Volatile compounds formation in alcoholic fermentation from grapes collected at

two maturation stages: Influence of nitrogen compounds and grape variety

Ana M. Martínez-Gila, Teresa Garde-Cerdána, b, Cándida Lorenzoa, José Félix Laraa,

Francisco Pardoc, M. Rosario Salinasa,*

a Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha,

Campus Universitario, 02071 Albacete, Spain. Tel: +34 967 599310.

Fax: +34 967 599238. *e-mail: Rosario.Salinas@uclm.es

b Servicio de Investigación y Desarrollo Tecnológico Agroalimentario (CIDA). Instituto de

Ciencias de la Vid y del Vino (CSIC-Universidad de La Rioja-Gobierno de La Rioja). Ctra.

Mendavia-Logroño NA 134, Km. 90. 26071 Logroño, La Rioja, Spain

c Bodega San Isidro (BSI), Carretera de Murcia s/n, 30520 Jumilla, Murcia, Spain

Short version of title: Amino acids and wine volatile composition…

Desired section: Food Chemistry

Word count: 4989

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ABSTRACT: The aim of this work was to study the influence of nitrogen compounds

on the formation of volatile compounds during the alcoholic fermentation carried out

with four non-aromatic grape varieties collected at two different maturation stages. To

do this, Monastrell, Merlot, Syrah, and Petit Verdot grapes were collected one week

before harvest and at harvest. Then, the musts were inoculated with the same S.

cerevisiae yeast strain and were fermented in the same winemaking conditions. Amino

acids that showed the highest and the lowest concentration inn the must were the same,

regardless of the grape variety and maturation stage. Moreover, the consumption of

amino acids during the fermentation increased with their concentration in the must. The

formation of volatile compounds was not nitrogen composition dependent. However,

the concentration of amino acids in the must from grapes collected one week before

harvest can be used as a parameter to estimate the concentration of esters in wines from

grapes collected at harvest and therefore to have more information to know the grape

oenological capacity. Application of principal components analysis (PCA) confirmed

the possibility to estimate the concentration of esters in the wines with the concentration

of nitrogen compounds in the must.

Keywords: amino acids, ammonium, grape variety, ripening, volatile composition,

wine

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Introduction

Higher alcohols, fatty acids, and esters are important compounds for wine aroma

(Ribéreau-Gayon and others 2006), especially from neutral varieties. These varieties

present low concentrations of varietal aromas, compounds found primarily as glycosides

and these can be released aglycones during the fermentation contributing to wine aroma,

so their aroma quality is principally related to the volatile compounds produced during

the alcoholic fermentation (Lambrechts and Pretorius 2000). The fermentative volatile

compounds mainly come from sugar and amino acids metabolism of yeasts.

Saccharomyces cerevisiae yeast produces different quantities of aroma compounds in

relation with the fermentation conditions and must treatments, for example, yeast strain,

temperature, grape variety, micronutrients, vitamins, additives and nitrogen composition

of must (Ruiz-Larrea and others 1998; Carrau and others 2008; Garde-Cerdán and

others 2008; Lorenzo and others 2008). Initial studies have attempted to relate the yeast

nitrogen demand with the profile of aroma compounds in wines (Carrau and others

2008). Paradoxically, various studies reported to characterize the yeast aroma

compounds of wines made with various wine yeasts, have not considered the

importance of grape initial concentrations as for example nitrogen compound levels

(Antonelli and others 1999; Romano and others 2003; Vila and others 2000).

Additionally, slow, sluggish and stuck fermentations have often been related to

nitrogen deficiency (Arias-Gil and others 2007; Bisson 1999). Therefore, it is common

practice in enology to supplement the must with diammonium phosphate (DAP) to

prevent problems related to this nitrogen deficiency. However, in the wineries there

should be analyzed the ammonium and amino acids in the grapes in order to determine

the nitrogen needs for a correct alcoholic fermentation development. Besides, the

concentration of amino acids from must could influence the wine aroma in a decisive

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way, due to the fact that the main groups of volatile compounds that form this aroma are

influenced by the nitrogen source (Rapp and Versini 1991). So, it would be interesting

to the wineries to have quantitative data of grape composition related to wine quality,

such as nitrogen compounds, previously to the harvest day. In this way, the oenologists

could know, in advance, the grapes oenological capacity to produce fermentative

volatile compounds, and so to decide the most adequate winemaking process.

For all these reasons, the aim of this work was to study the effect of nitrogen

compounds on the formation of volatile compounds during the alcoholic fermentation

from different non-aromatic grape varieties (Monastrell, Merlot, Syrah, and Petit

Verdot) collected at two different maturation stages (one week before harvest and at

harvest). In addition, The relationship between the amino acid composition of the

grapes collected one week before the harvest time, and the volatile composition of

wines made from those collected at harvest time was studied.

Materials and methods

Samples

For this study Monastrell, Merlot, Syrah, and Petit Verdot red grape varieties

cultivated in Origin Appellation Jumilla (SE of Spain) during the year 2007, under

optimum sanitary conditions, were used. Jumilla is a warm region with average

temperature of 15°C, average sunshine of 3,000 h year-1, and annual rainfall of 300 mm.

The vines were cultivated in trellis fitted with a drip irrigation system and treated with a

liquid fertilizer NPK 8-4-10 (%, w/w) (Agribeco, Spain), applied at 250 g per vine in

total. Grapes were supplied for the BSI winery, being collected for Monastrell on

September 27 (MO1) and October 8 (MO2), for Syrah on September 12 (SY1) and 19

(SY2), for Merlot on September 5 (ME1) and 12 (ME2), and for Petit Verdot on

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September 19 (PV1) and 27 (PV2). For each variety, the first sample corresponded to

the pre-harvest sample (one week approximately before the harvest day) and the second

sample corresponded to the harvest day (day in which the ratio reducing sugars/total

acidity was the highest).

Vinification

The grapes were destemmed and crushed to obtain the must. For each sample,

400 ml was used, which was divided into 2 aliquots, as they were fermented in

duplicate. Musts were inoculated with active dry S. cerevisiae subsp. cerevisiae

(U.C.L.M. S325, Springer Oenologie, France) in a proportion of 0.2 g l-1. According to

commercial recommendations, 0.65 g of dry yeast was rehydrated in 7.5 ml of distilled

water with 0.07 g of sucrose (number of viable cells ml-1 ≥ 2 x 109); it was kept in this

medium for 30 min at 35ºC. The musts were inoculated while being mixed to obtain a

homogeneous distribution. Before the fermentation, the must was sulphited with

potassium metabisulfite to a final total SO2 concentration of 70 mg l-1. The

fermentations took place in glass fermenters of 250 ml with 2 outlets, one for sample

extractions and the other to allow the CO2 escape. The orifice through which samples

were extracted was covered with a septum during the fermentation. The fermenters were

placed over magnetic stirrers, to ensure a homogenous fermentation. The alcoholic

fermentations were carried out under controlled temperature (28ºC). The fermentation

evolution was followed by daily measurement of sugars by the refraction index, using a

refractometer CT (Sopelem, France). The samples were taken to analysis at the end of

the alcoholic fermentation (reducing sugars < 2.5 g l-1) and were frozen at -20ºC until

analysis. At the end of the alcoholic fermentation, wines were pressed manually and

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skins and seeds were removed, so the maceration with the grape skins occurred during

the alcoholic fermentation.

Oenological parameters analysis

Total acidity, volatile acidity, pH, reducing sugars, and alcohol were measured

using ECC (1990) methods. The phenolic ripeness of the grapes was measured

indirectly from the color intensity of the extract obtained by crushing 200 berries

without breaking seeds and then centrifugated at 3500 rpm. The color intensity was

determined by the sum of the absorbances at 420, 520, and 620 nm (Franco and Iñiguez

1999); this parameter is called the color index.

Analysis of volatile compounds by gas chromatography

The fermentative volatile compounds were extracted by stir bar sorptive

extraction (SBSE) according to Marín and others (2005) and these were analysed by

GC-MS. The volatile compounds were extracted from wines by introducing the

polydimethylsiloxane coated stir bar (0.5 mm film thickness, 10 mm length, Twister,

Gerstel, Mülheim and der Ruhr, Germany) into 10 ml of sample, to which 100 µl of

internal standards γ-hexalactone and 3-methyl-1-pentanol solution at 1 µl ml-1, both in

absolute ethanol (Merck, Damstard, Germany) was added. Samples were stirred at 500

rpm at room temperature for 60 min. The stir bar was then removed from the sample,

rinsed with distilled water and dried with a cellulose tissue, and later transferred into a

thermal desorption tube for GC–MS analysis. In the thermal desorption tube, the

volatile compounds were desorbed from the stir bar at the following conditions: oven

temperature at 330ºC; desorption time, 4 min; cold trap temperature, -30ºC; helium inlet

flow 45 ml min-1. The compounds were transferred into the Hewlett-Packard LC 3D

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GC-MS (Palo Alto, USA) with a fused silica capillary column (BP21 stationary phase

30 m length, 0.25 mm i.d., and 0.25 μm film thickness; SGE, Ringwood, Australia). The

chromatographic program was set at 40ºC (held for 5 min), raised to 230ºC at 10ºC min-

1 (held for 15 min). The total time analysis was 36 minutes. For mass spectrometry

analysis, electron impact mode (EI) at 70 eV was used. The mass range varied from 35

to 500 uma and the detector temperature was 150ºC. The analysis of volatile compounds

in the wines was done in duplicate, and as the fermentations were done in duplicate, the

results shown for these compounds were the mean of 4 analyses. Identification was

carried out using the NIST library and by comparison with the mass spectrum and

retention index of chromatographic standards designed by us and data found in the

bibliography. Quantification was based on five-point calibration curves of respective

standards (Aldrich, Gillingham, England) (R2 > 0.94) in a 12% ethanol (v/v) solution at

pH 3.6.

The odor activity value (OAV) of each volatile compound was calculated by

dividing its mean concentration in the wine by its perception threshold.

Analysis of amino acids and ammonium by HPLC

The analysis of amino acids presented in the wines was carried out using the

method described by Gómez-Alonso and others (2007). Briefly, the derivatization of

these compounds was carried out by reaction of 1.75 ml of borate buffer 1 M (pH = 9),

750 l of methanol (Merck), 1 ml of sample (previously filtered), 20 l of internal

standard (2-aminoadipic acid, 1 g l-1) (Aldrich) and 30 l 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

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then heated at 70-80ºC for 2 h to allow complete degradation of excess DEEMM and

reagent by-products.

Agilent 1100 HPLC (Palo Alto, USA) equipment, with a photodiode array

detector, was used. Chromatographic separation was performed in an ACE HPLC

column (C18-HL) (Aberdeen, Scotland) particle size 5 m (250 mm x 4.6 mm). Amino

acids and ammonium were eluted under the following conditions: 0.9 ml min-1 flow

rate, 10% B during 20 minutes, then elution with linear gradients from 10% to 17% B in

10 minutes, from 17% to 19% B in 0.01 minutes, held during 0.99 minutes, from 19%

to 19.5% B in 0.01 minutes, from 19.5% to 23% B in 8.5 minutes, from 23% to 29.4%

B in 20.6 minutes, from 29.4% to 72% B in 8 minutes, from 72% to 82% B in 5

minutes, from 82% to 100% B in 4 minutes, held during 3 minutes, followed by

washing and reconditioning the column. Phase A was 25 mM acetate buffer, pH = 5.8,

with 0.4 g of sodium azide; phase B was 80:20 (v/v) mixture of acetonitrile and

methanol (Merck). A photodiode array detector monitored at 280, 269 and 300 nm was

used for detection. The volume of sample injected was 50 l. The analysis of amino

acids and ammonium was done in duplicate, and as the fermentations were done in

duplicate, the results for these compounds in the samples were the mean of 4 analyses.

The target compounds were identified according to the retention times and UV-vis

spectral characteristics of corresponding standards (Aldrich) derivatizated.

Quantification was done using the calibration graphs of the respective standards (R2 >

0.98) in 0.1 M HCl, which underwent the same process of derivatization as the samples.

The results of amino acids and ammonium in the grapes were taken from a previous

work (Garde-Cerdán and others 2009).

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Statistical analysis

Volatile and nitrogen compounds data were analyzed statistically by Principal

Component Analysis (PCA), which involves a mathematical procedure that attempts

identify underlying variables or factors that explain the pattern of correlations within a

set of observed variables (Kallithraka and others 2001), besides to seek similarities and

differences between data (Granato and others 2010). PCA was carried out with InfoStat

(www.infostat.com.ar).

Results and discussion

Kinetics of fermentation and oenological parameters

Fermentation kinetics are shown in Figure 1. The fermentations for the same

variety showed similar kinetics, for both samples collected pre-harvest and at harvest.

Among all the fermentations carried out, it was observed that those from the Merlot

variety were the slowest to reach the end of the alcoholic fermentation process. This

could be because the must of this grape variety showed the highest sugar content (Table

1) and low nitrogen content (Table 2). The first variety to reach the end of the alcoholic

fermentation was Monastrell, probably because it was the one with the lowest sugar

content (Table 1) and suitable nitrogen content for sugar consumption (Table 2). The

other two varieties (Syrah and Petit Verdot) needed the same days to reach the end of

alcoholic fermentation.

The oenological parameters can be observed in Table 1. The total acidity

increased during the alcoholic fermentation, except for SY1 and PV2. These two

samples showed the highest total acidity in the must. The wines obtained from the

slowest fermentations (Figure 1) had the highest volatile acidity (Table 1), probably due

to, among other causes, their longer contact time with oxygen (Ribéreau-Gayon and

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others 2006). The volatile acidity increased according to the number of fermenting days.

pH of wine was higher than the corresponding pH of the must in all the cases. The

values of pH were high but are considered normal for wines from warm areas, like

Jumilla region. In the musts, Monastrell variety had the lowest sugar content and Merlot

variety the highest (Table 1). The wines had sugar concentration lower than 2.57 g l-1,

with the exception of the wine obtained from the Merlot variety collected at harvest

(ME2). The color index value of the wines was positively correlated to the color index

value of the must. This interesting oenological feature suggests the color index in musts

as a predictor of the color quality of the wine.

Amino acids and ammonium content in the must

Table 2 shows amino acids and ammonium concentration in musts from grapes

of the studied varieties (Monastrell, Merlot, Petit Verdot, and Syrah) one week before

harvest and at harvest. The four amino acids which showed the highest concentration

were arginine, alanine, serine, and threonine in all varieties at both stages of maturation,

except in Petit Verdot collected at harvest, which showed more concentration in

glutamic acid than in threonine. These amino acids are good nitrogen sources for S.

cerevisiae. Moreover, in all samples the amino acids which showed the lowest

concentrations were glycine and lysine, both considered as poor nitrogen sources for S.

cerevisiae. These results are in accordance with those found by other authors about the

amino acids which shows the highest and the lowest concentrations in musts from

different varieties (Valero and others 2003; Garde-Cerdán and others 2007).

Ammonium concentration was between 18.2 mg l-1, in Petit Verdot pre-harvest,

and 33 mg l-1, in Monastrell pre-harvest (Table 2). The concentration of this ion was

low; this fact could promote an increase of higher alcohols because the yeasts are forced

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to use the amino acids of must as nitrogen source (Usseglio-Tomasset 1998). The

concentration of all the amino acids increased significantly at the end of grape ripening,

except for aspartic acid, tyrosine, and methionine in Syrah, histidine in Monastrell, and

alanine in Merlot. For this reason, the total amino acids concentration was higher in

musts from grapes collected at harvest than in those from grapes collected one week

before harvest (Table 2). These increases were 59% in Monastrell, 54% in Merlot, 57%

in Petit Verdot, and 21% in Syrah. These results are in accordance with other authors, as

they observed an increase in nitrogen composition throughout grape ripening

(Hernández-Orte and others 1999; Bell and Henschke 2005; Garde-Cerdán and others

2009). This is very important, since amino acids are necessary to the fermentation

progress and they influence on wine quality as we commented above. The variety which

showed the highest amino acid concentrations was Syrah, while that showing the lowest

was Merlot for both stages of ripening. Therefore, we can say that amino acid

concentration depended on the variety more than on the harvest moment.

Amino acids and ammonium consumption during the alcoholic fermentation

In Table 3 we can see shows the mean consumption of amino acids and

ammonium during the alcoholic fermentation in the four varieties studied one week

before harvest and at harvest. The four amino acids more consumed were those that

showed more concentration in the initial must, i.e. arginine, alanine, serine and

threonine, except alanine in the case of grapes collected at harvest from Merlot. The

amino acids less consumed were not exactly the same in all varieties and both stages of

ripening, with the exception of glycine that was not only low consumed but released,

since, as we write above this amino acid is considered poor nitrogen source to S.

cerevisiae. Others amino acids were also released in some varieties, as glutamic acid,

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valine and lysine, probably due to the yeast autolysis (Martínez-Rodríguez and others

2001). The total amino acids consumed during the alcoholic fermentation was higher

when the grapes were collected at harvest for all varieties (Table 3), which confirm one

more time that the higher concentration, the higher consumption. Moreover, Syrah was

the variety which showed the highest consumption of amino acids, and Merlot the least,

in close relation with their concentrations in the musts (Table 2).

Formation of volatile compounds during the alcoholic fermentation

Table 4 shows the concentration of volatile compounds formed during the

alcoholic fermentation of the different samples Monastrell, Merlot, Petit Verdot, and

Syrah collected one week before harvest and at harvest.

The synthesis of n-propanol was higher in musts with low concentration and

therefore low consumption of amino acids, i.e., Merlot and Petit Verdot, than in those

with high concentration and thus high consumption of amino acids, i.e., Monastrell and

Syrah. This result is according to Garde-Cerdán and Ancín-Azpilicueta (2008), who

observed that the formation of this compound is inversely proportional to the quantity of

amino acids; however, Rapp and Versini (1991) found that the production of this

alcohol does not seem to be influenced by the concentration of the precursor amino

acid. The highest formation of isobutanol was in the fermentation of ME2 and SY1

samples, while in the fermentation of MO2 and ME1 samples its synthesis was the

lowest (Table 4). The concentration of isoamyl alcohols, 2-methyl-1-butanol and 3-

methyl-1-butanol, was high in all cases, being always higher the concentration of 2-

methyl-1-butanol than the concentration of 3-methyl-1-butanol. The sum of both

alcohols was in some cases above the limit (400 mg l-1) considered by some authors as

the point where the higher alcohols deteriorate the aroma of wine (Rapp and Versini

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1991). This high formation of alcohols could be due to, as mentioned above, the low

concentration of ammonium in the musts (Table 2). The 2-phenylethanol was the

alcohol that had the lowest concentrations (Table 4). Moreover, the wines from the

Monastrell variety were the ones that showed the lowest concentration of this

compound. According with other authors (González-Marco and others 2010), in

general, it was no relation between the formation of alcohols and the content of amino

acids in the must and/or the amino acids consumed during the different fermentations,

either those carried out with pre-harvest grapes or those with grapes at harvest. This can

be due to fermentative alcohols are formed anabolically from sugars as well as

catabolically from amino acids way by the Ehrlich pathway (Äyräpää 1971). Therefore,

the synthesis of the higher alcohols during the alcoholic fermentation showed no pattern

in terms of the grapes harvest time.

Table 5 shows the ratios between the mean alcohol concentration in the wines

and the mean consumption of the corresponding precursor amino acid during the

alcoholic fermentation. It can be observed that the ratio between the concentration of the

alcohol and its corresponding precursor amino acid was over 1, with the exception of n-

propanol/threonine, in some cases, since this alcohol can also come from -

aminobutyric acid, amino acid that was not quantified in this study. Regarding to

isobutanol/valine, the negative value could be due to valine excretion (Garde-Cerdán

and others 2011). In the case of isoamyl alcohols this ratio was particularly high, that

could be due to their formation were mainly produced anabolically from sugar, as

previously mentioned.

On the other hand, the formation of isoamyl acetate was higher in must from

Monastrell and Syrah varieties (Table 4), with high nitrogen content, than in those with

low nitrogen content. However, there was not the same for 2-phenylethyl acetate,

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unable to establish any relationship between its formation and the total nitrogen content.

The principal esters of wine are synthesised enzymatically by yeast during alcoholic

fermentation from alcohols and acids. Thus, acetyl-CoA is condensed with higher

alcohols by the enzyme alcohol acetyltransferase to form acetate esters (Peddie 1990).

Acetic esters of higher alcohols are present in moderated quantities, but have intense,

rather unusual odors (banana, acid drops, and apple) and they contributed to the aroma

complexity of wines (Ribéreau-Gayon and others 2006). The ethyl esters are formed by

the enzymatically catalyzed reaction between ethanol and activated medium and long

chain fatty acids (Lambrechts and Pretorius 2000). Among the ethyl esters studied,

Ethyl hexanoate and ethyl dodecanoate showed a higher concentration in wines

obtained from fermentations carried out with varieties with the highest nitrogen content,

Monastrell and Syrah than in varieties with the lowest, Merlot and Petit Verdot.

Nevertheless, in the case of ethyl octanoate and decanoate, wines from Syrah variety

were the ones that showed the highest concentrations of these esters (Table 4). When

comparing ester composition of wines from grapes collected one week before harvest

and at harvest, we can see that Monastrell grapes showed lower composition at harvest

than one week before, in Merlot and Petit Verdot was similar and Syrah showed higher

composition at harvest than one week before. However, as we have seen before, both

consumption and the initial concentration of amino acids were always higher in samples

collected at harvest, so the synthesis of esters was mainly function of the variety. This

fact is very important because esters are some of the most important compounds in wine

aroma (Jackson 2008; Ribéreau-Gayon and others 2006).

Regarding to the acid concentration in wine, Merlot wines presented the lowest

octanoic and total acids concentration, as it was the variety with the lowest amino acid

concentration, however showed little differences between varieties (Table 4). The

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synthesis of fatty acids by yeast is related to metabolism of carbohydrates, since glucose

is the main source of its precursor, acetyl-CoA (Lambrechts and Pretorius 2000). In

addition, for each of the four varieties studied, few differences were observed between

the concentration of these compounds in wines from the samples collected one week

before harvest and at harvest.

PCA analysis of the initial ammonium and amino acid concentration in the must

and the formation of volatile compounds during the alcoholic fermentation

The samples were separated along the first principal component (PC1), which

explain the 42.5% of the variance, by differences observed mainly in arginine, total

amino acids, ethyl hexanoate, ethyl octanoate, and total esters. PC2 separated the

samples on the basis of explaining 60.7% of all the variation in the data, being the

principal components responsible ammonium, alanine, total alcohols and 2-

phenylethanol. The right part of the Figure 1 shows that most of the amino acids and

most of the esters showed the same trend. The Syrah variety was located on the right of

the figure showing the highest concentration of both groups of compounds studied.

However, higher concentrations of alanine and ammonium showed a negative

correlation with 2-phenylethanol levels and total alcohols, so Monastrell variety

presented lower concentration of these aromatic compounds. The n-propanol was

inversely proportional to the content of total amino acids and arginine (one of the most

abundant amino acids in the grapes), among others. So, the samples of the different

grape variety and maturation stages were clearly distinguished for the concentration of

the amino acids and volatile compounds.

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Influence of nitrogen composition of pre-harvest must on wine volatile composition

of samples collected at harvest

Moreover would be interesting to know the effect of total amino acid content in

pre-harvest grapes on the concentration of volatile compounds in wines obtained from

grapes at harvest. In this sense, wineries would have a way to predict wine aroma before

the harvest moment, as they have not enough analytic tools to know it. So, the analysis

of principal components was done with the total amino acid content in pre-harvest

grapes and the concentration of volatile compounds in wines obtained from grapes at

harvest (Figure 2). The samples were separated along the first principal component

(PC1), which explain the 61.4% of the variance, by differences observed mainly in

esters and acids. PC2 separated the samples on the basis of higher alcohols and one

ester, diethyl succinate, explaining 81.7% of all the variation in the data. In this way,

four sample groups (Syrah, Monastrell, Petit Verdot, and Merlot) were made, from the

highest to the lowest amino acid content. That is, Syrah corresponding to musts with the

highest content of amino acids in the pre-harvest grapes, and their wines from harvest

grapes presented the highest concentration of esters and acids. The total amino acid

concentration in pre-harvest grapes of Monastrell and Petit Verdot was similar (Figure

2). Finally, the furthest group from the two components (esters and acids) was Merlot,

with the lowest concentration of amino acids although is nearer to the n-propanol and

isobutanol. For these reasons, the correlation between esters and amino acids existed,

but the correlation among amino acids and higher alcohols was not so sure.

Odor activity values of the different volatile compounds in the wines

In relation to the contribution of fermentative volatile compounds to wine

aroma, Table 6 shows their odor activity values (OAV). It was observed that, in general,

1616

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

alcohols such as isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-

phenylethanol presented OAV higher than 1 in all wines and so they contribute

significantly to wine aroma. Between the esters, isoamyl acetate, ethyl hexanoate and

ethyl octanoate presented an OAV superior to 1 in all wines and regarding ethyl

decanoate, in almost all wines. Esters are an important factor in wine quality since their

concentration in wine is usually found above their threshold level adding floral and

fruity aroma. Fatty acids contribute to wine fresh flavor, or an unpleasant flavor if they

are in excess and they also help to modify the perception of other taste sensations

(Ribéreau-Gayon and others 2006). Octanoic acid showed also an OAV higher than 1.

In all the wines the esters contributed in the greatest proportion to total OAV (Table 6).

In Petit Verdot and Merlot varieties, which had the lowest concentration of amino acids

in the must, it was observed a lower contribution of esters to the global aroma of the

wine than in those wines obtained from Monastrell and Syrah varieties (Table 6), which

had the highest concentration of amino acids in the must. Therefore, all the results

obtained drive us to affirm that the ester contribution to wine global aroma is closely

related to the initial concentration of amino acids, which is, in its turn, a function of

grape ripeness and variety.

Conclusions

Samples collected at their optimum maturation stage had higher concentration of amino

acids than those collected one week before harvest. In addition, the highest

concentration of amino acids resulted in an increased consumption of these compounds

during fermentation. In general, it was no relation between the formation of alcohols

and the nitrogen compounds, being the concentration of higher alcohols high. Regarding

to esters, the variety with the highest concentration of nitrogen compounds in the must

1717

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400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

was also that with the highest concentration of esters in wine. Finally, the concentration

of acids in wine was not function of the nitrogen composition. Moreover, the synthesis

of volatile compounds during the alcoholic fermentation showed no pattern in terms of

grape harvest moment, depending mainly on the variety. The PCA showed that samples

of different grape variety and maturation stages are clearly distinguished by the

concentration of the amino acids and volatile compounds. On the other hand, the

concentration of total amino acids in the must form pre-harvest grapes was mainly

related to the concentration of esters in wines obtained from the grapes collected at

harvest. The PCA confirmed the possibility to estimate the concentration of esters in the

wines with the concentration of nitrogen compounds in the musts. Since esters are some

of the most important compounds for wine aroma, as we write above, we can used the

concentration of amino acids in the pre-harvest grapes can used as a tool to estimate the

wine aroma quality.

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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.

Thanks also to the Ministerio de Educación y Ciencia, Ministerio de Ciencia e

Innovación and Consejo Superior de Investigaciones Científicas for the Juan de la

Cierva, project AGL2009-08950 and JAE-Doc contracts for T.G.-C. We wish to express

our gratitude to Kathy Walsh for proofreading the English manuscript.

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FIGURE CAPTION

Figure 1. Fermentation kinetics of the different samples. 1 Pre-harvest grapes. 2.

Grapes collected at harvest.

Figure 1. Principal component analysis between the initial amino acid and ammonium

concentration and the volatile composition of wines. See Table 1 for the definition of

sample abbreviations.

Figure 2. Principal component analysis of the amino acid composition of the grapes

collected one week before the harvest time and the volatile composition of wines made

from those collected at harvest.

2323

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549

550

551

552

553

554

555

556

557

558

Table 1. Oenological parameters of must and wine

Sample Total acidity (g

l-1)a

Volatile acidity (g

l-1)b

pH Reducing sugars (g l-1)

Alcohol (v/v %)

Color index (I420+I520+I620)

Pre-harvestMust Monastrell (MO1) 5.4 - 3.3 161.0 - 2.8 Syrah (SY1) 6.7 - 3.2 192.5 - 9.4 Merlot (ME1) 5.4 - 3.5 210.0 - 5.5 Petit Verdot (PV1) 5.7 - 3.4 175.0 - 8.4Wine Monastrell (MO1) 5.6 0.2 3.8 0.5 10.2 2.7 Syrah (SY1) 6.5 0.3 3.9 1.5 11.8 8.4 Merlot (ME1) 6.2 0.4 4.0 2.6 12.9 5.3 Petit Verdot (PV1) 7.1 0.3 3.9 1.1 10.7 6.0

At harvestMust Monastrell (MO2) 5.0 - 3.5 168.0 - 4.2 Syrah (SY2) 4.5 - 3.6 227.5 - 9.6 Merlot (ME2) 5.1 - 3.6 234.5 - 9.2 Petit Verdot (PV2) 5.5 - 3.2 175.0 - 7.0Wine Monastrell (MO2) 5.5 0.2 3.8 0.6 10.6 2.2 Syrah (SY2) 6.3 0.3 4.1 0.4 13.1 8.5 Merlot (ME2) 6.6 0.5 4.0 8.3 14.1 6.8 Petit Verdot (PV2) 5.3 0.2 3.7 0.9 8.3 4.9a As g l-1 tartaric acid. b As g l-1 acetic acid.

2424

559

560561

562563564565

Table 2. Amino acids and ammonium concentration (mg l-1) in the musts obtained from the different samples.MO1 MO2 ME1 ME2 PV1 PV2 SY1 SY2

Aspartic acid 13.1 ± 0.7 20.1 ± 0.1 12.8 ± 0.0 18.2 ± 0.2 8.0 ± 2.0 19.6 ± 0.4 32.5 ± 0.8 20.1 ± 0.3Glutamic acid 25.2 ± 0.7 27.6 ± 0.5 23.5 ± 0.2 31.1 ± 0.4 30.3 ± 0.1 50.0 ± 2.0 36.5 ± 0.8 51.0 ± 1.0Serine 39.0 ± 1.0 57.0 ± 1.0 38.5 ± 0.1 55.5 ± 0.1 38.8 ± 0.0 73.0 ± 1.0 37.0 ± 0.4 59.5 ± 0.4Histidine 17.0 ± 0.2 37.9 ± 0.0 9.4 ± 0.2 28.6 ± 0.0 10.7 ± 0.0 16.7 ± 0.6 19.7 ± 0.4 49.4 ± 0.4Glycine 5.0 ± 0.3 9.1 ± 0.8 3.4 ± 0.1 4.8 ± 0.3 4.4 ± 0.2 7.2 ± 0.4 4.5 ± 0.0 7.8 ± 0.1Threonine 35.0 ± 1.0 69.4 ± 0.0 26.9 ± 0.1 67.6 ± 0.3 35.7 ± 0.1 43.1 ± 0.8 53.0 ± 1.0 86.8 ± 0.5Arginine 250.0 ± 8.0 403.0 ± 1.0 109.1 ± 0.7 203.7 ± 0.8 180.8 ± 0.2 327.0 ± 7.0 383.0 ± 6.0 409.0 ± 5.0Alanine 107.0 ± 4.0 138.3 ± 0.5 61.8 ± 0.6 40.6 ± 0.0 61.9 ± 0.0 74.8 ± 0.9 64.4 ± 0.8 72.1 ± 0.2Tyrosine 6.8 ± 0.1 14.7 ± 0.1 5.7 ± 0.1 6.4 ± 0.3 9.2 ± 0.0 11.8 ± 0.1 22.4 ± 0.1 15.8 ± 0.1Ammonium 33.0 ± 1.0 30.2 ± 0.1 22.7 ± 0.2 22.1 ± 0.1 18.2 ± 0.2 26.2 ± 0.6 27.4 ± 0.5 22.7 ± 0.2Valine 11.6 ± 0.5 23.9 ± 0.0 17.4 ± 0.1 25.5 ± 0.2 18.5 ± 0.5 21.9 ± 0.3 24.6 ± 0.5 44.3 ± 0.7Methionine 5.6 ± 0.1 7.1 ± 0.0 4.6 ± 0.3 5.5 ± 0.6 7.9 ± 0.2 10.3 ± 0.4 8.1 ± 0.0 6.4 ± 0.2Isoleucine 12.3 ± 0.1 22.2 ± 0.0 11.3 ± 0.3 16.6 ± 0.0 20.1 ± 0.1 23.2 ± 0.5 20.2 ± 0.5 29.8 ± 0.6Leucine 9.5 ± 0.1 19.6 ± 0.0 16.1 ± 0.2 24.1 ± 0.0 19.3 ± 0.1 21.6 ± 0.2 25.1 ± 0.5 36.0 ± 0.3Phenylalanine 10.9 ± 0.2 18.2 ± 0.0 9.6 ± 0.0 12.0 ± 0.0 9.2 ± 0.0 13.5 ± 0.2 14.4 ± 0.3 15.2 ± 0.1Lysine 4.3 ± 0.1 9.2 ± 0.0 4.0 ± 0.0 5.7 ± 0.1 4.4 ± 0.0 7.3 ± 0.2 9.3 ± 0.2 9.7 ± 0.1Total amino acids 553.0 ± 17.0 877.6 ± 0.2 353.9 ± 0.4 546.0 ± 1.0 459.0 ± 2.0 720.0 ± 14.0 755.0 ± 13.0 913.0 ± 9.0All parameters are given with their standard deviation.

2525

566

567

568

569570

571

572

573

574

Table 3. Amino acids and ammonium consumption (mg l-1) during the alcoholic fermentations carried out with the different samples. The consumption of amino acids corresponds to the difference between the concentration in the must and in the wine, negative values indicate that there was excretion of amino acids to the medium.

All parameters are given with their standard deviation.

MO1 MO2 ME1 ME2 PV1 PV2 SY1 SY2

Aspartic acid 7.0 ± 2.0 13.9 ± 0.4 8.0 ± 2.0 12.3 ± 0.3 0.8 ± 0.2 16.4 ± 0.5 24.0 ± 4.0 13.0 ± 2.0Glutamic acid -2.9 ± 0.4 -18.0 ± 5.0 5.0 ± 2.0 23.0 ± 2.0 -11.8 ± 0.6 19.0 ± 3.0 25.0 ± 7.0 39.0 ± 5.0Serine 35.0 ± 1.0 52.0 ± 2.0 33.3 ± 0.8 49.0 ± 1.0 32.2 ± 0.1 67.0 ± 1.0 29.0 ± 2.0 54.0 ± 0.4Histidine 10.6 ± 0.2 31.5 ± 0.3 2.3 ± 0.3 21.0 ± 0.3 4.5 ± 0.1 11.3 ± 0.7 13.0 ± 2.0 42.1 ± 0.9Glycine -3.8 ± 0.8 0.4 ± 0.1 -7.0 ± 1.0 -7.0 ± 1.0 -6.9 ± 0.3 -1.8 ± 0.5 -4.0 ± 1.0 0.2 ± 0.0Threonine 31.0 ± 1.0 65.3 ± 0.6 23.2 ± 0.1 63.9 ± 0.4 32.4 ± 0.1 37.2 ± 0.9 49.0 ± 1.0 83.3 ± 0.5Arginine 240.0 ± 8.0 392.0 ± 2.0 101.4 ± 0.7 195.2 ± 0.9 172.0 ± 0.2 318.0 ± 7.0 370.0 ± 7.0 396.0 ± 5.0Alanine 89.0 ± 5.0 120.0 ± 2.0 32.8 ± 0.8 13.0 ± 4.0 38.9 ± 0.5 58.0 ± 1.0 49.0 ± 10.0 57.0 ± 7.0Tyrosine 2.2 ± 0.5 9.8 ± 0.8 1.9 ± 0.2 6.4 ± 0.3 4.4 ± 0.1 7.9 ± 0.3 18.0 ± 1.0 11.6 ± 0.1Ammonium 33.0 ± 1.0 30.2 ± 0.1 20.4 ± 0.2 20.4 ± 0.6 15.0 ± 0.2 23.7 ± 0.9 24.5 ± 0.8 21.6 ± 0.2Valine -7.0 ± 3.0 10.0 ± 1.0 14.4 ± 0.2 21.0 ± 1.0 16.0 ± 0.5 19.5 ± 0.3 21.5 ± 0.9 41.5 ± 0.8Methionine 1.7 ± 0.1 2.9 ± 0.1 0.3 ± 0.1 0.9 ± 0.1 3.8 ± 0.2 6.3 ± 0.4 3.8 ± 0.3 6.4 ± 0.2Isoleucine 9.6 ± 0.1 19.9 ± 0.1 8.8 ± 0.3 13.7 ± 0.2 20.1 ± 0.1 20.2 ± 0.5 15.7 ± 0.6 27.4 ± 0.6Leucine 6.0 ± 0.2 15.0 ± 0.1 11.9 ± 0.2 19.0 ± 0.3 15.7 ± 0.2 17.5 ± 0.3 19.6 ± 0.9 32.1 ± 0.3Phenylalanine 6.0 ± 0.3 14.0 ± 0.0 6.0 ± 0.2 12.0 ± 0.0 4.0 ± 1.0 9.4 ± 0.4 8.7 ± 0.7 10.0 ± 0.4Lysine 0.5 ± 0.0 1.6 ± 0.0 1.1 ± 0.2 0.9 ± 0.0 0.4 ± 0.0 5.2 ± 0.6 -0.4 ± 0.0 2.3 ± 0.1Total amino acids 434.0 ± 18.0 738.0 ± 11.0 246.0 ± 1.0 445.0 ± 1.0 326.0 ± 2.0 612.0 ± 15.0 644.0 ± 45.0 820.0 ± 29.0

2626

575

576577578579580581582583584585586587588589590591592593594595596597598599600

601

602

603

Table 4. Concentration of fermentative volatile compounds (mg l-1) in the wines elaborated with the different samples.MO1 MO2 ME1 ME2 PV1 PV2 SY1 SY2

Alcoholsn-Propanol 29.0 ± 1.0 13.0 ± 2.0 146.0 ± 4.0 189.0 ± 15.0 64.0 ± 4.0 60.0 ± 10.0 23.0 ± 4.0 14.0 ± 3.0Isobutanol 50.0 ± 1.0 19.0 ± 2.0 16.0 ± 3.0 88.0 ± 9.0 54.0 ± 10.0 41.0 ± 5.0 88.0 ± 15.0 45.0 ± 4.02-Methyl-1-butanol 229.0 ± 14.0 231.0 ± 8.0 226.0 ± 28.0 182.0 ± 40.0 415.0 ± 27.0 520.0 ± 75.0 237.0 ± 15.0 315.0 ± 44.03-Methyl-1-butanol 90.0 ± 10.0 104.0 ± 16.0 133.0 ± 18.0 100.0 ± 10.0 75.0 ± 1.0 37.0 ± 3.0 149.0 ± 13.0 194.0 ± 18.02-Phenylethanol 7.0 ± 1.0 6.0 ± 1.0 12.0 ± 1.0 13.0 ± 1.0 13.0 ± 1.0 13.0 ± 1.0 10.0 ± 1.0 13.0 ± 1.0

EstersIsoamyl acetate 1.1 ± 0.2 0.8 ± 0.1 0.5 ± 0.0 0.5 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 1.7 ± 0.2 2.6 ± 0.12-Phenylethyl acetate 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.0Ethyl hexanoate 0.6 ± 0.0 0.5 ± 0.1 0.4 ± 0.0 0.3 ± 0.0 0.3 ± 0.1 0.4 ± 0.1 0.7 ± 0.0 0.9 ± 0.1Ethyl octanoate 0.4 ± 0.1 0.4 ± 0.0 0.4 ± 0.1 0.3 ± 0.0 0.3 ± 0.0 0.5 ± 0.0 0.6 ± 0.0 0.7 ± 0.1Ethyl decanoate 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.4 ± 0.1Ethyl dodecanoate 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0Diethyl succinate 0.6 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.7 ± 0.0 0.8 ± 0.0 0.2 ± 0.0

AcidsOctanoic acid 1.2 ± 0.1 0.9 ± 0.0 0.7 ± 0.1 0.6 ± 0.1 1.2 ± 0.1 0.9 ± 0.1 1.0 ± 0.1 1.1 ± 0.1Decanoic acid 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.1 0.4 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.4 ± 0.1

All parameters are given with their standard deviation.

2727

604

605

606

607608

Table 5. Ratios between the mean alcohol concentration in the wines (mmol/l) and the mean consumption (mmol/l) of the corresponding precursor amino acid during the alcoholic fermentation

Pre-harvestMonastrell Merlot Syrah Petit Verdot

3-Methyl-1-butanol/leucine 21.8 16.6 11.3 6.22-Methyl-1-butanol/isoleucine 35.5 38.1 22.5 30.72-Phenylethanol/phenylalanine 1.6 2.8 2.0 3.4

Isobutanol/valine 12.0 1.8 6.5 5.3n-Propanol/threonine 1.8 12.5 1.0 3.9

At harvestMonastrell Merlot Syrah Petit Verdot

3-Methyl-1-butanol/leucine 10.3 7.8 9.0 3.12-Methyl-1-butanol/isoleucine 17.3 19.8 17.1 38.32-Phenylethanol/phenylalanine 5.4 1.5 1.7 1.9

Isobutanol/valine 3.1 6.5 1.7 3.3n-Propanol/threonine 0.4 5.9 0.3 3.2

2828

609

610

611

612613614

615616

617

618

619

620

621

622

623

624

625

626

627

628

629

Table 6. Perception threshold and odor activity values for volatile compounds of wines obtained from grapes collected pre-harvest and at harvest

Volatile compoundsPerception

threshold (mg l-

1)

Odor activity value (OAV)a

Pre-harvest At harvest

MO1 ME1 PV1 SY1 MO2 ME2 PV2 SY2

Alcohols n-Propanol Isobutanol 2-Methyl-1-butanol 3-Methyl-1-butanol 2-Phenylethanol Esters Isoamyl acetate 2-Phenylethyl acetate Ethyl hexanoate Ethyl octanoate Ethyl decanoate Ethyl dodecanoate Diethyl succinate Acids Otanoic acid Decanoic acid

306b

40c

30d

30d

7.5b

0.03c

0.25d

0.014c

0.005c

0.2c

0.8e

6f

0.5c

1c

0.11.37.62.90.8

36.10.541.189.31.20.10.1

2.40.3

0.50.47.54.41.6

15.90.225.969.61.00.00.0

1.40.2

0.21.413.82.51.9

9.70.421.266.10.90.00.0

2.40.4

0.12.27.94.51.3

58.20.946.7117.21.40.10.1

2.00.3

0.00.57.73.50.8

26.20.237.371.90.90.00.1

1.80.2

0.62.26.13.31.6

17.10.223.965.00.70.00.0

1.20.3

0.21.017.31.21.6

8.10.831.092.81.00.00.1

1.80.3

0.11.19.76.51.6

86.91.264.5143.81.90.10.0

2.20.4

aThe odor activity values were calculated by dividing the mean concentration by the perception threshold of the compound. bEtiévant (1991). cFerreira and others (2000). dGuth (1997). eLi and others (2008).fFerreira and others (1993).

2929

630

631

632

633634635

636637638

3030

639

Figure 2

3131

640

Figure 3

3232

641642643