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Eur Food Res Technol (2008) 226:1479–1484 DOI 10.



Characterisation of the volatile fraction of Andalusian sweet wines
Raquel Márquez · Remedios Castro · Ramon Natera · Carmelo García-Barroso

Received: 24 January 2007 / Revised: 14 May 2007 / Accepted: 28 May 2007 / Published online: 23 June 2007 © Springer-Verlag 2007

Abstract The characterization of the volatile composition that could have relevance on the organoleptic proWle of Andalusian sweet wines obtained from Muscat and Pedro Ximenez grapes has been carried out. Twenty commercial samples, ten from Pedro Ximenez grapes and ten for Muscat grapes, have been studied. Various diVerent chemometric tools have been applied in the analysis of the results. Using Cluster analysis and principal component analysis the samples have been grouped according to grape variety. Linear discriminate analysis allowed a 100% diVerentiation according to grape variety. Keywords Andalusian sweet wines · Volatile compounds · Multivariate analysis Abbreviations SPME Solid phase microextraction GC Gas chromatography ANOVA Analysis of variance CA Cluster analysis PCA Principal component analysis LDA Linear discriminate analysis TDN 1,1,6-trimethyl-1,2-dihydro naphthalene

Introduction The volatile fraction of wine is determined by several hundreds of chemically diVerent compounds. Alcohols,

R. Márquez · R. Castro (&) · R. Natera · C. García-Barroso Analytical Chemistry Department, Faculty of Sciences, University of Cádiz, P.O. Box 40, E-11510. Puerto Real, Cádiz, Spain e-mail:

aldehydes, esters, acids, monoterpenes, and other minor compounds usually constitute the volatile fraction of this product. Their presence or absence in a particular wine depends on several factors including the environment (climate and soil), ripeness and grape variety, fermentation and other conditions in winemaking, and ageing. Some of these compounds are already present in the grapes; others are formed during the processes of fermentation and ageing [1–3]. In many cases the variety of grape employed in making a particular wine completely determines the aroma of that wine. This is because certain compounds present in the grape persist throughout the entire process of viniWcation. Many of these compounds are of the terpenic type, although there are also others that are speciWc to certain varieties. The extent to which these compounds persist from the grape through to the Wnished wine will be inXuenced by the conditions of its viniWcation and the ageing [4]. However, the process of viniWcation itself and particularly the alcoholic fermentation carried out by the yeasts generates the greater part of the aromatic compounds present in the wine [5, 6]. In most wines all of these compounds are subjected to the subsequent process of ageing in wood. During this process, important reactions occur and new compounds are extracted from oak wood [7]. The contact of the wine with the wood favors the transfer of ellagiotannins and other molecules to the wine. Additionally, new volatile compounds are extracted, modifying the wine’s “bouquet”. On the other hand, fruity and fermentative volatile compounds decrease during the process of ageing in wood [8]. Futhermore during ageing, wines are constantly oxygenated and this oxygen enhances oxidation reactions in which phenolic compounds are involved. These reactions modiWed the colour and the organoleptic characteristics of the wine.


musts with high sugar content and consequently. A small magnetic stirring bar was also added. The GC was equipped with a DB-Wax capillary column 60 m £ 25 mm I. which constitute the free aroma composition and could be relevant for determining the organoleptic proWle of Andalusian sweet wines obtained from Muscat and Pedro Ximenez grapes. USA). a diYcult fermentation.u. After completion of sampling.0 g of NaCl. MO. they are reWlled with younger wine from the row immediately above. 4-methyl-2-pentanol was 123 . The GC oven was programmed as follows: held at 35 °C for 10 min. Chemicals and reagents All standards used in this study were supplied by SigmaAldrich (St Louis. The young sweet wine is then. Working solutions used in further studies were prepared by diluting diVerent amounts of the global standard solution in the synthetic wine solution. The carrier gas was helium at a Xow rate of 1. the SPME Wber (CAR/PDMS (85 m)) was inserted into the headspace. Ethanol. in Milli-Q water (Millipore. Each compound was quantiWed by comparison with a calibration curve. Italy) was used. Both grape varieties are aromatic and the persistence of certain varietal compounds in the Wnal wines should depend on the exacting conditions of their viniWcation and the ageing. Material and Methods Samples Twenty Andalusian sweet wines were analyzed. The grapes attain sugar levels above 300 g/L and are crushed and pressed employing vertical presses. All of them were commercial samples purchased in Spain. In turn. after this. and lower in number. CA. in the highest row number. During the sampling time (45 min). Grapes of these varieties. USA). For the desorption of the analytes inside the GC injection port. being numbered from the ground up. The new wine is introduced into the solera system at the top row. This methodology had previously been optimized by the authors [10].m. with a 0. generally. The sample was equilibrated for 15 min at sampling temperature (40 °C) and. Folsom. GC analysis The injection was made in the splitless mode for 2 min.1 mL min¡1. NaCl and tartaric acid were purchased from Scharlau (Barcelona. and FID. The detector temperature was 250 °C. the sample was stirred at constant speed. MA. IdentiWcation was performed by mass spectrometric analysis. special sweet wines are obtained using dried grapes by direct exposition to the sun. submitted to a system of dynamic oxidative ageing. the Wrst level of the solera. Individual stock standard solutions of each aroma compound were prepared by weight in ethanol. The mass detector operated in EI+ mode at 70 eV in the range 30–450 a. Spain). employed as internal standard. each level is a row of the system. The signal was recorded and processed with Masslab software supplied with the Wiley 6. a GC 8000 coupled to a MD 800 mass detector (Fisons Instruments. Then it was raised to 210 °C at 3 °C min¡1 and held for 40 min.1480 Eur Food Res Technol (2008) 226:1479–1484 In Andalusia. After this. then ramped at 5 °C min¡1 to 100 °C. ten from Muscat grape and ten from the Pedro Ximenez variety. USA).0 MS library. and 15% v/v of ethanol. with a proportion of their content being added to that of the row immediately below. so that the Wrst row is that one of barrels resting on the ground in the wine cellar [9].. The solera consists of rows of barrels (of American oak) stacked one upon the other. Milan. Montilla-Moriles and Jerez in a traditional method dating many years ago using two grapes varieties. Each sample was spiked with 75 L of a solution of 4-methyl-2-pentanol (2. obtained using the relative peak area in relation to that of 4-methyl-2-pentanol. in bunches. Bedford. In these analyses. Peak identiWcation was carried out by analogy of mass spectra and conWrmed by retention times of standards. Several diVerent chemometric tools have been applied for the analysis of the results.D.516 g L¡1 in Milli-Q water containing 15% v/v of ethanol). All these solutions were stored at 4 °C. The objective of this study is to characterise the main chemical compounds. the barrels of each row are partially emptied in the “saque”. A global stock standard solution containing all the analytes was prepared in a synthetic wine matrix (3 g/L of tartaric acid. the internal standard. Every four months or so. The aged wine required for bottling is drawn from the lowermost row. SPME 25 mL of wine was pipetted and placed into a 50 mL glass vial with 3. They are elaborated in Malaga. the temperature was 280 °C. The ageing system is very traditional and is termed “ageing in solera”.25 m coating (J and W ScientiWc. Muscat and Pedro Ximénez. The vial was tightly capped with a PTFE-faced silicone septum and placed in a thermostatted block on a stirrer. are spread out on esparto grass mats to toast in the sun before pressing. are fortiWed with ethanol to ensure that the wine will contain at least 15–18% alcohol. the Wber was removed from the sample vial and inserted into the GC injection port. The samples were divided in two categories according to grape variety. The bunches are turned over on a regular basis and covered at night to avoid the adverse eVects of high air moisture.

7913 0.0121* 0. n = number of samples for each category.28) 0.52 4.07 39.019 (0.253) 0.108) 0.79 9. Table 1 Means (mg/L) and standard deviations of volatile compounds found for the two grape varieties studied.12) 0.76 Hexyl acetate Ethyl lactate Hexanal** 3-Hexen-1-ol Methyl octanoate Ethyl octanoate Acetic acid 2-Furaldehyde 2.157 (0. Riesling and several Muscat cultivars [11].74) 2.61 20.378 (0.92 31.76 (3.14 13.071 (0.090) 0.033) F value p value Ethyl acetate Ethyl butanoate Ethyl pentanoate 4-Methyl-2-pentanol acetate Isoamyl acetate Limonene Isoamyl alcohols p-Cymene ** 2.91 0.332 (0.45 6.08 (5.4) 0.016) 0.12) 2.01 48.181 (0.0) 0.047) nd 0.074 (0.189 (0. ANOVA for grape variety Compound Retention time (min) 10. The major volatile compounds quantiWed were ethyl acetate.03 (0.034 (0.332 (0.6608 0.334) 0.79 58.0283* 0.336) 0.299) 0.0000** 0.025 (0. High standard deviations were found for some compounds (Table 1).35 29.098) nd 0.724 (30.30 50.78 48. -terpineol and limonene.0103* 0.45 43.99 (1. 12] found an increase in the content of terpenols in musts from Muscat grapes submitted to carbonic anaerobiosis at 32 °C.257) 22.60 25.93 17. isoamyl alcohols.131) 0.009 (0. [11.048 (0.681) nd 0.6640 0.506 (0.041) 0.008) 1.1”.63 47.18 (14.005 (0.803) 0.20 5.174 (0.1537 0.502) 0.087) 61.00 7.0165* 0.021 (0.0043** 0.033 (0.83 19.444 (0.0088** 0.85) 0.131 (0.026) Pedro Ximenez (n = 10) 28.61 29.386 (0.88 2.028 (0.015 (0.0004** 0.78 41. Results and discussion Thirty one volatile compounds were identiWed in the samples studied.62 (1.139) 0.098) 0.560) <LOQ 31.45 8.0344* 0.14 7.19 0.84 0.64 0.6519 0.01 4-Ethylphenol Decanoic acid 123 .29) 0.84 (1.06 33.38) nd 1.74) 0.167) 0.86 36.31 (4.230 (0.1543 0.787) < LOQ 0.0105* 0.27 (4.716 (0.769 (1.23 61.46 5.0031** 0.133) 0. 2-furaldehyde.017 (0.1062 0.013) 0.0349* 0.98 7. p < 0.50 2.0009** 20.136) 4. linalool.710 (0.14 2.003) 0.09 39.01 54.46 0.39 10.0000** 0.335 (0.5087 0.0330* 0.071 (0. ethyl lactate.144) 0.580 (0.47) 1.3) 0.1022 0.13 23.6 (22.0070** 0.21 4. This could be explained on the basis of a close relationship between the content of these compounds and the particular grape varieties employed together with the speciWc conditions of the winemaking process (viniWcation and ageing processes).3720 0. acetic acid.004 (0.141) 3. principal component analysis (PCA) and linear discriminate analysis (LDA) were performed using the Statgraphics Statistical Computer Package “Statgraphics Plus 5.3-Butanediol Linalool 5-Methyl-2-furaldehyde Ethyl decanoate Diethyl succinate -Terpineol TDN a a 38.8 (73.0096** 0.034) 0.054) nd 0.000) 0.77 4.60 36.07 5.251) nd 93.23) nd 0.190 (0.20 32.8 (49. Table 1 shows the mean values and standard deviations calculated for each volatile compound of the two grape varieties considered.0206* 0.05. -terpineol and 2phenylethanol. These authors propose that the migration of both free and bound terpenols from the solid to the liquid part of the berries could be the origin of this increase.33 0.0) 0. diethyl succinate.81 26.58 44.11 21. ** p < 0.0001** 0.000 (0.0399* 0. Bitteur et al.71 24.91 33.001 (0.84 0. These varietal compounds are responsible for the distinctive Xavor of grape cultivars such as Gewurztraminer.1 (38.10 ** Muscat (n = 10) 42.173 (0.121 (0.74 Hexanoic acid Ethyl-2-phenyl acetate Benzyl alcohol 2-Phenylethanol Octanoic acid a * mean relative areas.31 7.61 42.82 15.055) 0.Eur Food Res Technol (2008) 226:1479–1484 1481 Statistical analysis Univariate analysis and multivariate analysis of data included cluster analysis (CA). Muscat wines showed a high content of linalool.73 30.19 7.102) 0.003) 3.

decanoic acid. Gerdes et al.3 butanediol and 4-ethylphenol. and two clusters for Muscat sweet wines. These compounds have also been found by Franco et al. M: Muscat wine. Fisher’s weight was calculated to establish the discriminant capacity of each volatile compound. Univariate analysis An one-way ANOVA was carried out according to grape variety. -butyrolactone. two factors. hexanoic acid. The dendrogram obtained is shown in Fig. 2-Furaldehyde and 5-methyl-2-furaldehyde were present in Pedro Ximenez wines. degree of toasting) and the process of production (with an initial stage of sun drying of the grapes) should explain the high content in 2-furaldehyde found for these particular wines [15. 1. multivariate analysis methods were used. play an important role in white wine aroma [2. Three main clusters can be appreciated: one cluster for Pedro Ximenez sweet wines.1. In this study. these high concentrations in the Muscat wines could be explained by the anaerobic metabolism of the grapes during drying by exposure to the sun. The most discriminate variables (from the univariate point of view) were: -terpineol. As can be seen. limonene. although these decreases can be counteracted by hydrolysis of glycosilated terpenes during the same period. present only in some wines. ethyl-2-phenyl acetate. A C13 norisoprenoid. 1500 1200 Distance 900 600 300 0 M1a M1b M2a M2b M4a M4b M8a M8b M9a M9b M10b M10 a M3a M3b PX1a PX1b PX2a Px2b PX4a PX4b PX5a PX5b PX8a PX8b PX6a PX6b PX7a PX7b PX9a PX9b PX10a PX10 b PX3a PX3b M5a M5b M7a M7b M6a M6b Muscat Pedro Ximénez Muscat Fig. Franco et al. isobutanol and 2-phenylethanol were the major compounds in must from Pedro Ximenez grapes dried by the sun. hexanoic acid and 4-methyl-2-pentanol acetate. [13] found that the free concentrations of linalool. octanoic acid and decanoic acid were found in both types of wines. Cluster analysis The data matrix was subjected to a hierarchical agglomerative cluster analysis of cases. has been identiWed in all samples. 3]. They are by-products of yeast fermentation and their amounts in wine depend on many factors. TDN. However. wines with a high content of this type of volatile compounds. two of them from the same wine producer. one of these last clusters groups only three samples. in concentrations above 20 g/L causes an unpleasant kerosene note. 1 Dendrogram obtained after a hierarchical agglomerative CA performed on volatile compounds of all samples studied. [23]. 5-methyl-2-furaldehyde. 2-furfural is produced when pentoses (xylose) are heated and 5-methyl-2-furaldehyde arises from rhamnose. ethyl acetate and ethyl lactate. isoamyl alcohols. Some authors have found a clear increase of these compounds during ageing in wood [17–19]. the ageing in wood (age of barrel. Muscat wines showed a higher content in TDN than Pedro Ximenez wines. 1. Therefore. citronellol. ethyl acetate.6-trimethyl-1. 2. The occurrence of this compound in wines is principally related to the maturation process by carotenoid-degradation [21]. Furanic compounds are formed by degradation of carbohydrates during toasting of the barrel. [22] established that certain viticultural practices. both monovarietal wines showed a high content in isoamyl alcohols. [16] found that acetoin. isoamyl alcohols. such as yeast strain. grape must. PX: Pedro Ximenez wine 123 . were not considered for these studies. 20]. Hence. Hexyl acetate. several authors have pointed out a positive inXuence in the wine aroma when TDN is present at concentrations lower than its threshold limit [4]. taking the Euclidean distance as metric and the Ward method as amalgamation rule. later. In this study. nerol and geraniol decrease during prolonged ageing in wood. methyl octanoate. fermentation conditions [14] and ageing in wood [15]. [16] in musts from Pedro Ximenez grapes after their sun drying. which aVect fruit exposure to sunlight may inXuence on the norisoprenoid composition of grapes and wines. A linear behaviour of these compounds over time of ageing has been found for Madeira wines [15]. These authors ascribed their presence to the anaerobic metabolism of grapes during the drying. In order to diVerentiate between both grape varieties.2 dihydro naphthalene (TDN). Silva et al. linalool. This fact should produce musts and. Higher alcohols and esters. whose concentrations vary over a wide range. Among the fatty acids. Sunlight and storage time and temperature have an important eVect on the content of this compound. in our case.1482 Eur Food Res Technol (2008) 226:1479–1484 Karagiannis et al. in a forced ageing study (7 days at 60 °C under saturation with oxygen) found a high concentration for this compound in the wines studied.

in order to obtain suitable classiWcation rules for assigning categories to samples.37 Component 1 0.2 Component 2 1. The possible category classiWcation considered is the grape variety used. This was performed according to the Wilks’ lambda statistic [24] in order to choose the descriptors that best distinguish the diVerent classes. it is clear that. supervised learning pattern recognition methods were applied.27 -0. The groups obtained demonstrate that the variables possess suYcient explanatory power to detect the grape variety employed.8 -4.8 -2. the particular conditions under which these two processes were carried out modiWed their presence in the Wnal wines.13 linalool -0.09.39 -0. It should mean that in methylfuraldehyde methyl pentanol acetate isoamyl acetate isoamyl alcohols ethyl pentanoate terpineol 0. 2 PCA.8 -3 0 3 6 Component 1 Muscat Pedro Ximenez 9 12 Clusters were formed exactly according to the grape variety. hexanal and p-cymene. Principal component analysis When a principal components analysis (PCA) was performed employing all the variables determined in this study. It can be observed that two types of wines are identiWed according to their aromatic composition (Fig.03 a 0.41 Fig.54 of the overall variance whereas Component 2 explains the 10.17 0. Component 1 explains the 60. A partial lambda is computed for each variable as the ratio of Wilks’ lambda after adding the respective variable over the Wilks’ lambda before adding the variable.2 -0. Figs. 3 were obtained. the volatile compounds selected are descriptors that can be used to diVerentiate sweet wine samples according to the grape variety.35 ethyl octanoate octanoic diethyl succinate hexanoic ethyl acetate 3-hexenol 2-ethyl-phenyl acet. 3): Wrst there are the wines from Pedro Ximenez variety (negative values of PC1 and positive values of PC2).25 0.17 Component 1 ethyl butanoate 0. 5methyl-2-furaldehyde. a broad group constituted by the wines obtained from Muscat grapes (negative values of PC2). The Wilks’ lambda statistic for the overall discrimination is computed as the ratio of the determinant of the within-group variance/covariance matrix over the determinant of the total variance/covariance matrix. 2.Eur Food Res Technol (2008) 226:1479–1484 1483 5. Muscat and Pedro Ximenez. 2-phenyletanol TDN benzaldehyde p-cymene limonene 0. The loadings of each compound on PC1 and PC2 show clearly that ethyl esters and fatty acids are mainly responsible for PC1.37 0. By “leave one out” method 100% of the samples were correctly classiWed in the checking process. A high dispersion is observed in PC1 for sweet wines from Muscat grape. 2). isoamyl alcohols.15 Fig. and then. (a) Loadings of each compound on PC1 and PC2.27 0. isoamyl alcohols and 5-methyl-2-furaldehyde are mainly responsible for PC2 (Fig. Zoom of the loadings of each compound on PC1 and PC2 Component 2 acetic 123 . Thus.19 octanoic ethyl octanoate hexanoic ethyl 3-hexenol ethyl acetate decanoate benzyl alcohol diethyl succinate hexanal 2-ethyl-phenyl acet. Linear discriminant analysis A forward stepwise linear discriminant analysis was carried out. the variables included in the discriminant function obtained (cited according to their discriminant power) were: -terpineol. A F-statistic is computed from the partial lambda values leading to a p-level.21 acetic ethyl lactate Component 2 0. decanoic furaldeh.07 0. Grouping the samples according to this factor. whereas some terpenic compounds (limonene. Since that the volatile compounds responsible for this PC are mainly produced during the 0.2 3.05 decanoic 2-phenylethanol ethyl butanoate b TDN -0. The maximum discriminatory power corresponds to minimum p-level values. The so-called “leave one out” method has been employed [25]. ethyl decanoate. ethyl decanoate -0. for this grape variety. p-cymene and linalool). These methods assume an a priori knowledge of the number of classes and the sample class memberships.01 -0. (b). In conclusion. 3 Score plot on the Wrst and second principal components for the samples studied alcoholic fermentation and/or during the sun drying.

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