Food Chemistry 124 (2011) 106116

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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-Cerdn a, Ana M. Martnez-Gil a, Cndida Lorenzo a, Jos Flix Lara a, Francisco Pardo b, M. Rosario Salinas a,*
a b

Ctedra de Qumica Agrcola, E.T.S.I. Agrnomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain 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
The aim of this work was to study the inuence of grape variety, cultivation system and stage of grape maturation on nitrogen compounds evolution during alcoholic fermentation. To do this, four grape varieties, Monastrell, Merlot, Syrah and Petit Verdot, traditionally cultivated and Monastrell cultivated using 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 consumption of nitrogen compounds was directly proportional (R2 > 0.7) to their concentration in natural musts. This is very important as it is the rst time that these results were obtained with natural musts without external addition of nitrogen compounds. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 9 February 2010 Received in revised form 8 April 2010 Accepted 26 May 2010

Keywords: Amino acids Grape varieties Wine Must Maturation stages

1. Introduction Nitrogen compounds of must are essential in the metabolism of yeast because the nitrogen is, after carbon, the second element utilised during its growth. The content of these compounds affects the kinetics of fermentation, as nitrogen-decient musts can cause slow and stuck fermentations (Arias-Gil, Garde-Cerdn, & AncnAzpilicueta, 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 (Soueros, Bouloumpasi, Tsarchopoulos, & Biliaderis, 2003; Vilanova 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, Surez Lepe, Lombardero, & Garca 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 composition of the must, on yeast and on the fermentation temperature, among other factors (Barbosa, Falco, Mendes-Faia, & Mendes-Ferreira, 2009; Bell & Henschke, 2005; Bouloumpasi, Souerops, Tsarchopoulos, & Biliaderis, 2002; Henschke & Jiranek,
* Corresponding author. E-mail address: Rosario.Salinas@uclm.es (M.R. Salinas). 0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.05.112

1993; Hberger, Csoms, & Simon-Sarkadi, 2003; Valero, Milln, 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, Rozs, Mas, & Guillamn, 2005; Carrau et al., 2008; Hernndez-Orte, Cacho, & Ferreira, 2002; Manginot, Roustan, & Sablayrolles, 1998; Martnez-Rodrguez & Polo, 2000; Mendes-Ferreira, Barbosa, Falco, Leo, & Mendes-Faia, 2009; Mendes-Ferreira, Mendes-Faia, & Leo, 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-Cerdn & Ancn-Azpilicueta, 2008; Hernndez-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 previous 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-Cerdn et al., 2009). So, the two last samples of grape maturation were selected in order to do the alcoholic fermentation and to study the possible effect of stage grape maturation on the evolution of nitrogen compounds during alcoholic

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107

fermentation. This different stage of maturation is determined by the Baum/total acidity ratio, among other parameters. This stage of maturation inuences 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 inuence of grape variety, cultivation system and stage of grape maturation on nitrogen compounds evolution during alcoholic fermentation.

2.2. Enological parameters Titratable acidity, pH, volatile acidity, reducing sugars, and alcohol 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 & Iiguez, 1999), and this parameter is called the colour index.

2. Material and methods 2.1. Samples and vinication For this study, Monastrell, Syrah, Merlot, and Petit Verdot nonorganic and Monastrell organic, red grape varieties cultivated in O.D. Jumilla (SE of Spain) and harvested in 2007 were used. Environmental 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.winesfromspain.com). In the conventional agriculture system (Monastrell non-organic, Syrah, Merlot, and Petit Verdot), the vineyards were cultivated in trellis and were tted 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 cultivated in vessel and was treated with fertilizer cultivit ecolgico (Agribeco), consisting of dried granulated sheep manure, the composition of which was NPK 1.55-1.21-2.35 (%, w/w), applying in total 200 g per vine. The organic system was not irrigated. The Monastrell non-organic grapes were collected on September 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 September 5 (ME1) and 12 (ME2), and the Petit Verdot grapes on September 19 (PV1) and 27 (PV2). For each variety, the rst sample corresponded to the pre-harvest sample (about a week before harvest) and the second sample corresponded to the complete ripeness 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 fermented in duplicate. Must were inoculated with active dry Saccharomyces cerevisiae subsp. cerevisiae (U.C.L.M. S325, Springer Oenologie, Argentina) in a proportion of 0.2 g/l according to commercial recommendations. For this, 0.65 g of dry yeast was rehydrated in a sterile ask 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 fermentation, potassium metabisulte was added to the musts to give a nal total SO2 concentration of 70 mg/l. The fermentations took place in glass fermenters with a capacity 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 prevent the entrance of air during fermentation. The orice 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 nished (reducing sugars < 2.5 g/l). 2.3. Analysis of amino acids and ammonium by HPLC The analysis of amino acids and ammonium of the half fermentation samples and of the nal wines were made using the method described by Gmez-Alonso, Hermosn-Gutirrez, and GarcaRomero (2007). The results for initial musts used for this study were taken from a previous work (Garde-Cerdn 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 (previously ltered), 20 ll of internal standard (2-aminoadipic acid, 1 g/l) (Aldrich, Gillingham, England) and 30 ll of derivatization reagent 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 7080 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) (Aberdeen, Scotland) particle size 5 lm (250 mm 4.6 mm). Amino acids were eluted under the conditions described by Gmez-Alonso 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 gures do not include standard deviation; however, the coefcients of variation for amino acids concentration were between 1% and 15%. The target compounds were identied according to the retention times and the UVvis spectral characteristics of corresponding standards (Aldrich) derivatizated. Quantication was carried out by using the calibration graphs of the respective standards (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.

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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 acidity 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 (Ribreau-Gayon, Peynaud, Sudraud, & Ribreau-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 (Ribreau-Gayon,

Table 1 Enological parameters during the alcoholic fermentation of the different samples. Sample Monastrell non-organic (MnO) Must September 27 (MnO1)c Oct 8 (MnO2)d 50% of consumed sugars September 27 (MnO1) October 8 (MnO2) Wine September 27 (MnO1) October 8 (MnO2) Monastrell organic (MO) Must September 19 (MO1) September 27 (MO2) 50% of consumed sugars September 19 (MO1) September 27 (MO2) Wine September 19 (MO1) September 27 (MO2) Syrah (SY) Must September 12 (SY1) September 19 (SY2) 50% of consumed sugars September 12 (SY1) September 19 (SY2) Wine September 12 (SY1) September 19 (SY2) Merlot (ME) Must September 5 (ME1) September 12 (ME2) 50% of consumed sugars September 5 (ME1) September 12 (ME2) Wine September 5 (ME1) September 12 (ME2) Petit Verdot (PV) Must September 19 (PV1) September 27 (PV2) 50% of consumed sugars September 19 (PV1) September 27 (PV2) Wine September 19 (PV1) September 27 (PV2)
a b c d

Titratable acidity (g/l)a

Volatile acidity (g/l)b

pH

Reducing sugars (g/l)

Alcohol (v/v%)

Colour index

5.37 4.96 5.02 5.23 5.55 5.51

0.18 0.30 0.19 0.15

3.28 3.50 3.76 3.80 3.75 3.81

161 168 86.6 56.5 0.53 0.64

4.56 6.91 10.2 10.6

2.78 4.17 2.58 2.18 2.72 2.20

4.24 5.21 5.44 5.58 6.05 6.31

0.37 0.43 0.36 0.35

3.40 3.17 3.70 3.60 3.71 3.59

210 210 101 105 0.78 0.83

6.16 5.83 12.6 12.8

8.72 5.34 7.52 7.87 6.07 7.24

6.69 4.48 5.51 5.30 6.49 6.25

0.41 0.44 0.27 0.26

3.24 3.59 3.98 4.12 3.87 4.13

193 228 80.3 96.3 1.54 0.43

6.45 7.33 11.8 13.1

9.44 9.64 9.10 8.49 8.37 8.51

5.37 5.11 5.98 6.03 6.17 6.58

0.54 0.69 0.40 0.47

3.54 3.57 4.00 3.91 3.95 3.96

210 235 103 106 2.57 8.30

7.02 7.60 12.9 14.1

5.47 9.18 6.62 7.65 5.32 6.75

5.66 6.54 6.19 6.25 7.10 5.27

0.27 0.19 0.25 0.19

3.36 3.18 3.79 3.73 3.90 3.74

175 175 86.1 71.9 1.13 0.90

6.00 6.69 10.7 8.25

8.37 7.00 6.27 7.25 5.99 4.94

As g/l tartaric acid. As g/l acetic acid. Number 1 corresponded to pre-harvest samples. Number 2 corresponded to samples at harvest.

T. Garde-Cerdn et al. / Food Chemistry 124 (2011) 106116 Table 2 Features of the fermentation kinetics in the samples. dt550a (days) Mosnastrell non-organic (MnO) September 27 2 (MnO1)e f 2 October 8 (MnO2) Monastrell organic (MO) September 19 2 (MO1) September 27 2 (MO2) Syrah (SY) September 12 (SY1) September 19 (SY2) Merlot (ME) September 5 (ME1) September 12 (ME2) Petit Verdot (PV) September 19 (PV1) September 27 (PV2)
a b

109

(a)
Volatile acidity (g/l) of wines
Vf550c (%/ days) 22.5 22.5 22.5 22.5 Vf099d (%/ days) 24.75 24.75 16.5 16.5

0.5
ME2

dt099b (days) 4 4 6 6

0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 8 10 12

MnO2 MnO PV2 MO1 MO2 SY1 SY2 PV1 ME1

y = 0,0428x + 0,015 R2 = 0,7371

Days of fermentation
1 2 6 6 45 22.5 16.5 16.5

(b)
Color index of the wines

9
SY1 SY2

8
MO2

7 6
ME1 MO1 PV1 PV2

ME2

3 3

10 10

15 15

9.9 9.9

5 4 3 2 1 0
MnO1 MnO2

2 2

6 6

22.5 22.5

16.5 16.5

y = 0,7066x + 0,857 R2 = 0,6594

10

12

Days needed to ferment from 5% to 50% of the sugars. Days needed to ferment from 0% to 99% of the sugars. c Average percentage of sugar used daily during the fermentation time required from 5% to 50% of the total. d Average percentage of sugar used daily during the fermentation time required from 0% to 99% of the total. e Number 1 corresponded to pre-harvest samples. f Number 2 corresponded to samples at harvest.

Color index of the musts

Fig. 1. Relationship between the days of alcoholic fermentation and volatile acidity (g/l) of the obtained wines (Fig. 1a); relationship between consumed sugars (g/l) during the fermentation and the alcohol degree (v/v%) of the wines (Fig. 1b); and relationship between the colour index of the musts and the colour index of the wines (Fig. 1c).

Glories, Maujean, & Dubourdieu, 2006). There was a direct correlation (R2 > 0.7) between the volatile acidity of wines and the days of fermentations (Fig. 1a). Our wines included values of volatile acidity between 0.15 g/l for the Monastrell non-organic sample at harvest (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 Baum/total acidity ratio, which in our samples was between 1.97 and 3.15 for Petit Verdot and Syrah samples, respectively (Garde-Cerdn et al., 2009). The sugar content in the musts was between 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 collected 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 calculated from alcoholic fermentation curves, as an average percentage of the daily consumed sugar in the ranges of 550% (vf550) and 0 99% (vf099) of total sugars (Houtman & du Plessis, 1985). The data dt550 and dt099 are also shown; these data represent the days ta-

ken to consume from 5% to 50% and 0% to 99% of sugars, respectively. 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 because the musts of this variety showed the highest sugar content and low assimilable nitrogen content (Tables 1 and 3). The fermentation of Syrah variety collected before harvest (SY1) was the fastest 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 rst variety to reach the end of fermentation was Monastrell from non-organic cultivation system (Table 2), probably because it was the one with the lowest sugar content and suitable 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 assimilable 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 rst 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 uptake of all nitrogen fractions occurred in the rst half of the fermentation (Table 3), likely due to the exponential growth phase of yeast where nitrogen is used for biomass production (OConnor-Cox & Ingledew, 1989). The ammonium nitrogen was almost entirely consumed, being the consumption percentage during the

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Table 3 Nitrogenous fractions (mg N/l) and total amino acids without proline (mg/l) of the different samples during the alcoholic fermentation. Sample Monastrell non-organic (MnO) Must September 27 (MnO1)a October 8 (MnO2)b 50% of consumed sugars September 27 (MnO1) October 8 (MnO2) Wine September 27 (MnO1) October 8 (MnO2) Monastrell organic (MO) Must September 19 (MO1) September 27 (MO2) 50% of consumed sugars September 19 (MO1) September 27 (MO2) Wine September 19 (MO1) September 27 (MO2) Syrah (SY) Must September 12 (SY1) Sep 19 (SY2) 50% of consumed sugars September 12 (SY1) September 19 (SY2) Wine September 12 (SY1) September 19 (SY2) Merlot (ME) Must September 5 (ME1) September 12 (ME2) 50% of consumed sugars September 5 (ME1) September 12 (ME2) Wine September 5 (ME1) September 12 (ME2) Petit Verdot (PV) Must September 19 (PV1) September 27 (PV2) 50% of consumed sugars September 19 (PV1) September 27 (PV2) Wine September 19 (PV1) September 27 (PV2) Ammonium nitrogen Amino nitrogen Assimilable nitrogen Total amino acids without proline

25.0 1.0 23.5 0.0

123 4.0 201 0.1 20.5 6.8 38.2 1.8 58.5 4.1 73.4 7.4

148 5.0 220 0.2 10.0 2.0 9.7 1.5 17.5 0.4 20.3 1.7

553 17.1 878 0.2 64.2 9.9 58.6 11.2 119 4.4 140 10.9

28.5 0.4 15.7 0.0 1.7 0.2 1.6 0.1

155 2.0 65.1 0.1 16.6 0.6 8.6 1.9 48.1 2.7 26.4 1.6

183 2.0 78.8 0.1 8.0 0.2 8.0 0.1 16.7 1.4 16.1 0.5

713 6.9 330 0.1 49.3 1.2 34.0 3.1 100 7.0 85.1 1.7

21.3 0.4 17.6 0.2 1.5 0.2 0.6 0.3 2.3 0.5 0.8 0.1

172 3.0 201 2.0 60.2 4.2 44.7 13.0 214 72.4 176 6.3

192 3.0 218 2.0 11.3 0.1 9.7 1.5 20.4 7.3 16.7 3.9

755 12.8 913 9.1 54.5 1.0 49.3 5.5 112 42.8 92.5 27.7

17.6 0.1 17.2 0.1 1.6 0.0 1.2 0.0 1.7 0.1 1.3 0.4

67.8 0.1 114 0.2 22.0 6.6 31.1 19.5 263 111.3 374 90.2

84.9 0.3 128 0.1 8.0 0.1 7.4 0.5 18.3 0.3 18.0 0.1

354 0.4 546 1.0 37.6 1.4 32.7 2.7 108 1.0 101 0.7

14.2 0.2 20.3 0.5 1.6 0.4 1.8 0.2 2.5 0.0 1.9 0.5

95.4 0.2 157 3.0 76.2 8.8 49.8 0.6 214 3.2 123 3.4

108 0.3 176 4.0 7.9 0.9 9.8 0.4 21.4 0.1 17.5 0.1

459 1.8 720 14.3 36.7 2.8 48.9 1.4 133 1.0 108 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 samples (MnO1 and MnO2) and of Monastrell organic corresponding to the pre-harvest grape (MO1) (Table 3). During the rst half of fermentation, the amino nitrogen was reduced 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 during this phase of the alcoholic fermentation (Table 4). Thus, the total consumption of the assimilable nitrogen during the rst 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 preference amino acid for yeast (Bell & Henschke, 2005; Garde-Cerdn,

T. Garde-Cerdn et al. / Food Chemistry 124 (2011) 106116 Table 4 Proline content in the must and excreted during the rst and second half of the fermentation (mg/l). Sample Must Excreted during the rst halfof the fermentation Excreted during the second halfof the fermentation 251 50.5

111

3.3. Amino acids The most consumed amino acids during the rst half of fermentation in all fermentations were arginine, alanine, serine, and threonine (Fig. 2). These amino acids, especially arginine, are considered good nitrogen sources for S. cerevisiae (Bell & Henschke, 2005; Henschke & Jiranek, 1993). The least consumed amino acids were methionine, glycine, and lysine (Fig. 2). This could be because these two last amino acids are not metabolised by S. cerevisiae (Cooper, 1982), but they could be metabolised by non-Saccharomyces yeast, which could be present in the early stages of fermentation. Moreover, the most consumed amino acids were the amino acids found in a major concentration in the must; and the least consumed were the amino acids found in a much lesser concentration in the must (Fig. 2). In the rst half of fermentation, proline was released in all the fermentations, in concentrations that ranged from 3.0 mg/l for the fermentation of MO2 sample to 565 mg/l for the fermentation of PV1 sample (Table 4). The release of this amino acid was probably due to the fact that it is an intermediate product in the degradation of arginine (Garde-Cerdn et al., 2008; Moreno-Arribas & Polo, 2009). Direct correlation was observed (R2 > 0.7) between the concentration of each amino acid in the initial must and their consumption during the rst half of fermentation (Fig. 2), except for lysine and methionine for the fermentations carried out from the pre-harvest grapes (Figs. 2e and o). This indicates that the consumption of amino acids by inoculated yeast was related to their concentration in the must, regardless of other parameters, as the musts used for this study were of different grape varieties, collected at different maturation stages and presented different enological parameters. These results were different from those found by other authors, who have observed that the physicochemical properties of wine, the grape variety and the yeast strain are some of the factors affecting the consumption of nitrogen compounds during alcoholic fermentation (Ancn-Azpilicueta et al., 2005; Bell & Henschke, 2005; Jiranek, Langridge, & Henschke, 1990). For the traditional agriculture, the amino acids studied were found in higher concentration in the grapes at harvest than in the pre-harvest grapes, with the exception of alanine for Merlot (Fig. 2b) and tyrosine and aspartic acid for Syrah (Figs. 2c and n). Therefore, the consumption of amino acids were higher in the alcoholic fermentation carried out with the grapes collected at harvest than the grapes collected pre-harvest. This was because there was direct correlation, in general, between their concentration in the must and their consumption during the rst half of fermentation (Fig. 2). At the end of maturation, the Monastrell from organic cultivation system showed a different trend from the rest of varieties as glutamic acid, alanine, glycine, lysine, phenylalanine, arginine, histidine, threonine, serine, and aspartic acid were found in a higher concentration in MO1 than in MO2 musts. Therefore, these amino acids were consumed in higher quantity in the alcoholic fermentation carried out with the pre-harvest grapes than with harvest grapes (Fig. 2). This difference in the evolution of the amino acid content at the end of maturation between organic and non-organic systems could be due to the stress that plants undergo in an organic system. Fig. 3 shows the ve amino acids most excreted by the yeast during the second half of fermentation. Without taking into account proline, in all the fermentations, the two amino acids that were highly released by the yeasts during the second half of fermentation were glutamic acid and alanine (Fig. 3). This was according to results found by other authors who observed that these two amino acids are among the most release during yeast autolysis (Martnez-Rodrguez, Carrascosa, & Polo, 2001; Martnez-Rodrguez & Polo, 2000; Perrot, Charpentier, Charpentier, Feuillat, & Chassagne, 2002). As it was the case during the rst half

Monastrell non-organic (MnO) September 3.9 0.2 83.1 39.6 27 (MnO1)a October 8 42.4 2.7 193 16.4 (MnO2)b Monastrell organic (MO) September 6.8 0.1 19 (MO1) 16.1 0.1 September 27 (MO2) Syrah (SY) September 12 (SY1) Sep 19 (SY2) Merlot (ME) September 5 (ME1) September 12 (ME2) 8.5 0.5 7.8 1.4 63.8 5.9

202 70.9

188 11.8

3.0 13.1

78.6 15.7

407 .32.4 286 98.1

1201 543 1025 129

4.1 0.5 20.9 0.2

125 55.1 184 166

1901 917 2741 764

Petit Verdot (PV) September 10.7 0.6 19 (PV1) September 9.7 1.5 27 (PV2)

565 68.6 334 3.6

1029 73.1 541 31.1

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.

Marsells-Fontanet, Arias-Gil, Martn-Belloso, & Ancn-Azpilicueta, 2007), to the high sugar content in the initial must and to the high alcohol content in the wine (Table 1). A minimal concentration of more than 140 mg N/l is often quoted as necessary for the fermentation of musts of moderate sugar level (Bely, Sablayrolles, & Barre, 1990). The consumption of total amino acids without proline during the rst half of fermentation was from 88.4% in MnO1 sample to 94.6% in SY2 sample (Table 3). This least and greatest consumption of amino acids during the rst half of fermentation coincided with the lowest and highest concentration of amino acids in the initial must, respectively. Moreover, the must obtained from the pre-harvest grapes had lower concentration of total amino acids without proline than the must from the grapes collected at harvest, with the exception of Monastrell organic variety (Table 3). However, in all the fermentations observed, there was an increase of amino nitrogen, assimilable nitrogen and total amino acids without proline during the second half of the fermentation (Table 3). This could be due to yeast autolysis that occurs at the end of fermentation (Dizy & Polo, 1996; Fornairon-Bonnefond, Camarasa, Moutounet, & Salmon, 2002; Moreno-Arribas & Polo, 2009) and/or due to toxic effect of ethanol (Ancn-Azpilicueta, Fraile-Jimnez de Maquirriain, Garde-Cerdn, & Torrea-Goi, 2005; Ferreras, Iglesias, & Girbs, 1989). During the autolysis, different compounds are released into the medium as amino acids (Alexandre & Guilloux-Benatier, 2006), and the ethanol inhibits the amino acid transport systems and causing the release of these compounds into the medium. The increase of the concentration of total amino acids without proline in the second half of fermentation was between 43.2 mg/l in SY2 sample and 96.0 mg/l in PV1 sample (Table 3).

112
(a) Glutamic acid 60 50

T. Garde-Cerdn et al. / Food Chemistry 124 (2011) 106116

y1 = 1,2661x - 12,507 y 2= 0,8729x + 1,1232 R 2 = 0,8914 R 2 = 0,9204 (b) Alanine 160
MO1 SY2

y1 = 1,0052x - 3,7575 R 2 = 0,9999

y2 = 0,9966x - 3,1581 R2 = 1

mg/L consumed

mg/L consumed

MO1

120
MnO1

MnO2

40
ME2

SY1

PV2

30
MnO1 PV1 MnO2 ME1

80
PV1

SY2 SY1 ME1

PV2

20
MO2

40

MO2

10 0 0 10

ME2

0 20 30 40 50 60 0 50 100 150 200

mg/L in the must

(c) Tyrosine 25
SY1

mg/L in the must

y2 = x R2 = 1 (d) Glycine 6
MnO2

y1 = x R2 = 1

y1= 1 ,2373x - 4,595 R2 = 0,7039

y 2= 0,8229x - 2,3006 R2 = 0,9126

20

SY2

mg/L consumed

mg/L consumed

4
MO1

15
PV2

MnO2

SY2

3 2 1 0
ME1

PV2

10
MO2 ME2

PV1 MO1 MnO1

MO2 ME2 PV1 SY1 MnO1

ME1

0 0 5 10 15 20 25 -1

10

mg/L in the must

(e) Lysine 7 y1 = 0,1181x + 1,4179 y2 = 0,7829x - 1,2882 R 2 = 0,9606 R 2 = 0,0375 (f) Phenylalanine 20
MnO2

mg/L in the must

y1 = 1,1087x - 2,0363 R 2 = 0,697 y2 = 0,8748x + 1,0475 R 2 = 0,7714
MnO2

mg/L consumed

6 5 4 3 2 1
MO2 MnO1 ME1 MO1 ME2 PV2

mg/L consumed

SY2

16
MO1

SY1 ME2 MO2 ME1 PV1 MnO1 PV2

12

SY2

8 4 0

SY1 PV1

0 0 2 4 6 8 10 12

10

15

20

mg/L in the must

(g) Arginine y1 = 0,9886x - 3,5322 R 2 = 0,9999 y2 = 0,9893x - 4,4151 R 2 = 0,9999 (h) Isoleucine 30
SY2 SY1 MO1 MnO1

mg/L in the must

y1 = 0,7452x + 2,0692 R 2 = 0,913 y 2= 0,9533x - 0,0101 R 2 = 0,9412
SY2

450 375

mg/L consumed

mg/L consumed

MnO2

25
MnO2

PV2

300 225
ME2

20 15 10 5 0
ME1 MnO1

PV2

ME2

PV1 SY1

MO2

150 75 0 0 100
ME1 MO2

PV1

MO1

200

300

400

500

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 rst 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 rst 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 rst half of fermentation.

T. Garde-Cerdn et al. / Food Chemistry 124 (2011) 106116

(i) Histidine
50
SY2

113
y1 = 2,232x - 28,581 R 2 = 0,8643 y2 = 1,051x - 5,1573 R 2 = 0,823

y 1= 0,9074x - 2,8781 R 2 = 0,9947

y2 = 0,979x - 3,5179 R2 = 1

(j) Valine
50 40

40

mg/L consumed

SY2

30
ME2

mg/L consumed

MnO2

30
PV2 ME2 SY1

20 10
MO1

20 10
ME1 MO2 SY1 PV2 MO1 PV1 MnO1

PV1 ME1

MO2

MnO2

0 0 -10 10
MnO1

0 0 10 20 30 40 50 60

20

30

40

50

mg/L in the must

(k) Threonine
90
MnO2 SY2

-20

mg/L in the must

y1 = 1,0529x - 5,8598 R 2 = 0,9917 y2 = 0,9729x - 2,119 R 2 = 0,9999
PV2

y1 = 0,9078x + 1,0019 R 2 = 0,9825

y2 = 1,0279x - 5,4313 R 2 = 0,9948

(l) Serine
80

mg/L consumed

mg/L consumed

60

MO1 ME2

SY2

60
SY1 MO1 PV1

ME2

MnO2

40

MnO1 ME1 SY1 PV1

30
ME1

MO2 MnO1

PV2

20

MO2

0 0 20 40 60 80 100

0 0 20 40 60 80

mg/L in the must

(m) Leucine
40 35
SY2

mg/L in the must

y2 = 1,0102x - 2,9597 R2 = 0,9645

y1 = 1,0095x - 2,3642 R2 = 0,9105

(n) Aspartic acid

30 25

y1= 0,8901x - 3,5983 R2 = 0,9891

y2 = 0,94x - 3,8467 R2 = 0,9521

SY1

mg/L consumed

30 25 20 15 10 5 0 0 10 20 30 40
MO1 MnO1 ME1 MnO2 MO2 PV1 PV2 ME2

mg/L consumed

20
PV2

SY1

1 5 1 0 5
PV1 ME1 MO2

ME2 MO1

SY2 MnO2

MnO1

0 0 10 20 30 40

mg/L in the must

(o) Methionine
12
PV2

mg/L in the must

y1 = 1,597x - 5,5219 y2 = 0,9529x - 0,5331 R 2 = 0,553 R 2 = 0,5001

mg/L consumed

SY1

8
SY2 ME2 MO2

PV1

Fermentations made from the first samples Fermentations made from the second samples

ME1 MO1MnO2

MnO1

0 0 2 4 6 8 10 12

mg/L in the must

Fig. 2 (continued)

114
10 0

T. Garde-Cerdn et al. / Food Chemistry 124 (2011) 106116

Glutamic acid

A lanine

Tyro sine

Glycine

Lysine

1
-10 -20 -30 -40 -50 -60

mg/L

MnO1

MO1

ME1

SY1

PV1

MnO2

MO2

ME2

SY2

PV2

Fig. 3. The ve 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 fermentations (Table 4). To classify the different grape varieties (Monastrell, Syrah, Merlot, and Petit Verdot), the different cultivation systems (non-organic 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 concentration, 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 rst case (Fig. 4), function 1 explained 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 (function 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 contributed most to the discriminant model were alanine, lysine, glutamic acid, and valine (function 1) and valine, alanine, lysine, and aspartic acid (function 2). The two discriminant functions showed a good separation among the different wines, and allowed us to correctly classify 100% of the samples studied.

T. Garde-Cerdn et al. / Food Chemistry 124 (2011) 106116

115

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

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 preharvest 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 rst 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 cultivation system. This is important as it is the rst time that these results were obtained with different natural must collected at different stages of maturation without external addition of nitrogen compounds. During the second half of fermentation, the release 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 composition allowed discrimination between both the initial musts and the nal wines. Acknowledgements Many thanks for the nancial support given by the Junta de Comunidades de Castilla-La Mancha to the Project PII1I09-01579307 and to the FPI scholarship for A.M.M.-G and also to the Ministerio de Educacin y Ciencia for the Juan de la Cierva contract for T.G.-C. We wish to express our gratitude to Kathy Walsh for proofreading the English manuscript. References
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