Food Chemistry 124 (2011) 106–116

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

Implications of nitrogen compounds during alcoholic fermentation from some

grape varieties at different maturation stages and cultivation systems
Teresa Garde-Cerdán a, Ana M. Martínez-Gil a, Cándida Lorenzo a, José Félix Lara a,
Francisco Pardo b, M. Rosario Salinas a,*
a
Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain
b
Bodega San Isidro (BSI), Carretera de Murcia s/n, 30520 Jumilla, Murcia, Spain

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

Article history: The aim of this work was to study the influence of grape variety, cultivation system and stage of grape
Received 9 February 2010 maturation on nitrogen compounds evolution during alcoholic fermentation. To do this, four grape vari-
Received in revised form 8 April 2010 eties, Monastrell, Merlot, Syrah and Petit Verdot, traditionally cultivated and Monastrell cultivated using
Accepted 26 May 2010
organic agriculture, which were collected in two different stages of maturation, were used. The results
showed that, regardless of grape variety, cultivation system and stage of grape maturation, the consump-
tion of nitrogen compounds was directly proportional (R2 > 0.7) to their concentration in natural musts.
Keywords:
This is very important as it is the first time that these results were obtained with natural musts without
Amino acids
Grape varieties
external addition of nitrogen compounds.
Wine Ó 2010 Elsevier Ltd. All rights reserved.
Must
Maturation stages

1. Introduction 1993; Héberger, Csomós, & Simon-Sarkadi, 2003; Valero, Millán,

Nitrogen compounds of must are essential in the metabolism of
yeast because the nitrogen is, after carbon, the second element uti-
lised during its growth. The content of these compounds affects the
kinetics of fermentation, as nitrogen-deficient musts can cause
slow and stuck fermentations (Arias-Gil, Garde-Cerdán, & Ancín-
Azpilicueta, 2007; Bisson, 1991). Also, several amino acids undergo
a series of biotransformations, yielding higher alcohols, aldehydes,
esters and kenotic acids, compounds that contribute to wine aroma
(Soufleros, Bouloumpasi, Tsarchopoulos, & Biliaderis, 2003; Vilano-
va et al., 2007). However, wines with higher amounts of residual
nitrogen have more risk of microbiological instability, with the
possible formation of biogenic amines and ethyl carbamate, which
are negative compounds for wine quality (Moreno-Arribas & Polo,
2009; Uthurry, Suárez Lepe, Lombardero, & García del Hierro,
2007).
The consumption of nitrogen compounds during fermentation
mainly depends on the physicochemical properties of the must
(pH, acidity and sugars), on the grape variety, on the nitrogen com-
position of the must, on yeast and on the fermentation tempera-
ture, among other factors (Barbosa, Falco, Mendes-Faia, &
Mendes-Ferreira, 2009; Bell & Henschke, 2005; Bouloumpasi, Sou-
flerops, Tsarchopoulos, & Biliaderis, 2002; Henschke & Jiranek,
* Corresponding author.
E-mail address: Rosario.Salinas@uclm.es (M.R. Salinas).
Ortega, & Mauricio, 2003). Nevertheless, most of the work carried
out, until now, uses a model must in which the initial conditions
have been previously decided (Barbosa et al., 2009; Beltran,
Esteve-Zarzoso, Rozés, Mas, & Guillamón, 2005; Carrau et al.,
2008; Hernández-Orte, Cacho, & Ferreira, 2002; Manginot,
Roustan, & Sablayrolles, 1998; Martínez-Rodríguez & Polo, 2000;
Mendes-Ferreira, Barbosa, Falco, Leão, & Mendes-Faia, 2009;
Mendes-Ferreira, Mendes-Faia, & Leão, 2004; Taillandier, Portugal,
Fuster, & Strehaiano, 2007; Vilanova et al., 2007); in the cases of
using a real must, a single grape variety to which nitrogen
compounds are usually added, is used (Arias-Gil et al., 2007;
Garde-Cerdán & Ancín-Azpilicueta, 2008; Hernández-Orte, Ibarz,
Cacho, & Ferreira, 2005; Ugliano, Siebert, Mercurio, Capone, &
Henschke, 2008; Ugliano et al., 2009). In the majority of wineries
it is common practice to add diammonium phosphate (DAP) to
the must before the fermentation without having carried out pre-
vious studies of the nitrogen composition of the must. So, it would
be important to determine the initial concentration of nitrogen
compounds. This could avoid unnecessary additions of nitrogen,
thus avoiding possible negative effects in wines later on.
In previous work, different evolutions of the amino acid content
of the different grapes varieties were observed at the end of the
grape ripening (Garde-Cerdán et al., 2009). So, the two last samples
of grape maturation were selected in order to do the alcoholic fer-
mentation and to study the possible effect of stage grape matura-
tion on the evolution of nitrogen compounds during alcoholic

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.05.112
 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116
fermentation. This different stage of maturation is determined by
the °Baumé/total acidity ratio, among other parameters. This stage
of maturation influences the content of nitrogen compounds and
therefore affects the ability of must fermentation and quality of
wine. For these reasons, the aim of this work was to study the
influence of grape variety, cultivation system and stage of grape
maturation on nitrogen compounds evolution during alcoholic
fermentation.
2. Material and methods
2.1. Samples and vinification
For this study, Monastrell, Syrah, Merlot, and Petit Verdot non-
organic and Monastrell organic, red grape varieties cultivated in
O.D. Jumilla (SE of Spain) and harvested in 2007 were used. Envi-
ronmental conditions were identical for the different vineyards.
Jumilla is a warm region with a maximum temperature of 40 °C
and a minimum temperature of 0 °C, and an of average 3000 h of
sunshine and 300 mm of rainfall per year (www.winesfroms-
pain.com). In the conventional agriculture system (Monastrell
non-organic, Syrah, Merlot, and Petit Verdot), the vineyards were
cultivated in trellis and were fitted with a drip irrigation system.
They were fertilised with liquid fertilizer NPK 8-4-10 (%, w/w)
(Agribeco, Spain), applied in total 250 g per vine. In the case of
the organic system (Monastrell organic), the vineyard was culti-
vated in vessel and was treated with fertilizer ‘‘cultivit ecológico”
(Agribeco), consisting of dried granulated sheep manure, the com-
position of which was NPK 1.55-1.21-2.35 (%, w/w), applying in to-
tal 200 g per vine. The organic system was not irrigated.
The Monastrell non-organic grapes were collected on Septem-
ber 27 (MnO1) and October 8 (MnO2), the Monastrell organic
grapes on September 19 (MO1) and 27 (MO2), the Syrah grapes
on September 12 (SY1) and 19 (SY2), the Merlot grapes on Septem-
ber 5 (ME1) and 12 (ME2), and the Petit Verdot grapes on Septem-
ber 19 (PV1) and 27 (PV2). For each variety, the first sample
corresponded to the pre-harvest sample (about a week before har-
vest) and the second sample corresponded to the complete ripe-
ness at grape harvest.
The grape was destemmed and crushed and afterwards, it
underwent pressing to obtain the must. For each sample, 400 ml
was used, which was divided into two aliquots as they were fer-
mented in duplicate. Must were inoculated with active dry Saccha-
romyces cerevisiae subsp. cerevisiae (U.C.L.M. S325, Springer
Oenologie, Argentina) in a proportion of 0.2 g/l according to com-
mercial recommendations. For this, 0.65 g of dry yeast was rehy-
drated in a sterile flask in 7.5 ml of distilled water with 0.07 g of
sucrose (number of viable cells/ml P 2  109); it was kept in this
medium for 30 min at 35 °C. The musts were inoculated while
being mixed to obtain a homogeneous distribution. Before fermen-
tation, potassium metabisulfite was added to the musts to give a
final total SO2 concentration of 70 mg/l.
The fermentations took place in glass fermenters with a capac-
ity of 250 ml and with a lid with two outlets; one for sample
extractions and the other with a CO2 trap to allow its exit and pre-
vent the entrance of air during fermentation. The orifice for sample
extraction was covered with a septum during the fermentation.
The fermenters were placed over magnetic stirrers, to ensure a
homogenous fermentation. The fermentations were carried out in
a hot incubator at a controlled temperature of 28 °C. The evolution
of the fermentation was followed by the daily measurement of
sugars through the refraction index at 20 °C, using a refractometer
CT (Sopelem, France). The samples were taken in the middle of the
fermentation (when 50% of the sugars were consumed) and when
fermentation was finished (reducing sugars < 2.5 g/l).
107
2.2. Enological parameters
Titratable acidity, pH, volatile acidity, reducing sugars, and alco-
hol of different samples were measured following the methods
established by the ECC (1990). The phenolic ripeness of the grapes
was measured indirectly from the colour intensity of the extract
obtained by crushing 200 grains without breaking the seeds and
then it was centrifugated. The colour intensity was determined
by the sum of the absorbances at 420, 520, and 620 nm (Franco
& Iñiguez, 1999), and this parameter is called the colour index.
2.3. Analysis of amino acids and ammonium by HPLC
The analysis of amino acids and ammonium of the half fermen-
tation samples and of the final wines were made using the method
described by Gómez-Alonso, Hermosín-Gutiérrez, and García-
Romero (2007). The results for initial musts used for this study
were taken from a previous work (Garde-Cerdán et al., 2009).
The derivatization of amino acids and ammonium was carried
out by reaction of 1.75 ml of borate buffer 1 M (pH 9), 750 ll of
methanol (Merck, Darmstadt, Germany), 1 ml of sample (previ-
ously filtered), 20 ll of internal standard (2-aminoadipic acid,
1 g/l) (Aldrich, Gillingham, England) and 30 ll of derivatization re-
agent diethyl ethoxymethylenemalonate (DEEMM) (Aldrich). The
reaction of derivatization was done in a screw-cap test tube over
30 min in an ultrasound bath. The sample was then heated at
70–80 °C for 2 h to allow complete degradation of excess DEEMM
and reagent by-products.
The analyses were performed on an Agilent 1100 HPLC (Palo
Alto, USA), with a photodiode array detector. Chromatographic
separation was performed in an ACE HPLC column (C18-HL) (Aber-
deen, Scotland) particle size 5 lm (250 mm  4.6 mm). Amino
acids were eluted under the conditions described by Gómez-Alon-
so et al. (2007). Phase A, 25 mM acetate buffer, pH 5.8, with 0.4 g of
sodium azide; phase B, 80:20 (v/v) mixture of acetonitrile and
methanol (Merck). A photodiode array detector monitored at
280, 269 and 300 nm was used to detection. The volume of sample
injected was 50 ll. The analysis of amino acids and ammonium
was done in duplicate, and as the fermentations were done in
duplicate, the results shown for amino acids and ammonium in
the samples at the middle and the end of fermentations were the
mean of four analyses. To improve clarity, the results presented
in figures do not include standard deviation; however, the coeffi-
cients of variation for amino acids concentration were between
1% and 15%. The target compounds were identified according to
the retention times and the UV–vis spectral characteristics of cor-
responding standards (Aldrich) derivatizated. Quantification was
carried out by using the calibration graphs of the respective stan-
dards (R2 > 0.98) in 0.1 N HCl, which underwent the same process
of derivatization that the samples.
The amino nitrogen was calculated by determining free amino
acids by HPLC and the assimilable nitrogen was calculated as the
amount of ammonium and amino nitrogen without taking into
account the proline.
2.4. Statistical analysis
The statistical elaboration of the data was performed using the
SPSS Version 17.0 statistical package for Windows (SPSS, Chicago,
USA). Discriminant analyses were performed on data expressing
amino acids, ammonium and total amino acids concentration,
and proline/arginine ratio in the must samples and on the data
expressing amino acids, ammonium, total amino acids and total
amino acids without proline concentration in the wine samples.
108 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116

3. Results and discussion
3.1. Enological parameters and kinetics of fermentations
The titratable acidity increased in the alcoholic fermentation,
except for SY1 and PV2 samples (Table 1). These two samples
showed the highest titratable acidity in the musts, with values of
6.69 and 6.54 g/l, respectively. In the case of wines, titratable acid-
ity was between 5.27 and 7.10 g/l, referred to as the minimum and
maximum values in Petit Verdot, PV2 and PV1, respectively (Table
1). Volatile acidity, generally, decreased during the second half of
alcoholic fermentation (Table 1), results that coincided with those
found by other authors, who observed that the concentration of
acetic acid is usually at its maximum in mid-fermentation (Ribé-
reau-Gayon, Peynaud, Sudraud, & Ribéreau-Gayon, 1980). The
wines obtained from the slowest fermentations (Merlot variety)
were found to have high volatile acidity (Tables 1 and 2), probably
due to their longer contact time with oxygen (Ribéreau-Gayon,

Table 1
Enological parameters during the alcoholic fermentation of the different samples.

Sample Titratable acidity (g/l)a Volatile acidity (g/l)b pH Reducing sugars (g/l) Alcohol (v/v%) Colour index
Monastrell non-organic (MnO)
Must
September 27 (MnO1)c 5.37 – 3.28 161 – 2.78
Oct 8 (MnO2)d 4.96 – 3.50 168 – 4.17
50% of consumed sugars
September 27 (MnO1) 5.02 0.18 3.76 86.6 4.56 2.58
October 8 (MnO2) 5.23 0.30 3.80 56.5 6.91 2.18
Wine
September 27 (MnO1) 5.55 0.19 3.75 0.53 10.2 2.72
October 8 (MnO2) 5.51 0.15 3.81 0.64 10.6 2.20

Monastrell organic (MO)

Must
September 19 (MO1) 4.24 – 3.40 210 – 8.72
September 27 (MO2) 5.21 – 3.17 210 - 5.34
50% of consumed sugars
September 19 (MO1) 5.44 0.37 3.70 101 6.16 7.52
September 27 (MO2) 5.58 0.43 3.60 105 5.83 7.87
Wine
September 19 (MO1) 6.05 0.36 3.71 0.78 12.6 6.07
September 27 (MO2) 6.31 0.35 3.59 0.83 12.8 7.24

Syrah (SY)
Must
September 12 (SY1) 6.69 – 3.24 193 – 9.44
September 19 (SY2) 4.48 – 3.59 228 – 9.64
50% of consumed sugars
September 12 (SY1) 5.51 0.41 3.98 80.3 6.45 9.10
September 19 (SY2) 5.30 0.44 4.12 96.3 7.33 8.49
Wine
September 12 (SY1) 6.49 0.27 3.87 1.54 11.8 8.37
September 19 (SY2) 6.25 0.26 4.13 0.43 13.1 8.51

Merlot (ME)
Must
September 5 (ME1) 5.37 – 3.54 210 – 5.47
September 12 (ME2) 5.11 – 3.57 235 – 9.18
50% of consumed sugars
September 5 (ME1) 5.98 0.54 4.00 103 7.02 6.62
September 12 (ME2) 6.03 0.69 3.91 106 7.60 7.65
Wine
September 5 (ME1) 6.17 0.40 3.95 2.57 12.9 5.32
September 12 (ME2) 6.58 0.47 3.96 8.30 14.1 6.75

Petit Verdot (PV)

Must
September 19 (PV1) 5.66 – 3.36 175 – 8.37
September 27 (PV2) 6.54 – 3.18 175 – 7.00
50% of consumed sugars
September 19 (PV1) 6.19 0.27 3.79 86.1 6.00 6.27
September 27 (PV2) 6.25 0.19 3.73 71.9 6.69 7.25
Wine
September 19 (PV1) 7.10 0.25 3.90 1.13 10.7 5.99
September 27 (PV2) 5.27 0.19 3.74 0.90 8.25 4.94
a
As g/l tartaric acid.
b
As g/l acetic acid.
c
Number 1 corresponded to pre-harvest samples.
d
Number 2 corresponded to samples at harvest.
 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116 109

Table 2
Features of the fermentation kinetics in the samples.
(a)
0.5
ME2

Volatile acidity (g/l) of wines

0.45
dt5–50a dt0–99b Vf5–50c (%/ Vf0–99d (%/
(days) (days) days) days) 0.4 ME1
MO1
0.35 MO2
Mosnastrell non-organic (MnO)
0.3 SY1
September 27 2 4 22.5 24.75 SY2
(MnO1)e 0.25 PV1
f 0.2 MnO
October 8 (MnO2) 2 4 22.5 24.75 PV2
0.15 y = 0,0428x + 0,015
Monastrell organic (MO) MnO2
0.1 R2 = 0,7371
September 19 2 6 22.5 16.5
(MO1) 0.05
September 27 2 6 22.5 16.5 0
(MO2) 0 2 4 6 8 10 12

Syrah (SY) Days of fermentation

September 12 1 6 45 16.5
(SY1) (b) 9
September 19 2 6 22.5 16.5 SY1 SY2
(SY2) 8

Color index of the wines

MO2

Merlot (ME) 7 ME2

September 5 3 10 15 9.9 6
ME1
MO1

(ME1) 5
PV1

September 12 3 10 15 9.9 PV2

4
(ME2)
3
Petit Verdot (PV) MnO1 MnO2 y = 0,7066x + 0,857
2
September 19 2 6 22.5 16.5 R2 = 0,6594
(PV1) 1
September 27 2 6 22.5 16.5 0
(PV2) 0 2 4 6 8 10 12

a
Days needed to ferment from 5% to 50% of the sugars.
b
Days needed to ferment from 0% to 99% of the sugars.
c Fig. 1. Relationship between the days of alcoholic fermentation and volatile acidity
Average percentage of sugar used daily during the fermentation time required
from 5% to 50% of the total.
d during the fermentation and the alcohol degree (v/v%) of the wines (Fig. 1b); and
Average percentage of sugar used daily during the fermentation time required
from 0% to 99% of the total.
e wines (Fig. 1c).
Number 1 corresponded to pre-harvest samples.
f
Number 2 corresponded to samples at harvest.
Glories, Maujean, & Dubourdieu, 2006). There was a direct correla-
tion (R2 > 0.7) between the volatile acidity of wines and the days of
fermentations (Fig. 1a). Our wines included values of volatile acid-
ity between 0.15 g/l for the Monastrell non-organic sample at har-
vest (MnO2) and 0.47 g/l for the sample of Merlot at harvest (ME2)
(Table 1). The pH of wines was higher than the corresponding pH of
the must in all cases, the highest values being in the Syrah sample
at harvest (SY2), in both must and wine (Table 1). The pH of our
wines was between 3.59 and 4.13 for samples of MO2 and SY2,
respectively (Table 1). These values were high but are considered
normal for wines from warm areas.
To determine the harvest day, one of the parameters used was
the °Baumé/total acidity ratio, which in our samples was between
1.97 and 3.15 for Petit Verdot and Syrah samples, respectively
(Garde-Cerdán et al., 2009). The sugar content in the musts was be-
tween 161 and 235 g/l for MnO1 and ME2 samples, respectively
(Table 1). Wines had a sugar concentration lower than 2.57 g/l,
with the exception of the wine obtained from Merlot variety col-
lected at harvest (ME2). This wine had a reducing sugar content
of 8.3 g/l so, the yeast did not complete the alcoholic fermentation
(Table 1). There was an acceptable correlation (R2  0.7) (Fig. 1b)
between the colour index of the must and the colour index of the
wines (Table 1). This is of enological interest because it suggests
that by measuring this parameter in the musts, as can be carried
out in a simple way in the winery, could be predictable colour
quality of the wine.
To characterise the kinetics, the process rate has been calcu-
lated from alcoholic fermentation curves, as an average percentage
of the daily consumed sugar in the ranges of 5–50% (vf5–50) and 0–
99% (vf0–99) of total sugars (Houtman & du Plessis, 1985). The data
dt5–50 and dt0–99 are also shown; these data represent the days ta-
Color index of the musts
(g/l) of the obtained wines (Fig. 1a); relationship between consumed sugars (g/l)
relationship between the colour index of the musts and the colour index of the
ken to consume from 5% to 50% and 0% to 99% of sugars, respec-
tively. These results are shown in Table 2. In all of the
fermentations carried out, there was observed that those from
Merlot variety were the most different, being the slowest to reach
both the middle and the end of the fermentation. This could be be-
cause the musts of this variety showed the highest sugar content
and low assimilable nitrogen content (Tables 1 and 3). The fermen-
tation of Syrah variety collected before harvest (SY1) was the fast-
est to reach the half fermentation (Table 2), taking a day to
consume the half of its sugars; this could be explained because this
variety was the one that had the highest amount of amino acids
(Table 3). The first variety to reach the end of fermentation was
Monastrell from non-organic cultivation system (Table 2), proba-
bly because it was the one with the lowest sugar content and suit-
able nitrogen content for sugar consumption (Tables 1 and 3). The
other varieties (Monastrell organic, Syrah, and Petit Verdot)
showed the same kinetics of alcoholic fermentation (Table 2).
3.2. Nitrogenous fractions
The ammonium nitrogen of the initial must in term of assimila-
ble nitrogen was between 8.1% from Syrah must (SY2) to 20.7% in
Merlot must from pre-harvest (ME1) (Table 3). This is important as
the ammonium is the first inorganic source of nitrogen used by
yeast (Bell & Henschke, 2005). Moreover, low concentrations of
ammonium could promote an increase of higher alcohols because
the yeasts are forced to use the amino acids of must as a nitrogen
source (Usseglio-Tomasset, 1998). For all fermentations, the up-
take of all nitrogen fractions occurred in the first half of the fer-
mentation (Table 3), likely due to the exponential growth phase
of yeast where nitrogen is used for biomass production (O’Con-
nor-Cox & Ingledew, 1989). The ammonium nitrogen was almost
entirely consumed, being the consumption percentage during the
110 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116

Table 3
Nitrogenous fractions (mg N/l) and total amino acids without proline (mg/l) of the different samples during the alcoholic fermentation.

Sample Ammonium nitrogen Amino nitrogen Assimilable nitrogen Total amino acids without proline
Monastrell non-organic (MnO)
Must
September 27 (MnO1)a 25.0 ± 1.0 123 ± 4.0 148 ± 5.0 553 ± 17.1
October 8 (MnO2)b 23.5 ± 0.0 201 ± 0.1 220 ± 0.2 878 ± 0.2
50% of consumed sugars
September 27 (MnO1) – 20.5 ± 6.8 10.0 ± 2.0 64.2 ± 9.9
October 8 (MnO2) – 38.2 ± 1.8 9.7 ± 1.5 58.6 ± 11.2
Wine
September 27 (MnO1) – 58.5 ± 4.1 17.5 ± 0.4 119 ± 4.4
October 8 (MnO2) – 73.4 ± 7.4 20.3 ± 1.7 140 ± 10.9

Monastrell organic (MO)

Must
September 19 (MO1) 28.5 ± 0.4 155 ± 2.0 183 ± 2.0 713 ± 6.9
September 27 (MO2) 15.7 ± 0.0 65.1 ± 0.1 78.8 ± 0.1 330 ± 0.1
50% of consumed sugars
September 19 (MO1) – 16.6 ± 0.6 8.0 ± 0.2 49.3 ± 1.2
September 27 (MO2) 1.7 ± 0.2 8.6 ± 1.9 8.0 ± 0.1 34.0 ± 3.1
Wine
September 19 (MO1) – 48.1 ± 2.7 16.7 ± 1.4 100 ± 7.0
September 27 (MO2) 1.6 ± 0.1 26.4 ± 1.6 16.1 ± 0.5 85.1 ± 1.7

Syrah (SY)
Must
September 12 (SY1) 21.3 ± 0.4 172 ± 3.0 192 ± 3.0 755 ± 12.8
Sep 19 (SY2) 17.6 ± 0.2 201 ± 2.0 218 ± 2.0 913 ± 9.1
50% of consumed sugars
September 12 (SY1) 1.5 ± 0.2 60.2 ± 4.2 11.3 ± 0.1 54.5 ± 1.0
September 19 (SY2) 0.6 ± 0.3 44.7 ± 13.0 9.7 ± 1.5 49.3 ± 5.5
Wine
September 12 (SY1) 2.3 ± 0.5 214 ± 72.4 20.4 ± 7.3 112 ± 42.8
September 19 (SY2) 0.8 ± 0.1 176 ± 6.3 16.7 ± 3.9 92.5 ± 27.7

Merlot (ME)
Must
September 5 (ME1) 17.6 ± 0.1 67.8 ± 0.1 84.9 ± 0.3 354 ± 0.4
September 12 (ME2) 17.2 ± 0.1 114 ± 0.2 128 ± 0.1 546 ± 1.0
50% of consumed sugars
September 5 (ME1) 1.6 ± 0.0 22.0 ± 6.6 8.0 ± 0.1 37.6 ± 1.4
September 12 (ME2) 1.2 ± 0.0 31.1 ± 19.5 7.4 ± 0.5 32.7 ± 2.7
Wine
September 5 (ME1) 1.7 ± 0.1 263 ± 111.3 18.3 ± 0.3 108 ± 1.0
September 12 (ME2) 1.3 ± 0.4 374 ± 90.2 18.0 ± 0.1 101 ± 0.7

Petit Verdot (PV)

Must
September 19 (PV1) 14.2 ± 0.2 95.4 ± 0.2 108 ± 0.3 459 ± 1.8
September 27 (PV2) 20.3 ± 0.5 157 ± 3.0 176 ± 4.0 720 ± 14.3
50% of consumed sugars
September 19 (PV1) 1.6 ± 0.4 76.2 ± 8.8 7.9 ± 0.9 36.7 ± 2.8
September 27 (PV2) 1.8 ± 0.2 49.8 ± 0.6 9.8 ± 0.4 48.9 ± 1.4
Wine
September 19 (PV1) 2.5 ± 0.0 214 ± 3.2 21.4 ± 0.1 133 ± 1.0
September 27 (PV2) 1.9 ± 0.5 123 ± 3.4 17.5 ± 0.1 108 ± 4.3

All parameters are given with their standard deviation (n = 2 for the must and n = 4 for the samples at 50% of consumed sugars and for the wines).
a
Number 1 corresponded to pre-harvest samples.
b
Number 2 corresponded to samples at harvest.

fermentation from 82.4% in the fermentation of the PV1 sample to
100% in the fermentation of the two Monastrell non-organic sam-
ples (MnO1 and MnO2) and of Monastrell organic corresponding to
the pre-harvest grape (MO1) (Table 3).
During the first half of fermentation, the amino nitrogen was re-
duced in a high proportion that was between 65.0% in the case of
must from pre-harvest Syrah (SY1), and 89.3%, from pre-harvest
Monastrell organic variety (MO1). The only fermentation in which
the amino nitrogen was reduced to a low proportion was for PV1
must. In this fermentation, the amino nitrogen was reduced by
20.1% (Table 3), probably due to the high formation of proline dur-
ing this phase of the alcoholic fermentation (Table 4). Thus, the to-
tal consumption of the assimilable nitrogen during the first half of
fermentation was between 89.8% for the fermentation of MO2
sample and 95.6% for the fermentations of MnO2, MO1, and SY2
samples (Table 3). As we previously mentioned, the wines obtained
from the ME2 sample showed high residual sugar content (Table
1). This was probably due to low assimilable nitrogen content in
initial must (Table 3), especially arginine that is one of the prefer-
ence amino acid for yeast (Bell & Henschke, 2005; Garde-Cerdán,
 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116 111

Table 4 3.3. Amino acids

Proline content in the must and excreted during the first and second half of the
fermentation (mg/l).
The most consumed amino acids during the first half of fermen-
Sample Must Excreted during the first Excreted during the tation in all fermentations were arginine, alanine, serine, and thre-
halfof the fermentation second halfof the onine (Fig. 2). These amino acids, especially arginine, are considered
fermentation
good nitrogen sources for S. cerevisiae (Bell & Henschke, 2005;
Monastrell non-organic (MnO) Henschke & Jiranek, 1993). The least consumed amino acids were
September 3.9 ± 0.2 83.1 ± 39.6 251 ± 50.5
27
methionine, glycine, and lysine (Fig. 2). This could be because these
(MnO1)a two last amino acids are not metabolised by S. cerevisiae (Cooper,
October 8 42.4 ± 2.7 193 ± 16.4 202 ± 70.9 1982), but they could be metabolised by non-Saccharomyces yeast,
(MnO2)b which could be present in the early stages of fermentation. More-
Monastrell organic (MO) over, the most consumed amino acids were the amino acids found
September 6.8 ± 0.1 63.8 ± 5.9 188 ± 11.8 in a major concentration in the must; and the least consumed were
19
the amino acids found in a much lesser concentration in the must
(MO1)
September 16.1 ± 0.1 3.0 ± 13.1 78.6 ± 15.7 (Fig. 2). In the first half of fermentation, proline was released in
27 all the fermentations, in concentrations that ranged from 3.0 mg/l
(MO2) for the fermentation of MO2 sample to 565 mg/l for the fermenta-
Syrah (SY) tion of PV1 sample (Table 4). The release of this amino acid was
September 8.5 ± 0.5 407 ± .32.4 1201 ± 543 probably due to the fact that it is an intermediate product in the
12 (SY1) degradation of arginine (Garde-Cerdán et al., 2008; Moreno-Arribas
Sep 19 7.8 ± 1.4 286 ± 98.1 1025 ± 129
(SY2)
& Polo, 2009).
Direct correlation was observed (R2 > 0.7) between the concen-
Merlot (ME)
September 4.1 ± 0.5 125 ± 55.1 1901 ± 917
tration of each amino acid in the initial must and their consump-
5 (ME1) tion during the first half of fermentation (Fig. 2), except for
September 20.9 ± 0.2 184 ± 166 2741 ± 764 lysine and methionine for the fermentations carried out from the
12 pre-harvest grapes (Figs. 2e and o). This indicates that the con-
(ME2)
sumption of amino acids by inoculated yeast was related to their
Petit Verdot (PV) concentration in the must, regardless of other parameters, as the
September 10.7 ± 0.6 565 ± 68.6 1029 ± 73.1
musts used for this study were of different grape varieties, col-
19 (PV1)
September 9.7 ± 1.5 334 ± 3.6 541 ± 31.1 lected at different maturation stages and presented different eno-
27 (PV2) logical parameters. These results were different from those found
by other authors, who have observed that the physicochemical
All parameters are given with their standard deviation (n = 2 for the must and n = 4
for the samples at 50% of consumed sugars and for the wines).
properties of wine, the grape variety and the yeast strain are some
a
Number 1 corresponded to pre-harvest samples. of the factors affecting the consumption of nitrogen compounds
b
Number 2 corresponded to samples at harvest. during alcoholic fermentation (Ancín-Azpilicueta et al., 2005; Bell
& Henschke, 2005; Jiranek, Langridge, & Henschke, 1990). For the
Marsellés-Fontanet, Arias-Gil, Martín-Belloso, & Ancín-Azpilicueta, traditional agriculture, the amino acids studied were found in
2007), to the high sugar content in the initial must and to the high higher concentration in the grapes at harvest than in the pre-har-
alcohol content in the wine (Table 1). A minimal concentration of vest grapes, with the exception of alanine for Merlot (Fig. 2b) and
more than 140 mg N/l is often quoted as necessary for the fermen- tyrosine and aspartic acid for Syrah (Figs. 2c and n). Therefore, the
tation of musts of moderate sugar level (Bely, Sablayrolles, & Barre, consumption of amino acids were higher in the alcoholic fermen-
1990). The consumption of total amino acids without proline dur- tation carried out with the grapes collected at harvest than the
ing the first half of fermentation was from 88.4% in MnO1 sample grapes collected pre-harvest. This was because there was direct
to 94.6% in SY2 sample (Table 3). This least and greatest consump- correlation, in general, between their concentration in the must
tion of amino acids during the first half of fermentation coincided and their consumption during the first half of fermentation
with the lowest and highest concentration of amino acids in the (Fig. 2). At the end of maturation, the Monastrell from organic cul-
initial must, respectively. Moreover, the must obtained from the tivation system showed a different trend from the rest of varieties
pre-harvest grapes had lower concentration of total amino acids as glutamic acid, alanine, glycine, lysine, phenylalanine, arginine,
without proline than the must from the grapes collected at harvest, histidine, threonine, serine, and aspartic acid were found in a high-
with the exception of Monastrell organic variety (Table 3). er concentration in MO1 than in MO2 musts. Therefore, these ami-
However, in all the fermentations observed, there was an no acids were consumed in higher quantity in the alcoholic
increase of amino nitrogen, assimilable nitrogen and total amino fermentation carried out with the pre-harvest grapes than with
acids without proline during the second half of the fermentation harvest grapes (Fig. 2). This difference in the evolution of the amino
(Table 3). This could be due to yeast autolysis that occurs at the acid content at the end of maturation between organic and non-or-
end of fermentation (Dizy & Polo, 1996; Fornairon-Bonnefond, ganic systems could be due to the stress that plants undergo in an
Camarasa, Moutounet, & Salmon, 2002; Moreno-Arribas & Polo, organic system.
2009) and/or due to toxic effect of ethanol (Ancín-Azpilicueta, Fig. 3 shows the five amino acids most excreted by the yeast
Fraile-Jiménez de Maquirriain, Garde-Cerdán, & Torrea-Goñi, during the second half of fermentation. Without taking into ac-
2005; Ferreras, Iglesias, & Girbés, 1989). During the autolysis, count proline, in all the fermentations, the two amino acids that
different compounds are released into the medium as amino acids were highly released by the yeasts during the second half of fer-
(Alexandre & Guilloux-Benatier, 2006), and the ethanol inhibits the mentation were glutamic acid and alanine (Fig. 3). This was
amino acid transport systems and causing the release of these according to results found by other authors who observed that
compounds into the medium. The increase of the concentration these two amino acids are among the most release during yeast
of total amino acids without proline in the second half of fermen- autolysis (Martínez-Rodríguez, Carrascosa, & Polo, 2001; Martí-
tation was between 43.2 mg/l in SY2 sample and 96.0 mg/l in PV1 nez-Rodríguez & Polo, 2000; Perrot, Charpentier, Charpentier,
sample (Table 3). Feuillat, & Chassagne, 2002). As it was the case during the first half
112 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116

(a) Glutamic acid y1 = 1,2661x - 12,507 y 2= 0,8729x + 1,1232 (b) Alanine y1 = 1,0052x - 3,7575 y2 = 0,9966x - 3,1581
R 2 = 0,9204 R 2 = 0,8914 R 2 = 0,9999 R2 = 1
60 160
MO1

50 SY2
MO1

mg/L consumed
120
mg/L consumed

MnO2
40 SY1 PV2 MnO1
ME2
30 80
SY2 PV2
MnO1
PV1 PV1 SY1
20
MnO2 MO2 ME1
MO2 40
10 ME1 ME2

0 0
0 10 20 30 40 50 60 0 50 100 150 200
mg/L in the must mg/L in the must

(c) Tyrosine y1 = x y2 = x (d) Glycine y1= 1,2373x - 4,595 y 2= 0,8229x - 2,3006

R2 = 1 R2 = 1 R2 = 0,7039 R2 = 0,9126
25 6
SY1 MnO2
5 SY2
20

mg/L consumed
4
mg/L consumed

15 MnO2 SY2 MO1

3 PV2
PV2
10 PV1
2
MO2 MO2
MO1
ME2
MnO1 ME2
5 ME1 1 PV1

ME1 MnO1
SY1
0
0 0 2 4 6 8 10
0 5 10 15 20 25 -1
mg/L in the must mg/L in the must

(e) Lysine y1 = 0,1181x + 1,4179 y2 = 0,7829x - 1,2882 (f) Phenylalanine y1 = 1,1087x - 2,0363 y2 = 0,8748x + 1,0475
R 2 = 0,0375 R 2 = 0,9606 R 2 = 0,697 R 2 = 0,7714
7 20
MnO2
MnO2
6 SY2
mg/L consumed

16
mg/L consumed

SY1
5 PV2
MO1
12 ME2 PV2
MO1 SY2
4 MO2
ME1
ME2
3 8 PV1
ME1 MnO1
2 SY1
PV1 4
1
MO2
MnO1
0 0
0 2 4 6 8 10 12 0 5 10 15 20
mg/L in the must mg/L in the must

(g) Arginine y1 = 0,9886x - 3,5322 y2 = 0,9893x - 4,4151 (h) Isoleucine y1 = 0,7452x + 2,0692 y 2= 0,9533x - 0,0101
R 2 = 0,9999 R 2 = 0,9999 R 2 = 0,913 R 2 = 0,9412
30
450
SY2 SY2
PV2
25
375 SY1 MnO2
MnO2
mg/L consumed
mg/L consumed

300 MO1 20 ME2

PV2 PV1
MnO1
225 15 MnO1 SY1

ME2 ME1 MO2

150 10
PV1
ME1 MO1
75 MO2 5

0 0
0 100 200 300 400 500 0 10 20 30 40
mg/L in the must mg/L in the must

Fermentations made from the first samples

Fermentations made from the second sam ples

Fig. 2. Relationship between the concentration (mg/l) of each amino acid in the must and their consumption (mg/l) during the first half of fermentation carried out for the
different fermentations done from the pre-harvest grapes (Monastrell non-organic, MnO1; Monastrell organic, MO1; Merlot, ME1; Syrah, SY1; Petit Verdot, PV1) and from the
grapes collected at harvest (Monastrell non-organic, MnO2; Monastrell organic, MO2; Merlot, ME2; Syrah, SY2; Petit Verdot, PV2). The equation 1 that is presented for each
compound is the correlation between its concentration in the pre-harvest grapes and its consumption during the first half of fermentation and the equation 2 is the
correlation between the concentration of each amino acid in the grapes collected at harvest and its consumption during the first half of fermentation.
 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116 113

(i) Histidine y 1= 0,9074x - 2,8781 y2 = 0,979x - 3,5179 (j) Valine y1 = 2,232x - 28,581 y2 = 1,051x - 5,1573
R 2 = 0,9947 R2 = 1 R 2 = 0,8643 R 2 = 0,823
50 50
SY2

40 40
SY2
mg/L consumed

MnO2
30

mg/L consumed
30 ME2
PV2
ME2
20 MO2 SY1
PV1
20
MO2 SY1 ME1
10
PV2 MO1 MnO2
10 PV1 MnO1 MO1
ME1 0
0 10 20 30 40 50
0
-10
0 10 20 30 40 50 60 MnO1

mg/L in the must -20 mg/L in the must

(k) Threonine y1 = 0,9078x + 1,0019 y2 = 1,0279x - 5,4313 (l) Serine y1 = 1,0529x - 5,8598 y2 = 0,9729x - 2,119
R 2 = 0,9825 R 2 = 0,9948 R 2 = 0,9917 R 2 = 0,9999
90 80
PV2
SY2
MnO2
mg/L consumed

mg/L consumed
60 MO1 SY2
60 ME2
SY1 ME2
MnO2
MO1 MnO1
40
ME1
PV1
30 MO2 PV2 PV1
SY1
ME1 MnO1 MO2
20

0 0
0 20 40 60 80 100 0 20 40 60 80

mg/L in the must mg/L in the must

(m) Leucine y1 = 1,0095x - 2,3642 y2 = 1,0102x - 2,9597 (n) Aspartic acid y1= 0,8901x - 3,5983 y2 = 0,94x - 3,8467
R2 = 0,9105 R2 = 0,9645 R2 = 0,9891 R2 = 0,9521
40 30

35 SY2
25 SY1
mg/L consumed
mg/L consumed

30
ME2 20
25
PV2
PV1
20 SY1 15 ME2 SY2
ME1 MO1 MnO2
15 PV2
MnO2 10 ME1
10 MO1 MO2 MO2
MnO1
5
5
MnO1
PV1
0 0
0 10 20 30 40 0 10 20 30 40
mg/L in the must mg/L in the must

(o) Methionine y1 = 1,597x - 5,5219 y2 = 0,9529x - 0,5331

R 2 = 0,553 R 2 = 0,5001
12
PV2
mg/L consumed

SY1
8 PV1
SY2 Fermentations made from the
ME2 first samples
MO2 Fermentations made from the
4 second samples
ME1
MO1MnO2

MnO1
0
0 2 4 6 8 10 12
mg/L in the must

Fig. 2 (continued)
114 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116

10 Glutamic acid A lanine Tyro sine Glycine Lysine

0
1 2 3 4 5
-10

-20

mg/L -30

-40

-50

-60
MnO1 MO1 ME1 SY1 PV1 MnO2 MO2 ME2 SY2 PV2

Fig. 3. The five amino acids (mg/l) most released during the second half of the fermentations carried out from the pre-harvest grapes (Monastrell non-organic, MnO1;
Monastrell organic, MO1; Merlot, ME1; Syrah, SY1; Petit Verdot, PV1) and with the grapes collected at harvest (Monastrell non-organic, MnO2; Monastrell organic, MO2;
Merlot, ME2; Syrah, SY2; Petit Verdot, PV2).

1 (MnO1)
2 (MnO2)
3 (MO1)
4 (MO2)
5 (ME1)
6 (ME2)
7 (SY1)
8 (SY2)
9 (PV1)
10 (PV2)

Fig. 4. Application of discriminant analysis to the data expressing as concentration (mg/l) of amino acids, ammonium, and total amino acids, and proline/arginine ratio of the
different must samples [1, Monastrell non-organic pre-harvest sample (MnO1); 2, Monastrell non-organic at harvest sample (MnO2); 3, Monastrell organic pre-harvest
sample (MO1); 4, Monastrell organic at harvest sample (MO2); 5, Merlot pre-harvest sample (ME1); 6, Merlot at harvest sample (ME2); 7, Syrah pre-harvest sample (SY1); 8,
Syrah at harvest sample (SY2); 9, Petit Verdot pre-harvest sample (PV1); 10, Petit Verdot at harvest sample (PV2)].

of fermentation, proline excretion was observed in all the fermen-
tations (Table 4).
To classify the different grape varieties (Monastrell, Syrah, Mer-
lot, and Petit Verdot), the different cultivation systems (non-organ-
ic and organic), and the different maturation stages and the
different wines obtained from these samples, discriminant analysis
was carried out. The discrimant analysis was performed on data
expressing amino acids, ammonium, total amino acids concentra-
tion, and proline/arginine ratio in the must samples and amino
acids, ammonium, total amino acids, and total amino acids without
proline concentration in the wine samples. The results are shown
in Figs. 4 and 5, respectively. In the first case (Fig. 4), function 1 ex-
plained 91.6% of the variance and function 2 explained 4.6% of the
variance, so the total variance explained by these two functions
was 96.2%. The variables that contributed most to the discriminant
model were threonine, glutamic acid, ammonium, and leucine
(function 1) and leucine, lysine, ammonium, and threonine (func-
tion 2). The two discriminant functions showed a good separation
among the different samples, except for MnO2 and MO2 samples,
which were found to be closer to each other (Fig. 4). This indicated
that it was not possible to discriminate the cultivate system for
Monastrell grapes collected at harvest. The discriminant functions
allowed us to correctly classify 100% of the studied samples. In the
case of the wines obtained from the different grape samples
(Fig. 5), function 1 explained 65.8% of the variance and function
2 explained 25.5% of the variance, the total explained by these
two functions being 91.3%. In this case, the variables that contrib-
uted most to the discriminant model were alanine, lysine, glutamic
acid, and valine (function 1) and valine, alanine, lysine, and aspar-
tic acid (function 2). The two discriminant functions showed a
good separation among the different wines, and allowed us to cor-
rectly classify 100% of the samples studied.
 T. Garde-Cerdán et al. / Food Chemistry 124 (2011) 106–116
Fig. 5. Application of discriminant analysis to the data expressing as concentration (mg/l) of amino acids, ammonium, total amino acids, and total amino acids without
proline in the different wine samples [1, Monastrell non-organic pre-harvest sample (MnO1); 2, Monastrell non-organic at harvest sample (MnO2); 3, Monastrell organic pre-
harvest sample (MO1); 4, Monastrell organic at harvest sample (MO2); 5, Merlot pre-harvest sample (ME1); 6, Merlot at harvest sample (ME2); 7, Syrah pre-harvest sample
(SY1); 8, Syrah at harvest sample (SY2); 9, Petit Verdot pre-harvest sample (PV1); 10, Petit Verdot at harvest sample (PV2)].
4. Conclusions
The consumption of the majority of nitrogen compounds during
the first half of fermentation showed a great correlation with the
concentration of these compounds in the musts, regardless of
grape variety, enological parameters, harvest moment and cultiva-
tion system. This is important as it is the first time that these re-
sults were obtained with different natural must collected at
different stages of maturation without external addition of nitro-
gen compounds. During the second half of fermentation, the re-
lease of amino acids was thought to be due to the yeast autolysis
and/or due to the toxic effect of ethanol. The concentration of
nitrogen compounds from the must had an effect on enological
parameters during the alcoholic fermentation. Also, nitrogen com-
position allowed discrimination between both the initial musts
and the final wines.
Acknowledgements
Many thanks for the financial support given by the Junta de
Comunidades de Castilla-La Mancha to the Project PII1I09-0157-
9307 and to the FPI scholarship for A.M.M.-G and also to the Min-
isterio de Educación y Ciencia for the Juan de la Cierva contract for
T.G.-C. We wish to express our gratitude to Kathy Walsh for proof-
reading the English manuscript.
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