4 ± 0.0028 b 12 ± 0.30 a 1186" 1.6 ± 0.056 y 2.5 ± 0.078 x 2.7 ± 0.067 x 66 " 332 ;
Geraniol 6.1 ± 0.60 a 2.7 ± 1.2 b 4.2 ± 1.0 ab 31 ; 0.96 ± 0.31 y 2.0 ± 0.87 xy 2.8 ± 0.13 x 188 " –
Geranic acid 4.8 ± 0.11c 9.5 ± 0.31 b 11 ± 0.068 a 137 ; 35 ± 17 x 33 ± 0.12 x 68 ± 28 x – –
trans, trans-Farnesol

(continued on next page)

120 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124

Table 4 (continued)

Tips % D31B Shoulders % D31B % DabC

1st Sampling 2nd Sampling 3rd Sampling 1st Sampling 2nd Sampling 3rd Sampling

SubTOTAL 17 30 42 151 " 49 57 98 98 "

% 27 31 51 52 55 68

C13-Norisoprenoids
Theaspirane A
Theaspirane B
b-Damascone
b-Ionone 0.023 ± 0.0030x 0.029 ± 0.000076x 0.027 ± 0.0018x –
SubTOTAL 0.023 0.029 0.027 19 "
% 0.024 0.028 0.019

Aldehydes
Hexanal 13 ± 2.0 a 23 ± 6.4 a 18 ± 4.1 a – 15 ± 4.3 x 14 ± 2.0 x 17 ± 3.6 x – –
Heptanal
trans-2-Hexenal 2.4 ± 0.63 b 3.3 ± 1.1 ab 5.2 ± 0.51 a 116 " 2.9 ± 0.89 x 3.0 ± 0.14 x 2.6 ± 0.33 x – 97 ;
Benzaldehyde 0.92 ± 0.029 a 0.58 ± 0.43 a 0.21 ± 0.00070a – 0.38 ± 0.019 x 0.30 ± 0.068 x 0.31 ± 0.015 x – 31 "
SubTOTAL 17 27 24 41 " 18 17 20 11 "
% 27 28 29 19 16 14

Alcohols C6
1-Hexanol 5.4 ± 0.32 ab 6.9 ± 1.1 a 4.2 ± 0.13 b – 5.5 ± 1.9 x 5.3 ± 1.0 x 5.7 ± 1.7 x – –
trans-3-Hexen-1-ol
cis-3-Hexen-1-ol 0.61 ± 0.0091a 0.65 ± 0.037a –
trans-2-Hexen-1-ol 0.99 ± 0.084 a 1.3 ± 0.21 a 0.53 ± 0.038 b 47 ; 1.6 ± 0.29 x 1.7 ± 0.25 x 1.7 ± 0.47 x – –
SubTOTAL 7.0 8.9 4.7 34 7.1 7.0 7.4 5"
% 11 9.2 5.7 7.4 6.7 5.2

Aromatic alcohols
Benzyl alcohol 13 ± 1.7 ab 20 ± 6.8 a 6.3 ± 0.62 b – 14 ± 1.0 x 14 ± 3.8 x 10 ± 0.48 x – 40 "
2-Phenylethanol 3.9 ± 0.29 a 3.5 ± 0.23 a 1.5 ± 0.18 b 62 ; 2.2 ± 0.30 x 2.6 ± 0.62 x 2.2 ± 0.28 x – –
SubTOTAL 17 24 7.8 55 ; 16 17 13 22 ;
% 28 24 9.5 17 16 8.8

Volatile phenols
4-Ethylguaiacol
Eugenol 1.2 ± 0.048 a 1.1 ± 0.072ab 0.98 ± 0.036 b 18 ; 1.3 ± 0.067 x 1.4 ± 0.16 x 1.5 ± 0.12 x – 36 "
4-Ethyl-phenol
4-Vinylguaiacol 0.33 ± 0.054 b 1.7 ± 0.051 a 0.35 ± 0.098 b – 0.23 ± 0.14 x 0.53 ± 0.13 x 0.23 ± 0.082 x – –
Syringol 0.57 ± 0.051 a 0.71 ± 0.056 a 0.58 ± 0.040 a – 0.52 ± 0.095 x 0.69 ± 0.0035 x 0.62 ± 0.083 x – –
Vanillin 0.57 ± 0.26 a 1.0 ± 0.080 a 0.92 ± 0.17 a – 1.1 ± 0.13 y 1.4 ± 0.028 x 1.5 ± 0.15 x 39 " –
Acetovainillone 1.3 ± 0.66 ab 2.7 ± 0.47 a 1.2 ± 0.011 b – 1.9 ± 0.19 x 2.2 ± 0.33 x 2.6 ± 0.18 x – 55 "
SubTOTAL 4.0 7.3 4.0 0.2 ; 5.0 6.3 6.4 27 "
% 6.5 7.5 4.9 5.3 6.1 4.4

Miscellaneous
Hexyl acetate
Pantolactone
SubTOTAL
%
A
Values are mean ± standard error (n = 2). Different letters within rows indicate statistical differences according to ANOVA (p < 0.05): a, b, c for tips positions and x, y, z for
shoulders positions.
B
Increase percentage from the 1st sampling to the 3rd sampling calculated as (((concentration of 3rd sampling – concentration of 1st sampling)/concentration 1st
sampling) * 100).
C
Increase percentage between the tips and shoulders berries at 3rd sampling calculated as (((concentration of tips berries at 3rd sampling – concentration of shoulders
berries at 3rd sampling)/concentration of shoulders berries at 3rd sampling) * 100).

those from the tips. The concentration of free pantolactone was
similar in both cluster positions, whereas the shoulders contained
a larger concentration of bound pantolactone.
To sum up, the concentration of free volatiles was very similar
in both positions within the cluster, and the main differences be-
tween tips and shoulders were regarding the bound volatiles. The
total concentration of glycosides increased with ripening, espe-
cially monoterpenes (Table 4). This demonstrated that a higher
concentration of flavour compounds could be established in the
berries by leaving the fruit on the vine for extended periods and
confirmed Hardy’s (1970) conclusion that sugar and acid values
do not give an accurate indication of ripeness from an aroma point
of view. Of the total monoterpenes there was a greater concentra-
tion present as flavourless glycosides than as free compounds. This
clearly indicates that juice processing techniques, such as pH
adjustment and heating to hydrolyse these flavourless forms, are
appropriate to induce flavour in the must.
3.4. Ratios (bound/free and skin/flesh) found for chemical classes of
volatile compounds
In the berries from the tips of the clusters (Fig. 3a), most of vol-
atiles were found in the flesh, except in the case of aldehydes,
which were dominant in the skin. Hexanal and cis-3-hexenal were
produced by oxidation of the linoleic and linolenic acids catalysed
by the lipoxygenase enzyme, followed by the action of a peroxidase
 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124 121

Monoterpenes Norisoprenoids
(a) 400 (b) 3.0
350
2.5
300
ng/g of berry

ng/g of berry
250 2.0
200 1.5
150
1.0
100
50 0.5
0 0.0
flesh skin flesh skin flesh skin flesh skin

tips shoulders tips shoulders

Free volatile compounds Bound volatile compounds Free volatile compounds Bound volatile compounds

(c) Aldehydes (d) Alcoholes C6

4000 2500
3500
3000 2000
ng/g of berry

ng/g of berry
2500 1500
2000
1500 1000
1000
500
500
0 0
flesh skin flesh skin flesh skin flesh skin

tips shoulders tips shoulders

Free volatile compounds Bound volatile compounds Free volatile compounds Bound volatile compounds

Aromatic alcohols Volatile phenols

(e) 4000 (f) 180
3500 160
3000 140
ng/g of berry

ng/g of berry

2500 120
2000 100
80
1500
60
1000
40
500 20
0 0
flesh skin flesh skin flesh skin flesh skin

tips shoulders tips shoulders

Free volatile compounds Bound volatile compounds Free volatile compounds Bound volatile compounds

Miscellaneous
(g)
ng/g of berry

flesh skin flesh skin

tips shoulders
Free volatile compounds Bound volatile compounds

Fig. 2. Total concentration (ng/g of berry) of different chemical classes of volatile compounds obtained in the 3rd sampling (a–g) split by two different berry position within
the cluster (tips/shoulders), flesh/skin content, and free/bound volatile form.

that causes the rupture of the hydroperoxides in C-13 (Drawert, skin. The C6 alcohols are products from the reduction of their
1966). trans-2-Hexenal is a product of the isomerization of cis-3- respective aldehydes by the alcohol-dehydrogenase. An isomerase
hexenal (Crouzet, 1999). In the berries from the shoulders enzyme may also be present which catalyses the reaction from cis-
(Fig. 3b), most of the volatile chemical classes (monoterpenes, nor- 3-hexenol to trans-2-hexenol (Crouzet, 1999). The dominance of
isoprenoids, aldehydes, and C6 alcohols) were mainly found in the alcohols in the late stages of berry development, preceded by
122 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124

aldehydes, offers an opportunity for alcohols to aldehydes ratios to
be used in the prediction of harvest timing for enhanced grape and
wine aroma. This dominance is desirable as alcohols usually have
higher herbaceous odour thresholds than related aldehydes. Fur-
thermore, alcohols possess a higher propensity to form fruity es-
ters in the presence of carboxylic acids during vinification than
aldehydes, thereby minimising the herbaceous character from
aldehydes and maximising the fruity characters from esters (Kalua
& Boss, 2009). On the other hand, the flesh was slightly richer in
aromatic alcohols, volatile phenols and pantolactone.
As regards the volatile chemical form (free or bound), both ber-
ry positions within the cluster showed a very similar trend. Aro-
matic alcohols, volatile phenols and miscellaneous compounds
(hexyl acetate and pantolactone) were mainly in the bound form,
whereas aldehydes and C6 alcohols can be mainly found in the free
form. For monoterpenes and norisoprenoids, free and bound forms
appeared practically at the same level.
3.5. Changes found in volatile odour descriptors in grapes with bunch
position
In order to assess the influence of the compounds studied on
overall wine aroma, OAVs were calculated by dividing the concen-
tration of each compound by its perception threshold. Of all the
compounds analysed, only those displaying OAVs higher than 1
were deemed to contribute to wine aroma (Guth, 1997). Neverthe-
less, recent studies have reported the relevance to the overall aro-
ma of substances present at concentrations representing at least
20% of their threshold value (OAV > 0.2) (Gómez-Míguez, Gómez-
Míguez, Vicario, & Heredia, 2007). Table 1 shows the OAVs for
the compounds, together with sensory descriptors and the percep-
tion threshold taken from the literature. The terpene displaying the
highest OAV value was linalool (orange flowers); similar findings
have been reported for other aromatic varieties including Albariño
and Gewürztraminer (Falqué, Fernández, & Dubourdieu, 2001; Ong
& Acree, 1999). C13-norisoprenoids were present at concentrations
below their odour thresholds, with the exception of b-ionone (vio-
let descriptor). Aldehydes with major sensory impact were hexanal
and trans-2-hexenal (grass descriptor). Volatile phenols tend to
present spicy nuances and may play a major sensory role, espe-
cially in neutral grapevine varieties containing negligible amounts
of terpenes. In this way, eugenol (clove descriptor), 4-vinylguaiacol
(phenolic descriptor) and vanillin (vanilla descriptor) displayed
higher OAVs. According to the threshold value of OAV > 0.2
(Table 1), floral odours to anise (trans, trans-farnesol) and rose
(2-phenylethanol), herbaceous odours to grass (1-hexanol) and
green (cis-3-hexen-1-ol), and the spicy odour to walnut (benzyl
alcohol) could contribute to the final aroma at the end of ripeness.
The main odour descriptors in the berries were floral and herba-
ceous (Table 1). The berries from the shoulders of the clusters were
richer in floral and herbaceous nuances than those from the tips
(Fig. 3c). The spicy nuances can be found at higher levels in the tips,
especially at longer maturation stages. b-Ionone, despite having lit-
tle quantitative importance in terms of concentration, showed a
very large OAV because of its low odour threshold (0.007 lg/l).
When its influence is removed from the overall floral nuance
(Fig. 3d), the berries from the tips became richer in the rest of floral
odours together with those that were spicy.
In general, on the basis of OAVs (Table 1), linalool (orange flow-
ers descriptor) was the most floral-active odorant in the tips of the
clusters, whereas b-ionone (violet descriptor) was in the shoulders.
Other volatiles potentially contributing to the herbaceous aroma
were trans-2-hexenal in the tips and hexanal in the shoulders, both
with a grass descriptor. As regards spicy odours, eugenol (clove

(a) (b) 10
12
11 Tips 9 Shoulders
10 8
9 7
8
6
Ratio
Ratio

7
6 5
5 4
4 3
3
2 2
1 1
0 0

Bound/Free
Bound/Free
Skin/Flesh Skin/Flesh

(c) 2 (d) 2
Shoulders/Tips Ratio
Shoulders/Tips Ratio

1
1

0
0 FLORAL HERBACEOUS SPICY
(without -Ionone)
FLORAL HERBACEOUS SPICY

1st sampling 2nd sampling 3rd sampling 1st sampling 2nd sampling 3rd sampling

Fig. 3. Bound:free and skin:flesh ratios of total concentrations of different chemical classes of volatile compounds in the 3rd sampling for (a) tips and (b) shoulders, together
with total OAVs of different odour descriptors for shoulders:tips ratios (c and d). In (d) the floral ratio was calculated without taking into consideration b-ionone.
 R. Noguerol-Pato et al. / Food Chemistry 132 (2012) 112–124
descriptor) and 4-vinylguaiacol (phenolic descriptor) had higher
OAVs in the berries from the tips.
4. Conclusions
The variability between shoulders and tips of the clusters for
the probable alcoholic degree and total acidity parameters was lit-
tle or not significant during the samplings, whereas important dif-
ferences, with respect to the aromatic composition, were observed
in Brancellao berries. These results could be very useful for those
wineries that are considering the possibility of separating shoul-
ders and tips, with the aim of making wines of different character-
istics and qualities. The fact of finding great differences at an
aromatic level (in skin or flesh), between shoulders and tips, and
hardly any variability with respect to probable alcoholic degree
and total acidity, open the door to any winery being able to com-
bine the selection of tips or shoulders berries with different times
for the maceration of peels and must based on the type of aroma
that is desirable to promote.
In Brancellao berries, aromatic alcohols were mainly in bound
form, whereas aldehydes and C6 alcohols can be mainly found in
free form. This makes interesting the use of glycolytic enzymes
during maceration to liberate the bound floral nuances for the ber-
ries coming from either tips or shoulders.
For the berries from the tips of the clusters, most of the volatiles
were found in the flesh (except aldehydes). Spicy and floral nuan-
ces (with the only exception of b-ionone), those with a higher im-
pact, were in higher levels.
In conclusion, from a flavour point of view, it can be recom-
mended that the berries from the tips have the berry skins sepa-
rated before enzymatic maceration of the berry flesh must. The
wines obtained from the tips of Brancellao clusters have a bigger
chance for very typical and peculiar nuances, and are what give
an extra-distinction to high quality wines.
For the berries from the shoulders of the clusters, most of the
volatiles were found in the skin (monoterpenes, norisoprenoids,
aldehydes, and C6 alcohols), with the flesh being slightly richer in
aromatic alcohols, volatile phenols and pantolactone; violet (odour
descriptor of b-ionone) and herbaceous nuances (non-characteris-
tic odours) were in higher proportions.
In conclusion, from a flavour point of view, it can be recom-
mended for the berries from the shoulders to maintain berry skins
during enzymatic maceration of the must. The wines obtained
from shoulders could have a bigger chance for very common
nuances.
By comparing the time-evolution of Brancellao berries at two
different positions within the cluster, it seems that those from the
tips are ahead in developing variety-linked aroma compounds. Free
monoterpenes in berry flesh increased around 40% both in tips and
shoulders in the 3rd sampling with regards to the 1st sampling. This
increase was mainly conditioned by a 60% increase in geraniol, with
a typical geranium descriptor. Bound monoterpenes experienced an
important augment both in skin and flesh of berries, being their
flesh richer in these compounds. Linalool was the bound monoter-
pene with the highest increase in all situations. Therefore, the
screening of geraniol and linalool during ripening could be used
to define the evolution profile of the varietal volatile compounds.
Acknowledgements
This work was Granted by EU FEDER funds, by Ministry of
Science and Technology (Spain) INIA RF-2008-00002-C02 and by
the INCITE09PXIB383322 PR (Autonomous Community Govern-
ment in N.W. Spain) contracts. C. Gonzalez-Barreiro acknowledges
the grant from the Galician Human Resources Program known as
‘‘Isidro Parga Pondal’’, whereas R. Noguerol-Pato acknowledges
123
the Grant from the Spanish Human Resources Program known as
‘‘FPU’’. Elena Zubiaurre and Mª Soledad Taboada are thanked for
providing technical assistance in the plot.
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